Plant pattern recognition receptor complexes at the plasma membrane.

PubMed

Monaghan, Jacqueline; Zipfel, Cyril

2012-08-01

A key feature of innate immunity is the ability to recognize and respond to potential pathogens in a highly sensitive and specific manner. In plants, the activation of pattern recognition receptors (PRRs) by pathogen-associated molecular patterns (PAMPs) elicits a defense programme known as PAMP-triggered immunity (PTI). Although only a handful of PAMP-PRR pairs have been defined, all known PRRs are modular transmembrane proteins containing ligand-binding ectodomains. It is becoming clear that PRRs do not act alone but rather function as part of multi-protein complexes at the plasma membrane. Recent studies describing the molecular interactions and protein modifications that occur between PRRs and their regulatory proteins have provided important mechanistic insight into how plants avoid infection and achieve immunity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Subcellular localisations of the CPTI collection of YFP-tagged proteins in Drosophila embryos

PubMed Central

Lye, Claire M.; Naylor, Huw W.; Sanson, Bénédicte

2014-01-01

A key challenge in the post-genomic area is to identify the function of the genes discovered, with many still uncharacterised in all metazoans. A first step is transcription pattern characterisation, for which we now have near whole-genome coverage in Drosophila. However, we have much more limited information about the expression and subcellular localisation of the corresponding proteins. The Cambridge Protein Trap Consortium generated, via piggyBac transposition, over 600 novel YFP-trap proteins tagging just under 400 Drosophila loci. Here, we characterise the subcellular localisations and expression patterns of these insertions, called the CPTI lines, in Drosophila embryos. We have systematically analysed subcellular localisations at cellularisation (stage 5) and recorded expression patterns at stage 5, at mid-embryogenesis (stage 11) and at late embryogenesis (stages 15-17). At stage 5, 31% of the nuclear lines (41) and 26% of the cytoplasmic lines (67) show discrete localisations that provide clues on the function of the protein and markers for organelles or regions, including nucleoli, the nuclear envelope, nuclear speckles, centrosomes, mitochondria, the endoplasmic reticulum, Golgi, lysosomes and peroxisomes. We characterised the membranous/cortical lines (102) throughout stage 5 to 10 during epithelial morphogenesis, documenting their apico-basal position and identifying those secreted in the extracellular space. We identified the tricellular vertices as a specialized membrane domain marked by the integral membrane protein Sidekick. Finally, we categorised the localisation of the membranous/cortical proteins during cytokinesis. PMID:25294944

GSL-enriched membrane microdomains in innate immune responses.

PubMed

Nakayama, Hitoshi; Ogawa, Hideoki; Takamori, Kenji; Iwabuchi, Kazuhisa

2013-06-01

Many pathogens target glycosphingolipids (GSLs), which, together with cholesterol, GPI-anchored proteins, and various signaling molecules, cluster on host cell membranes to form GSL-enriched membrane microdomains (lipid rafts). These GSL-enriched membrane microdomains may therefore be involved in host-pathogen interactions. Innate immune responses are triggered by the association of pathogens with phagocytes, such as neutrophils, macrophages and dendritic cells. Phagocytes express a diverse array of pattern-recognition receptors (PRRs), which sense invading microorganisms and trigger pathogen-specific signaling. PRRs can recognize highly conserved pathogen-associated molecular patterns expressed on microorganisms. The GSL lactosylceramide (LacCer, CDw17), which binds to various microorganisms, including Candida albicans, is expressed predominantly on the plasma membranes of human mature neutrophils and forms membrane microdomains together with the Src family tyrosine kinase Lyn. These LacCer-enriched membrane microdomains can mediate superoxide generation, migration, and phagocytosis, indicating that LacCer functions as a PRR in innate immunity. Moreover, the interactions of GSL-enriched membrane microdomains with membrane proteins, such as growth factor receptors, are important in mediating the physiological properties of these proteins. Similarly, we recently found that interactions between LacCer-enriched membrane microdomains and CD11b/CD18 (Mac-1, CR3, or αMβ2-integrin) are significant for neutrophil phagocytosis of non-opsonized microorganisms. This review describes the functional role of LacCer-enriched membrane microdomains and their interactions with CD11b/CD18.

Characterization of mechanical unfolding intermediates of membrane proteins by coarse grained molecular dynamics simulation

NASA Astrophysics Data System (ADS)

Yamada, Tatsuya; Mitaku, Shigeki; Yamato, Takahisa

2018-01-01

Single-molecule force spectroscopy by atomic force microscopy allows us to get insight into the mechanical unfolding of membrane proteins, and a typical experiment exhibits characteristic patterns on the force distance curves. The origin of these patterns, however, has not been fully understood yet. We performed coarse-grained simulation of the forced unfolding of halorodopsin, reproduced the characteristic features of the experimental force distance curves. A further examination near the membrane-water interface indicated the existence of a motif for the force peak formation, i.e., the occurrence of hydrophobic residues in the upper interface region and hydrophilic residues below the lower interface region.

Network based approaches reveal clustering in protein point patterns

NASA Astrophysics Data System (ADS)

Parker, Joshua; Barr, Valarie; Aldridge, Joshua; Samelson, Lawrence E.; Losert, Wolfgang

2014-03-01

Recent advances in super-resolution imaging have allowed for the sub-diffraction measurement of the spatial location of proteins on the surfaces of T-cells. The challenge is to connect these complex point patterns to the internal processes and interactions, both protein-protein and protein-membrane. We begin analyzing these patterns by forming a geometric network amongst the proteins and looking at network measures, such the degree distribution. This allows us to compare experimentally observed patterns to models. Specifically, we find that the experimental patterns differ from heterogeneous Poisson processes, highlighting an internal clustering structure. Further work will be to compare our results to simulated protein-protein interactions to determine clustering mechanisms.

Membrane-bound MinDE complex acts as a toggle switch that drives Min oscillation coupled to cytoplasmic depletion of MinD.

PubMed

Vecchiarelli, Anthony G; Li, Min; Mizuuchi, Michiyo; Hwang, Ling Chin; Seol, Yeonee; Neuman, Keir C; Mizuuchi, Kiyoshi

2016-03-15

The Escherichia coli Min system self-organizes into a cell-pole to cell-pole oscillator on the membrane to prevent divisions at the cell poles. Reconstituting the Min system on a lipid bilayer has contributed to elucidating the oscillatory mechanism. However, previous in vitro patterns were attained with protein densities on the bilayer far in excess of those in vivo and failed to recapitulate the standing wave oscillations observed in vivo. Here we studied Min protein patterning at limiting MinD concentrations reflecting the in vivo conditions. We identified "burst" patterns--radially expanding and imploding binding zones of MinD, accompanied by a peripheral ring of MinE. Bursts share several features with the in vivo dynamics of the Min system including standing wave oscillations. Our data support a patterning mechanism whereby the MinD-to-MinE ratio on the membrane acts as a toggle switch: recruiting and stabilizing MinD on the membrane when the ratio is high and releasing MinD from the membrane when the ratio is low. Coupling this toggle switch behavior with MinD depletion from the cytoplasm drives a self-organized standing wave oscillator.

Wood cell-wall structure requires local 2D-microtubule disassembly by a novel plasma membrane-anchored protein.

PubMed

Oda, Yoshihisa; Iida, Yuki; Kondo, Yuki; Fukuda, Hiroo

2010-07-13

Plant cells have evolved cortical microtubules, in a two-dimensional space beneath the plasma membrane, that regulate patterning of cellulose deposition. Although recent studies have revealed that several microtubule-associated proteins facilitate self-organization of transverse cortical microtubules, it is still unknown how diverse patterns of cortical microtubules are organized in different xylem cells, which are the major components of wood. Using our newly established in vitro xylem cell differentiation system, we found that a novel microtubule end-tracking protein, microtubule depletion domain 1 (MIDD1), was anchored to distinct plasma membrane domains and promoted local microtubule disassembly, resulting in pits on xylem cell walls. The introduction of RNA interference for MIDD1 resulted in the failure of local microtubule depletion and the formation of secondary walls without pits. Conversely, the overexpression of MIDD1 reduced microtubule density. MIDD1 has two coiled-coil domains for the binding to microtubules and for the anchorage to plasma membrane domains, respectively. Combination of the two coils caused end tracking of microtubules during shrinkage and suppressed their rescue events. Our results indicate that MIDD1 integrates spatial information in the plasma membrane with cortical microtubule dynamics for determining xylem cell wall pattern. Copyright 2010 Elsevier Ltd. All rights reserved.

Deciphering membrane-associated molecular processes in target tissue of autoimmune uveitis by label-free quantitative mass spectrometry.

PubMed

Hauck, Stefanie M; Dietter, Johannes; Kramer, Roxane L; Hofmaier, Florian; Zipplies, Johanna K; Amann, Barbara; Feuchtinger, Annette; Deeg, Cornelia A; Ueffing, Marius

2010-10-01

Autoimmune uveitis is a blinding disease presenting with autoantibodies against eye-specific proteins as well as autoagressive T cells invading and attacking the immune-privileged target tissue retina. The molecular events enabling T cells to invade and attack the tissue have remained elusive. Changes in membrane protein expression patterns between diseased and healthy stages are especially interesting because initiating events of disease will most likely occur at membranes. Since disease progression is accompanied with a break-down of the blood-retinal barrier, serum-derived proteins mask the potential target tissue-related changes. To overcome this limitation, we used membrane-enriched fractions derived from retinas of the only available spontaneous animal model for the disease equine recurrent uveitis, and compared expression levels by a label-free LC-MSMS-based strategy to healthy control samples. We could readily identify a total of 893 equine proteins with 57% attributed to the Gene Ontology project term "membrane." Of these, 179 proteins were found differentially expressed in equine recurrent uveitis tissue. Pathway enrichment analyses indicated an increase in proteins related to antigen processing and presentation, TNF receptor signaling, integrin cell surface interactions and focal adhesions. Additionally, loss of retina-specific proteins reflecting decrease of vision was observed as well as an increase in Müller glial cell-specific proteins indicating glial reactivity. Selected protein candidates (caveolin 1, integrin alpha 1 and focal adhesion kinase) were validated by immunohistochemistry and tissue staining pattern pointed to a significant increase of these proteins at the level of the outer limiting membrane which is part of the outer blood-retinal barrier. Taken together, the membrane enrichment in combination with LC-MSMS-based label-free quantification greatly increased the sensitivity of the comparative tissue profiling and resulted in detection of novel molecular pathways related to equine recurrent uveitis.

Characterization of a major 31-kilodalton peptidoglycan-bound protein of Legionella pneumophila

DOE Office of Scientific and Technical Information (OSTI.GOV)

Butler, C.A.; Hoffman, P.S.

1990-05-01

A 31-kilodalton (kDa) protein was solubilized from the peptidoglycan (PG) fraction of Legionella pneumophila after treatment with either N-acetylmuramidase from the fungus Chalaropsis sp. or with mutanolysin from Streptomyces globisporus. The protein exhibited a ladderlike banding pattern by autoradiography when radiolabeled ((35S)cysteine or (35S)methionine) PG material was extensively treated with hen lysozyme. The banding patterns ranging between 31 and 45 kDa and between 55 and 60 kDa resolved as a single 31-kDa protein when the material was subsequently treated with N-acetylmuramidase. Analysis of the purified 31-kDa protein for diaminopimelic acid by gas chromatography revealed 1 mol of diaminopimelic acid permore » mol of protein. When outer membrane PG material containing the major outer membrane porin protein was treated with N-acetylmuramidase or mutanolysin, both the 28.5-kDa major outer membrane protein and the 31-kDa protein were solubilized from the PG material under reducing conditions. In the absence of 2-mercaptoethanol, a high-molecular-mass complex (100 kDa) was resolved. The results of this study indicate that a 31-kDa PG-bound protein is a major component of the cell wall of L. pneumophila whose function may be to anchor the major outer membrane protein to PG. Finally, a survey of other Legionella species and other serogroups of L. pneumophila suggested that PG-bound proteins may be a common feature of this genus.« less

Nanoscopic compartmentalization of membrane protein motion at the axon initial segment.

PubMed

Albrecht, David; Winterflood, Christian M; Sadeghi, Mohsen; Tschager, Thomas; Noé, Frank; Ewers, Helge

2016-10-10

The axon initial segment (AIS) is enriched in specific adaptor, cytoskeletal, and transmembrane molecules. During AIS establishment, a membrane diffusion barrier is formed between the axonal and somatodendritic domains. Recently, an axonal periodic pattern of actin, spectrin, and ankyrin forming 190-nm-spaced, ring-like structures has been discovered. However, whether this structure is related to the diffusion barrier function is unclear. Here, we performed single-particle tracking time-course experiments on hippocampal neurons during AIS development. We analyzed the mobility of lipid-anchored molecules by high-speed single-particle tracking and correlated positions of membrane molecules with the nanoscopic organization of the AIS cytoskeleton. We observe a strong reduction in mobility early in AIS development. Membrane protein motion in the AIS plasma membrane is confined to a repetitive pattern of ∼190-nm-spaced segments along the AIS axis as early as day in vitro 4, and this pattern alternates with actin rings. Mathematical modeling shows that diffusion barriers between the segments significantly reduce lateral diffusion along the axon. © 2016 Albrecht et al.

Steric exclusion and protein conformation determine the localization of plasma membrane transporters.

PubMed

Bianchi, Frans; Syga, Łukasz; Moiset, Gemma; Spakman, Dian; Schavemaker, Paul E; Punter, Christiaan M; Seinen, Anne-Bart; van Oijen, Antoine M; Robinson, Andrew; Poolman, Bert

2018-02-05

The plasma membrane (PM) of Saccharomyces cerevisiae contains membrane compartments, MCC/eisosomes and MCPs, named after the protein residents Can1 and Pma1, respectively. Using high-resolution fluorescence microscopy techniques we show that Can1 and the homologous transporter Lyp1 are able to diffuse into the MCC/eisosomes, where a limited number of proteins are conditionally trapped at the (outer) edge of the compartment. Upon addition of substrate, the immobilized proteins diffuse away from the MCC/eisosomes, presumably after taking a different conformation in the substrate-bound state. Our data indicate that the mobile fraction of all integral plasma membrane proteins tested shows extremely slow Brownian diffusion through most of the PM. We also show that proteins with large cytoplasmic domains, such as Pma1 and synthetic chimera of Can1 and Lyp1, are excluded from the MCC/eisosomes. We hypothesize that the distinct localization patterns found for these integral membrane proteins in S. cerevisiae arises from a combination of slow lateral diffusion, steric exclusion, and conditional trapping in membrane compartments.

Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots

DOEpatents

Zhang, Jian-Shi; Giometti, Carol S.; Tollaksen, Sandra L.

1989-01-01

After two-dimensional electrophoresis of proteins or the like, resulting in a polyacrylamide gel slab having a pattern of protein gel spots thereon, an individual protein gel spot is cored out from the slab, to form a gel spot core which is placed in an extraction tube, with a dialysis membrane across the lower end of the tube. Replicate gel spots can be cored out from replicate gel slabs and placed in the extraction tube. Molten agarose gel is poured into the extraction tube where the agarose gel hardens to form an immobilizing gel, covering the gel spot cores. The upper end portion of the extraction tube is filled with a volume of buffer solution, and the upper end is closed by another dialysis membrane. Upper and lower bodies of a buffer solution are brought into contact with the upper and lower membranes and are provided with electrodes connected to the positive and negative terminals of a DC power supply, thereby producing an electrical current which flows through the upper membrane, the volume of buffer solution, the agarose, the gel spot cores and the lower membrane. The current causes the proteins to be extracted electrophoretically from the gel spot cores, so that the extracted proteins accumulate and are contained in the space between the agarose gel and the upper membrane. A high percentage extraction of proteins is achieved. The extracted proteins can be removed and subjected to partial digestion by trypsin or the like, followed by two-dimensional electrophoresis, resulting in a gel slab having a pattern of peptide gel spots which can be cored out and subjected to electrophoretic extraction to extract individual peptides.

Topography of the Dictyostelium discoideum plasma membrane: analysis of membrane asymmetry and intermolecular disulfide bonds.

PubMed

Shiozawa, J A; Jelenska, M M; Jacobson, B S

1987-07-28

Through the application of a unique method for isolating plasma membranes, it was possible to specifically iodinate cytoplasm-exposed plasma membrane proteins in vegetative cells of the cellular slime mold Dictyostelium discoideum. The original procedure [Chaney, L. K., & Jacobson, B. S. (1983) J. Biol. Chem. 258, 10062] which involved coating cells with colloidal silica has been modified to yield a more pure preparation. The presence of the continuous and dense silica pellicle on the outside surface of the isolated plasma membrane permitted the specific labeling of cytoplasm-exposed membrane proteins. Lactoperoxidase-catalyzed iodination was employed to label cell-surface and cytoplasm-exposed membrane proteins. The isolated and radioiodinated membranes were then compared and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cell-surface and cytoplasmic face labeling patterns were distinct. A total of 65 proteins were found to be accessible to at least one surface of the membrane. Sixteen intermolecular disulfide bond complexes were observed in the plasma membrane of Dictyostelium; most of these complexes involved glycoproteins and, hence, were exposed to the cell surface.

Analyzing Protein Clusters on the Plasma Membrane: Application of Spatial Statistical Analysis Methods on Super-Resolution Microscopy Images.

PubMed

Paparelli, Laura; Corthout, Nikky; Pavie, Benjamin; Annaert, Wim; Munck, Sebastian

2016-01-01

The spatial distribution of proteins within the cell affects their capability to interact with other molecules and directly influences cellular processes and signaling. At the plasma membrane, multiple factors drive protein compartmentalization into specialized functional domains, leading to the formation of clusters in which intermolecule interactions are facilitated. Therefore, quantifying protein distributions is a necessity for understanding their regulation and function. The recent advent of super-resolution microscopy has opened up the possibility of imaging protein distributions at the nanometer scale. In parallel, new spatial analysis methods have been developed to quantify distribution patterns in super-resolution images. In this chapter, we provide an overview of super-resolution microscopy and summarize the factors influencing protein arrangements on the plasma membrane. Finally, we highlight methods for analyzing clusterization of plasma membrane proteins, including examples of their applications.

Mechanistic insights of the Min oscillator via cell-free reconstitution and imaging

NASA Astrophysics Data System (ADS)

Mizuuchi, Kiyoshi; Vecchiarelli, Anthony G.

2018-05-01

The MinD and MinE proteins of Escherichia coli self-organize into a standing-wave oscillator on the membrane to help align division at mid-cell. When unleashed from cellular confines, MinD and MinE form a spectrum of patterns on artificial bilayers—static amoebas, traveling waves, traveling mushrooms, and bursts with standing-wave dynamics. We recently focused our cell-free studies on bursts because their dynamics recapitulate many features of Min oscillation observed in vivo. The data unveiled a patterning mechanism largely governed by MinE regulation of MinD interaction with membrane. We proposed that the MinD to MinE ratio on the membrane acts as a toggle switch between MinE-stimulated recruitment and release of MinD from the membrane. In this review, we summarize cell-free data on the Min system and expand upon a molecular mechanism that provides a biochemical explanation as to how these two ‘simple’ proteins can form the remarkable spectrum of patterns.

Proteome Analysis of the Plasma Membrane of Mycobacterium Tuberculosis

PubMed Central

Arora, Shalini; Kosalai, K.; Namane, Abdelkader; Pym, Alex S.; Cole, Stewart T.

2002-01-01

The plasma membrane of Mycobacterium tuberculosis is likely to contain proteins that could serve as novel drug targets, diagnostic probes or even components of a vaccine against tuberculosis. With this in mind, we have undertaken proteome analysis of the membrane of M. tuberculosis H37Rv. Isolated membrane vesicles were extracted with either a detergent (Triton X114) or an alkaline buffer (carbonate) following two of the protocols recommended for membrane protein enrichment. Proteins were resolved by 2D-GE using immobilized pH gradient (IPG) strips, and identified by peptide mass mapping utilizing the M. tuberculosis genome database. The two extraction procedures yielded patterns with minimal overlap. Only two proteins, both HSPs, showed a common presence. MALDI–MS analysis of 61 spots led to the identification of 32 proteins, 17 of which were new to the M. tuberculosis proteome database. We classified 19 of the identified proteins as ‘membrane-associated’; 14 of these were further classified as ‘membrane-bound’, three of which were lipoproteins. The remaining proteins included four heat-shock proteins and several enzymes involved in energy or lipid metabolism. Extraction with Triton X114 was found to be more effective than carbonate for detecting ‘putative’ M. tuberculosis membrane proteins. The protocol was also found to be suitable for comparing BCG and M. tuberculosis membranes, identifying ESAT-6 as being expressed selectively in M. tuberculosis. While this study demonstrates for the first time some of the membrane proteins of M. tuberculosis, it also underscores the problems associated with proteomic analysis of a complex membrane such as that of a mycobacterium. PMID:18629250

Pex11mediates peroxisomal proliferation by promoting deformation of the lipid membrane

PubMed Central

Yoshida, Yumi; Niwa, Hajime; Honsho, Masanori; Itoyama, Akinori; Fujiki, Yukio

2015-01-01

Pex11p family proteins are key players in peroxisomal fission, but their molecular mechanisms remains mostly unknown. In the present study, overexpression of Pex11pβ caused substantial vesiculation of peroxisomes in mammalian cells. This vesicle formation was dependent on dynamin-like protein 1 (DLP1) and mitochondrial fission factor (Mff), as knockdown of these proteins diminished peroxisomal fission after Pex11pβ overexpression. The fission-deficient peroxisomes exhibited an elongated morphology, and peroxisomal marker proteins, such as Pex14p or matrix proteins harboring peroxisomal targeting signal 1, were discernible in a segmented staining pattern, like beads on a string. Endogenous Pex11pβ was also distributed a striped pattern, but which was not coincide with Pex14p and PTS1 matrix proteins. Altered morphology of the lipid membrane was observed when recombinant Pex11p proteins were introduced into proteo-liposomes. Constriction of proteo-liposomes was observed under confocal microscopy and electron microscopy, and the reconstituted Pex11pβ protein localized to the membrane constriction site. Introducing point mutations into the N-terminal amphiphathic helix of Pex11pβ strongly reduced peroxisomal fission, and decreased the oligomer formation. These results suggest that Pex11p contributes to the morphogenesis of the peroxisomal membrane, which is required for subsequent fission by DLP1. PMID:25910939

Evolutionary Influenced Interaction Pattern as Indicator for the Investigation of Natural Variants Causing Nephrogenic Diabetes Insipidus

PubMed Central

Labudde, Dirk

2015-01-01

The importance of short membrane sequence motifs has been shown in many works and emphasizes the related sequence motif analysis. Together with specific transmembrane helix-helix interactions, the analysis of interacting sequence parts is helpful for understanding the process during membrane protein folding and in retaining the three-dimensional fold. Here we present a simple high-throughput analysis method for deriving mutational information of interacting sequence parts. Applied on aquaporin water channel proteins, our approach supports the analysis of mutational variants within different interacting subsequences and finally the investigation of natural variants which cause diseases like, for example, nephrogenic diabetes insipidus. In this work we demonstrate a simple method for massive membrane protein data analysis. As shown, the presented in silico analyses provide information about interacting sequence parts which are constrained by protein evolution. We present a simple graphical visualization medium for the representation of evolutionary influenced interaction pattern pairs (EIPPs) adapted to mutagen investigations of aquaporin-2, a protein whose mutants are involved in the rare endocrine disorder known as nephrogenic diabetes insipidus, and membrane proteins in general. Furthermore, we present a new method to derive new evolutionary variations within EIPPs which can be used for further mutagen laboratory investigations. PMID:26180540

Evolutionary Influenced Interaction Pattern as Indicator for the Investigation of Natural Variants Causing Nephrogenic Diabetes Insipidus.

PubMed

Grunert, Steffen; Labudde, Dirk

2015-01-01

The importance of short membrane sequence motifs has been shown in many works and emphasizes the related sequence motif analysis. Together with specific transmembrane helix-helix interactions, the analysis of interacting sequence parts is helpful for understanding the process during membrane protein folding and in retaining the three-dimensional fold. Here we present a simple high-throughput analysis method for deriving mutational information of interacting sequence parts. Applied on aquaporin water channel proteins, our approach supports the analysis of mutational variants within different interacting subsequences and finally the investigation of natural variants which cause diseases like, for example, nephrogenic diabetes insipidus. In this work we demonstrate a simple method for massive membrane protein data analysis. As shown, the presented in silico analyses provide information about interacting sequence parts which are constrained by protein evolution. We present a simple graphical visualization medium for the representation of evolutionary influenced interaction pattern pairs (EIPPs) adapted to mutagen investigations of aquaporin-2, a protein whose mutants are involved in the rare endocrine disorder known as nephrogenic diabetes insipidus, and membrane proteins in general. Furthermore, we present a new method to derive new evolutionary variations within EIPPs which can be used for further mutagen laboratory investigations.

Neuroacanthocytosis associated with a defect of the 4.1R membrane protein

PubMed Central

Orlacchio, Antonio; Calabresi, Paolo; Rum, Adriana; Tarzia, Anna; Salvati, Anna Maria; Kawarai, Toshitaka; Stefani, Alessandro; Pisani, Antonio; Bernardi, Giorgio; Cianciulli, Paolo; Caprari, Patrizia

2007-01-01

Background Neuroacanthocytosis (NA) denotes a heterogeneous group of diseases that are characterized by nervous system abnormalities in association with acanthocytosis in the patients' blood. The 4.1R protein of the erythrocyte membrane is critical for the membrane-associated cytoskeleton structure and in central neurons it regulates the stabilization of AMPA receptors on the neuronal surface at the postsynaptic density. We report clinical, biochemical, and genetic features in four patients from four unrelated families with NA in order to explain the cause of morphological abnormalities and the relationship with neurodegenerative processes. Case presentation All patients were characterised by atypical NA with a novel alteration of the erythrocyte membrane: a 4.1R protein deficiency. The 4.1R protein content was significantly lower in patients (3.40 ± 0.42) than in controls (4.41 ± 0.40, P < 0.0001), reflecting weakened interactions of the cytoskeleton with the membrane. In patients IV:1 (RM23), IV:3 (RM15), and IV:6 (RM16) the 4.1 deficiency seemed to affect the horizontal interactions of spectrin and an impairment of the dimer self-association into tetramers was detected. In patient IV:1 (RM16) the 4.1 deficiency seemed to affect the skeletal attachment to membrane and the protein band 3 was partially reduced. Conclusion A decreased expression pattern of the 4.1R protein was observed in the erythrocytes from patients with atypical NA, which might reflect the expression pattern in the central nervous system, especially basal ganglia, and might lead to dysfunction of AMPA-mediated glutamate transmission. PMID:17298666

Characterisation of detergent-insoluble membranes in pollen tubes of Nicotiana tabacum (L.)

PubMed Central

Moscatelli, Alessandra; Gagliardi, Assunta; Maneta-Peyret, Lilly; Bini, Luca; Stroppa, Nadia; Onelli, Elisabetta; Landi, Claudia; Scali, Monica; Idilli, Aurora Irene; Moreau, Patrick

2015-01-01

ABSTRACT Pollen tubes are the vehicle for sperm cell delivery to the embryo sac during fertilisation of Angiosperms. They provide an intriguing model for unravelling mechanisms of growing to extremes. The asymmetric distribution of lipids and proteins in the pollen tube plasma membrane modulates ion fluxes and actin dynamics and is maintained by a delicate equilibrium between exocytosis and endocytosis. The structural constraints regulating polarised secretion and asymmetric protein distribution on the plasma membrane are mostly unknown. To address this problem, we investigated whether ordered membrane microdomains, namely membrane rafts, might contribute to sperm cell delivery. Detergent insoluble membranes, rich in sterols and sphingolipids, were isolated from tobacco pollen tubes. MALDI TOF/MS analysis revealed that actin, prohibitins and proteins involved in methylation reactions and in phosphoinositide pattern regulation are specifically present in pollen tube detergent insoluble membranes. Tubulins, voltage-dependent anion channels and proteins involved in membrane trafficking and signalling were also present. This paper reports the first evidence of membrane rafts in Angiosperm pollen tubes, opening new perspectives on the coordination of signal transduction, cytoskeleton dynamics and polarised secretion. PMID:25701665

Evaluation of stereo-array isotope labeling (SAIL) patterns for automated structural analysis of proteins with CYANA.

PubMed

Ikeya, Teppei; Terauchi, Tsutomu; Güntert, Peter; Kainosho, Masatsune

2006-07-01

Recently we have developed the stereo-array isotope labeling (SAIL) technique to overcome the conventional molecular size limitation in NMR protein structure determination by employing complete stereo- and regiospecific patterns of stable isotopes. SAIL sharpens signals and simplifies spectra without the loss of requisite structural information, thus making large classes of proteins newly accessible to detailed solution structure determination. The automated structure calculation program CYANA can efficiently analyze SAIL-NOESY spectra and calculate structures without manual analysis. Nevertheless, the original SAIL method might not be capable of determining the structures of proteins larger than 50 kDa or membrane proteins, for which the spectra are characterized by many broadened and overlapped peaks. Here we have carried out simulations of new SAIL patterns optimized for minimal relaxation and overlap, to evaluate the combined use of SAIL and CYANA for solving the structures of larger proteins and membrane proteins. The modified approach reduces the number of peaks to nearly half of that observed with uniform labeling, while still yielding well-defined structures and is expected to enable NMR structure determinations of these challenging systems.

Appearance and distribution of surface proteins of the human erythrocyte membrane. An electron microscope and immunochemical labeling study

PubMed Central

Shotton, D.; Thompson, K.; Wofsy, L.; Branton, D.

1978-01-01

We have used freeze-etching, before and after immunoferritin labeling, to visualize spectrin molecules and other surface proteins of the human erythrocyte membrane. After intramembrane particle aggregation was induced, spectrin molecules, identified by labeling with ferritin-conjugated antispectrin, were clustered on the cytoplasmic surface of the membrane in patches directly underlying the particle clusters. This labeling pattern confirms the involvement of spectrin in such particle aggregates, as previously inferred from indirect evidence. Ferritin-conjugated antihapten molecules, directed against external and cytoplasmic surface proteins of the erythrocyte membrane which had been covalently labeled nonspecifically with the hapten p-diazoniumphenyl-beta-D-lactoside, were similarly found in direct association with such intramembrane particle aggregates. This indicates that when spectrin and the intramembrane particles are aggregated, all the major proteins of the erythrocyte membrane are constrained to coaggregate with them. Although giving no direct information concerning the freedom of translational movement of proteins in the unperturbed erythrocyte membrane, these experiments suggest that a close dynamic association may exist between the integral and peripheral protein components of the membrane, such that immobilization of one component can restrict the lateral mobility of others. PMID:10605454

The immunohistochemical detection of involucrin in denture induced fibrous inflammatory hyperplasia of oral mucous membrane.

PubMed

Thomas, G A

1991-01-01

Involucrin is a major structural protein specific to the cross-linked cell envelope found in the stratum corneum of stratified squamous epithelium. This protein is considered to be an excellent immunohistochemical marker of normal squamous differentiation. Detection of variations to the patterns of immunostaining for involucrin may also be of value in the differential diagnosis between benign and malignant lesions. Previous studies of involucrin expression in oral mucosa have failed to clarify the effect of chronic inflammatory change upon the patterns of immunoreactivity. This study investigated involucrin staining patterns in fibrous inflammatory hyperplasia of oral mucous membrane (FIH). The results suggest that in FIH an altered pattern of involucrin immunostain occurs in areas of severe inflammatory change. This may reflect changes to the pattern of squamous differentiation in this tissue.

Neutrophilic leukocyte membrane proteins. I. Isolation.

PubMed

Hawkins, D; Sauvé, M

1978-03-01

Rabbit exudate-derived PMN were homogenized and the cell membranes isolated on a two-phase aqueous system. Glycoproteins were extracted from cell membranes with lithium diiodosalicylate. SDS polyacrylamide gel electrophoretic analysis showed a consistent pattern of three major glycoprotein entities. Cells radioiodinated supravitally showed most of the radioactivity associated with larger glycoprotein entities whereas PMN membranes radiolabeled after isolation yielded a single major peak of radioactivity associated with a much smaller protein entity. Heterologous antisera against rabbit PMN, PMN membranes, and membrane glycoproteins were all cytotoxic for PMN in the presence of complement, and all bound to the PMN surface as demonstrated with immunocolloidal gold on electron microscopy. The data suggest that one or more glycoprotein entities are membrane-associated ectoglycoproteins which can be radiolabeled supravitally.

Trafficking of plant plasma membrane aquaporins: multiple regulation levels and complex sorting signals.

PubMed

Chevalier, Adrien S; Chaumont, François

2015-05-01

Aquaporins are small channel proteins which facilitate the diffusion of water and small neutral molecules across biological membranes. Compared with animals, plant genomes encode numerous aquaporins, which display a large variety of subcellular localization patterns. More specifically, plant aquaporins of the plasma membrane intrinsic protein (PIP) subfamily were first described as plasma membrane (PM)-resident proteins, but recent research has demonstrated that the trafficking and subcellular localization of these proteins are complex and highly regulated. In the past few years, PIPs emerged as new model proteins to study subcellular sorting and membrane dynamics in plant cells. At least two distinct sorting motifs (one cytosolic, the other buried in the membrane) are required to direct PIPs to the PM. Hetero-oligomerization and interaction with SNAREs (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptors) also influence the subcellular trafficking of PIPs. In addition to these constitutive processes, both the progression of PIPs through the secretory pathway and their dynamics at the PM are responsive to changing environmental conditions. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Membrane-bound MinDE complex acts as a toggle switch that drives Min oscillation coupled to cytoplasmic depletion of MinD

PubMed Central

Vecchiarelli, Anthony G.; Li, Min; Mizuuchi, Michiyo; Hwang, Ling Chin; Seol, Yeonee; Neuman, Keir C.; Mizuuchi, Kiyoshi

2016-01-01

The Escherichia coli Min system self-organizes into a cell-pole to cell-pole oscillator on the membrane to prevent divisions at the cell poles. Reconstituting the Min system on a lipid bilayer has contributed to elucidating the oscillatory mechanism. However, previous in vitro patterns were attained with protein densities on the bilayer far in excess of those in vivo and failed to recapitulate the standing wave oscillations observed in vivo. Here we studied Min protein patterning at limiting MinD concentrations reflecting the in vivo conditions. We identified “burst” patterns—radially expanding and imploding binding zones of MinD, accompanied by a peripheral ring of MinE. Bursts share several features with the in vivo dynamics of the Min system including standing wave oscillations. Our data support a patterning mechanism whereby the MinD-to-MinE ratio on the membrane acts as a toggle switch: recruiting and stabilizing MinD on the membrane when the ratio is high and releasing MinD from the membrane when the ratio is low. Coupling this toggle switch behavior with MinD depletion from the cytoplasm drives a self-organized standing wave oscillator. PMID:26884160

Cellulose membranes are more effective in holding back vital proteins and exhibit less interaction with plasma proteins during hemodialysis.

PubMed

Pešić, Ivana; Müller, Gerhard A; Baumann, Cosima; Dihazi, Gry H; Koziolek, Michael J; Eltoweissy, Marwa; Bramlage, Carsten; Asif, Abdul R; Dihazi, Hassan

2013-04-01

The vast majority of patients with end-stage renal disease are treated with intermittent hemodialysis as a form of renal replacement therapy. To investigate the impact of hemodialysis membrane material on vital protein removal, dialysates from 26 well-characterized hemodialysis patients were collected 5 min after beginning, during 5h of treatment, as well as 5 min before ending of the dialysis sessions. Dialysis sessions were performed using either modified cellulose (n=12) (low-flux and high flux) or synthetic Polyflux (n=14) (low-flux and high-flux) dialyzer. Protein removal during hemodialysis was quantified and the dialysate proteome patterns were analyzed by 2-DE, MS and Western blot. There was a clear correlation between the type of membrane material and the amount of protein removed. Synthetic Polyflux membranes exhibit strong interaction with plasma proteins resulting in a significantly higher protein loss compared to modified cellulosic membrane. Moreover, the proteomics analysis showed that the removed proteins represented different molecular weight range and different functional groups: transport proteins, protease inhibitors, proteins with role in immune response and regulations, constructive proteins and as a part of HLA immune complex. The effect of this protein removal on hemodialysis treatment outcome should be investigated in further studies. Copyright © 2013 Elsevier B.V. All rights reserved.

The molecular mechanisms of plant plasma membrane intrinsic proteins trafficking and stress response.

PubMed

Wang, Xing; Zhang, Ji-long; Feng, Xiu-xiu; Li, Hong-jie; Zhang, Gen-fa

2017-04-20

Plasma membrane intrinsic proteins (PIPs) are plant channel proteins located on the plasma membrane. PIPs transfer water, CO 2 and small uncharged solutes through the plasma membrane. PIPs have high selectivity to substrates, suggestive of a central role in maintaining cellular water balance. The expression, activity and localization of PIPs are regulated at the transcriptional and post-translational levels, and also affected by environmental factors. Numerous studies indicate that the expression patterns and localizations of PIPs can change in response to abiotic stresses. In this review, we summarize the mechanisms of PIP trafficking, transcriptional and post-translational regulations, and abiotic stress responses. Moreover, we also discuss the current research trends and future directions on PIPs.

Molecular modeling of biomembranes and their complexes with protein transmembrane α-helices

NASA Astrophysics Data System (ADS)

Kuznetsov, Andrey S.; Smirnov, Kirill V.; Antonov, Mikhail Yu.; Nikolaev, Ivan N.; Efremov, Roman G.

2017-11-01

Helical segments are common structural elements of membrane proteins. Dimerization and oligomerization of transmembrane (TM) α-helices provides the framework for spatial structure formation and protein-protein interactions. The membrane itself also takes part in the protein functioning. There are some examples of the mutual influence of the lipid bilayer properties and embedded membrane proteins. This work aims at the detail investigation of protein-lipid interactions using model systems: TM peptides corresponding to native protein segments. Three peptides were considered corresponding to TM domains of human glycophorin A (GpA), epidermal growth factor receptor (EGFR) and proposed TM-segment of human neuraminidase-1 (Neu1). A computational analysis of structural and dynamical properties was performed using molecular dynamics method. Monomers of peptides were considered incorporated into hydrated lipid bilayers. It was confirmed, that all these TM peptides have stable helical conformation in lipid environment, and the mutual adaptation of peptides and membrane was observed. It was shown that incorporation of the peptide into membrane results in the modulation of local and mean structural properties of the bilayer. Each peptide interacts with lipid acyl chains having special binding sites on the surface of central part of α-helix that exist for at least 200 ns. However, lipid acyl chains substitute each other faster occupying the same site. The formation of a special pattern of protein-lipid interactions may modulate the association of TM domains of membrane proteins, so membrane environment should be considered when proposing new substances targeting cell receptors.

Quantitative Proteomics Reveals Membrane Protein-Mediated Hypersaline Sensitivity and Adaptation in Halophilic Nocardiopsis xinjiangensis.

PubMed

Zhang, Yao; Li, Yanchang; Zhang, Yongguang; Wang, Zhiqiang; Zhao, Mingzhi; Su, Na; Zhang, Tao; Chen, Lingsheng; Wei, Wei; Luo, Jing; Zhou, Yanxia; Xu, Yongru; Xu, Ping; Li, Wenjun; Tao, Yong

2016-01-04

The genus Nocardiopsis is one of the most dominant Actinobacteria that survives in hypersaline environments. However, the adaptation mechanisms for halophilism are still unclear. Here, we performed isobaric tags for relative and absolute quantification based quantitative proteomics to investigate the functions of the membrane proteome after salt stress. A total of 683 membrane proteins were identified and quantified, of which 126 membrane proteins displayed salt-induced changes in abundance. Intriguingly, bioinformatics analyses indicated that these differential proteins showed two expression patterns, which were further validated by phenotypic changes and functional differences. The majority of ABC transporters, secondary active transporters, cell motility proteins, and signal transduction kinases were up-regulated with increasing salt concentration, whereas cell differentiation, small molecular transporter (ions and amino acids), and secondary metabolism proteins were significantly up-regulated at optimum salinity, but down-regulated or unchanged at higher salinity. The small molecule transporters and cell differentiation-related proteins acted as sensing proteins that played a more important biological role at optimum salinity. However, the ABC transporters for compatible solutes, Na(+)-dependent transporters, and cell motility proteins acted as adaptive proteins that actively counteracted higher salinity stress. Overall, regulation of membrane proteins may provide a major protection strategy against hyperosmotic stress.

Differential affinities of MinD and MinE to anionic phospholipid influence Min Patterning dynamics in vitro

PubMed Central

Vecchiarelli, Anthony G.; Li, Min; Mizuuchi, Michiyo; Mizuuchi, Kiyoshi

2014-01-01

The E. coli Min system forms a cell-pole-to-cell-pole oscillator that positions the divisome at mid-cell. The MinD ATPase binds the membrane and recruits the cell division inhibitor MinC. MinE interacts with and releases MinD (and MinC) from the membrane. The chase of MinD by MinE creates the in vivo oscillator that maintains a low level of the division inhibitor at mid-cell. In vitro reconstitution and visualization of Min proteins on a supported lipid bilayer has provided significant advances in understanding Min patterns in vivo. Here we studied the effects of flow, lipid composition, and salt concentration on Min patterning. Flow and no-flow conditions both supported Min protein patterns with somewhat different characteristics. Without flow, MinD and MinE formed spiraling waves. MinD and, to a greater extent MinE, have stronger affinities for anionic phospholipid. MinD-independent binding of MinE to anionic lipid resulted in slower and narrower waves. MinE binding to the bilayer was also more susceptible to changes in ionic strength than MinD. We find that modulating protein diffusion with flow, or membrane binding affinities with changes in lipid composition or salt concentration, can differentially affect the retention time of MinD and MinE, leading to spatiotemporal changes in Min patterning. PMID:24930948

Accurate Prediction of Contact Numbers for Multi-Spanning Helical Membrane Proteins

PubMed Central

Li, Bian; Mendenhall, Jeffrey; Nguyen, Elizabeth Dong; Weiner, Brian E.; Fischer, Axel W.; Meiler, Jens

2017-01-01

Prediction of the three-dimensional (3D) structures of proteins by computational methods is acknowledged as an unsolved problem. Accurate prediction of important structural characteristics such as contact number is expected to accelerate the otherwise slow progress being made in the prediction of 3D structure of proteins. Here, we present a dropout neural network-based method, TMH-Expo, for predicting the contact number of transmembrane helix (TMH) residues from sequence. Neuronal dropout is a strategy where certain neurons of the network are excluded from back-propagation to prevent co-adaptation of hidden-layer neurons. By using neuronal dropout, overfitting was significantly reduced and performance was noticeably improved. For multi-spanning helical membrane proteins, TMH-Expo achieved a remarkable Pearson correlation coefficient of 0.69 between predicted and experimental values and a mean absolute error of only 1.68. In addition, among those membrane protein–membrane protein interface residues, 76.8% were correctly predicted. Mapping of predicted contact numbers onto structures indicates that contact numbers predicted by TMH-Expo reflect the exposure patterns of TMHs and reveal membrane protein–membrane protein interfaces, reinforcing the potential of predicted contact numbers to be used as restraints for 3D structure prediction and protein–protein docking. TMH-Expo can be accessed via a Web server at www.meilerlab.org. PMID:26804342

Dynamic association with donor cell filopodia and lipid-modification are essential features of Wnt8a during patterning of the zebrafish neuroectoderm.

PubMed

Luz, Marta; Spannl-Müller, Stephanie; Özhan, Günes; Kagermeier-Schenk, Birgit; Rhinn, Muriel; Weidinger, Gilbert; Brand, Michael

2014-01-01

Wnt proteins are conserved signaling molecules that regulate pattern formation during animal development. Many Wnt proteins are post-translationally modified by addition of lipid adducts. Wnt8a provides a crucial signal for patterning the anteroposterior axis of the developing neural plate in vertebrates. However, it is not clear how this protein propagates from its source, the blastoderm margin, to the target cells in the prospective neural plate, and how lipid-modifications might influence Wnt8a propagation and activity. We have dynamically imaged biologically active, fluorescently tagged Wnt8a in living zebrafish embryos. We find that Wnt8a localizes to membrane-associated, punctate structures in live tissue. In Wnt8a expressing cells, these puncta are found on filopodial cellular processes, from where the protein can be released. In addition, Wnt8a is found colocalized with Frizzled receptor-containing clusters on signal receiving cells. Combining in vitro and in vivo assays, we compare the roles of conserved Wnt8a residues in cell and non-cell-autonomous signaling activity and secretion. Non-signaling Wnt8 variants show these residues can regulate Wnt8a distribution in producing cell membranes and filopodia as well as in the receiving tissue. Together, our results show that Wnt8a forms dynamic clusters found on filopodial donor cell and on signal receiving cell membranes. Moreover, they demonstrate a differential requirement of conserved residues in Wnt8a protein for distribution in producing cells and receiving tissue and signaling activity during neuroectoderm patterning.

Dynamic Association with Donor Cell Filopodia and Lipid-Modification Are Essential Features of Wnt8a during Patterning of the Zebrafish Neuroectoderm

PubMed Central

Luz, Marta; Spannl-Müller, Stephanie; Özhan, Günes; Kagermeier-Schenk, Birgit; Rhinn, Muriel; Weidinger, Gilbert; Brand, Michael

2014-01-01

Background Wnt proteins are conserved signaling molecules that regulate pattern formation during animal development. Many Wnt proteins are post-translationally modified by addition of lipid adducts. Wnt8a provides a crucial signal for patterning the anteroposterior axis of the developing neural plate in vertebrates. However, it is not clear how this protein propagates from its source, the blastoderm margin, to the target cells in the prospective neural plate, and how lipid-modifications might influence Wnt8a propagation and activity. Results We have dynamically imaged biologically active, fluorescently tagged Wnt8a in living zebrafish embryos. We find that Wnt8a localizes to membrane-associated, punctate structures in live tissue. In Wnt8a expressing cells, these puncta are found on filopodial cellular processes, from where the protein can be released. In addition, Wnt8a is found colocalized with Frizzled receptor-containing clusters on signal receiving cells. Combining in vitro and in vivo assays, we compare the roles of conserved Wnt8a residues in cell and non-cell-autonomous signaling activity and secretion. Non-signaling Wnt8 variants show these residues can regulate Wnt8a distribution in producing cell membranes and filopodia as well as in the receiving tissue. Conclusions Together, our results show that Wnt8a forms dynamic clusters found on filopodial donor cell and on signal receiving cell membranes. Moreover, they demonstrate a differential requirement of conserved residues in Wnt8a protein for distribution in producing cells and receiving tissue and signaling activity during neuroectoderm patterning. PMID:24427298

Self-organizing actin patterns shape membrane architecture but not cell mechanics

NASA Astrophysics Data System (ADS)

Fritzsche, M.; Li, D.; Colin-York, H.; Chang, V. T.; Moeendarbary, E.; Felce, J. H.; Sezgin, E.; Charras, G.; Betzig, E.; Eggeling, C.

2017-02-01

Cell-free studies have demonstrated how collective action of actin-associated proteins can organize actin filaments into dynamic patterns, such as vortices, asters and stars. Using complementary microscopic techniques, we here show evidence of such self-organization of the actin cortex in living HeLa cells. During cell adhesion, an active multistage process naturally leads to pattern transitions from actin vortices over stars into asters. This process is primarily driven by Arp2/3 complex nucleation, but not by myosin motors, which is in contrast to what has been theoretically predicted and observed in vitro. Concomitant measurements of mechanics and plasma membrane fluidity demonstrate that changes in actin patterning alter membrane architecture but occur functionally independent of macroscopic cortex elasticity. Consequently, tuning the activity of the Arp2/3 complex to alter filament assembly may thus be a mechanism allowing cells to adjust their membrane architecture without affecting their macroscopic mechanical properties.

Self-organizing actin patterns shape membrane architecture but not cell mechanics

PubMed Central

Fritzsche, M.; Li, D.; Colin-York, H.; Chang, V. T.; Moeendarbary, E.; Felce, J. H.; Sezgin, E.; Charras, G.; Betzig, E.; Eggeling, C.

2017-01-01

Cell-free studies have demonstrated how collective action of actin-associated proteins can organize actin filaments into dynamic patterns, such as vortices, asters and stars. Using complementary microscopic techniques, we here show evidence of such self-organization of the actin cortex in living HeLa cells. During cell adhesion, an active multistage process naturally leads to pattern transitions from actin vortices over stars into asters. This process is primarily driven by Arp2/3 complex nucleation, but not by myosin motors, which is in contrast to what has been theoretically predicted and observed in vitro. Concomitant measurements of mechanics and plasma membrane fluidity demonstrate that changes in actin patterning alter membrane architecture but occur functionally independent of macroscopic cortex elasticity. Consequently, tuning the activity of the Arp2/3 complex to alter filament assembly may thus be a mechanism allowing cells to adjust their membrane architecture without affecting their macroscopic mechanical properties. PMID:28194011

Electrical manipulation of glycan-phosphatidyl inositol-tethered proteins in planar supported bilayers.

PubMed Central

Groves, J T; Wülfing, C; Boxer, S G

1996-01-01

Electric fields have been used to manipulate and concentrate glycan-phosphatidyl inositol (GPI)-tethered proteins in planar supported bilayers. Naturally GPI-linked CD48, along with engineered forms of I-Ek and B7-2, in which their transmembrane domains have been genetically replaced with the GPI linkage, were studied. The proteins were labeled with fluorescently tagged antibodies, allowing the electric field-induced behavior to be followed by epifluorescence microscopy. All three protein complexes were observed to migrate toward the cathode with the B7-2 and CD48, each tethered to the membrane by a single GPI linker, moving significantly faster than the I-Ek, which has two GPI linkers. Patterns scratched into the membrane function as barriers to lateral diffusion and were used to isolate the proteins into highly concentrated corrals. All field-induced concentration profiles were completely reversible, indicating that the supported bilayer provides a stable, fluid environment in which GPI-tethered proteins can be manipulated. The ability to electrically control the spatial distribution of membrane-tethered proteins provides new opportunities for the study of biological membranes and the development of membrane-based devices. Images FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 PMID:8913608

Hepatitis C Virus-Induced Cytoplasmic Organelles Use the Nuclear Transport Machinery to Establish an Environment Conducive to Virus Replication

PubMed Central

Neufeldt, Christopher J.; Joyce, Michael A.; Levin, Aviad; Steenbergen, Rineke H.; Pang, Daniel; Shields, Justin; Tyrrell, D. Lorne J.; Wozniak, Richard W.

2013-01-01

Hepatitis C virus (HCV) infection induces formation of a membranous web structure in the host cell cytoplasm where the viral genome replicates and virions assemble. The membranous web is thought to concentrate viral components and hide viral RNA from pattern recognition receptors. We have uncovered a role for nuclear pore complex proteins (Nups) and nuclear transport factors (NTFs) in the membranous web. We show that HCV infection leads to increased levels of cytoplasmic Nups that accumulate at sites enriched for HCV proteins. Moreover, we detected interactions between specific HCV proteins and both Nups and NTFs. We hypothesize that cytoplasmically positioned Nups facilitate formation of the membranous web and contribute to the compartmentalization of viral replication. Accordingly, we show that transport cargo proteins normally targeted to the nucleus are capable of entering regions of the membranous web, and that depletion of specific Nups or Kaps inhibits HCV replication and assembly. PMID:24204278

Live cell imaging of phosphoinositide dynamics during Legionella infection.

PubMed

Weber, Stephen; Hilbi, Hubert

2014-01-01

The "accidental" pathogen Legionella pneumophila replicates intracellularly in a distinct compartment, the Legionella-containing vacuole (LCV). To form this specific pathogen vacuole, the bacteria translocate via the Icm/Dot type IV secretion system approximately 300 different effector proteins into the host cell. Several of these secreted effectors anchor to the cytoplasmic face of the LCV membrane by binding to phosphoinositide (PI) lipids. L. pneumophila thus largely controls the localization of secreted bacterial effectors and the recruitment of host factors to the LCV through the modulation of the vacuole membrane PI pattern. The LCV PI pattern and its dynamics can be studied in real-time using fluorescently labeled protein probes stably produced by the soil amoeba Dictyostelium discoideum. In this chapter, we describe a protocol to (1) construct and handle amoeba model systems as a tool for observing PIs in live cell imaging, (2) capture rapid changes in membrane PI patterning during uptake events, and (3) observe the dynamics of LCV PIs over the course of a Legionella infection.

Dynamic Partitioning of a GPI-Anchored Protein in Glycosphingolipid-Rich Microdomains Imaged by Single-Quantum Dot Tracking

PubMed Central

Pinaud, Fabien; Michalet, Xavier; Iyer, Gopal; Margeat, Emmanuel; Moore, Hsiao-Ping; Weiss, Shimon

2009-01-01

Recent experimental developments have led to a revision of the classical fluid mosaic model proposed by Singer and Nicholson 35 years ago. In particular, it is now well established that lipids and proteins diffuse heterogeneously in cell plasma membranes. Their complex motion patterns reflect the dynamic structure and composition of the membrane itself, as well as the presence of the underlying cytoskeleton scaffold and that of the extracellular matrix. How the structural organization of plasma membranes influences the diffusion of individual proteins remains a challenging, yet central question for cell signaling and its regulation. Here we have developed a raft-associated glycosylphosphatidyl Inositol-anchored avidin test probe (Av-GPI), whose diffusion patterns indirectly reports on the structure and dynamics of putative raft microdomains in the membrane of HeLa cells. Labeling with quantum dots (qdots) allowed high-resolution and long-term tracking of individual Av-GPI and the classification of their various diffusive behaviors. Using dual-color total internal reflection fluorescence (TIRF) microscopy, we studied the correlation between the diffusion of individual Av-GPI and the location of glycosphingolipid GM1-rich microdomains and caveolae. We show that Av-GPI exhibit a fast and a slow diffusion regime in different membrane regions, and that slowing down of their diffusion is correlated with entry in GM1-rich microdomains located in close proximity to, but distinct, from caveolae. We further show that Av-GPI dynamically partition in and out of these microdomains in a cholesterol-dependent manner. Our results provide direct evidence that cholesterol/sphingolipid-rich microdomains can compartmentalize the diffusion of GPI-anchored proteins in living cells and that the dynamic partitioning raft model appropriately describes the diffusive behavior of some raft-associated proteins across the plasma membrane. PMID:19416475

Dynamic partitioning of a glycosyl-phosphatidylinositol-anchored protein in glycosphingolipid-rich microdomains imaged by single-quantum dot tracking.

PubMed

Pinaud, Fabien; Michalet, Xavier; Iyer, Gopal; Margeat, Emmanuel; Moore, Hsiao-Ping; Weiss, Shimon

2009-06-01

Recent experimental developments have led to a revision of the classical fluid mosaic model proposed by Singer and Nicholson more than 35 years ago. In particular, it is now well established that lipids and proteins diffuse heterogeneously in cell plasma membranes. Their complex motion patterns reflect the dynamic structure and composition of the membrane itself, as well as the presence of the underlying cytoskeleton scaffold and that of the extracellular matrix. How the structural organization of plasma membranes influences the diffusion of individual proteins remains a challenging, yet central, question for cell signaling and its regulation. Here we have developed a raft-associated glycosyl-phosphatidyl-inositol-anchored avidin test probe (Av-GPI), whose diffusion patterns indirectly report on the structure and dynamics of putative raft microdomains in the membrane of HeLa cells. Labeling with quantum dots (qdots) allowed high-resolution and long-term tracking of individual Av-GPI and the classification of their various diffusive behaviors. Using dual-color total internal reflection fluorescence (TIRF) microscopy, we studied the correlation between the diffusion of individual Av-GPI and the location of glycosphingolipid GM1-rich microdomains and caveolae. We show that Av-GPI exhibit a fast and a slow diffusion regime in different membrane regions, and that slowing down of their diffusion is correlated with entry in GM1-rich microdomains located in close proximity to, but distinct, from caveolae. We further show that Av-GPI dynamically partition in and out of these microdomains in a cholesterol-dependent manner. Our results provide direct evidence that cholesterol-/sphingolipid-rich microdomains can compartmentalize the diffusion of GPI-anchored proteins in living cells and that the dynamic partitioning raft model appropriately describes the diffusive behavior of some raft-associated proteins across the plasma membrane.

Differential Expression and Immunolocalization of Antioxidant Enzymes in Entamoeba histolytica Isolates during Metronidazole Stress

PubMed Central

Iyer, Lakshmi Rani; Singh, Nishant; Verma, Anil Kumar; Paul, Jaishree

2014-01-01

Entamoeba histolytica infections are endemic in the Indian subcontinent. Five to eight percent of urban population residing under poor sanitary conditions suffers from Entamoeba infections. Metronidazole is the most widely prescribed drug used for amoebiasis. In order to understand the impact of metronidazole stress on the parasite, we evaluated the expression of two antioxidant enzymes, peroxiredoxin and FeSOD, in Entamoeba histolytica isolates during metronidazole stress. The results reveal that, under metronidazole stress, the mRNA expression levels of these enzymes did not undergo any significant change. Interestingly, immunolocalization studies with antibodies targeting peroxiredoxin indicate differential localization of the protein in the cell during metronidazole stress. In normal conditions, all the Entamoeba isolates exhibit presence of peroxiredoxin in the nucleus as well as in the membrane; however with metronidazole stress the protein localized mostly to the membrane. The change in the localization pattern was more pronounced when the cells were subjected to short term metronidazole stress compared to cells adapted to metronidazole. The protein localization to the cell membrane could be the stress response mechanism in these isolates. Colocalization pattern of peroxiredoxin with CaBp1, a cytosolic protein, revealed that the membrane and nuclear localization was specific to peroxiredoxin during metronidazole stress. PMID:25013795

Large-scale purification and biochemical characterization of crystallization-grade porin protein P from Pseudomonas aeruginosa.

PubMed

Worobec, E A; Martin, N L; McCubbin, W D; Kay, C M; Brayer, G D; Hancock, R E

1988-04-07

A large-scale purification scheme was developed for lipopolysaccharide-free protein P, the phosphate-starvation-inducible outer-membrane porin from Pseudomonas aeruginosa. This highly purified protein P was used to successfully form hexagonal crystals in the presence of n-octyl-beta-glucopyranoside. Amino-acid analysis indicated that protein P had a similar composition to other bacterial outer membrane proteins, containing a high percentage (50%) of hydrophilic residues. The amino-terminal sequence of this protein, although not homologous to either outer membrane protein, PhoE or OmpF, of Escherichia coli, was found to have an analogous protein-folding pattern. Protein P in the native trimer form was capable of maintaining a stable functional trimer after proteinase cleavage. This suggested the existence of a strongly associated tertiary and quaternary structure. Circular dichroism studies confirmed these results in that a large proportion of the protein structure was determined to be beta-sheet and resistant to acid pH and heating in 0.1% sodium dodecyl sulphate.

Dynamic Cholesterol-Conditioned Dimerization of the G Protein Coupled Chemokine Receptor Type 4

PubMed Central

Kranz, Franziska

2016-01-01

G protein coupled receptors (GPCRs) allow for the transmission of signals across biological membranes. For a number of GPCRs, this signaling was shown to be coupled to prior dimerization of the receptor. The chemokine receptor type 4 (CXCR4) was reported before to form dimers and their functionality was shown to depend on membrane cholesterol. Here, we address the dimerization pattern of CXCR4 in pure phospholipid bilayers and in cholesterol-rich membranes. Using ensembles of molecular dynamics simulations, we show that CXCR4 dimerizes promiscuously in phospholipid membranes. Addition of cholesterol dramatically affects the dimerization pattern: cholesterol binding largely abolishes the preferred dimer motif observed for pure phospholipid bilayers formed mainly by transmembrane helices 1 and 7 (TM1/TM5-7) at the dimer interface. In turn, the symmetric TM3,4/TM3,4 interface is enabled first by intercalating cholesterol molecules. These data provide a molecular basis for the modulation of GPCR activity by its lipid environment. PMID:27812115

Monte Carlo simulations of protein micropatterning in biomembranes: effects of immobile sticky obstacles

NASA Astrophysics Data System (ADS)

Arnold, Andreas M.; Sevcsik, Eva; Schütz, Gerhard J.

2016-09-01

Single molecule trajectories of lipids and proteins can yield valuable information about the nanoscopic organization of the plasma membrane itself. The interpretation of such trajectories, however, is complicated, as the mobility of molecules can be affected by the presence of immobile obstacles, and the transient binding of the tracers to these obstacles. We have previously developed a micropatterning approach that allows for immobilizing a plasma membrane protein and probing the diffusional behavior of a putative interaction partner in living cells. Here, we provide guidelines on how this micropatterning approach can be extended to quantify interaction parameters between plasma membrane constituents in their natural environment. We simulated a patterned membrane system and evaluated the effect of different surface densities of patterned immobile obstacles on the relative mobility as well as the surface density of diffusing tracers. In the case of inert obstacles, the size of the obstacle can be assessed from its surface density at the percolation threshold, which in turn can be extracted from the diffusion behavior of the tracer. For sticky obstacles, 2D dissociation constants can be determined from the tracer diffusion or surface density.

Bioelectric patterning during oogenesis: stage-specific distribution of membrane potentials, intracellular pH and ion-transport mechanisms in Drosophila ovarian follicles.

PubMed

Krüger, Julia; Bohrmann, Johannes

2015-01-16

Bioelectric phenomena have been found to exert influence on various developmental and regenerative processes. Little is known about their possible functions and the cellular mechanisms by which they might act during Drosophila oogenesis. In developing follicles, characteristic extracellular current patterns and membrane-potential changes in oocyte and nurse cells have been observed that partly depend on the exchange of protons, potassium ions and sodium ions. These bioelectric properties have been supposed to be related to various processes during oogenesis, e. g. pH-regulation, osmoregulation, cell communication, cell migration, cell proliferation, cell death, vitellogenesis and follicle growth. Analysing in detail the spatial distribution and activity of the relevant ion-transport mechanisms is expected to elucidate the roles that bioelectric phenomena play during oogenesis. To obtain an overview of bioelectric patterning along the longitudinal and transversal axes of the developing follicle, the spatial distributions of membrane potentials (Vmem), intracellular pH (pHi) and various membrane-channel proteins were studied systematically using fluorescent indicators, fluorescent inhibitors and antisera. During mid-vitellogenic stages 9 to 10B, characteristic, stage-specific Vmem-patterns in the follicle-cell epithelium as well as anteroposterior pHi-gradients in follicle cells and nurse cells were observed. Corresponding distribution patterns of proton pumps (V-ATPases), voltage-dependent L-type Ca(2+)-channels, amiloride-sensitive Na(+)-channels and Na(+),H(+)-exchangers (NHE) and gap-junction proteins (innexin 3) were detected. In particular, six morphologically distinguishable follicle-cell types are characterized on the bioelectric level by differences concerning Vmem and pHi as well as specific compositions of ion channels and carriers. Striking similarities between Vmem-patterns and activity patterns of voltage-dependent Ca(2+)-channels were found, suggesting a mechanism for transducing bioelectric signals into cellular responses. Moreover, gradients of electrical potential and pH were observed within single cells. Our data suggest that spatial patterning of Vmem, pHi and specific membrane-channel proteins results in bioelectric signals that are supposed to play important roles during oogenesis, e. g. by influencing spatial coordinates, regulating migration processes or modifying the cytoskeletal organization. Characteristic stage-specific changes of bioelectric activity in specialized cell types are correlated with various developmental processes.

A highly efficient, cell-free translation/translocation system prepared from Xenopus eggs.

PubMed Central

Matthews, G; Colman, A

1991-01-01

We describe the use of a Xenopus laevis egg extract for the in vitro translation and post translational modification of membrane and secretory proteins. This extract is capable of the translation and segregation into membranes of microgram per millilitre levels of protein from added mRNAs. Signal sequences of segregated proteins are efficiently cleaved and appropriate N-linked glycosylation patterns are produced. The extract also supports the quantitative assembly of murine immunoglobulin heavy and light chains into tetramers, and two events which take place beyond the endoplasmic reticulum, mannose 6 phosphorylation of murine cathepsin D and O-linked glycosylation of coronavirus E1 protein, also occur, but at reduced efficiency. The stability of the membranes allows protease protection studies and quantitative centrifugal fractionation of segregated and unsegregated proteins to be performed. Conditions for the use of stored extract have also been determined. Images PMID:1754376

Biofunctional paper via the covalent modification of cellulose.

PubMed

Yu, Arthur; Shang, Jing; Cheng, Fang; Paik, Bradford A; Kaplan, Justin M; Andrade, Rodrigo B; Ratner, Daniel M

2012-07-31

Paper-based analytical devices are the subject of growing interest for the development of low-cost point-of-care diagnostics, environmental monitoring technologies, and research tools for limited-resource settings. However, there are limited chemistries available for the conjugation of biomolecules to cellulose for use in biomedical applications. Herein, divinyl sulfone (DVS) chemistry was demonstrated to immobilize small molecules, proteins, and DNA covalently onto the hydroxyl groups of cellulose membranes through nucleophilic addition. Assays on modified cellulose using protein-carbohydrate and protein-glycoprotein interactions as well as oligonucleotide hybridization showed that the membrane's bioactivity was specific, dose-dependent, and stable over a long period of time. The use of an inkjet printer to form patterns of biomolecules on DVS-activated cellulose illustrates the adaptability of the DVS functionalization technique to pattern sophisticated designs, with potential applications in cellulose-based lateral flow devices.

Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots

DOEpatents

Zhang, Jian-Shi; Giometti, C.S.; Tollaksen, S.L.

1987-09-04

After two-dimensional electrophoresis of proteins or the like, resulting in a polyacrylamide gel slab having a pattern of protein gel spots thereon, an individual protein gel spot is cored out from the slab, to form a gel spot core which is placed in an extraction tube, with a dialysis membrane across the lower end of the tube. Replicate gel spots can be cored out from replicate gel slabs and placed in the extraction tube. Molten agarose gel is poured into the extraction tube where the agarose gel hardens to form an immobilizing gel, covering the gel spot cores. The upper end portion of the extraction tube is filled with a volume of buffer solution, and the upper end is closed by another dialysis membrane. Upper and lower bodies of a buffer solution are brought into contact with the upper and lower membranes and are provided with electrodes connected to the positive and negative terminals of a dc power supply, thereby producing an electrical current which flows through the upper membrane, the volume of buffer solution, the agarose, the gel spot cores and the lower membrane. The current causes the proteins to be extracted electrophoretically from the gel spot cores, so that the extracted proteins accumulate and are contained in the space between the agarose gel and the upper membrane. 8 figs.

Mapping the Extracellular and Membrane Proteome Associated with the Vasculature and the Stroma in the Embryo*

PubMed Central

Soulet, Fabienne; Kilarski, Witold W.; Roux-Dalvai, Florence; Herbert, John M. J.; Sacewicz, Izabela; Mouton-Barbosa, Emmanuelle; Bicknell, Roy; Lalor, Patricia; Monsarrat, Bernard; Bikfalvi, Andreas

2013-01-01

In order to map the extracellular or membrane proteome associated with the vasculature and the stroma in an embryonic organism in vivo, we developed a biotinylation technique for chicken embryo and combined it with mass spectrometry and bioinformatic analysis. We also applied this procedure to implanted tumors growing on the chorioallantoic membrane or after the induction of granulation tissue. Membrane and extracellular matrix proteins were the most abundant components identified. Relative quantitative analysis revealed differential protein expression patterns in several tissues. Through a bioinformatic approach, we determined endothelial cell protein expression signatures, which allowed us to identify several proteins not yet reported to be associated with endothelial cells or the vasculature. This is the first study reported so far that applies in vivo biotinylation, in combination with robust label-free quantitative proteomics approaches and bioinformatic analysis, to an embryonic organism. It also provides the first description of the vascular and matrix proteome of the embryo that might constitute the starting point for further developments. PMID:23674615

Dynamic caveolae exclude bulk membrane proteins and are required for sorting of excess glycosphingolipids

PubMed Central

Shvets, Elena; Bitsikas, Vassilis; Howard, Gillian; Hansen, Carsten Gram; Nichols, Benjamin J.

2015-01-01

Caveolae have long been implicated in endocytosis. Recent data question this link, and in the absence of specific cargoes the potential cellular function of caveolar endocytosis remains unclear. Here we develop new tools, including doubly genome-edited cell lines, to assay the subcellular dynamics of caveolae using tagged proteins expressed at endogenous levels. We find that around 5% of the cellular pool of caveolae is present on dynamic endosomes, and is delivered to endosomes in a clathrin-independent manner. Furthermore, we show that caveolae are indeed likely to bud directly from the plasma membrane. Using a genetically encoded tag for electron microscopy and ratiometric light microscopy, we go on to show that bulk membrane proteins are depleted within caveolae. Although caveolae are likely to account for only a small proportion of total endocytosis, cells lacking caveolae show fundamentally altered patterns of membrane traffic when loaded with excess glycosphingolipid. Altogether, these observations support the hypothesis that caveolar endocytosis is specialized for transport of membrane lipid. PMID:25897946

The Complete Set of Genes Encoding Major Intrinsic Proteins in Arabidopsis Provides a Framework for a New Nomenclature for Major Intrinsic Proteins in Plants1

PubMed Central

Johanson, Urban; Karlsson, Maria; Johansson, Ingela; Gustavsson, Sofia; Sjövall, Sara; Fraysse, Laure; Weig, Alfons R.; Kjellbom, Per

2001-01-01

Major intrinsic proteins (MIPs) facilitate the passive transport of small polar molecules across membranes. MIPs constitute a very old family of proteins and different forms have been found in all kinds of living organisms, including bacteria, fungi, animals, and plants. In the genomic sequence of Arabidopsis, we have identified 35 different MIP-encoding genes. Based on sequence similarity, these 35 proteins are divided into four different subfamilies: plasma membrane intrinsic proteins, tonoplast intrinsic proteins, NOD26-like intrinsic proteins also called NOD26-like MIPs, and the recently discovered small basic intrinsic proteins. In Arabidopsis, there are 13 plasma membrane intrinsic proteins, 10 tonoplast intrinsic proteins, nine NOD26-like intrinsic proteins, and three small basic intrinsic proteins. The gene structure in general is conserved within each subfamily, although there is a tendency to lose introns. Based on phylogenetic comparisons of maize (Zea mays) and Arabidopsis MIPs (AtMIPs), it is argued that the general intron patterns in the subfamilies were formed before the split of monocotyledons and dicotyledons. Although the gene structure is unique for each subfamily, there is a common pattern in how transmembrane helices are encoded on the exons in three of the subfamilies. The nomenclature for plant MIPs varies widely between different species but also between subfamilies in the same species. Based on the phylogeny of all AtMIPs, a new and more consistent nomenclature is proposed. The complete set of AtMIPs, together with the new nomenclature, will facilitate the isolation, classification, and labeling of plant MIPs from other species. PMID:11500536

Three-dimensional electron diffraction of plant light-harvesting complex

PubMed Central

Wang, Da Neng; Kühlbrandt, Werner

1992-01-01

Electron diffraction patterns of two-dimensional crystals of light-harvesting chlorophyll a/b-protein complex (LHC-II) from photosynthetic membranes of pea chloroplasts, tilted at different angles up to 60°, were collected to 3.2 Å resolution at -125°C. The reflection intensities were merged into a three-dimensional data set. The Friedel R-factor and the merging R-factor were 21.8 and 27.6%, respectively. Specimen flatness and crystal size were critical for recording electron diffraction patterns from crystals at high tilts. The principal sources of experimental error were attributed to limitations of the number of unit cells contributing to an electron diffraction pattern, and to the critical electron dose. The distribution of strong diffraction spots indicated that the three-dimensional structure of LHC-II is less regular than that of other known membrane proteins and is not dominated by a particular feature of secondary structure. ImagesFIGURE 1FIGURE 2 PMID:19431817

Protein Adsorption to In-Line Filters of Intravenous Administration Sets.

PubMed

Besheer, Ahmed

2017-10-01

Ensuring compatibility of administered therapeutic proteins with intravenous administration sets is an important regulatory requirement. A low-dose recovery during administration of low protein concentrations is among the commonly observed incompatibilities, and it is mainly due to adsorption to in-line filters. To better understand this phenomenon, we studied the adsorption of 4 different therapeutic proteins (2 IgG1s, 1 IgG4, and 1 Fc fusion protein) diluted to 0.01 mg/mL in 5% glucose (B. Braun EcoFlac; B. Braun Melsungen AG, Melsungen, Germany) or 0.9% sodium chloride (NaCl; Freeflex; Fresenius Kabi, Friedberg, Germany) solutions to 8 in-line filters (5 positively charged and 3 neutral filters made of different polymers and by different suppliers). The results show certain patterns of protein adsorption, which depend to a large extent on the dilution solution and filter material, and to a much lower extent on the proteins' biophysical properties. Investigation of the filter membranes' zeta potential showed a correlation between the observed adsorption pattern in 5% glucose solution and the filter's surface charge, with higher protein adsorption for the strongly negatively charged membranes. In 0.9% NaCl solution, the surface charges are masked, leading to different adsorption patterns. These results contribute to the general understanding of the protein adsorption to IV infusion filters and allow the design of more efficient compatibility studies. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Identification of lipopolysaccharide-interacting plasma membrane-type proteins in Arabidopsis thaliana.

PubMed

Vilakazi, Cornelius S; Dubery, Ian A; Piater, Lizelle A

2017-02-01

Lipopolysaccharide (LPS) is an amphiphatic bacterial glycoconjugate found on the external membrane of Gram-negative bacteria. This endotoxin is considered as a microbe-associated molecular pattern (MAMP) molecule and has been shown to elicit defense responses in plants. Here, LPS-interacting proteins from Arabidopsis thaliana plasma membrane (PM)-type fractions were captured and identified in order to investigate those involved in LPS perception and linked to triggering of innate immune responses. A novel proteomics-based affinity-capture strategy coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed for the enrichment and identification of LPS-interacting proteins. As such, LPS isolated from Burkholderia cepacia (LPS B.cep. ) was immobilized on three independent and distinct affinity-based matrices to serve as bait for interacting proteins from A. thaliana leaf and callus tissue. These were resolved by 1D electrophoresis and identified by mass spectrometry. Proteins specifically bound to LPS B.cep. have been implicated in membrane structure (e.g. COBRA-like and tubulin proteins), membrane trafficking and/or transport (e.g. soluble NSF attachment protein receptor (SNARE) proteins, patellin, aquaporin, PM instrinsic proteins (PIP) and H + -ATPase), signal transduction (receptor-like kinases and calcium-dependent protein kinases) as well as defense/stress responses (e.g. hypersensitive-induced response (HIR) proteins, jacalin-like lectin domain-containing protein and myrosinase-binding proteins). The novel affinity-capture strategy for the enrichment of LPS-interacting proteins proved to be effective, especially in the binding of proteins involved in plant defense responses, and can thus be used to elucidate LPS-mediated molecular recognition and disease mechanism(s). Copyright © 2016 Elsevier Masson SAS. All rights reserved.

A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kalra, Rajkumar S., E-mail: renu-wadhwa@aist.go.jp; Wadhwa, Renu, E-mail: renu-wadhwa@aist.go.jp

2015-02-27

Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylationmore » target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing and transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein.« less

Disruption of Ankyrin B and Caveolin-1 Interaction Sites Alters Na+,K+-ATPase Membrane Diffusion.

PubMed

Junghans, Cornelia; Vukojević, Vladana; Tavraz, Neslihan N; Maksimov, Eugene G; Zuschratter, Werner; Schmitt, Franz-Josef; Friedrich, Thomas

2017-11-21

The Na + ,K + -ATPase is a plasma membrane ion transporter of high physiological importance for ion homeostasis and cellular excitability in electrically active tissues. Mutations in the genes coding for Na + ,K + -ATPase α-subunit isoforms lead to severe human pathologies including Familial Hemiplegic Migraine type 2, Alternating Hemiplegia of Childhood, Rapid-onset Dystonia Parkinsonism, or epilepsy. Many of the reported mutations lead to change- or loss-of-function effects, whereas others do not alter the functional properties, but lead to, e.g., reduced protein stability, reduced protein expression, or defective plasma membrane targeting. Na + ,K + -ATPase frequently assembles with other membrane transporters or cellular matrix proteins in specialized plasma membrane microdomains, but the effects of these interactions on targeting or protein mobility are elusive so far. Mutation of established interaction motifs of the Na + ,K + -ATPase with ankyrin B and caveolin-1 are expected to result in changes in plasma membrane targeting, changes of the localization pattern, and of the diffusion behavior of the enzyme. We studied the consequences of mutations in these binding sites by monitoring diffusion of eGFP-labeled Na + ,K + -ATPase constructs in the plasma membrane of HEK293T cells by fluorescence correlation spectroscopy as well as fluorescence recovery after photobleaching or photoswitching, and observed significant differences compared to the wild-type enzyme, with synergistic effects for combinations of interaction site mutations. These measurements expand the possibilities to study the consequences of Na + ,K + -ATPase mutations and provide information about the interaction of Na + ,K + -ATPase α-isoforms with cellular matrix proteins, the cytoskeleton, or other membrane protein complexes. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The use of holographic interferometry and electron speckle pattern interferometry for diffusion measurement in biochemical and pharmaceutical engineering applications

NASA Astrophysics Data System (ADS)

Axelsson, Anders; Marucci, Mariagrazia

2008-12-01

In this review holographic interferometry and electron speckle pattern interferometry are discussed as efficient techniques for diffusion measurements in biochemical and pharmaceutical applications. Transport phenomena can be studied, quantitatively and qualitatively, in gels, liquids and membranes. Detailed information on these phenomena is required to design effective chromatography bioseparation processes using gel beads or ultrafiltration membranes, and in the design of controlled-release pharmaceuticals using membrane-coated pellets or tablets. The influence of gel concentration, ion strength in the liquid and the size of diffusing protein molecules can easily be studied with good accuracy. When studying membranes, the resistance can be quantified, and it is also possible to discriminate between permeable and semi-permeable membranes. In this review the influence of temperature, natural convection and light deflection on the accuracy of the diffusion measurements is also discussed.

Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells

PubMed Central

1975-01-01

The enzymatic iodination technique has been utilized in a study of the externally disposed membrane proteins of the mouse L cell. Iodination of cells in suspension results in lactoperoxidase-specific iodide incorporation with no loss of cell viability under the conditions employed, less than 3% lipid labeling, and more than 90% of the labeled species identifiable as monoiodotyrosine. 90% of the incorporated label is localized to the cell surface by electron microscope autoradiography, with 5-10% in the centrosphere region and postulated to represent pinocytic vesicles. Sodium dodecylsulfate-polyacrylamide gels of solubilized L-cell proteins reveals five to six labeled peaks ranging from 50,000 to 200,000 daltons. Increased resolution by use of gradient slab gels reveals 15-20 radioactive bands. Over 60% of the label resides in approximately nine polypeptides of 80,000 to 150,000 daltons. Various controls indicate that the labeling pattern reflects endogenous membrane proteins, not serum components. The incorporated 125-I, cholesterol, and one plasma membrane enzyme marker, alkaline phosphodiesterase I, are purified in parallel when plasma membranes are isolated from intact, iodinated L cells. The labeled components present in a plasma membrane-rich fraction from iodinated cells are identical to those of the total cell, with a 10- to 20-fold enrichment in specific activity of each radioactive peak in the membrane. PMID:163833

Variable-angle epifluorescence microscopy characterizes protein dynamics in the vicinity of plasma membrane in plant cells.

PubMed

Chen, Tong; Ji, Dongchao; Tian, Shiping

2018-03-14

The assembly of protein complexes and compositional lipid patterning act together to endow cells with the plasticity required to maintain compositional heterogeneity with respect to individual proteins. Hence, the applications for imaging protein localization and dynamics require high accuracy, particularly at high spatio-temporal level. We provided experimental data for the applications of Variable-Angle Epifluorescence Microscopy (VAEM) in dissecting protein dynamics in plant cells. The VAEM-based co-localization analysis took penetration depth and incident angle into consideration. Besides direct overlap of dual-color fluorescence signals, the co-localization analysis was carried out quantitatively in combination with the methodology for calculating puncta distance and protein proximity index. Besides, simultaneous VAEM tracking of cytoskeletal dynamics provided more insights into coordinated responses of actin filaments and microtubules. Moreover, lateral motility of membrane proteins was analyzed by calculating diffusion coefficients and kymograph analysis, which represented an alternative method for examining protein motility. The present study presented experimental evidence on illustrating the use of VAEM in tracking and dissecting protein dynamics, dissecting endosomal dynamics, cell structure assembly along with membrane microdomain and protein motility in intact plant cells.

Cholesterol removal from adult skeletal muscle impairs excitation–contraction coupling and aging reduces caveolin-3 and alters the expression of other triadic proteins

PubMed Central

Barrientos, Genaro; Llanos, Paola; Hidalgo, Jorge; Bolaños, Pura; Caputo, Carlo; Riquelme, Alexander; Sánchez, Gina; Quest, Andrew F. G.; Hidalgo, Cecilia

2015-01-01

Cholesterol and caveolin are integral membrane components that modulate the function/location of many cellular proteins. Skeletal muscle fibers, which have unusually high cholesterol levels in transverse tubules, express the caveolin-3 isoform but its association with transverse tubules remains contentious. Cholesterol removal impairs excitation–contraction (E–C) coupling in amphibian and mammalian fetal skeletal muscle fibers. Here, we show that treating single muscle fibers from adult mice with the cholesterol removing agent methyl-β-cyclodextrin decreased fiber cholesterol by 26%, altered the location pattern of caveolin-3 and of the voltage dependent calcium channel Cav1.1, and suppressed or reduced electrically evoked Ca2+ transients without affecting membrane integrity or causing sarcoplasmic reticulum (SR) calcium depletion. We found that transverse tubules from adult muscle and triad fractions that contain ~10% attached transverse tubules, but not SR membranes, contained caveolin-3 and Cav1.1; both proteins partitioned into detergent-resistant membrane fractions highly enriched in cholesterol. Aging entails significant deterioration of skeletal muscle function. We found that triad fractions from aged rats had similar cholesterol and RyR1 protein levels compared to triads from young rats, but had lower caveolin-3 and glyceraldehyde 3-phosphate dehydrogenase and increased Na+/K+-ATPase protein levels. Both triad fractions had comparable NADPH oxidase (NOX) activity and protein content of NOX2 subunits (p47phox and gp91phox), implying that NOX activity does not increase during aging. These findings show that partial cholesterol removal impairs E–C coupling and alters caveolin-3 and Cav1.1 location pattern, and that aging reduces caveolin-3 protein content and modifies the expression of other triadic proteins. We discuss the possible implications of these findings for skeletal muscle function in young and aged animals. PMID:25914646

The Hepatitis C Virus-Induced Membranous Web and Associated Nuclear Transport Machinery Limit Access of Pattern Recognition Receptors to Viral Replication Sites

PubMed Central

Neufeldt, Christopher J.; Joyce, Michael A.; Van Buuren, Nicholas; Levin, Aviad; Kirkegaard, Karla; Gale Jr., Michael; Tyrrell, D. Lorne J.; Wozniak, Richard W.

2016-01-01

Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family and a major cause of liver disease worldwide. HCV replicates in the cytoplasm, and the synthesis of viral proteins induces extensive rearrangements of host cell membranes producing structures, collectively termed the membranous web (MW). The MW contains the sites of viral replication and assembly, and we have identified distinct membrane fractions derived from HCV-infected cells that contain replication and assembly complexes enriched for viral RNA and infectious virus, respectively. The complex membrane structure of the MW is thought to protect the viral genome limiting its interactions with cytoplasmic pattern recognition receptors (PRRs) and thereby preventing activation of cellular innate immune responses. Here we show that PRRs, including RIG-I and MDA5, and ribosomes are excluded from viral replication and assembly centers within the MW. Furthermore, we present evidence that components of the nuclear transport machinery regulate access of proteins to MW compartments. We show that the restricted assess of RIG-I to the MW can be overcome by the addition of a nuclear localization signal sequence, and that expression of a NLS-RIG-I construct leads to increased immune activation and the inhibition of viral replication. PMID:26863439

Inhomogeneity Based Characterization of Distribution Patterns on the Plasma Membrane

PubMed Central

Paparelli, Laura; Corthout, Nikky; Wakefield, Devin L.; Sannerud, Ragna; Jovanovic-Talisman, Tijana; Annaert, Wim; Munck, Sebastian

2016-01-01

Cell surface protein and lipid molecules are organized in various patterns: randomly, along gradients, or clustered when segregated into discrete micro- and nano-domains. Their distribution is tightly coupled to events such as polarization, endocytosis, and intracellular signaling, but challenging to quantify using traditional techniques. Here we present a novel approach to quantify the distribution of plasma membrane proteins and lipids. This approach describes spatial patterns in degrees of inhomogeneity and incorporates an intensity-based correction to analyze images with a wide range of resolutions; we have termed it Quantitative Analysis of the Spatial distributions in Images using Mosaic segmentation and Dual parameter Optimization in Histograms (QuASIMoDOH). We tested its applicability using simulated microscopy images and images acquired by widefield microscopy, total internal reflection microscopy, structured illumination microscopy, and photoactivated localization microscopy. We validated QuASIMoDOH, successfully quantifying the distribution of protein and lipid molecules detected with several labeling techniques, in different cell model systems. We also used this method to characterize the reorganization of cell surface lipids in response to disrupted endosomal trafficking and to detect dynamic changes in the global and local organization of epidermal growth factor receptors across the cell surface. Our findings demonstrate that QuASIMoDOH can be used to assess protein and lipid patterns, quantifying distribution changes and spatial reorganization at the cell surface. An ImageJ/Fiji plugin of this analysis tool is provided. PMID:27603951

Cytoskeleton-mediated templating of complex cellulose-scaffolded extracellular structure and its association with oikosins in the urochordate Oikopleura.

PubMed

Sagane, Yoshimasa; Hosp, Julia; Zech, Karin; Thompson, Eric M

2011-05-01

Oriented cellulose deposition is critical to plant patterning and models suggest microtubules constrain cellulose synthase movements through the plasma membrane. Though widespread in plants, urochordates are the only animals that synthesize cellulose. We characterized the distinctive cellulose microfibril scaffold of the larvacean house and its interaction with house structural proteins (oikosins). Targeted disruption of cytoskeletal elements, secretory pathways, and plasma membrane organization, suggested a working model for templating extracellular cellulose microfibrils from animal cells that shows both convergence and differences to plant models. Specialized cortical F-actin arrays template microfibril orientation and glycosylphosphatidylinositol-anchored proteins in lipid rafts may act as scaffolding proteins in microfibril elongation. Microtubules deliver and maintain cellulose synthase complexes to specific cell membrane sites rather than orienting their movement through the membrane. Oikosins are incorporated into house compartments directly above their corresponding cellular field of expression and interact with the cellulose scaffold to a variable extent.

Non-Brownian diffusion in lipid membranes: Experiments and simulations.

PubMed

Metzler, R; Jeon, J-H; Cherstvy, A G

2016-10-01

The dynamics of constituents and the surface response of cellular membranes-also in connection to the binding of various particles and macromolecules to the membrane-are still a matter of controversy in the membrane biophysics community, particularly with respect to crowded membranes of living biological cells. We here put into perspective recent single particle tracking experiments in the plasma membranes of living cells and supercomputing studies of lipid bilayer model membranes with and without protein crowding. Special emphasis is put on the observation of anomalous, non-Brownian diffusion of both lipid molecules and proteins embedded in the lipid bilayer. While single component, pure lipid bilayers in simulations exhibit only transient anomalous diffusion of lipid molecules on nanosecond time scales, the persistence of anomalous diffusion becomes significantly longer ranged on the addition of disorder-through the addition of cholesterol or proteins-and on passing of the membrane lipids to the gel phase. Concurrently, experiments demonstrate the anomalous diffusion of membrane embedded proteins up to macroscopic time scales in the minute time range. Particular emphasis will be put on the physical character of the anomalous diffusion, in particular, the occurrence of ageing observed in the experiments-the effective diffusivity of the measured particles is a decreasing function of time. Moreover, we present results for the time dependent local scaling exponent of the mean squared displacement of the monitored particles. Recent results finding deviations from the commonly assumed Gaussian diffusion patterns in protein crowded membranes are reported. The properties of the displacement autocorrelation function of the lipid molecules are discussed in the light of their appropriate physical anomalous diffusion models, both for non-crowded and crowded membranes. In the last part of this review we address the upcoming field of membrane distortion by elongated membrane-binding particles. We discuss how membrane compartmentalisation and the particle-membrane binding energy may impact the dynamics and response of lipid membranes. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Expression patterns of genes encoding plasma membrane aquaporins during fruit development in cucumber (Cucumis sativus L.).

PubMed

Shi, Jin; Wang, Jinfang; Li, Ren; Li, Dianbo; Xu, Fengfeng; Sun, Qianqian; Zhao, Bin; Mao, Ai-Jun; Guo, Yang-Dong

2015-11-01

Aquaporins are membrane channels precisely regulating water movement through cell membranes in most living organisms. Despite the advances in the physiology of fruit development, their participation during fruit development in cucumber still barely understood. In this paper, the expressions of 12 genes encoding plasma membrane intrinsic proteins (PIPs) were analyzed during cucumber fruit development in our work. Based on the homology search with known PIPs from rice, Arabidopsis and strawberry, 12 cucumber PIP genes subfamily members were identified. Cellular localization assays indicated that CsPIPs were localized in the plasma membrane. The qRT-PCR analysis of CsPIPs showed that 12 CsPIPs were differentially expressed during fruit development. These results suggest that 12 genes encoding plasma membrane intrinsic proteins (CsPIPs) play very important roles in cucumber life cycle and the data generated will be helpful in understanding their precise roles during fruit development in cucumber. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

The periciliary ring in polarized epithelial cells is a hot spot for delivery of the apical protein gp135

PubMed Central

Stoops, Emily H.; Hull, Michael; Olesen, Christina; Mistry, Kavita; Harder, Jennifer L.; Rivera-Molina, Felix; Toomre, Derek

2015-01-01

In polarized epithelial cells, newly synthesized cell surface proteins travel in carrier vesicles from the trans Golgi network to the apical or basolateral plasma membrane. Despite extensive research on polarized trafficking, the sites of protein delivery are not fully characterized. Here we use the SNAP tag system to examine the site of delivery of the apical glycoprotein gp135. We show that a cohort of gp135 is delivered to a ring surrounding the base of the primary cilium, followed by microtubule-dependent radial movement away from the cilium. Delivery to the periciliary ring was specific to newly synthesized and not recycling protein. A subset of this newly delivered protein traverses the basolateral membrane en route to the apical membrane. Crumbs3a, another apical protein, was not delivered to the periciliary region, instead making its initial apical appearance in a pattern that resembled its steady-state distribution. Our results demonstrate a surprising “hot spot” for gp135 protein delivery at the base of the primary cilium and suggest the existence of a novel microtubule-based directed movement of a subset of apical surface proteins. PMID:26504168

Plantaricin A, a cationic peptide produced by Lactobacillus plantarum, permeabilizes eukaryotic cell membranes by a mechanism dependent on negative surface charge linked to glycosylated membrane proteins.

PubMed

Sand, Sverre L; Nissen-Meyer, Jon; Sand, Olav; Haug, Trude M

2013-02-01

Lactobacillus plantarum C11 releases plantaricin A (PlnA), a cationic peptide pheromone that has a membrane-permeabilizing, antimicrobial effect. We have previously shown that PlnA may also permeabilize eukaryotic cells, with a potency that differs between cell types. It is generally assumed that cationic antimicrobial peptides exert their effects through electrostatic attraction to negatively charged phospholipids in the membrane. The aim of the present study was to investigate if removal of the negative charge linked to glycosylated proteins at the cell surface reduces the permeabilizing potency of PlnA. The effects of PlnA were tested on clonal rat anterior pituitary cells (GH(4) cells) using patch clamp and microfluorometric techniques. In physiological extracellular solution, GH(4) cells are highly sensitive to PlnA, but the sensitivity was dramatically reduced in solutions that partly neutralize the negative surface charge of the cells, in agreement with the notion that electrostatic interactions are probably important for the PlnA effects. Trypsination of cells prior to PlnA exposure also rendered the cells less sensitive to the peptide, suggesting that negative charges linked to membrane proteins are involved in the permeabilizing action. Finally, pre-exposure of cells to a mixture of enzymes that split carbohydrate residues from the backbone of glycosylated proteins also impeded the PlnA-induced membrane permeabilization. We conclude that electrostatic attraction between PlnA and glycosylated membrane proteins is probably an essential first step before PlnA can interact with membrane phospholipids. Deviating glycosylation patterns may contribute to the variation in PlnA sensitivity of different cell types, including cancerous cells and their normal counterparts. Copyright © 2012 Elsevier B.V. All rights reserved.

A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways*

PubMed Central

Lecat, Sandra; Matthes, Hans W.D.; Pepperkok, Rainer; Simpson, Jeremy C.; Galzi, Jean-Luc

2015-01-01

Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening. PMID:25759509

A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways.

PubMed

Lecat, Sandra; Matthes, Hans W D; Pepperkok, Rainer; Simpson, Jeremy C; Galzi, Jean-Luc

2015-05-01

Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Membrane Driven Spatial Organization of GPCRs

NASA Astrophysics Data System (ADS)

Mondal, Sayan; Johnston, Jennifer M.; Wang, Hao; Khelashvili, George; Filizola, Marta; Weinstein, Harel

2013-10-01

Spatial organization of G-protein coupled receptors (GPCRs) into dimers and higher order oligomers has been demonstrated in vitro and in vivo. The pharmacological readout was shown to depend on the specific interfaces, but why particular regions of the GPCR structure are involved, and how ligand-determined states change them remains unknown. Here we show why protein-membrane hydrophobic matching is attained upon oligomerization at specific interfaces from an analysis of coarse-grained molecular dynamics simulations of the spontaneous diffusion-interaction of the prototypical beta2-adrenergic (β2AR) receptors in a POPC lipid bilayer. The energy penalty from mismatch is significantly reduced in the spontaneously emerging oligomeric arrays, making the spatial organization of the GPCRs dependent on the pattern of mismatch in the monomer. This mismatch pattern is very different for β2AR compared to the highly homologous and structurally similar β1AR, consonant with experimentally observed oligomerization patterns of β2AR and β1AR. The results provide a mechanistic understanding of the structural context of oligomerization.

Live-cell topology assessment of URG7, MRP6{sub 102} and SP-C using glycosylatable green fluorescent protein in mammalian cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Hunsang; Lara, Patricia; Ostuni, Angela

2014-08-08

Highlights: • Glycosylatable GFP (gGFP) is developed for the use in mammalian cells. • gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen. • Differential fluorescence/glycosylation pattern probes membrane protein topology. • Membrane topology of URG7, MRP6{sub 102}, and SP-C was determined by gGFP tagging in vivo. - Abstract: Experimental tools to determine membrane topology of a protein are rather limited in higher eukaryotic organisms. Here, we report the use of glycosylatable GFP (gGFP) as a sensitive and versatile membrane topology reporter in mammalian cells. gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen.more » Thus, positive fluorescence signal assigns location of gGFP to the cytosol whereas no fluorescence signal and a glycosylated status of gGFP map the location of gGFP to the ER lumen. By using mammalian gGFP, the membrane topology of disease-associated membrane proteins, URG7, MRP6{sub 102}, SP-C(Val) and SP-C(Leu) was confirmed. URG7 is partially targeted to the ER, and inserted in C{sub in} form. MRP6{sub 102} and SP-C(Leu/Val) are inserted into the membrane in C{sub out} form. A minor population of untargeted SP-C is removed by proteasome dependent quality control system.« less

Electron crystallography of PhoE porin, an outer membrane, channel- forming protein from E. coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walian, P.J.

1989-11-01

One approach to studying the structure of membrane proteins is the use of electron crystallography. Dr. Bing Jap has crystallized PhoE pore-forming protein (porin) from the outer membrane of escherichia coli (E. coli) into monolayer crystals. The findings of this research and those of Jap (1988, 1989) have determined these crystals to be highly ordered, yielding structural information to a resolution of better than 2.8 angstroms. The task of this thesis has been to collect and process the electron diffraction patterns necessary to generate a complete three-dimensional set of high resolution structure factor amplitudes of PhoE porin. Fourier processing ofmore » these amplitudes when combined with the corresponding phase data is expected to yield the three-dimensional structure of PhoE porin at better than 3.5 angstroms resolution. 92 refs., 33 figs., 3 tabs. (CBS)« less

Biochemical characteristics of thylakoid membranes in chloroplasts of dark-grown pine cotyledons.

PubMed

Shinohara, K; Murakami, A; Fujita, Y

1992-01-01

Japanese black pine (Pinus thunbergii) cotyledons were found to synthesize chlorophylls in complete darkness during germination, although the synthesis was not as great as that in the light. The compositions of thylakoid components in plastids of cotyledons grown in the dark and light were compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of polypeptides and spectroscopic determination of membrane redox components. All thylakoid membrane proteins found in preparations from light-grown cotyledons were also present in preparations from dark-grown cotyledons. However, levels of photosystem I, photosystem II, cytochrome b([ill])/f, and light-harvesting chlorophyll-protein complexes in dark-grown cotyledons were only one-fourth of those in light-grown cotyledons, on a fresh weight basis. These results suggest that the low abundance of thylakoid components in dark-grown cotyledons is associated with the limited supply of chlorophyll needed to assemble the two photosystem complexes and the light-harvesting chlorophyll-protein complex.

Directed formation of micro- and nanoscale patterns of functional light-harvesting LH2 complexes.

PubMed

Reynolds, Nicholas P; Janusz, Stefan; Escalante-Marun, Maryana; Timney, John; Ducker, Robert E; Olsen, John D; Otto, Cees; Subramaniam, Vinod; Leggett, Graham J; Hunter, C Neil

2007-11-28

The precision placement of the desired protein components on a suitable substrate is an essential prelude to any hybrid "biochip" device, but a second and equally important condition must also be met: the retention of full biological activity. Here we demonstrate the selective binding of an optically active membrane protein, the light-harvesting LH2 complex from Rhodobacter sphaeroides, to patterned self-assembled monolayers at the micron scale and the fabrication of nanometer-scale patterns of these molecules using near-field photolithographic methods. In contrast to plasma proteins, which are reversibly adsorbed on many surfaces, the LH2 complex is readily patterned simply by spatial control of surface polarity. Near-field photolithography has yielded rows of light-harvesting complexes only 98 nm wide. Retention of the native optical properties of patterned LH2 molecules was demonstrated using in situ fluorescence emission spectroscopy.

Differential protein expression in ovaries of uninfected and Babesia-infected southern cattle ticks, Rhipicephalus (Boophilus) microplus.

PubMed

Rachinsky, Anna; Guerrero, Felix D; Scoles, Glen A

2007-12-01

We used gel electrophoresis and mass spectrometry to investigate differences in protein expression in ovarian tissues from Babesia bovis-infected and uninfected southern cattle tick, Rhipicephalus (Boophilus) microplus. Soluble and membrane proteins were extracted from ovaries of adult female ticks, and analyzed by isoelectric focusing (IEF) and one-dimensional or two-dimensional (2-D) gel electrophoresis. Protein patterns were analyzed for differences in expression between infected and uninfected ticks. 2-D separation of proteins revealed a number of proteins that appeared to be up- or down-regulated in response to infection with Babesia, in particular membrane/membrane-associated proteins and proteins in a low molecular mass range between 6 and 36kDa. A selection of differentially expressed proteins was subjected to analysis by capillary-HPLC-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). Among the ovarian proteins that were up-regulated in infected ticks were calreticulin, two myosin subunits, an endoplasmic reticulum protein, a peptidyl-prolyl cis-trans isomerase (PPIase), a cytochrome c oxidase subunit, a glutamine synthetase, and a family of Kunitz-type serine protease inhibitors. Among the down-regulated ovarian proteins were another PPIase, a hemoglobin subunit, and a lysozyme. This study is part of an ongoing effort to establish a proteome database that can be utilized to investigate specific proteins involved in successful pathogen transmission.

Uroplakins, specific membrane proteins of urothelial umbrella cells, as histological markers of metastatic transitional cell carcinomas.

PubMed Central

Moll, R.; Wu, X. R.; Lin, J. H.; Sun, T. T.

1995-01-01

Uroplakins (UPs) Ia, Ib, II, and III, transmembrane proteins constituting the asymmetrical unit membrane of urothelial umbrella cells, are the first specific urothelial differentiation markers described. We investigated the presence and localization patterns of UPs in various human carcinomas by applying immunohistochemistry (avidin-biotin-peroxidase complex method), using rabbit antibodies against UPs II and III, to paraffin sections. Positive reactions for UP III (sometimes very focal) were noted in 14 of the 16 papillary noninvasive transitional cell carcinomas (TCCs) (88%), 29 of the 55 invasive TCCs (53%), and 23 of the 35 TCC metastases (66%). Different localization patterns of UPs could be distinguished, including superficial membrane staining like that found in normal umbrella cells (in papillary carcinoma), luminal (microluminal) membrane staining (in papillary and invasive carcinoma), and, against expectations, peripheral membrane staining (in invasive carcinoma). Non-TCC carcinomas of various origins (n = 177) were consistently negative for UPs. The presence of UPs in metastatic TCCs represents a prime example of even advanced tumor progression being compatible with the (focal) expression of highly specialized differentiation repertoires. Although of only medium-grade sensitivity, UPs do seem to be highly specific urothelial lineage markers, thus operating up interesting histodiagnostic possibilities in cases of carcinoma metastases of uncertain origin. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:7485401

Microdomains of endoplasmic reticulum within the sarcoplasmic reticulum of skeletal myofibers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaakinen, Mika; Papponen, Hinni; Metsikkoe, Kalervo

2008-01-15

The relationship between the endoplasmic reticulum (ER) and the sarcoplasmic reticulum (SR) of skeletal muscle cells has remained obscure. In this study, we found that ER- and SR-specific membrane proteins exhibited diverse solubility properties when extracted with mild detergents. Accordingly, the major SR-specific protein Ca{sup 2+}-ATPase (SERCA) remained insoluble in Brij 58 and floated in sucrose gradients while typical ER proteins were partially or fully soluble. Sphingomyelinase treatment rendered SERCA soluble in Brij 58. Immunofluorescence staining for resident ER proteins revealed dispersed dots over I bands contrasting the continuous staining pattern of SERCA. Infection of isolated myofibers with enveloped virusesmore » indicated that interfibrillar protein synthesis occurred. Furthermore, we found that GFP-tagged Dad1, able to incorporate into the oligosaccharyltransferase complex, showed the dot-like structures but the fusion protein was also present in membranes over the Z lines. This behaviour mimics that of cargo proteins that accumulated over the Z lines when blocked in the ER. Taken together, the results suggest that resident ER proteins comprised Brij 58-soluble microdomains within the insoluble SR membrane. After synthesis and folding in the ER-microdomains, cargo proteins and non-incorporated GFP-Dad1 diffused into the Z line-flanking compartment which likely represents the ER exit sites.« less

Developmental distribution of the plasma membrane-enriched proteome in the maize primary root growth zone.

PubMed

Zhang, Zhe; Voothuluru, Priyamvada; Yamaguchi, Mineo; Sharp, Robert E; Peck, Scott C

2013-01-01

Within the growth zone of the maize primary root, there are well-defined patterns of spatial and temporal organization of cell division and elongation. However, the processes underlying this organization remain poorly understood. To gain additional insights into the differences amongst the defined regions, we performed a proteomic analysis focusing on fractions enriched for plasma membrane (PM) proteins. The PM is the interface between the plant cell and the apoplast and/or extracellular space. As such, it is a key structure involved in the exchange of nutrients and other molecules as well as in the integration of signals that regulate growth and development. Despite the important functions of PM-localized proteins in mediating these processes, a full understanding of dynamic changes in PM proteomes is often impeded by low relative concentrations relative to total proteins. Using a relatively simple strategy of treating microsomal fractions with Brij-58 detergent to enrich for PM proteins, we compared the developmental distribution of proteins within the root growth zone which revealed a number of previously known as well as novel proteins with interesting patterns of abundance. For instance, the quantitative proteomic analysis detected a gradient of PM aquaporin proteins similar to that previously reported using immunoblot analyses, confirming the veracity of this strategy. Cellulose synthases increased in abundance with increasing distance from the root apex, consistent with expected locations of cell wall deposition. The similar distribution pattern for Brittle-stalk-2-like protein implicates that this protein may also have cell wall related functions. These results show that the simplified PM enrichment method previously demonstrated in Arabidopsis can be successfully applied to completely unrelated plant tissues and provide insights into differences in the PM proteome throughout growth and development zones of the maize primary root.

Characterization of Bifunctional Spin Labels for Investigating the Structural and Dynamic Properties of Membrane Proteins Using EPR Spectroscopy.

PubMed

Sahu, Indra D; Craig, Andrew F; Dunagum, Megan M; McCarrick, Robert M; Lorigan, Gary A

2017-10-05

Site-directed spin labeling (SDSL) coupled with electron paramagnetic resonance (EPR) spectroscopy is a very powerful technique to study structural and dynamic properties of membrane proteins. The most widely used spin label is methanthiosulfonate (MTSL). However, the flexibility of this spin label introduces greater uncertainties in EPR measurements obtained for determining structures, side-chain dynamics, and backbone motion of membrane protein systems. Recently, a newer bifunctional spin label (BSL), 3,4-bis(methanethiosulfonylmethyl)-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxy, has been introduced to overcome the dynamic limitations associated with the MTSL spin label and has been invaluable in determining protein backbone dynamics and inter-residue distances due to its restricted internal motion and fewer size restrictions. While BSL has been successful in providing more accurate information about the structure and dynamics of several proteins, a detailed characterization of the spin label is still lacking. In this study, we characterized BSLs by performing CW-EPR spectral line shape analysis as a function of temperature on spin-labeled sites inside and outside of the membrane for the integral membrane protein KCNE1 in POPC/POPG lipid bilayers and POPC/POPG lipodisq nanoparticles. The experimental data revealed a powder pattern spectral line shape for all of the KCNE1-BSL samples at 296 K, suggesting the motion of BSLs approaches the rigid limit regime for these series of samples. BSLs were further utilized to report for the first time the distance measurement between two BSLs attached on an integral membrane protein KCNE1 in POPC/POPG lipid bilayers at room temperature using dipolar line broadening CW-EPR spectroscopy. The CW dipolar line broadening EPR data revealed a 15 ± 2 Å distance between doubly attached BSLs on KCNE1 (53/57-63/67) which is consistent with molecular dynamics modeling and the solution NMR structure of KCNE1 which yielded a distance of 17 Å. This study demonstrates the utility of investigating the structural and dynamic properties of membrane proteins in physiologically relevant membrane mimetics using BSLs.

FRAP to Characterize Molecular Diffusion and Interaction in Various Membrane Environments.

PubMed

Pincet, Frédéric; Adrien, Vladimir; Yang, Rong; Delacotte, Jérôme; Rothman, James E; Urbach, Wladimir; Tareste, David

2016-01-01

Fluorescence recovery after photobleaching (FRAP) is a standard method used to study the dynamics of lipids and proteins in artificial and cellular membrane systems. The advent of confocal microscopy two decades ago has made quantitative FRAP easily available to most laboratories. Usually, a single bleaching pattern/area is used and the corresponding recovery time is assumed to directly provide a diffusion coefficient, although this is only true in the case of unrestricted Brownian motion. Here, we propose some general guidelines to perform FRAP experiments under a confocal microscope with different bleaching patterns and area, allowing the experimentalist to establish whether the molecules undergo Brownian motion (free diffusion) or whether they have restricted or directed movements. Using in silico simulations of FRAP measurements, we further indicate the data acquisition criteria that have to be verified in order to obtain accurate values for the diffusion coefficient and to be able to distinguish between different diffusive species. Using this approach, we compare the behavior of lipids in three different membrane platforms (supported lipid bilayers, giant liposomes and sponge phases), and we demonstrate that FRAP measurements are consistent with results obtained using other techniques such as Fluorescence Correlation Spectroscopy (FCS) or Single Particle Tracking (SPT). Finally, we apply this method to show that the presence of the synaptic protein Munc18-1 inhibits the interaction between the synaptic vesicle SNARE protein, VAMP2, and its partner from the plasma membrane, Syn1A.

Reverse and forward engineering of protein pattern formation.

PubMed

Kretschmer, Simon; Harrington, Leon; Schwille, Petra

2018-05-26

Living systems employ protein pattern formation to regulate important life processes in space and time. Although pattern-forming protein networks have been identified in various prokaryotes and eukaryotes, their systematic experimental characterization is challenging owing to the complex environment of living cells. In turn, cell-free systems are ideally suited for this goal, as they offer defined molecular environments that can be precisely controlled and manipulated. Towards revealing the molecular basis of protein pattern formation, we outline two complementary approaches: the biochemical reverse engineering of reconstituted networks and the de novo design, or forward engineering, of artificial self-organizing systems. We first illustrate the reverse engineering approach by the example of the Escherichia coli Min system, a model system for protein self-organization based on the reversible and energy-dependent interaction of the ATPase MinD and its activating protein MinE with a lipid membrane. By reconstituting MinE mutants impaired in ATPase stimulation, we demonstrate how large-scale Min protein patterns are modulated by MinE activity and concentration. We then provide a perspective on the de novo design of self-organizing protein networks. Tightly integrated reverse and forward engineering approaches will be key to understanding and engineering the intriguing phenomenon of protein pattern formation.This article is part of the theme issue 'Self-organization in cell biology'. © 2018 The Author(s).

Plasma Membrane Proteomics of Human Breast Cancer Cell Lines Identifies Potential Targets for Breast Cancer Diagnosis and Treatment

PubMed Central

Ziegler, Yvonne S.; Moresco, James J.; Tu, Patricia G.; Yates, John R.; Nardulli, Ann M.

2014-01-01

The use of broad spectrum chemotherapeutic agents to treat breast cancer results in substantial and debilitating side effects, necessitating the development of targeted therapies to limit tumor proliferation and prevent metastasis. In recent years, the list of approved targeted therapies has expanded, and it includes both monoclonal antibodies and small molecule inhibitors that interfere with key proteins involved in the uncontrolled growth and migration of cancer cells. The targeting of plasma membrane proteins has been most successful to date, and this is reflected in the large representation of these proteins as targets of newer therapies. In view of these facts, experiments were designed to investigate the plasma membrane proteome of a variety of human breast cancer cell lines representing hormone-responsive, ErbB2 over-expressing and triple negative cell types, as well as a benign control. Plasma membranes were isolated by using an aqueous two-phase system, and the resulting proteins were subjected to mass spectrometry analysis. Overall, each of the cell lines expressed some unique proteins, and a number of proteins were expressed in multiple cell lines, but in patterns that did not always follow traditional clinical definitions of breast cancer type. From our data, it can be deduced that most cancer cells possess multiple strategies to promote uncontrolled growth, reflected in aberrant expression of tyrosine kinases, cellular adhesion molecules, and structural proteins. Our data set provides a very rich and complex picture of plasma membrane proteins present on breast cancer cells, and the sorting and categorizing of this data provides interesting insights into the biology, classification, and potential treatment of this prevalent and debilitating disease. PMID:25029196

Reconstituted TOM core complex and Tim9/Tim10 complex of mitochondria are sufficient for translocation of the ADP/ATP carrier across membranes.

PubMed

Vasiljev, Andreja; Ahting, Uwe; Nargang, Frank E; Go, Nancy E; Habib, Shukry J; Kozany, Christian; Panneels, Valérie; Sinning, Irmgard; Prokisch, Holger; Neupert, Walter; Nussberger, Stephan; Rapaport, Doron

2004-03-01

Precursor proteins of the solute carrier family and of channel forming Tim components are imported into mitochondria in two main steps. First, they are translocated through the TOM complex in the outer membrane, a process assisted by the Tim9/Tim10 complex. They are passed on to the TIM22 complex, which facilitates their insertion into the inner membrane. In the present study, we have analyzed the function of the Tim9/Tim10 complex in the translocation of substrates across the outer membrane of mitochondria. The purified TOM core complex was reconstituted into lipid vesicles in which purified Tim9/Tim10 complex was entrapped. The precursor of the ADP/ATP carrier (AAC) was found to be translocated across the membrane of such lipid vesicles. Thus, these components are sufficient for translocation of AAC precursor across the outer membrane. Peptide libraries covering various substrate proteins were used to identify segments that are bound by Tim9/Tim10 complex upon translocation through the TOM complex. The patterns of binding sites on the substrate proteins suggest a mechanism by which portions of membrane-spanning segments together with flanking hydrophilic segments are recognized and bound by the Tim9/Tim10 complex as they emerge from the TOM complex into the intermembrane space.

Two-dimensional protein crystals (S-layers): fundamentals and applications.

PubMed

Sleytr, U B; Sára, M; Messner, P; Pum, D

1994-10-01

Two-dimensional crystalline surface layers (S-layers) composed of protein or glycoprotein subunits are one of the most commonly observed prokaryotic cell envelope structures. Isolated S-layer subunits are endowed with the ability to assemble into monomolecular arrays in suspension, on surfaces or interfaces by an entropy-driven process. S-layer lattices are isoporous structures with functional groups located on the surface in an identical position and orientation. These characteristic features have already led to applications of S-layers as (1) ultrafiltration membranes with well-defined molecular weight cut-offs and excellent antifouling characteristics, (2) immobilization matrices for functional molecules as required for affinity and enzyme membranes, affinity microcarriers and biosensors, (3) conjugate vaccines, (4) carriers for Langmuir-Blodgett films and reconstituted biological membranes, and (5) patterning elements in molecular nanotechnology.

Distinct subcellular patterns of neprilysin protein and activity in the brains of Alzheimer’s disease patients, transgenic mice and cultured human neuronal cells

PubMed Central

Zhou, Li; Wei, Chunsheng; Huang, Wei; Bennett, David A; Dickson, Dennis W; Wang, Rui; Wang, Dengshun

2013-01-01

We investigated the subcellular distribution of NEP protein and activity in brains of human individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI) and AD dementia, as well as double transgenic mice and human neuronal cell line treated with Aβ and 4-hydroxy-2-nonenal (HNE). Total cortical neuronal-related NEP was significantly increased in MCI compared to NCI brains. NeuN was decreased in both MCI and AD, consistent with neuronal loss occurring in MCI and AD. Negative relationship between NEP protein and NeuN in MCI brains, and positive correlation between NEP and pan-cadherin in NCI and MCI brains, suggesting the increased NEP expression in NCI and MCI might be due to membrane associated NEP in non-neuronal cells. In subcellular extracts, NEP protein decreased in cytoplasmic fractions in MCI and AD, but increased in membrane fractions, with a significant increase in the membrane/cytoplasmic ratio of NEP protein in AD brains. By contrast, NEP activity was decreased in AD. Similar results were observed in AD-mimic transgenic mice. Studies of SH-SY5Y neuroblastoma showed an up-regulation of NEP protein in the cytoplasmic compartment induced by HNE and Aβ; however, NEP activity decreased in cytoplasmic fractions. Activity of NEP in membrane fractions increased at 48 hours and then significantly decreased after treatment with HNE and Aβ. The cytoplasmic/membrane ratio of NEP protein increased at 24 hours and then decreased in both HNE and Aβ treated cells. Both HNE and Aβ up-regulate NEP expression, but NEP enzyme activity did not show the same increase, possibly indicating immature cytoplasmic NEP is less active than membrane associated NEP. These observations indicate that modulation of NEP protein levels and its subcellular location influence the net proteolytic activity and this complex association might participate in deficiency of Aβ degradation that is associated with amyloid deposition in AD. PMID:24093058

Membrane microdomains in immunity: glycosphingolipid-enriched domain-mediated innate immune responses.

PubMed

Iwabuchi, Kazuhisa; Nakayama, Hitoshi; Masuda, Hiromi; Kina, Katsunari; Ogawa, Hideoki; Takamori, Kenji

2012-01-01

Over the last 30 years, many studies have indicated that glycosphingolipids (GSLs) expressed on the cell surface may act as binding sites for microorganisms. Based on their physicochemical characteristics, GSLs form membrane microdomains with cholesterol, sphingomyelin, glycosylphosphatidylinositol (GPI)-anchored proteins, and various signaling molecules, and GSL-enriched domains have been shown to be involved in these defense responses. Among the GSLs, lactosylceramide (LacCer, CDw17) can bind to various microorganisms. LacCer is expressed at high levels on the plasma membrane of human neutrophils, and forms membrane microdomains associated with the Src family tyrosine kinase Lyn. LacCer-enriched membrane microdomains mediate superoxide generation, chemotaxis, and non-opsonic phagocytosis. Therefore, LacCer-enriched membrane microdomains are thought to function as pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) expressed on microorganisms. In contrast, several pathogens have developed infection mechanisms using membrane microdomains. In addition, some pathogens have the ability to avoid degradation by escaping from the vacuolar compartment or preventing phagosome maturation, utilizing membrane microdomains, such as LacCer-enriched domains, of host cells. The detailed molecular mechanisms of these membrane microdomain-associated host-pathogen interactions remain to be elucidated. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

Localization of caveolin-1 and c-src in mature and differentiating photoreceptors: raft proteins co-distribute with rhodopsin during development

PubMed Central

Berta, Ágnes I.; Boesze-Battaglia, Kathleen; Magyar, Attila; Szél, Ágoston; Kiss, Anna L.

2014-01-01

Numerous biochemical and morphological studies have provided insight into the distribution pattern of caveolin-1 and the presence of membrane rafts in the vertebrate retina. To date however, studies have not addressed the localization profile of raft specific proteins during development. Therefore the purpose of our studies was to follow the localization pattern of caveolin-1, phosphocaveolin-1 and c-src in the developing retina and compare it to that observed in adults. Specific antibodies were used to visualize the distribution of caveolin-1, c-src, a kinase phosphorylating caveolin-1, and phospho-caveolin-1. The labeling pattern of this scaffolded complex was compared to those of rhodopsin and rhodopsin kinase. Samples were analyzed at various time points during postnatal development and compared to adult retinas. The immunocytochemical studies were complemented with immunoblots and immunoprecipitation studies. In the mature retina caveolin-1 and c-src localized mainly to the cell body and IS of photoreceptors, with only very weakly labeled OS. In contrast, phospho-caveolin-1 was only detectable in the OS of photoreceptors. During development we followed the expression and distribution profile of these proteins in a temporal sequence with special attention to the period when OS formation is most robust. Double labeling immunocytochemistry and immunoprecipitation showed rhodopsin to colocalize and co-immunoprecipitate with caveolin-1 and c-src. Individual punctate structures between the outer limiting membrane and the outer plexiform layer were seen at P10 to be labeled by both rhodopsin and caveolin-1 as well as by rhodopsin and c-src, respectively. These studies suggest that membrane raft specific proteins are co-distributed during development, thereby pointing to a role for such complexes in OS formation. In addition, the presence of small punctate structures containing caveolin-1, c-src and rhodopsin raise the possibility that these proteins may transport together to OS during development and that caveolin-1 exists predominantly in a phosphorylated form in the OS. PMID:21938483

Biomimetic surface patterning for long-term transmembrane access

PubMed Central

VanDersarl, Jules J.; Renaud, Philippe

2016-01-01

Here we present a planar patch clamp chip based on biomimetic cell membrane fusion. This architecture uses nanometer length-scale surface patterning to replicate the structure and function of membrane proteins, creating a gigaohm seal between the cell and a planar electrode array. The seal is generated passively during cell spreading, without the application of a vacuum to the cell surface. This interface can enable cell-attached and whole-cell recordings that are stable to 72 hours, and generates no visible damage to the cell. The electrodes can be very small (<5 μm) and closely packed, offering a high density platform for cellular measurement. PMID:27577519

Biomimetic surface patterning for long-term transmembrane access.

PubMed

VanDersarl, Jules J; Renaud, Philippe

2016-08-31

Here we present a planar patch clamp chip based on biomimetic cell membrane fusion. This architecture uses nanometer length-scale surface patterning to replicate the structure and function of membrane proteins, creating a gigaohm seal between the cell and a planar electrode array. The seal is generated passively during cell spreading, without the application of a vacuum to the cell surface. This interface can enable cell-attached and whole-cell recordings that are stable to 72 hours, and generates no visible damage to the cell. The electrodes can be very small (<5 μm) and closely packed, offering a high density platform for cellular measurement.

MDC9, a widely expressed cellular disintegrin containing cytoplasmic SH3 ligand domains

PubMed Central

1996-01-01

Cellular disintegrins are a family of proteins that are related to snake venom integrin ligands and metalloproteases. We have cloned and sequenced the mouse and human homologue of a widely expressed cellular disintegrin, which we have termed MDC9 (for metalloprotease/disintegrin/cysteine-rich protein 9). The deduced mouse and human protein sequences are 82% identical. MDC9 contains several distinct protein domains: a signal sequence is followed by a prodomain and a domain with sequence similarity to snake venom metalloproteases, a disintegrin domain, a cysteine-rich region, an EGF repeat, a membrane anchor, and a cytoplasmic tail. The cytoplasmic tail of MDC9 has two proline-rich sequences which can bind the SH3 domain of Src, and may therefore function as SH3 ligand domains. Western blot analysis shows that MDC9 is an approximately 84-kD glycoprotein in all mouse tissues examined, and in NIH 3T3 fibroblast and C2C12 myoblast mouse cell lines. MDC9 can be both cell surface biotinylated and 125I-labeled in NIH 3T3 mouse fibroblasts, indicating that the protein is present on the plasma membrane. Expression of MDC9 in COS-7 cells yields an 84-kD protein, and immunofluorescence analysis of COS-7 cells expressing MDC9 shows a staining pattern that is consistent with a plasma membrane localization. The apparent molecular mass of 84 kD suggests that MDC9 contains a membrane-anchored metalloprotease and disintegrin domain. We propose that MDC9 might function as a membrane-anchored integrin ligand or metalloprotease, or that MDC9 may combine both activities in one protein. PMID:8647900

Rapid micropatterning of cell lines and human pluripotent stem cells on elastomeric membranes.

PubMed

Paik, Isha; Scurr, David J; Morris, Bryan; Hall, Graham; Denning, Chris; Alexander, Morgan R; Shakesheff, Kevin M; Dixon, James E

2012-10-01

Tissue function during development and in regenerative medicine completely relies on correct cell organization and patterning at micro and macro scales. We describe a rapid method for patterning mammalian cells including human embryonic stem cells (HESCs) and induced pluripotent stem cells (iPSCs) on elastomeric membranes such that micron-scale control of cell position can be achieved over centimeter-length scales. Our method employs surface engineering of hydrophobic polydimethylsiloxane (PDMS) membranes by plasma polymerization of allylamine. Deposition of plasma polymerized allylamine (ppAAm) using our methods may be spatially restricted using a micro-stencil leaving faithful hydrophilic ppAAm patterns. We employed airbrushing to create aerosols which deposit extracellular matrix (ECM) proteins (such as fibronectin and Matrigel™) onto the same patterned ppAAm rich regions. Cell patterns were created with a variety of well characterized cell lines (e.g., NIH-3T3, C2C12, HL1, BJ6, HESC line HUES7, and HiPSC line IPS2). Individual and multiple cell line patterning were also achieved. Patterning remains faithful for several days and cells are viable and proliferate. To demonstrate the utility of our technique we have patterned cells in a variety of configurations. The ability to rapidly pattern cells at high resolution over macro scales should aid future tissue engineering efforts for regenerative medicine applications and in creating in vitro stem cell niches. Copyright © 2012 Wiley Periodicals, Inc.

Relating proton pumps with gap junctions: colocalization of ductin, the channel-forming subunit c of V-ATPase, with subunit a and with innexins 2 and 3 during Drosophila oogenesis.

PubMed

Lautemann, Julia; Bohrmann, Johannes

2016-07-13

Ion-transport mechanisms and gap junctions are known to cooperate in creating bioelectric phenomena, like pH gradients, voltage gradients and ion fluxes within single cells, tissues, organs, and whole organisms. Such phenomena have been shown to play regulatory roles in a variety of developmental and regenerative processes. Using Drosophila oogenesis as a model system, we aim at characterizing in detail the mechanisms underlying bioelectric phenomena in order to reveal their regulatory functions. We, therefore, investigated the stage-specific distribution patterns of V-ATPase components in relation to gap-junction proteins. We analysed the localization of the V-ATPase components ductin (subunit c) and subunit a, and the gap-junction components innexins 2 and 3, especially in polar cells, border cells, stalk cells and centripetally migrating cells. These types of follicle cells had previously been shown to exhibit characteristic patterns of membrane channels as well as membrane potential and intracellular pH. Stage-specifically, ductin and subunit a were found either colocalized or separately enriched in different regions of soma and germ-line cells. While ductin was often more prominent in plasma membranes, subunit a was more prominent in cytoplasmic and nuclear vesicles. Particularly, ductin was enriched in polar cells, stalk cells, and nurse-cell membranes, whereas subunit a was enriched in the cytoplasm of border cells, columnar follicle cells and germ-line cells. Comparably, ductin and both innexins 2 and 3 were either colocalized or separately enriched in different cellular regions. While ductin often showed a continuous membrane distribution, the distribution of both innexins was mostly punctate. Particularly, ductin was enriched in polar cells and stalk cells, whereas innexin 2 was enriched in the oolemma, and innexin 3 in centripetally migrating follicle cells. In lateral follicle-cell membranes, the three proteins were found colocalized as well as separately concentrated in presumed gap-junction plaques. Our results support the notion of a large variety of gap junctions existing in the Drosophila ovary. Moreover, since ductin is the channel-forming part of a proton pump and, like the innexins, is able to form junctional as well as non-junctional membrane channels, a plethora of cellular functions could be realized by using these proteins. The distribution and activity patterns of such membrane channels are expected to contribute to developmentally important bioelectric signals.

Solubilization methods and reference 2-DE map of cow milk fat globules.

PubMed

Bianchi, Laura; Puglia, Michele; Landi, Claudia; Matteoni, Silvia; Perini, Daniele; Armini, Alessandro; Verani, Margherita; Trombetta, Claudia; Soldani, Patrizia; Roncada, Paola; Greppi, Gianfranco; Pallini, Vitaliano; Bini, Luca

2009-07-21

Milk fat globules (MFGs) are secretory vesicles assembled and secreted by mammary epithelial cells during lactation. They consist of fat globules surrounded by a lipid bilayer membrane which is derived from the apical membrane of the lactating cells. MFGs contain, besides lipids, proteins from the apical plasma membrane and from the cytoplasmatic material. Their peculiar vesicle nature makes them a suitable and easily available source of biological material in monitoring the physiopathological state of the mammary gland. Unfortunately, the conspicuous lipidic component of MFGs consistently limits protein extraction and purification for MFG proteomic investigations. This work deals with the development of a suitable procedure for protein extraction from the cow MFGs in order to qualitatively and quantitatively improve 2-D electropherograms of the MFG. MFGs were purified from raw milk by centrifugation and then delipidated/precipitated. The resulting protein pellets were solubilised using four different 2-D SDS PAGE compatible lysis buffers. Applied methodological procedures for protein extraction and evaluation of the resulting 2-D protein-pattern are presented and discussed. Using these procedures a reference 2-D map of cow milk fat globules is also reported. The majority of the obtained identifications was represented by proteins involved in lipid synthesis or in fat globule secretion.

Food proteins and maturation of small intestinal microvillus membranes (MVM). III. Food protein binding and MVM proteins in rats from newborn to young adult age.

PubMed

Stern, M; Gellermann, B; Wieser, H

1990-10-01

To investigate postnatal maturational profiles of functional and biochemical properties of rat small intestinal microvillus membranes (MVM), we did a longitudinal study in rats from birth to the age of 12 weeks. In parallel, we studied binding of cow's milk proteins and of the wheat gliadin peptide B 3142, as well as MVM proteins (SDS-PAGE). Changes in MVM fluidity and lipid composition exhibited early (0-4 weeks) and intermediate and late (6-12 weeks) patterns, as has been published earlier. Postnatal changes of food protein and peptide binding occurred early during the observation period, not related to weaning. There was not much further change in binding after 6-8 weeks. Developmental profiles of MVM protein and some lipid changes resembled, but did not equal, changes in food protein binding. We conclude that changes in MVM biochemical composition affect MVM binding characteristics. In particular, high molecular weight MVM proteins (susceptible to trypsin treatment) appear to play a role in postnatal maturational differences in MVM food protein binding.

Subcellular localization and expression pattern of the neurofibromatosis type 2 protein merlin/schwannomin.

PubMed

Schmucker, B; Ballhausen, W G; Kressel, M

1997-01-01

To elucidate the physiological function of the neurofibromatosis type 2 (NF2) tumor suppressor protein merlin/schwannomin, we studied the expression pattern and subcellular localization in human fibroblasts by Western blot analyses and immunofluorescence using a polyclonal antibody raised against the C-terminus of merlin. Three of the six merlin isoforms identified in this study (75 kDa, 58 kDa, 45 kDa) have been reported earlier and can be explained by alternative splicing. In addition, we detected higher molecular weight bands of about 110 kDa, 100 kDa and 84 kDa. Although the merlin bands of 100 kDa and 110 kDa may represent homo- or heterodimers, oligomerization due to formation of disulfide bonds was excluded. Furthermore, the isoforms of 84 kDa and 58 kDa were quantitatively extractable in Lubrol WX, indicating a localization in or close to the plasma membrane. The 45 kDa band, however, was not soluble in Lubrol WX compatible with a localization of this NF2 isoform in the endoplasmic reticulum. Applying confocal laser scanning microscopy, merlin was shown to be located in four subcellular compartments: (i) perinuclear in a compartment resembling endoplasmic reticulum, (ii) in ruffling membranes and at the leading edges, (iii) in filopodia, and (iv) at cell/substrate adhesion points. Codistribution of merlin and F-actin filaments was found in filopodia, ruffling membranes and at the insertion points of stress fibers at cell/substrate adhesion junctions as shown by phalloidin-rhodamine staining. Double immunofluorescence analyses of merlin and moesin revealed a colocalization in filopodia and ruffling membranes. The localization of merlin in the actin-rich cortical cytoskeleton corresponds to the ezrin-radixin-moesin family of proteins suggesting the NF2 protein to contribute to the regulation of cell growth by interaction with cytoskeleton-associated proteins.

The solubilisation of boar sperm membranes by different detergents - a microscopic, MALDI-TOF MS, 31P NMR and PAGE study on membrane lysis, extraction efficiency, lipid and protein composition

PubMed Central

2009-01-01

Background Detergents are often used to isolate proteins, lipids as well as "detergent-resistant membrane domains" (DRMs) from cells. Different detergents affect different membrane structures according to their physico-chemical properties. However, the effects of different detergents on membrane lysis of boar spermatozoa and the lipid composition of DRMs prepared from the affected sperm membranes have not been investigated so far. Results Spermatozoa were treated with the selected detergents Pluronic F-127, sodium cholate, CHAPS, Tween 20, Triton X-100 and Brij 96V. Different patterns of membrane disintegration were observed by light and electron microscopy. In accordance with microscopic data, different amounts of lipids and proteins were released from the cells by the different detergents. The biochemical methods to assay the phosphorus and cholesterol contents as well as 31P NMR to determine the phospholipids were not influenced by the presence of detergents since comparable amounts of lipids were detected in the organic extracts from whole cell suspensions after exposure to each detergent. However, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry applied to identify phospholipids was essentially disturbed by the presence of detergents which exerted particular suppression effects on signal intensities. After separation of the membrane fractions released by detergents on a sucrose gradient only Triton X-100 and sodium cholate produced sharp turbid DRM bands. Only membrane solubilisation by Triton X-100 leads to an enrichment of cholesterol, sphingomyelin, phosphatidylinositol and phosphatidylethanolamine in a visible DRM band accompanied by a selective accumulation of proteins. Conclusion The boar sperm membranes are solubilised to a different extent by the used detergents. Particularly, the very unique DRMs isolated after Triton X-100 exposure are interesting candidates for further studies regarding the architecture of sperm. PMID:19906304

Sulfation of Eggshell Proteins by Pipe Defines Dorsal-Ventral Polarity in the Drosophila embryo

PubMed Central

Zhang, Zhenyu; Stevens, Leslie M.; Stein, David

2009-01-01

Summary Drosophila embryonic dorsal-ventral (DV) polarity is controlled by a group of sequentially acting serine proteases located in the fluid-filled perivitelline space between the embryonic membrane and the eggshell, which generate the ligand for the Toll receptor on the ventral side of the embryo [1, 2, 3]. Spatial control of the protease cascade relies on the Pipe sulfotransferase, a fly homologue of vertebrate glycosaminoglycan modifying enzymes [4, 5, 6], which is expressed in ventral cells of the follicular epithelium surrounding the developing oocyte. The identification of the Pipe enzymatic target has remained a major gap in our understanding of the mechanism controlling the perivitelline protease cascade, and hence embryonic DV patterning. Here we show that the protein Vitelline Membrane-Like (VML) [7] undergoes Pipe-dependent sulfation and, consistent with a role in conveying positional information from the egg chamber to the embryo, becomes incorporated into the eggshell at a position corresponding to the location of the follicle cells from which it was secreted. Although VML influences embryonic DV pattern in a sensitized genetic background, VML is not essential for DV axis formation, suggesting that there is redundancy in the composition of the Pipe enzymatic target. Correspondingly, we find that additional structural components of the vitelline membrane undergo Pipe-dependent sulfation. In identifying the elusive targets of Pipe, this ork points to the vitelline membrane as the source of signals that generate the Drosophila DV axis and provides a framework for understanding the mechanism controlling spatially-specific activation of serine protease activity during embryonic pattern formation. PMID:19540119

Sphingolipid hydroxylation in mammals, yeast and plants - An integrated view.

PubMed

Marquês, Joaquim Trigo; Susana Marinho, H; de Almeida, Rodrigo Freire Martins

2018-05-07

This review is focused on sphingolipid backbone hydroxylation, a small but widespread structural feature, with profound impact on membrane biophysical properties. We start by summarizing sphingolipid metabolism in mammalian cells, yeast and plants, focusing on how distinct hydroxylation patterns emerge in different eukaryotic kingdoms. Then, a comparison of the biophysical properties in membrane model systems and cellular membranes from diverse organisms is made. From an integrative perspective, these results can be rationalized considering that superficial hydroxyl groups in the backbone of sphingolipids (by intervening in the H-bond network) alter the balance of favorable interactions between membrane lipids. They may strengthen the bonding or compete with other hydroxyl groups, in particular the one of membrane sterols. Different sphingolipid hydroxylation patterns can stabilize/disrupt specific membrane domains or change whole plasma membrane properties, and therefore be important in the control of protein distribution, function and lateral diffusion and in the formation and overtime stability of signaling platforms. The recent examples explored throughout this review unveil a potentially key role for sphingolipid backbone hydroxylation in both physiological and pathological situations, as they can be of extreme importance for the proper organization of cell membranes in mammalian cells, yeast and, most likely, also in plants. Copyright © 2017. Published by Elsevier Ltd.

Expression of bovine non-classical major histocompatibility complex class I proteins in mouse P815 and human K562 cells.

PubMed

Parasar, Parveen; Wilhelm, Amanda; Rutigliano, Heloisa M; Thomas, Aaron J; Teng, Lihong; Shi, Bi; Davis, William C; Suarez, Carlos E; New, Daniel D; White, Kenneth L; Davies, Christopher J

2016-08-01

Major histocompatibility complex class I (MHC-I) proteins can be expressed as cell surface or secreted proteins. To investigate whether bovine non-classical MHC-I proteins are expressed as cell surface or secreted proteins, and to assess the reactivity pattern of monoclonal antibodies with non-classical MHC-I isoforms, we expressed the MHC proteins in murine P815 and human K562 (MHC-I deficient) cells. Following antibiotic selection, stably transfected cell lines were stained with H1A or W6/32 antibodies to detect expression of the MHC-I proteins by flow cytometry. Two non-classical proteins (BoLA-NC1*00501 and BoLA-NC3*00101) were expressed on the cell surface in both cell lines. Surprisingly, the BoLA-NC4*00201 protein was expressed on the cell membrane of human K562 but not mouse P815 cells. Two non-classical proteins (BoLA-NC1*00401, which lacks a transmembrane domain, and BoLA-NC2*00102) did not exhibit cell surface expression. Nevertheless, Western blot analyses demonstrated expression of the MHC-I heavy chain in all transfected cell lines. Ammonium-sulfate precipitation of proteins from culture supernatants showed that BoLA-NC1*00401 was secreted and that all surface expressed proteins where shed from the cell membrane by the transfected cells. Interestingly, the surface expressed MHC-I proteins were present in culture supernatants at a much higher concentration than BoLA-NC1*00401. This comprehensive study shows that bovine non-classical MHC-I proteins BoLA-NC1*00501, BoLA-NC3*00101, and BoLA-NC4*00201 are expressed as surface isoforms with the latter reaching the cell membrane only in K562 cells. Furthermore, it demonstrated that BoLA-NC1*00401 is a secreted isoform and that significant quantities of membrane associated MHC-I proteins can be shed from the cell membrane. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Characterization of a Biomimetic Mesophase Composed of Nonionic Surfactants and an Aqueous Solvent.

PubMed

Adrien, V; Rayan, G; Reffay, M; Porcar, L; Maldonado, A; Ducruix, A; Urbach, W; Taulier, N

2016-10-11

We have investigated the physical and biomimetic properties of a sponge (L 3 ) phase composed of pentaethylene glycol monododecyl ether (C 12 E 5 ), a nonionic surfactant, an aqueous solvent, and a cosurfactant. The following cosurfactants, commonly used for solubilizing membrane proteins, were incorporated: n-octyl-β-d-glucopyranoside (β-OG), n-dodecyl-β-d-maltopyranoside (DDM), 4-cyclohexyl-1-butyl-β-d-maltoside (CYMAL-4), and 5-cyclohexyl-1-pentyl-β-d-maltoside (CYMAL-5). Partial phase diagrams of these systems were created. The L 3 phase was characterized using crossed polarizers, diffusion of a fluorescent probe by fluorescence recovery after pattern photobleaching (FRAPP), and freeze fracture electron microscopy (FFEM). By varying the hydration of the phase, we were able to tune the distance between adjacent bilayers. The characteristic distance (d b ) of the phase was obtained from small angle scattering (SAXS/SANS) as well as from FFEM, which yielded complementary d b values. These d b values were neither affected by the nature of the cosurfactant nor by the addition of membrane proteins. These findings illustrate that a biomimetic surfactant sponge phase can be created in the presence of several common membrane protein-solubilizing detergents, thus making it a versatile medium for membrane protein studies.

Complex regulation of Arabidopsis AGR1/PIN2-mediated root gravitropic response and basipetal auxin transport by cantharidin-sensitive protein phosphatases

NASA Technical Reports Server (NTRS)

Shin, Heungsop; Shin, Hwa-Soo; Guo, Zibiao; Blancaflor, Elison B.; Masson, Patrick H.; Chen, Rujin

2005-01-01

Polar auxin transport, mediated by two distinct plasma membrane-localized auxin influx and efflux carrier proteins/complexes, plays an important role in many plant growth and developmental processes including tropic responses to gravity and light, development of lateral roots and patterning in embryogenesis. We have previously shown that the Arabidopsis AGRAVITROPIC 1/PIN2 gene encodes an auxin efflux component regulating root gravitropism and basipetal auxin transport. However, the regulatory mechanism underlying the function of AGR1/PIN2 is largely unknown. Recently, protein phosphorylation and dephosphorylation mediated by protein kinases and phosphatases, respectively, have been implicated in regulating polar auxin transport and root gravitropism. Here, we examined the effects of chemical inhibitors of protein phosphatases on root gravitropism and basipetal auxin transport, as well as the expression pattern of AGR1/PIN2 gene and the localization of AGR1/PIN2 protein. We also examined the effects of inhibitors of vesicle trafficking and protein kinases. Our data suggest that protein phosphatases, sensitive to cantharidin and okadaic acid, are likely involved in regulating AGR1/PIN2-mediated root basipetal auxin transport and gravitropism, as well as auxin response in the root central elongation zone (CEZ). BFA-sensitive vesicle trafficking may be required for the cycling of AGR1/PIN2 between plasma membrane and the BFA compartment, but not for the AGR1/PIN2-mediated root basipetal auxin transport and auxin response in CEZ cells.

Convergent and Divergent Signaling in PAMP-Triggered Immunity and Effector-Triggered Immunity.

PubMed

Peng, Yujun; van Wersch, Rowan; Zhang, Yuelin

2018-04-01

Plants use diverse immune receptors to sense pathogen attacks. Recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors localized on the plasma membrane leads to PAMP-triggered immunity (PTI). Detection of pathogen effectors by intracellular or plasma membrane-localized immune receptors results in effector-triggered immunity (ETI). Despite the large variations in the magnitude and duration of immune responses triggered by different PAMPs or pathogen effectors during PTI and ETI, plasma membrane-localized immune receptors activate similar downstream molecular events such as mitogen-activated protein kinase activation, oxidative burst, ion influx, and increased biosynthesis of plant defense hormones, indicating that defense signals initiated at the plasma membrane converge at later points. On the other hand, activation of ETI by immune receptors localized to the nucleus appears to be more directly associated with transcriptional regulation of defense gene expression. Here, we review recent progress in signal transductions downstream of different groups of plant immune receptors, highlighting the converging and diverging molecular events.

Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis

PubMed Central

Carroll, James A.; Olano, L. Rennee; Sturdevant, Daniel E.; Rosa, Patricia A.

2015-01-01

The saprophyte Leptospira biflexa is an excellent model for studying the physiology of the medically important Leptospira genus, the pathogenic members of which are more recalcitrant to genetic manipulation and have significantly slower in vitro growth. However, relatively little is known regarding the proteome of L. biflexa, limiting its utility as a model for some studies. Therefore, we have generated a proteomic map of both soluble and membrane-associated proteins of L. biflexa during exponential growth and in stationary phase. Using these data, we identified abundantly produced proteins in each cellular fraction and quantified the transcript levels from a subset of these genes using quantitative reverse transcription-PCR (RT-PCR). These proteins should prove useful as cellular markers and as controls for gene expression studies. We also observed a significant number of L. biflexa membrane-associated proteins with multiple isoforms, each having unique isoelectric focusing points. L. biflexa cell lysates were examined for several posttranslational modifications suggested by the protein patterns. Methylation and acetylation of lysine residues were predominately observed in the proteins of the membrane-associated fraction, while phosphorylation was detected mainly among soluble proteins. These three posttranslational modification systems appear to be conserved between the free-living species L. biflexa and the pathogenic species Leptospira interrogans, suggesting an important physiological advantage despite the varied life cycles of the different species. PMID:26655756

Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis.

PubMed

Stewart, Philip E; Carroll, James A; Olano, L Rennee; Sturdevant, Daniel E; Rosa, Patricia A

2016-02-15

The saprophyte Leptospira biflexa is an excellent model for studying the physiology of the medically important Leptospira genus, the pathogenic members of which are more recalcitrant to genetic manipulation and have significantly slower in vitro growth. However, relatively little is known regarding the proteome of L. biflexa, limiting its utility as a model for some studies. Therefore, we have generated a proteomic map of both soluble and membrane-associated proteins of L. biflexa during exponential growth and in stationary phase. Using these data, we identified abundantly produced proteins in each cellular fraction and quantified the transcript levels from a subset of these genes using quantitative reverse transcription-PCR (RT-PCR). These proteins should prove useful as cellular markers and as controls for gene expression studies. We also observed a significant number of L. biflexa membrane-associated proteins with multiple isoforms, each having unique isoelectric focusing points. L. biflexa cell lysates were examined for several posttranslational modifications suggested by the protein patterns. Methylation and acetylation of lysine residues were predominately observed in the proteins of the membrane-associated fraction, while phosphorylation was detected mainly among soluble proteins. These three posttranslational modification systems appear to be conserved between the free-living species L. biflexa and the pathogenic species Leptospira interrogans, suggesting an important physiological advantage despite the varied life cycles of the different species. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Glucose-independent inhibition of yeast plasma-membrane H+-ATPase by calmodulin antagonists.

PubMed

Romero, I; Maldonado, A M; Eraso, P

1997-03-15

Glucose metabolism causes activation of the yeast plasma-membrane H+-ATPase. The molecular mechanism of this regulation is not known, but it is probably mediated by phosphorylation of the enzyme. The involvement in this process of several kinases has been suggested but their actual role has not been proved. The physiological role of a calmodulin-dependent protein kinase in glucose-induced activation was investigated by studying the effect of specific calmodulin antagonists on the glucose-induced ATPase kinetic changes in wild-type and two mutant strains affected in the glucose regulation of the enzyme. Preincubation of the cells with calmidazolium or compound 48/80 impeded the increase in ATPase activity by reducing the Vmax of the enzyme without modifying the apparent affinity for ATP in the three strains. In one mutant, pma1-T912A, the putative calmodulin-dependent protein kinase-phosphorylatable Thr-912 was eliminated, and in the other, pma1-P536L, H+-ATPase was constitutively activated, suggesting that the antagonistic effect was not mediated by a calmodulin-dependent protein kinase and not related to glucose regulation. This was corroborated when the in vitro effect of the calmodulin antagonists on H+-ATPase activity was tested. Purified plasma membranes from glucose-starved or glucose-fermenting cells from both pma1-P890X, another constitutively activated ATPase mutant, and wild-type strains were preincubated with calmidazolium or melittin. In all cases, ATP hydrolysis was inhibited with an IC50 of approximately 1 microM. This inhibition was reversed by calmodulin. Analysis of the calmodulin-binding protein pattern in the plasma-membrane fraction eliminates ATPase as the calmodulin target protein. We conclude that H+-ATPase inhibition by calmodulin antagonists is mediated by an as yet unidentified calmodulin-dependent membrane protein.

The bovine seminal plasma protein PDC-109 extracts phosphorylcholine-containing lipids from the outer membrane leaflet.

PubMed

Tannert, Astrid; Kurz, Anke; Erlemann, Karl-Rudolf; Müller, Karin; Herrmann, Andreas; Schiller, Jürgen; Töpfer-Petersen, Edda; Manjunath, Puttaswamy; Müller, Peter

2007-04-01

The bovine seminal plasma protein PDC-109 modulates the maturation of bull sperm cells by removing lipids, mainly phosphatidylcholine and cholesterol, from their cellular membrane. Here, we have characterized the process of extraction of endogenous phospholipids and of their respective analogues. By measuring the PDC-109-mediated release of fluorescent phospholipid analogues from lipid vesicles and from biological membranes (human erythrocytes, bovine epididymal sperm cells), we showed that PDC-109 extracts phospholipids with a phosphorylcholine headgroup mainly from the outer leaflet of these membranes. The ability of PDC-109 to extract endogenous phospholipids from epididymal sperm cells was followed by mass spectrometry, which allowed us to characterize the fatty acid pattern of the released lipids. From these cells, PDC-109 extracted phosphatidylcholine and sphingomyelin that contained an enrichment of mono- and di-unsaturated fatty acids as well as short-chain and lyso-phosphatidylcholine species. Based on the results, a model explaining the phospholipid specificity of PDC-109-mediated lipid release is presented.

Genome-wide identification, characterization, and expression profile of aquaporin gene family in flax (Linum usitatissimum)

PubMed Central

Shivaraj, S. M.; Deshmukh, Rupesh K.; Rai, Rhitu; Bélanger, Richard; Agrawal, Pawan K.; Dash, Prasanta K.

2017-01-01

Membrane intrinsic proteins (MIPs) form transmembrane channels and facilitate transport of myriad substrates across the cell membrane in many organisms. Majority of plant MIPs have water transporting ability and are commonly referred as aquaporins (AQPs). In the present study, we identified aquaporin coding genes in flax by genome-wide analysis, their structure, function and expression pattern by pan-genome exploration. Cross-genera phylogenetic analysis with known aquaporins from rice, arabidopsis, and poplar showed five subgroups of flax aquaporins representing 16 plasma membrane intrinsic proteins (PIPs), 17 tonoplast intrinsic proteins (TIPs), 13 NOD26-like intrinsic proteins (NIPs), 2 small basic intrinsic proteins (SIPs), and 3 uncharacterized intrinsic proteins (XIPs). Amongst aquaporins, PIPs contained hydrophilic aromatic arginine (ar/R) selective filter but TIP, NIP, SIP and XIP subfamilies mostly contained hydrophobic ar/R selective filter. Analysis of RNA-seq and microarray data revealed high expression of PIPs in multiple tissues, low expression of NIPs, and seed specific expression of TIP3 in flax. Exploration of aquaporin homologs in three closely related Linum species bienne, grandiflorum and leonii revealed presence of 49, 39 and 19 AQPs, respectively. The genome-wide identification of aquaporins, first in flax, provides insight to elucidate their physiological and developmental roles in flax. PMID:28447607

Genome-wide identification, characterization, and expression profile of aquaporin gene family in flax (Linum usitatissimum).

PubMed

Shivaraj, S M; Deshmukh, Rupesh K; Rai, Rhitu; Bélanger, Richard; Agrawal, Pawan K; Dash, Prasanta K

2017-04-27

Membrane intrinsic proteins (MIPs) form transmembrane channels and facilitate transport of myriad substrates across the cell membrane in many organisms. Majority of plant MIPs have water transporting ability and are commonly referred as aquaporins (AQPs). In the present study, we identified aquaporin coding genes in flax by genome-wide analysis, their structure, function and expression pattern by pan-genome exploration. Cross-genera phylogenetic analysis with known aquaporins from rice, arabidopsis, and poplar showed five subgroups of flax aquaporins representing 16 plasma membrane intrinsic proteins (PIPs), 17 tonoplast intrinsic proteins (TIPs), 13 NOD26-like intrinsic proteins (NIPs), 2 small basic intrinsic proteins (SIPs), and 3 uncharacterized intrinsic proteins (XIPs). Amongst aquaporins, PIPs contained hydrophilic aromatic arginine (ar/R) selective filter but TIP, NIP, SIP and XIP subfamilies mostly contained hydrophobic ar/R selective filter. Analysis of RNA-seq and microarray data revealed high expression of PIPs in multiple tissues, low expression of NIPs, and seed specific expression of TIP3 in flax. Exploration of aquaporin homologs in three closely related Linum species bienne, grandiflorum and leonii revealed presence of 49, 39 and 19 AQPs, respectively. The genome-wide identification of aquaporins, first in flax, provides insight to elucidate their physiological and developmental roles in flax.

Guided by curvature: shaping cells by coupling curved membrane proteins and cytoskeletal forces.

PubMed

Gov, N S

2018-05-26

Eukaryote cells have flexible membranes that allow them to have a variety of dynamical shapes. The shapes of the cells serve important biological functions, both for cells within an intact tissue, and during embryogenesis and cellular motility. How cells control their shapes and the structures that they form on their surface has been a subject of intensive biological research, exposing the building blocks that cells use to deform their membranes. These processes have also drawn the interest of theoretical physicists, aiming to develop models based on physics, chemistry and nonlinear dynamics. Such models explore quantitatively different possible mechanisms that the cells can employ to initiate the spontaneous formation of shapes and patterns on their membranes. We review here theoretical work where one such class of mechanisms was investigated: the coupling between curved membrane proteins, and the cytoskeletal forces that they recruit. Theory indicates that this coupling gives rise to a rich variety of membrane shapes and dynamics, while experiments indicate that this mechanism appears to drive many cellular shape changes.This article is part of the theme issue 'Self-organization in cell biology'. © 2018 The Author(s).

Choice of modality with the use of high-performance membrane and evaluation for clinical effects.

PubMed

Masakane, Ikuto

2011-01-01

The golden target for dialysis therapy should be to guarantee longer survival and to give a higher quality of life without dialysis-related complications. In order to achieve this target, the choice of dialysis modality and membrane is essential but we have not yet established what the best choice for a dialysis modality and membrane are. Generally, we choose a dialysis modality for better solute removal and better biocompatibility. In this issue we would like to propose that the patients' preference for dialysis therapy is a useful parameter in prescribing the dialysis modality. In our recent experience, chronic dialysis patients have had preferences on a dialysis modality and membrane, those being PMMA, EVAL, AN-69 and pre-dilution online HDF. These modalities could relieve them of uncomfortable dialysis-related symptoms such as insomnia, itchiness, irritability, and so on. Other characteristics of these modalities are of a nutritional advantage, a broad removal pattern of uremic toxins including low-molecular-weight protein and protein-bound uremic toxins, and good biocompatibility free from chemical components of dialysis membrane. In conclusion, patients' symptoms could be a useful parameter to choose a dialysis modality and membrane. Copyright © 2011 S. Karger AG, Basel.

Matrix-specific protein kinase A signaling regulates p21 activated kinase activation by flow in endothelial cells

PubMed Central

Funk, Steven Daniel; Yurdagul, Arif; Green, Jonette M.; Jhaveri, Krishna A.; Schwartz, Martin Alexander; Orr, A. Wayne

2010-01-01

Rationale Atherosclerosis is initiated by blood flow patterns that activate inflammatory pathways in endothelial cells. Activation of inflammatory signaling by fluid shear stress is highly dependent on the composition of the subendothelial extracellular matrix. The basement membrane proteins laminin and collagen found in normal vessels suppress flow-induced p21 activated kinase (PAK) and NF-κB activation. By contrast, the provisional matrix proteins fibronectin and fibrinogen found in wounded or inflamed vessels support flow-induced PAK and NF-κB activation. PAK mediates both flow-induced permeability and matrix-specific activation of NF-κB. Objective To elucidate the mechanisms regulating matrix-specific PAK activation. Methods and Results We now show that matrix composition does not affect the upstream pathway by which flow activates PAK (integrin activation, Rac). Instead basement membrane proteins enhance flow-induced protein kinase A (PKA) activation, which suppresses PAK. Inhibiting PKA restored flow-induced PAK and NF-κB activation in cells on basement membrane proteins, whereas stimulating PKA inhibited flow-induced activation of inflammatory signaling in cells on fibronectin. PKA suppressed inflammatory signaling through PAK inhibition. Activating PKA by injection of the PGI2 analog iloprost reduced PAK activation and inflammatory gene expression at sites of disturbed flow in vivo, whereas inhibiting PKA by PKI injection enhanced PAK activation and inflammatory gene expression. Inhibiting PAK prevented the enhancement of inflammatory gene expression by PKI. Conclusions Basement membrane proteins inhibit inflammatory signaling in endothelial cells via PKA-dependent inhibition of PAK. PMID:20224042

Cell-free system for synthesizing membrane proteins cell free method for synthesizing membrane proteins

DOEpatents

Laible, Philip D; Hanson, Deborah K

2013-06-04

The invention provides an in vitro method for producing proteins, membrane proteins, membrane-associated proteins, and soluble proteins that interact with membrane-associated proteins for assembly into an oligomeric complex or that require association with a membrane for proper folding. The method comprises, supplying intracytoplasmic membranes from organisms; modifying protein composition of intracytoplasmic membranes from organism by modifying DNA to delete genes encoding functions of the organism not associated with the formation of the intracytoplasmic membranes; generating appropriate DNA or RNA templates that encode the target protein; and mixing the intracytoplasmic membranes with the template and a transcription/translation-competent cellular extract to cause simultaneous production of the membrane proteins and encapsulation of the membrane proteins within the intracytoplasmic membranes.

Plasmodium knowlesi Skeleton-Binding Protein 1 Localizes to the 'Sinton and Mulligan' Stipplings in the Cytoplasm of Monkey and Human Erythrocytes.

PubMed

Lucky, Amuza Byaruhanga; Sakaguchi, Miako; Katakai, Yuko; Kawai, Satoru; Yahata, Kazuhide; Templeton, Thomas J; Kaneko, Osamu

2016-01-01

The malaria parasite, Plasmodium, exports protein products to the infected erythrocyte to introduce modifications necessary for the establishment of nutrient acquisition and surface display of host interaction ligands. Erythrocyte remodeling impacts parasite virulence and disease pathology and is well documented for the human malaria parasite Plasmodium falciparum, but has been less described for other Plasmodium species. For P. falciparum, the exported protein skeleton-binding protein 1 (PfSBP1) is involved in the trafficking of erythrocyte surface ligands and localized to membranous structures within the infected erythrocyte, termed Maurer's clefts. In this study, we analyzed SBP1 orthologs across the Plasmodium genus by BLAST analysis and conserved gene synteny, which were also recently described by de Niz et al. (2016). To evaluate the localization of an SBP1 ortholog, we utilized the zoonotic malaria parasite, Plasmodium knowlesi. Immunofluorescence assay of transgenic P. knowlesi parasites expressing epitope-tagged recombinant PkSBP1 revealed a punctate staining pattern reminiscent of Maurer's clefts, following infection of either monkey or human erythrocytes. The recombinant PkSBP1-positive puncta co-localized with Giemsa-stained structures, known as 'Sinton and Mulligan' stipplings. Immunoelectron microscopy also showed that recombinant PkSBP1 localizes within or on the membranous structures akin to the Maurer's clefts. The recombinant PkSBP1 expressed in P. falciparum-infected erythrocytes co-localized with PfSBP1 at the Maurer's clefts, indicating an analogous trafficking pattern. A member of the P. knowlesi 2TM protein family was also expressed and localized to membranous structures in infected monkey erythrocytes. These results suggest that the trafficking machinery and induced erythrocyte cellular structures of P. knowlesi are similar following infection of both monkey and human erythrocytes, and are conserved with P. falciparum.

Speed Controls in Translating Secretory Proteins in Eukaryotes - an Evolutionary Perspective

PubMed Central

Mahlab, Shelly; Linial, Michal

2014-01-01

Protein translation is the most expensive operation in dividing cells from bacteria to humans. Therefore, managing the speed and allocation of resources is subject to tight control. From bacteria to humans, clusters of relatively rare tRNA codons at the N′-terminal of mRNAs have been implicated in attenuating the process of ribosome allocation, and consequently the translation rate in a broad range of organisms. The current interpretation of “slow” tRNA codons does not distinguish between protein translations mediated by free- or endoplasmic reticulum (ER)-bound ribosomes. We demonstrate that proteins translated by free- or ER-bound ribosomes exhibit different overall properties in terms of their translation efficiency and speed in yeast, fly, plant, worm, bovine and human. We note that only secreted or membranous proteins with a Signal peptide (SP) are specified by segments of “slow” tRNA at the N′-terminal, followed by abundant codons that are considered “fast.” Such profiles apply to 3100 proteins of the human proteome that are composed of secreted and signal peptide (SP)-assisted membranous proteins. Remarkably, the bulks of the proteins (12,000), or membranous proteins lacking SP (3400), do not have such a pattern. Alternation of “fast” and “slow” codons was found also in proteins that translocate to mitochondria through transit peptides (TP). The differential clusters of tRNA adapted codons is not restricted to the N′-terminal of transcripts. Specifically, Glycosylphosphatidylinositol (GPI)-anchored proteins are unified by clusters of low adapted tRNAs codons at the C′-termini. Furthermore, selection of amino acids types and specific codons was shown as the driving force which establishes the translation demands for the secretory proteome. We postulate that “hard-coded” signals within the secretory proteome assist the steps of protein maturation and folding. Specifically, “speed control” signals for delaying the translation of a nascent protein fulfill the co- and post-translational stages such as membrane translocation, proteins processing and folding. PMID:24391480

Highly stable and self-repairing membrane-mimetic 2D nanomaterials assembled from lipid-like peptoids

PubMed Central

Jin, Haibao; Jiao, Fang; Daily, Michael D.; Chen, Yulin; Yan, Feng; Ding, Yan-Huai; Zhang, Xin; Robertson, Ellen J.; Baer, Marcel D.; Chen, Chun-Long

2016-01-01

An ability to develop sequence-defined synthetic polymers that both mimic lipid amphiphilicity for self-assembly of highly stable membrane-mimetic 2D nanomaterials and exhibit protein-like functionality would revolutionize the development of biomimetic membranes. Here we report the assembly of lipid-like peptoids into highly stable, crystalline, free-standing and self-repairing membrane-mimetic 2D nanomaterials through a facile crystallization process. Both experimental and molecular dynamics simulation results show that peptoids assemble into membranes through an anisotropic formation process. We further demonstrated the use of peptoid membranes as a robust platform to incorporate and pattern functional objects through large side-chain diversity and/or co-crystallization approaches. Similar to lipid membranes, peptoid membranes exhibit changes in thickness upon exposure to external stimuli; they can coat surfaces in single layers and self-repair. We anticipate that this new class of membrane-mimetic 2D nanomaterials will provide a robust matrix for development of biomimetic membranes tailored to specific applications. PMID:27402325

Molecular self-assembly for biological investigations and nanoscale lithography

NASA Astrophysics Data System (ADS)

Cheunkar, Sarawut

Small, diffusible molecules when recognized by their binding partners, such as proteins and antibodies, trigger enzymatic activity, cell communication, and immune response. Progress in analytical methods enabling detection, characterization, and visualization of biological dynamics at the molecular level will advance our exploration of complex biological systems. In this dissertation, analytical platforms were fabricated to capture membrane-associated receptors, which are essential proteins in cell signaling pathways. The neurotransmitter serotonin and its biological precursor were immobilized on gold substrates coated with self-assembled monolayers (SAMs) of oligo(ethylene glycol)alkanethiols and their reactive derivatives. The SAM-coated substrates present the biologically selective affinity of immobilized molecules to target native membrane-associated receptors. These substrates were also tested for biospecificity using antibodies. In addition, small-molecule-functionalized platforms, expressing neurotransmitter pharmacophores, were employed to examine kinetic interactions between G-protein-coupled receptors and their associated neurotransmitters. The binding interactions were monitored using a quartz crystal microbalance equipped with liquid-flow injection. The interaction kinetics of G-protein-coupled serotonin 1A receptor and 5-hydroxytyptophan-functionalized surfaces were studied in a real-time, label-free environment. Key binding parameters, such as equilibrium dissociation constants, binding rate constants, and dissociative half-life, were extracted. These parameters are critical for understanding and comparing biomolecular interactions in modern biomedical research. By integrating self-assembly, surface functionalization, and nanofabrication, small-molecule microarrays were created for high-throughput screening. A hybrid soft-lithography, called microcontact insertion printing, was used to pattern small molecules at the dilute scales necessary for highly selective biorecognition. By carefully tuning the polar surface energy of polymeric stamps, problems associated with patterning hydrophilic tether molecules inserted into hydrophilic preformed SAMs are surmounted. The patterned substrates presenting neurotransmitter precursors selectively capture membrane-associated receptors. These advances provide new avenues for fabricating small-molecule arrays. Furthermore, a novel strategy based on a conventional microcontact printing, called chemical lift-off lithography, was invented to overcome the micrometer-scale resolution limits of molecular ink diffusion in soft lithography. Self-assembled monolayers of hydroxyl-terminated alkanethiols, preformed on gold substrates, were selectively removed by oxygen-plasma-treated polymeric stamps in a subtractive stamping process with high pattern fidelity. The covalent interactions formed at the stamp-substrate interface are believed to be responsible for removing not only alkanethiol molecules but also a monolayer of gold atoms from the substrates. A variety of high-resolution patterned features were fabricated, and stamps were cleaned and reused many times without feature deterioration. The remaining SAMs acted as resists for etching exposed gold features. Monolayer backfilling into lifted-off areas enabled patterned protein capture, and 40-nanometer chemical patterns were achieved.

Structures formed by a cell membrane-associated arabinogalactan-protein on graphite or mica alone and with Yariv phenylglycosides

PubMed Central

Zhou, Li Hong; Weizbauer, Renate A.; Singamaneni, Srikanth; Xu, Feng; Genin, Guy M.; Pickard, Barbara G.

2014-01-01

Background Certain membrane-associated arabinogalactan-proteins (AGPs) with lysine-rich sub-domains participate in plant growth, development and resistance to stress. To complement fluorescence imaging of such molecules when tagged and introduced transgenically to the cell periphery and to extend the groundwork for assessing molecular structure, some behaviours of surface-spread AGPs were visualized at the nanometre scale in a simplified electrostatic environment. Methods Enhanced green fluorescent protein (EGFP)-labelled LeAGP1 was isolated from Arabidopsis thaliana leaves using antibody-coated magnetic beads, deposited on graphite or mica, and examined with atomic force microscopy (AFM). Key Results When deposited at low concentration on graphite, LeAGP can form independent clusters and rings a few nanometres in diameter, often defining deep pits; the aperture of the rings depends on plating parameters. On mica, intermediate and high concentrations, respectively, yielded lacy meshes and solid sheets that could dynamically evolve arcs, rings, ‘pores’ and ‘co-pores’, and pits. Glucosyl Yariv reagent combined with the AGP to make very large and distinctive rings. Conclusions Diverse cell-specific nano-patterns of native lysine-rich AGPs are expected at the wall–membrane interface and, while there will not be an identical patterning in different environmental settings, AFM imaging suggests protein tendencies for surficial organization and thus opens new avenues for experimentation. Nanopore formation with Yariv reagents suggests how the reagent might bind with AGP to admit Ca2+ to cells and hints at ways in which AGP might be structured at some cell surfaces. PMID:25164699

HMPAS: Human Membrane Protein Analysis System

PubMed Central

2013-01-01

Background Membrane proteins perform essential roles in diverse cellular functions and are regarded as major pharmaceutical targets. The significance of membrane proteins has led to the developing dozens of resources related with membrane proteins. However, most of these resources are built for specific well-known membrane protein groups, making it difficult to find common and specific features of various membrane protein groups. Methods We collected human membrane proteins from the dispersed resources and predicted novel membrane protein candidates by using ortholog information and our membrane protein classifiers. The membrane proteins were classified according to the type of interaction with the membrane, subcellular localization, and molecular function. We also made new feature dataset to characterize the membrane proteins in various aspects including membrane protein topology, domain, biological process, disease, and drug. Moreover, protein structure and ICD-10-CM based integrated disease and drug information was newly included. To analyze the comprehensive information of membrane proteins, we implemented analysis tools to identify novel sequence and functional features of the classified membrane protein groups and to extract features from protein sequences. Results We constructed HMPAS with 28,509 collected known membrane proteins and 8,076 newly predicted candidates. This system provides integrated information of human membrane proteins individually and in groups organized by 45 subcellular locations and 1,401 molecular functions. As a case study, we identified associations between the membrane proteins and diseases and present that membrane proteins are promising targets for diseases related with nervous system and circulatory system. A web-based interface of this system was constructed to facilitate researchers not only to retrieve organized information of individual proteins but also to use the tools to analyze the membrane proteins. Conclusions HMPAS provides comprehensive information about human membrane proteins including specific features of certain membrane protein groups. In this system, user can acquire the information of individual proteins and specified groups focused on their conserved sequence features, involved cellular processes, and diseases. HMPAS may contribute as a valuable resource for the inference of novel cellular mechanisms and pharmaceutical targets associated with the human membrane proteins. HMPAS is freely available at http://fcode.kaist.ac.kr/hmpas. PMID:24564858

Exposures of Sus scrofa to a TASER(®) conducted electrical weapon: no effects on 2-dimensional gel electrophoresis patterns of plasma proteins.

PubMed

Jauchem, James R; Cerna, Cesario Z; Lim, Tiffany Y; Seaman, Ronald L

2014-12-01

In an earlier study, we found significant changes in red-blood-cell, leukocyte, and platelet counts, and in red-blood-cell membrane proteins, following exposures of anesthetized pigs to a conducted electrical weapon. In the current study, we examined potential changes in plasma proteins [analyzed via two-dimensional gel electrophoresis (2-DGE)] following two 30 s exposures of anesthetized pigs (Sus scrofa) to a TASER (®) C2 conducted electrical weapon. Patterns of proteins, separated by 2-DGE, were consistent and reproducible between animals and between times of sampling. We determined that the blood plasma collection, handling, storage, and processing techniques we used are suitable for swine blood. There were no statistically significant changes in plasma proteins following the conducted-electrical-weapon exposures. Overall gel patterns of fibrinogen were similar to results of other studies of both pigs and humans (in control settings, not exposed to conducted electrical weapons). The lack of significant changes in plasma proteins may be added to the body of evidence regarding relative safety of TASER C2 device exposures.

Self-organization and positioning of bacterial protein clusters

NASA Astrophysics Data System (ADS)

Murray, Seán M.; Sourjik, Victor

2017-10-01

Many cellular processes require proteins to be precisely positioned within the cell. In some cases this can be attributed to passive mechanisms such as recruitment by other proteins in the cell or by exploiting the curvature of the membrane. However, in bacteria, active self-positioning is likely to play a role in multiple processes, including the positioning of the future site of cell division and cytoplasmic protein clusters. How can such dynamic clusters be formed and positioned? Here, we present a model for the self-organization and positioning of dynamic protein clusters into regularly repeating patterns based on a phase-locked Turing pattern. A single peak in the concentration is always positioned at the midpoint of the model cell, and two peaks are positioned at the midpoint of each half. Furthermore, domain growth results in peak splitting and pattern doubling. We argue that the model may explain the regular positioning of the highly conserved structural maintenance of chromosomes complexes on the bacterial nucleoid and that it provides an attractive mechanism for the self-positioning of dynamic protein clusters in other systems.

Modification of structure and pattern of lipid monolayer on water and solid surfaces in presence of globular protein

NASA Astrophysics Data System (ADS)

Sah, Bijay Kumar; Kundu, Sarathi

2017-05-01

Langmuir monolayers of phospholipids at the air-water interface are well-established model systems for mimicking biological membranes and hence are useful for studying lipid-protein interactions. In the present work, phases and phase transformations occurring in the lipid (DMPA) monolayer in the presence of globular protein (BSA) at neutral subphase pH (≈7.0) are highlighted and the corresponding in-plane pattern and morphology are explored from the surface pressure (π) - specific molecular area (A) isotherm, Brewster angle microscopy (BAM) and atomic force microscopy (AFM) both at air-water and air-solid interfaces. Films of pure lipid and lipid-protein complexes are deposited on solid surfaces by Langmuir-Blodgett method. Due to the presence of BSA molecules, phases and domain pattern changes in comparison with that of the pure DMPA. Moreover, accumulations of globular proteins in between lipid domains are also visible through BAM. AFM shows that the mixed film has relatively bigger globular-like morphology in comparison with that of pure DMPA domains. Combination of electrostatic and hydrophobic interactions between protein and lipid are responsible for such modifications.

Chemical crosslinking and mass spectrometry studies of the structure and dynamics of membrane proteins and receptors.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haskins, William E.; Leavell, Michael D.; Lane, Pamela

2005-03-01

Membrane proteins make up a diverse and important subset of proteins for which structural information is limited. In this study, chemical cross-linking and mass spectrometry were used to explore the structure of the G-protein-coupled photoreceptor bovine rhodopsin in the dark-state conformation. All experiments were performed in rod outer segment membranes using amino acid 'handles' in the native protein sequence and thus minimizing perturbations to the native protein structure. Cysteine and lysine residues were covalently cross-linked using commercially available reagents with a range of linker arm lengths. Following chemical digestion of cross-linked protein, cross-linked peptides were identified by accurate mass measurementmore » using liquid chromatography-fourier transform mass spectrometry and an automated data analysis pipeline. Assignments were confirmed and, if necessary, resolved, by tandem MS. The relative reactivity of lysine residues participating in cross-links was evaluated by labeling with NHS-esters. A distinct pattern of cross-link formation within the C-terminal domain, and between loop I and the C-terminal domain, emerged. Theoretical distances based on cross-linking were compared to inter-atomic distances determined from the energy-minimized X-ray crystal structure and Monte Carlo conformational search procedures. In general, the observed cross-links can be explained by re-positioning participating side-chains without significantly altering backbone structure. One exception, between C3 16 and K325, requires backbone motion to bring the reactive atoms into sufficient proximity for cross-linking. Evidence from other studies suggests that residues around K325 for a region of high backbone mobility. These findings show that cross-linking studies can provide insight into the structural dynamics of membrane proteins in their native environment.« less

Cassava root membrane proteome reveals activities during storage root maturation.

PubMed

Naconsie, Maliwan; Lertpanyasampatha, Manassawe; Viboonjun, Unchera; Netrphan, Supatcharee; Kuwano, Masayoshi; Ogasawara, Naotake; Narangajavana, Jarunya

2016-01-01

Cassava (Manihot esculenta Crantz) is one of the most important crops of Thailand. Its storage roots are used as food, feed, starch production, and be the important source for biofuel and biodegradable plastic production. Despite the importance of cassava storage roots, little is known about the mechanisms involved in their formation. This present study has focused on comparison of the expression profiles of cassava root proteome at various developmental stages using two-dimensional gel electrophoresis and LC-MS/MS. Based on an anatomical study using Toluidine Blue, the secondary growth was confirmed to be essential during the development of cassava storage root. To investigate biochemical processes occurring during storage root maturation, soluble and membrane proteins were isolated from storage roots harvested from 3-, 6-, 9-, and 12-month-old cassava plants. The proteins with differential expression pattern were analysed and identified to be associated with 8 functional groups: protein folding and degradation, energy, metabolism, secondary metabolism, stress response, transport facilitation, cytoskeleton, and unclassified function. The expression profiling of membrane proteins revealed the proteins involved in protein folding and degradation, energy, and cell structure were highly expressed during early stages of development. Integration of these data along with the information available in genome and transcriptome databases is critical to expand knowledge obtained solely from the field of proteomics. Possible role of identified proteins were discussed in relation with the activities during storage root maturation in cassava.

beta. -Amyloid precursor protein of Alzheimer disease occurs as 110- to 135-kilodalton membrane-associated proteins in neural and nonneural tissues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Selkoe, D.J.; Podlisny, M.B.; Joachim, C.L.

1988-10-01

Progressive cerebral deposition of extracellular filaments composed of the {beta}-amyloid protein ({beta}AP) is a constant feature of Alzheimer disease (AD). Since the gene on chromosome 21 encoding the {beta}AP precursor ({beta}APP) is not known to be altered in AD, transcriptional or posttranslational changes may underlie accelerated {beta}AP deposition. Using two antibodies to the predicted carboxyl terminus of {beta}APP, the authors have identified the native {beta}APP in brain and nonneural human tissues as a 110- to 135-kDa protein complex that is insoluble in buffer and found in various membrane-rich subcellular fractions. These proteins are relatively uniformly distributed in adult brain, abundantmore » in fetal brain, and detected in nonneural tissues that contain {beta}APP mRNA. Similarly sized proteins occur in rat, cow, and monkey brain and in cultured human HL-60 and HeLa cells; the precise patterns in the 110- to 135-kDa range are heterogeneous among various tissues and cell lines. They conclude that the highly conserved {beta}APP molecule occurs in mammalian tissues as a heterogeneous group of membrane-associated proteins of {approx} 120 kDa. Detection of the nonamyloidogenic carboxyl terminus within plaques suggests that proteolytic processing of the {beta}APP into insoluble filaments occurs locally in cortical regions that develop {beta}-amyloid deposits with age.« less

Reconstitution of a kidney chloride channel and its identification by covalent labeling.

PubMed

Breuer, W

1990-02-28

The basolateral membrane of the thick ascending loop of Henle (TALH) of the mammalian kidney is characterized by its high content of Na+/K(+)-ATPase and a Cl- conductance, which function in parallel in salt reabsorption. In order to reconstitute the Cl- channels, TALH membrane vesicles were solubilized in 1% sodium cholate in buffer containing 200 mM KCl, followed by dilution with soybean lipids (final ratio of protein/detergent/lipid of 1:3:15 in mg) and removal of the detergent by gel filtration on Sephadex G-50. Cl- channel activity in the liposomes was determined by a 36Cl- uptake assay where the accumulation of the radioactive tracer against its chemical gradient is driven by the membrane potential (positive inside) generated by an outward Cl- gradient. The 36Cl- uptake by the KCl-loaded liposomes was dependent on the inclusion of membrane protein and was abolished by valinomycin, indicating the involvement of a conductive pathway. It was also inhibited by 36% by 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). Solubilization of the Cl- channels in cholate was optimal in the presence of 200 mm KCl, but was found to decrease markedly at low ionic strength. SDS-PAGE analysis of the proteins extracted by cholate at high and low salt concentrations showed that the Cl- channel-containing high KCl extract was enriched in the 96 and 55 kDa alpha- and beta-subunits of the Na+/K(+)-ATPase (the major proteins in the membrane preparation) and several minor protein bands. Treatment of the membrane vesicles with the radioactive analogue of DIDS, [3H]2DIDS, labeled primarily a 65 and a 31 kDa protein. The solubilization of the 31 kDa protein by cholate depended markedly on the ionic strength and thus paralleled the solubilization pattern of Cl- channel activity. Furthermore, the labeling of the 31 kDa protein was prevented by nonradioactive DIDS and by NPPB but not by other compounds, indicating that it may be a Cl- channel component.

Tracking single Kv2.1 channels in live cells reveals anomalous subdiffusion and ergodicity breaking

NASA Astrophysics Data System (ADS)

Weigel, Aubrey; Simon, Blair; Tamkun, Michael; Krapf, Diego

2011-03-01

The dynamic organization of the plasma membrane is responsible for essential cellular processes, such as receptor trafficking and signaling. By studying the dynamics of transmembrane proteins a greater understanding of these processes as a whole can be achieved. It is broadly observed that the diffusion pattern of membrane protein displays anomalous subdiffusion. However, the mechanisms responsible for this behavior are not yet established. We explore the dynamics of the voltage gated potassium channel Kv2.1 by using single-particle tracking. We analyze Kv2.1 channel trajectories in terms of the time and ensemble distributions of square displacements. Our results reveal that all Kv2.1 channels experience anomalous subdiffusion and we observe that the Kv2.1 diffusion pattern is non-ergodic. We further investigated the role of the actin cytoskeleton in these channel dynamics by applying actin depolymerizing drugs. It is seen that with the breakdown of the actin cytoskeleton the Kv2.1 channel trajectories recover ergodicity.

Membrane Bending by Protein Crowding

NASA Astrophysics Data System (ADS)

Stachowiak, Jeanne

2014-03-01

From endosomes and synaptic vesicles to the cristae of the mitochondria and the annulus of the nuclear pore, highly curved membranes are fundamental to the structure and physiology of living cells. The established view is that specific families of proteins are able to bend membranes by binding to them. For example, inherently curved proteins are thought to impose their structure on the membrane surface, while membrane-binding proteins with hydrophobic motifs are thought to insert into the membrane like wedges, driving curvature. However, computational models have recently revealed that these mechanisms would require specialized membrane-bending proteins to occupy nearly 100% of a curved membrane surface, an improbable physiological situation given the immense density and diversity of membrane-bound proteins, and the low expression levels of these specialized proteins within curved regions of the membrane. How then does curvature arise within the complex and crowded environment of cellular membranes? Our recent work using proteins involved in clathrin-mediated endocytosis, as well as engineered protein-lipid interactions, has suggested a new hypothesis - that lateral pressure generated by collisions between membrane-bound proteins can drive membrane bending. Specifically, by correlating membrane bending with quantitative optical measurements of protein density on synthetic membrane surfaces and simple physical models of collisions among membrane-bound proteins, we have demonstrated that protein-protein steric interactions can drive membrane curvature. These findings suggest that a simple imbalance in the concentration of membrane-bound proteins across a membrane surface can drive a membrane to bend, providing an efficient mechanism by which essentially any protein can contribute to shaping membranes.

Genome-wide analysis and expression profiling of the Solanum tuberosum aquaporins.

PubMed

Venkatesh, Jelli; Yu, Jae-Woong; Park, Se Won

2013-12-01

Aquaporins belongs to the major intrinsic proteins involved in the transcellular membrane transport of water and other small solutes. A comprehensive genome-wide search for the homologues of Solanum tuberosum major intrinsic protein (MIP) revealed 41 full-length potato aquaporin genes. All potato aquaporins are grouped into five subfamilies; plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), NOD26-like intrinsic proteins (NIPs), small basic intrinsic proteins (SIPs) and x-intrinsic proteins (XIPs). Functional predictions based on the aromatic/arginine (ar/R) selectivity filters and Froger's positions showed a remarkable difference in substrate transport specificity among subfamilies. The expression pattern of potato aquaporins, examined by qPCR analysis, showed distinct expression profiles in various organs and tuber developmental stages. Furthermore, qPCR analysis of potato plantlets, subjected to various abiotic stresses revealed the marked effect of stresses on expression levels of aquaporins. Taken together, the expression profiles of aquaporins imply that aquaporins play important roles in plant growth and development, in addition to maintaining water homeostasis in response to environmental stresses. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Heat shock protein-containing exosomes in mid-trimester amniotic fluids.

PubMed

Asea, Alexzander; Jean-Pierre, Claudel; Kaur, Punit; Rao, Preethi; Linhares, Iara M; Skupski, Daniel; Witkin, Steven S

2008-10-01

Exosomes are multivesicular bodies formed by inverse membrane budding into the lumen of an endocytic compartment. Fusion with the plasma membrane leads to their release into the external milieu. The incorporation of heat shock proteins into exosomes has been associated with immune regulatory activity. We have examined whether heat shock protein-containing exosomes are present in mid-trimester amniotic fluid. Exosomes were isolated from mid-trimester amniotic fluids by sequential low-speed and high-speed centrifugation followed by sucrose density gradient centrifugation. Biochemical characterization included floatation pattern in sucrose gradients, acetylcholinesterase (AChE) activity and Western blot analysis for exosome-containing proteins. Exosomes were present in each of 23 amniotic fluids tested. They banded at a density of 1.17g/ml in sucrose gradients, were positive for AChE activity and contained tubulin, the inducible 72kDa heat shock protein, Hsp72 and the constitutively expressed heat shock protein, Hsc73; they were negative for calnexin. Exosome concentrations correlated positively with the number of pregnancies. Heat shock protein-containing exosomes are constituents of mid-trimester amniotic fluids and may contribute to immune regulation within the amniotic cavity.

Membrane-Mediated Cooperativity of Proteins

NASA Astrophysics Data System (ADS)

Weikl, Thomas R.

2018-04-01

Besides direct protein-protein interactions, indirect interactions mediated by membranes play an important role for the assembly and cooperative function of proteins in membrane shaping and adhesion. The intricate shapes of biological membranes are generated by proteins that locally induce membrane curvature. Indirect curvature-mediated interactions between these proteins arise because the proteins jointly affect the bending energy of the membranes. These curvature-mediated interactions are attractive for crescent-shaped proteins and are a driving force in the assembly of the proteins during membrane tubulation. Membrane adhesion results from the binding of receptor and ligand proteins that are anchored in the apposing membranes. The binding of these proteins strongly depends on nanoscale shape fluctuations of the membranes, leading to a fluctuation-mediated binding cooperativity. A length mismatch between receptor-ligand complexes in membrane adhesion zones causes repulsive curvature-mediated interactions that are a driving force for the length-based segregation of proteins during membrane adhesion.

Enemy at the gates: traffic at the plant cell pathogen interface.

PubMed

Hoefle, Caroline; Hückelhoven, Ralph

2008-12-01

The plant apoplast constitutes a space for early recognition of potentially harmful non-self. Basal pathogen recognition operates via dynamic sensing of conserved microbial patterns by pattern recognition receptors or of elicitor-active molecules released from plant cell walls during infection. Recognition elicits defence reactions depending on cellular export via SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex-mediated vesicle fusion or plasma membrane transporter activity. Lipid rafts appear also involved in focusing immunity-associated proteins to the site of pathogen contact. Simultaneously, pathogen effectors target recognition, apoplastic host proteins and transport for cell wall-associated defence. This microreview highlights most recent reports on the arms race for plant disease and immunity at the cell surface.

Role of Pseudomonas putida tol-oprL Gene Products in Uptake of Solutes through the Cytoplasmic Membrane

PubMed Central

Llamas, María A.; Rodríguez-Herva, José J.; Hancock, Robert E. W.; Bitter, Wilbert; Tommassen, Jan; Ramos, Juan L.

2003-01-01

Proteins of the Tol-Pal (Tol-OprL) system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. Here we describe an additional role for this system in the transport of various carbon sources across the cytoplasmic membrane. Growth of Pseudomonas putida tol-oprL mutant strains in minimal medium with glycerol, fructose, or arginine was impaired, and the growth rate with succinate, proline, or sucrose as the carbon source was lower than the growth rate of the parental strain. Assays with radiolabeled substrates revealed that the rates of uptake of these compounds by mutant cells were lower than the rates of uptake by the wild-type strain. The pattern and amount of outer membrane protein in the P. putida tol-oprL mutants were not changed, suggesting that the transport defect was not in the outer membrane. Consistently, the uptake of radiolabeled glucose and glycerol in spheroplasts was defective in the P. putida tol-oprL mutant strains, suggesting that there was a defect at the cytoplasmic membrane level. Generation of a proton motive force appeared to be unaffected in these mutants. To rule out the possibility that the uptake defect was due to a lack of specific transporter proteins, the PutP symporter was overproduced, but this overproduction did not enhance proline uptake in the tol-oprL mutants. These results suggest that the Tol-OprL system is necessary for appropriate functioning of certain uptake systems at the level of the cytoplasmic membrane. PMID:12896989

Predicting helix–helix interactions from residue contacts in membrane proteins

PubMed Central

Lo, Allan; Chiu, Yi-Yuan; Rødland, Einar Andreas; Lyu, Ping-Chiang; Sung, Ting-Yi; Hsu, Wen-Lian

2009-01-01

Motivation: Helix–helix interactions play a critical role in the structure assembly, stability and function of membrane proteins. On the molecular level, the interactions are mediated by one or more residue contacts. Although previous studies focused on helix-packing patterns and sequence motifs, few of them developed methods specifically for contact prediction. Results: We present a new hierarchical framework for contact prediction, with an application in membrane proteins. The hierarchical scheme consists of two levels: in the first level, contact residues are predicted from the sequence and their pairing relationships are further predicted in the second level. Statistical analyses on contact propensities are combined with other sequence and structural information for training the support vector machine classifiers. Evaluated on 52 protein chains using leave-one-out cross validation (LOOCV) and an independent test set of 14 protein chains, the two-level approach consistently improves the conventional direct approach in prediction accuracy, with 80% reduction of input for prediction. Furthermore, the predicted contacts are then used to infer interactions between pairs of helices. When at least three predicted contacts are required for an inferred interaction, the accuracy, sensitivity and specificity are 56%, 40% and 89%, respectively. Our results demonstrate that a hierarchical framework can be applied to eliminate false positives (FP) while reducing computational complexity in predicting contacts. Together with the estimated contact propensities, this method can be used to gain insights into helix-packing in membrane proteins. Availability: http://bio-cluster.iis.sinica.edu.tw/TMhit/ Contact: tsung@iis.sinica.edu.tw Supplementary information:Supplementary data are available at Bioinformatics online. PMID:19244388

The mammalian Ced-1 ortholog MEGF10/KIAA1780 displays a novel adhesion pattern

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suzuki, Emiko; Nakayama, Manabu

2007-07-01

Ced-1 protein is a Caenorhabditis elegans cell surface receptor involved in phagocytosis of dead cells. The gene encoding the mammalian ortholog of Ced-1 is yet to be identified. Here, we describe a potential candidate: human MEGF10. MEGF10 has the overall domain organization of Ced-1, containing a signal peptide, a EMI domain, 17 atypical EGF-like repeats, a transmembrane domain, and a cytoplasmic domain with NPXY and YXXL motifs. MEGF10-EGFP fusion protein expressed in HEK293 cells produced an irregular, mosaic-like pattern on the surface of coated glass. Protruded MEGF10 bound tightly to the glass, in effect 'pinning' the cytoplasmic membrane firmly ontomore » the glass, thereby restricting cell motility. These cells also took on a flat appearance. Although MEGF10-EGFP localized throughout the cytoplasmic membrane, no MEGF10-EGFP was found in lamellipodia. The MEGF10-EGFP signal was surrounded by a 1-2-{mu}m-wide dark strip lacking EGFP. Expression analyses of various MEGF10 deletion mutants revealed that the irregular, mosaic-like adhesion pattern characteristic of MEGF10 family members is due to concerted interactions between the EMI and 17 atypical EGF-like domains. Co-culturing of MEGF10-EGFP-expressing cells with apoptotic cells revealed that MEGF10 protein accumulated around the contact region during engulfment of apoptotic cells.« less

The Magnaporthe oryzae Alt A 1-like protein MoHrip1 binds to the plant plasma membrane.

PubMed

Zhang, Yi; Liang, Yingbo; Dong, Yijie; Gao, Yuhan; Yang, Xiufen; Yuan, Jingjing; Qiu, Dewen

2017-10-07

MoHrip1, a protein isolated from Magnaporthe oryzae, belongs to the Alt A 1 (AA1) family. mohrip1 mRNA levels showed inducible expression throughout the infection process in rice. To determine the location of MoHrip1 in M. oryzae, a mohrip1-gfp mutant was generated. Fluorescence microscopy observations and western blotting analysis showed that MoHrip1 was both present in the secretome and abundant in the fungal cell wall. To obtain MoHrip1 protein, we carried out high-yield expression of MoHrip1 in Pichia pastoris. Treatment of tobacco plants with MoHrip1 induced the formation of necrosis, accumulation of reactive oxygen species and expression of several defense-related genes, as well as conferred disease resistance. By fusion to green fluorescent protein, we showed that MoHrip1 was able to bind to the tobacco and rice plant plasma membrane, causing rapid morphological changes at the cellular level, such as cell shrinkage and chloroplast disorganization. These findings indicate that MoHrip1 is a microbe-associated molecular pattern that is perceived by the plant immune system. This is the first study on an AA1 family protein that can bind to the plant plasma membrane. Copyright © 2017 Elsevier Inc. All rights reserved.

IFT Proteins Accumulate during Cell Division and Localize to the Cleavage Furrow in Chlamydomonas

PubMed Central

Wood, Christopher R.; Wang, Zhaohui; Diener, Dennis; Zones, James Matt; Rosenbaum, Joel; Umen, James G.

2012-01-01

Intraflagellar transport (IFT) proteins are well established as conserved mediators of flagellum/cilium assembly and disassembly. However, data has begun to accumulate in support of IFT protein involvement in other processes elsewhere in the cell. Here, we used synchronous cultures of Chlamydomonas to investigate the temporal patterns of accumulation and localization of IFT proteins during the cell cycle. Their mRNAs showed periodic expression that peaked during S and M phase (S/M). Unlike most proteins that are synthesized continuously during G1 phase, IFT27 and IFT46 levels were found to increase only during S/M phase. During cell division, IFT27, IFT46, IFT72, and IFT139 re-localized from the flagella and basal bodies to the cleavage furrow. IFT27 was further shown to be associated with membrane vesicles in this region. This localization pattern suggests a role for IFT in cell division. PMID:22328921

Reversible binding kinetics of a cytoskeletal protein at the erythrocyte submembrane.

PubMed Central

Stout, A. L.; Axelrod, D.

1994-01-01

Reversible binding among components of the cellular submembrane cytoskeleton and reversible binding of some of these components with the plasma membrane likely play a role in nonelastic morphological changes and mechanoplastic properties of cells. However, relatively few studies have been devoted to investigating directly the kinetic aspects of the interactions of individual components of the membrane skeleton with the membrane. The experiments described here investigated whether one component of the erythrocyte membrane cytoskeleton, protein 4.1, binds to its sites on the membrane reversibly and if so, whether the different 4.1-binding sites display distinct kinetic behavior. Protein 4.1 is known to stabilize the membrane and to mediate the attachment of spectrin filaments to the membrane. Protein 4.1 previously has been shown to bind to integral membrane proteins band 3, glycophorin C, and to negatively charged phospholipids. To examine the kinetic rates of dissociation of carboxymethyl fluorescein-labeled 4.1 (CF-4.1) to the cytofacial surface of erythrocyte membrane, a special preparation of hemolyzed erythrocyte ghosts was used, in which the ghosts became flattened on a glass surface and exposed their cytofacial surfaces to the solution through a membrane rip in a distinctive characteristic pattern. This preparation was examined by the microscopy technique of total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP). Four different treatments were employed to help identify which membrane binding sites gave rise to the multiplicity of observed kinetic rates. The first treatment, the control, stripped off the native spectrin, actin, 4.1, and ankyrin. About 60% of the CF-4.1 bound to this control binded irreversibly (dissociation time > 20 min), but the remaining approximately 40% binded reversibly with a range of residency times averaging approximately 3 s. The second treatment subjected these stripped membranes to trypsin, which presumably removed most of the band 3. CF-4.1 binded significantly less to these trypsinized membranes and most of the decrease was a loss of the irreversibly binding sites. The third treatment simply preserved the native 4.1 and ankyrin. CF-4.1 binded less to this sample too, and the loss involved both the irreversible and reversible sites. The fourth treatment blocked the gycophorin C sites on the native 4.1-stripped membranes with an antibody. CF-4.1 again binded less to this sample than to a nonimmune serum control, and almost all of the decrease is a loss of irreversible sites. These rest suggest that 1) protein 4.1 binds to membrane or submembrane sites at least in part reversibly ; 2) the most reversible sites are probably not proteinaceous and not glycophorin C, but possibly are phospholipids (especially phosphatidylserine); and 3) TIWRFRAP can successfully examine the fast reversible dynamics of cytoskeletal components binding to biological membranes. Images FIGURE 2 FIGURE 3 FIGURE 4 PMID:7811947

Molecular characterization of cDNAs encoding G protein alpha and beta subunits and study of their temporal and spatial expression patterns in Nicotiana plumbaginifolia Viv.

PubMed

Kaydamov, C; Tewes, A; Adler, K; Manteuffel, R

2000-04-25

We have isolated cDNA sequences encoding alpha and beta subunits of potential G proteins from a cDNA library prepared from somatic embryos of Nicotiana plumbaginifolia Viv. at early developmental stages. The predicted NPGPA1 and NPGPB1 gene products are 75-98% identical to the known respective plant alpha and beta subunits. Southern hybridizations indicate that NPGPA1 is probably a single-copy gene, whereas at least two copies of NPGPB1 exist in the N. plumbaginifolia genome. Northern analyses reveal that both NPGPA1 and NPGPB1 mRNA are expressed in all embryogenic stages and plant tissues examined and their expression is obviously regulated by the plant hormone auxin. Immunohistological localization of NPGPalpha1 and NPGPbeta1 preferentially on plasma and endoplasmic reticulum membranes and their immunochemical detection exclusively in microsomal cell fractions implicate membrane association of both proteins. The temporal and spatial expression patterns of NPGPA1 and NPGPB1 show conformity as well as differences. This could account for not only cooperative, but also individual activities of both subunits during embryogenesis and plant development.

Expression of Pkd2l2 in testis is implicated in spermatogenesis.

PubMed

Chen, Ye; Zhang, Zheng; Lv, Xiao-Yan; Wang, Yi-Dong; Hu, Zhong-Guo; Sun, Huan; Tan, Rui-Zhi; Liu, Yu-Hang; Bian, Guo-Hui; Xiao, Yan; Li, Qin-Wei; Yang, Qiu-Tan; Ai, Jian-Zhong; Feng, Lu; Yang, Yang; Wei, Yu-Quan; Zhou, Qin

2008-08-01

Pkd2l2 is a novel member of the polycystic kidney disease (PKD) gene family in mammals. Prominently expressed in testis, this gene is still poorly understood. In this study, reverse transcription polymerase chain reaction (RT-PCR) results showed a time-dependent expression pattern of Pkd2l2 in postnatal mouse testis. Immunohistochemical analysis revealed that Pkd2l2 encoded a protein, polycystin-L2, which was predominantly detectable in the plasma membrane of spermatocytes and round spermatids, as well as in the head and tail of elongating spermatids within seminiferous tubules in mouse testis tissue sections of postnatal day 14 and adult mice. A green fluorescent fusion protein of Pkd2l2 resided in the plasma membrane of HEK 293 and MDCK cells, suggesting that it functions as a plasma membrane protein. Overexpression of Pkd2l2 increased the intracellular calcium concentration of MDCK cells, as detected by flow cytometry. Collectively, these data indicated that Pkd2l2 may be involved in the mid-late stage of spermatogenesis through modulation of the intracellular calcium concentration.

Resolution extension by image summing in serial femtosecond crystallography of two-dimensional membrane-protein crystals

DOE PAGES

Casadei, Cecilia M.; Tsai, Ching-Ju; Barty, Anton; ...

2018-01-01

Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 Å. The results show that two-dimensional serial crystallography atmore » X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump–probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals.« less

Resolution extension by image summing in serial femtosecond crystallography of two-dimensional membrane-protein crystals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Casadei, Cecilia M.; Tsai, Ching-Ju; Barty, Anton

Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 Å. The results show that two-dimensional serial crystallography atmore » X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump–probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals.« less

MyelStones: the executive roles of myelin basic protein in myelin assembly and destabilization in multiple sclerosis.

PubMed

Vassall, Kenrick A; Bamm, Vladimir V; Harauz, George

2015-11-15

The classic isoforms of myelin basic protein (MBP, 14-21.5 kDa) are essential to formation of the multilamellar myelin sheath of the mammalian central nervous system (CNS). The predominant 18.5-kDa isoform links together the cytosolic surfaces of oligodendrocytes, but additionally participates in cytoskeletal turnover and membrane extension, Fyn-mediated signalling pathways, sequestration of phosphoinositides and maintenance of calcium homoeostasis. All MBP isoforms are intrinsically disordered proteins (IDPs) that interact via molecular recognition fragments (MoRFs), which thereby undergo local disorder-to-order transitions. Their conformations and associations are modulated by environment and by a dynamic barcode of post-translational modifications, particularly phosphorylation by mitogen-activated and other protein kinases and deimination [a hallmark of demyelination in multiple sclerosis (MS)]. The MBPs are thus to myelin what basic histones are to chromatin. Originally thought to be merely structural proteins forming an inert spool, histones are now known to be dynamic entities involved in epigenetic regulation and diseases such as cancer. Analogously, the MBPs are not mere adhesives of compact myelin, but active participants in oligodendrocyte proliferation and in membrane process extension and stabilization during myelinogenesis. A central segment of these proteins is pivotal in membrane-anchoring and SH3 domain (Src homology 3) interaction. We discuss in the present review advances in our understanding of conformational conversions of this classic basic protein upon membrane association, including new thermodynamic analyses of transitions into different structural ensembles and how a shift in the pattern of its post-translational modifications is associated with the pathogenesis and potentially onset of demyelination in MS. © 2015 Authors; published by Portland Press Limited.

In Silico Pattern-Based Analysis of the Human Cytomegalovirus Genome

PubMed Central

Rigoutsos, Isidore; Novotny, Jiri; Huynh, Tien; Chin-Bow, Stephen T.; Parida, Laxmi; Platt, Daniel; Coleman, David; Shenk, Thomas

2003-01-01

More than 200 open reading frames (ORFs) from the human cytomegalovirus genome have been reported as potentially coding for proteins. We have used two pattern-based in silico approaches to analyze this set of putative viral genes. With the help of an objective annotation method that is based on the Bio-Dictionary, a comprehensive collection of amino acid patterns that describes the currently known natural sequence space of proteins, we have reannotated all of the previously reported putative genes of the human cytomegalovirus. Also, with the help of MUSCA, a pattern-based multiple sequence alignment algorithm, we have reexamined the original human cytomegalovirus gene family definitions. Our analysis of the genome shows that many of the coded proteins comprise amino acid combinations that are unique to either the human cytomegalovirus or the larger group of herpesviruses. We have confirmed that a surprisingly large portion of the analyzed ORFs encode membrane proteins, and we have discovered a significant number of previously uncharacterized proteins that are predicted to be G-protein-coupled receptor homologues. The analysis also indicates that many of the encoded proteins undergo posttranslational modifications such as hydroxylation, phosphorylation, and glycosylation. ORFs encoding proteins with similar functional behavior appear in neighboring regions of the human cytomegalovirus genome. All of the results of the present study can be found and interactively explored online (http://cbcsrv.watson.ibm.com/virus/). PMID:12634390

In silico pattern-based analysis of the human cytomegalovirus genome.

PubMed

Rigoutsos, Isidore; Novotny, Jiri; Huynh, Tien; Chin-Bow, Stephen T; Parida, Laxmi; Platt, Daniel; Coleman, David; Shenk, Thomas

2003-04-01

More than 200 open reading frames (ORFs) from the human cytomegalovirus genome have been reported as potentially coding for proteins. We have used two pattern-based in silico approaches to analyze this set of putative viral genes. With the help of an objective annotation method that is based on the Bio-Dictionary, a comprehensive collection of amino acid patterns that describes the currently known natural sequence space of proteins, we have reannotated all of the previously reported putative genes of the human cytomegalovirus. Also, with the help of MUSCA, a pattern-based multiple sequence alignment algorithm, we have reexamined the original human cytomegalovirus gene family definitions. Our analysis of the genome shows that many of the coded proteins comprise amino acid combinations that are unique to either the human cytomegalovirus or the larger group of herpesviruses. We have confirmed that a surprisingly large portion of the analyzed ORFs encode membrane proteins, and we have discovered a significant number of previously uncharacterized proteins that are predicted to be G-protein-coupled receptor homologues. The analysis also indicates that many of the encoded proteins undergo posttranslational modifications such as hydroxylation, phosphorylation, and glycosylation. ORFs encoding proteins with similar functional behavior appear in neighboring regions of the human cytomegalovirus genome. All of the results of the present study can be found and interactively explored online (http://cbcsrv.watson.ibm.com/virus/).

Identification of specific posttranslational O-mycoloylations mediating protein targeting to the mycomembrane.

PubMed

Carel, Clément; Marcoux, Julien; Réat, Valérie; Parra, Julien; Latgé, Guillaume; Laval, Françoise; Demange, Pascal; Burlet-Schiltz, Odile; Milon, Alain; Daffé, Mamadou; Tropis, Maryelle G; Renault, Marie A M

2017-04-18

The outer membranes (OMs) of members of the Corynebacteriales bacterial order, also called mycomembranes, harbor mycolic acids and unusual outer membrane proteins (OMPs), including those with α-helical structure. The signals that allow precursors of such proteins to be targeted to the mycomembrane remain uncharacterized. We report here the molecular features responsible for OMP targeting to the mycomembrane of Corynebacterium glutamicum , a nonpathogenic member of the Corynebacteriales order. To better understand the mechanisms by which OMP precursors were sorted in C. glutamicum , we first investigated the partitioning of endogenous and recombinant PorA, PorH, PorB, and PorC between bacterial compartments and showed that they were both imported into the mycomembrane and secreted into the extracellular medium. A detailed investigation of cell extracts and purified proteins by top-down MS, NMR spectroscopy, and site-directed mutagenesis revealed specific and well-conserved posttranslational modifications (PTMs), including O -mycoloylation, pyroglutamylation, and N -formylation, for mycomembrane-associated and -secreted OMPs. PTM site sequence analysis from C. glutamicum OMP and other O -acylated proteins in bacteria and eukaryotes revealed specific patterns. Furthermore, we found that such modifications were essential for targeting to the mycomembrane and sufficient for OMP assembly into mycolic acid-containing lipid bilayers. Collectively, it seems that these PTMs have evolved in the Corynebacteriales order and beyond to guide membrane proteins toward a specific cell compartment.

Identification of specific posttranslational O-mycoloylations mediating protein targeting to the mycomembrane

PubMed Central

Carel, Clément; Réat, Valérie; Parra, Julien; Latgé, Guillaume; Laval, Françoise; Burlet-Schiltz, Odile; Milon, Alain; Daffé, Mamadou; Tropis, Maryelle G.; Renault, Marie A. M.

2017-01-01

The outer membranes (OMs) of members of the Corynebacteriales bacterial order, also called mycomembranes, harbor mycolic acids and unusual outer membrane proteins (OMPs), including those with α-helical structure. The signals that allow precursors of such proteins to be targeted to the mycomembrane remain uncharacterized. We report here the molecular features responsible for OMP targeting to the mycomembrane of Corynebacterium glutamicum, a nonpathogenic member of the Corynebacteriales order. To better understand the mechanisms by which OMP precursors were sorted in C. glutamicum, we first investigated the partitioning of endogenous and recombinant PorA, PorH, PorB, and PorC between bacterial compartments and showed that they were both imported into the mycomembrane and secreted into the extracellular medium. A detailed investigation of cell extracts and purified proteins by top-down MS, NMR spectroscopy, and site-directed mutagenesis revealed specific and well-conserved posttranslational modifications (PTMs), including O-mycoloylation, pyroglutamylation, and N-formylation, for mycomembrane-associated and -secreted OMPs. PTM site sequence analysis from C. glutamicum OMP and other O-acylated proteins in bacteria and eukaryotes revealed specific patterns. Furthermore, we found that such modifications were essential for targeting to the mycomembrane and sufficient for OMP assembly into mycolic acid-containing lipid bilayers. Collectively, it seems that these PTMs have evolved in the Corynebacteriales order and beyond to guide membrane proteins toward a specific cell compartment. PMID:28373551

Glucose transporter 3 (GLUT3) protein expression in human placenta across gestation

PubMed Central

Brown, Kelecia; Heller, Debra S.; Zamudio, Stacy; Illsley, Nicholas P.

2012-01-01

Conflicting information regarding expression of GLUT3 protein in the human placenta has been reported and the localization and pattern of expression of GLUT3 protein across gestation has not been clearly defined. The objective of this study was characterization of syncytial GLUT3 protein expression across gestation. We hypothesized that GLUT3 protein is present in the syncytial microvillous membrane and that its expression decreases over gestation. GLUT3 protein was measured in samples from a range of gestational ages (first to third trimester), with human brain and human bowel used as a positive and negative control respectively. As an additional measure of specificity, we transfected BeWo choriocarcinoma cells, a trophoblast cell line expressing GLUT3, with siRNA directed against GLUT3 and analyzed expression by Western blotting. GLUT3 was detected in the syncytiotrophoblast at all gestational ages by immunohistochemistry. Using Western blotting GLUT3 was detected as an integral membrane protein at a molecular weight of ~50kDa in microvillous membranes from all trimesters but not in syncytial basal membranes. The identity of the primary antibody target was confirmed by demonstrating that expression of the immunoblotting signal in GLUT3 siRNA-treated BeWo was decreased to 18 ± 6% (mean ± SEM) of that seen in cells transfected with a non-targeting siRNA. GLUT3 expression in microvillous membranes detected by Western blot decreased through the trimesters such that expression in the second trimester (wks 14–26) was 48 ± 7% of that in the first trimester and by the third trimester (wks 31–40) only 34 ± 10% of first trimester expression. In addition, glucose uptake into BeWo cells treated with GLUT3 siRNA was reduced to 60% of that measured in cells treated with the non-targeting siRNA. This suggests that GLUT3-mediated uptake comprises approximately 50% of glucose uptake into BeWo cells. These results confirm the hypothesis that GLUT3 is present in the syncytial microvillous membrane early in gestation and decreases thereafter, supporting the idea that GLUT3 is of greater importance for glucose uptake early in gestation. PMID:22000473

Entrainment in solution of an oscillating NADH oxidase activity from the bovine milk fat globule membrane with a temperature-compensated period length suggestive of an ultradian time-keeping (clock) function

NASA Technical Reports Server (NTRS)

Morre, D. James; Lawler, Juliana; Wang, Sui; Keenan, Thomas W.; Morre, Dorothy M.

2002-01-01

Entrainment in solution of an oscillating activity with a temperature compensated period of 24 min is described for a NADH oxidase (NOX) activity of the bovine milk fat globule membrane, a derivative of the mammary epithelial cell plasma membrane. The period of 24 min remained unchanged at 17 degrees C, 27 degrees C and 37 degrees C whereas the amplitude approximately doubled with each 10 degree C rise in temperature (Q(10)congruent with 2). The periodicity was observed with both intact milk fat globule membranes and with detergent-solubilized membranes, demonstrating that the oscillations did not require an association with membranes. The periodicity was not the result of instrument variation or of chemical interactions among reactants in solution. Preparations with different periodicities entrained (autosynchronized) when mixed. Upon mixing, the preparations exhibited two oscillatory patterns but eventually a single pattern representing the mean of the farthest separated maxima of the two preparations analyzed separately emerged. The cell surface NOX protein is the first reported example of an entrainable biochemical entity with a temperature-compensated periodicity potentially capable of functioning as an ultradian or circadian clock driver.

The 'permeome' of the malaria parasite: an overview of the membrane transport proteins of Plasmodium falciparum

PubMed Central

Martin, Rowena E; Henry, Roselani I; Abbey, Janice L; Clements, John D; Kirk, Kiaran

2005-01-01

Background The uptake of nutrients, expulsion of metabolic wastes and maintenance of ion homeostasis by the intraerythrocytic malaria parasite is mediated by membrane transport proteins. Proteins of this type are also implicated in the phenomenon of antimalarial drug resistance. However, the initial annotation of the genome of the human malaria parasite Plasmodium falciparum identified only a limited number of transporters, and no channels. In this study we have used a combination of bioinformatic approaches to identify and attribute putative functions to transporters and channels encoded by the malaria parasite, as well as comparing expression patterns for a subset of these. Results A computer program that searches a genome database on the basis of the hydropathy plots of the corresponding proteins was used to identify more than 100 transport proteins encoded by P. falciparum. These include all the transporters previously annotated as such, as well as a similar number of candidate transport proteins that had escaped detection. Detailed sequence analysis enabled the assignment of putative substrate specificities and/or transport mechanisms to all those putative transport proteins previously without. The newly-identified transport proteins include candidate transporters for a range of organic and inorganic nutrients (including sugars, amino acids, nucleosides and vitamins), and several putative ion channels. The stage-dependent expression of RNAs for 34 candidate transport proteins of particular interest are compared. Conclusion The malaria parasite possesses substantially more membrane transport proteins than was originally thought, and the analyses presented here provide a range of novel insights into the physiology of this important human pathogen. PMID:15774027

Characterization of the motion of membrane proteins using high-speed atomic force microscopy

NASA Astrophysics Data System (ADS)

Casuso, Ignacio; Khao, Jonathan; Chami, Mohamed; Paul-Gilloteaux, Perrine; Husain, Mohamed; Duneau, Jean-Pierre; Stahlberg, Henning; Sturgis, James N.; Scheuring, Simon

2012-08-01

For cells to function properly, membrane proteins must be able to diffuse within biological membranes. The functions of these membrane proteins depend on their position and also on protein-protein and protein-lipid interactions. However, so far, it has not been possible to study simultaneously the structure and dynamics of biological membranes. Here, we show that the motion of unlabelled membrane proteins can be characterized using high-speed atomic force microscopy. We find that the molecules of outer membrane protein F (OmpF) are widely distributed in the membrane as a result of diffusion-limited aggregation, and while the overall protein motion scales roughly with the local density of proteins in the membrane, individual protein molecules can also diffuse freely or become trapped by protein-protein interactions. Using these measurements, and the results of molecular dynamics simulations, we determine an interaction potential map and an interaction pathway for a membrane protein, which should provide new insights into the connection between the structures of individual proteins and the structures and dynamics of supramolecular membranes.

Live-cell imaging of phosphoinositide dynamics and membrane architecture during Legionella infection.

PubMed

Weber, Stephen; Wagner, Maria; Hilbi, Hubert

2014-01-28

The causative agent of Legionnaires' disease, Legionella pneumophila, replicates in amoebae and macrophages in a distinct membrane-bound compartment, the Legionella-containing vacuole (LCV). LCV formation is governed by the bacterial Icm/Dot type IV secretion system that translocates ~300 different "effector" proteins into host cells. Some of the translocated effectors anchor to the LCV membrane via phosphoinositide (PI) lipids. Here, we use the soil amoeba Dictyostelium discoideum, producing fluorescent PI probes, to analyze the LCV PI dynamics by live-cell imaging. Upon uptake of wild-type or Icm/Dot-deficient L. pneumophila, PtdIns(3,4,5)P3 transiently accumulated for an average of 40 s on early phagosomes, which acquired PtdIns(3)P within 1 min after uptake. Whereas phagosomes containing ΔicmT mutant bacteria remained decorated with PtdIns(3)P, more than 80% of wild-type LCVs gradually lost this PI within 2 h. The process was accompanied by a major rearrangement of PtdIns(3)P-positive membranes condensing to the cell center. PtdIns(4)P transiently localized to early phagosomes harboring wild-type or ΔicmT L. pneumophila and was cleared within minutes after uptake. During the following 2 h, PtdIns(4)P steadily accumulated only on wild-type LCVs, which maintained a discrete PtdIns(4)P identity spatially separated from calnexin-positive endoplasmic reticulum (ER) for at least 8 h. The separation of PtdIns(4)P-positive and ER membranes was even more pronounced for LCVs harboring ΔsidC-sdcA mutant bacteria defective for ER recruitment, without affecting initial bacterial replication in the pathogen vacuole. These findings elucidate the temporal and spatial dynamics of PI lipids implicated in LCV formation and provide insight into host cell membrane and effector protein interactions. The environmental bacterium Legionella pneumophila is the causative agent of Legionnaires' pneumonia. The bacteria form in free-living amoebae and mammalian immune cells a replication-permissive compartment, the Legionella-containing vacuole (LCV). To subvert host cell processes, the bacteria secrete the amazing number of ~300 different proteins into host cells. Some of these proteins bind phosphoinositide (PI) lipids to decorate the LCV. PI lipids are crucial factors involved in host cell membrane dynamics and LCV formation. Using Dictyostelium amoebae producing one or two distinct fluorescent probes, we elucidated the dynamic LCV PI pattern in high temporal and spatial resolution. Notably, the endocytic PI lipid PtdIns(3)P was slowly cleared from LCVs, thus incapacitating the host cell's digestive machinery, while PtdIns(4)P gradually accumulated on the LCV, enabling critical interactions with host organelles. The LCV PI pattern underlies the spatiotemporal configuration of bacterial effector proteins and therefore represents a crucial aspect of LCV formation.

The Septate Junction Protein Caspr is Required for Structural Support and Retention of KCNQ4 at Calyceal Synapses of Vestibular Hair Cells

PubMed Central

Sousa, Aurea D.; Andrade, Leonardo R.; Salles, Felipe T.; Pillai, Anilkumar M.; Buttermore, Elizabeth; Bhat, Manzoor A.; Kachar, Bechara

2009-01-01

The afferent innervation contacting the type I hair cells of the vestibular sensory epithelia form distinct calyceal synapses. The apposed pre- and post-synaptic membranes at this large area of synaptic contact are kept at a remarkably regular distance. Here, we show by freeze-fracture electron microscopy that a patterned alignment of proteins at the calyceal membrane resembles a type of intercellular junction that is rare in vertebrates, the septate junction (SJ). We found that a core molecular component of SJs, Caspr, colocalizes with the K+ channel KCNQ4 at the post-synaptic membranes of these calyceal synapses. Immunolabeling and ultrastructural analyses of Caspr knockout mice reveal that, in the absence of Caspr, the separation between the membranes of the hair cells and the afferent neurons is conspicuously irregular and often increased by an order of magnitude. In these mutants, KCNQ4 fails to cluster at the post-synaptic membrane and appears diffused along the entire calyceal membrane. Our results indicate that a septate-like junction provides structural support to calyceal synaptic contact with the vestibular hair cell, and that Caspr is required for the recruitment or retention of KCNQ4 at these synapses. PMID:19279247

Analysis of the SDS-PAGE patterns of outer membrane proteins from Escherichia coli strains that have lost the ability to form K1 antigen and varied in the susceptibility to normal human serum.

PubMed

Cisowska, Agnieszka; Bugla-Płoskońska, Gabriela

2014-01-01

We used SDS-polyacrylamide gel electrophoresis to investigate the outer membrane proteins (OMPs) band composition of 19 Escherichia coli K1 strains that have spontaneously lost the ability to form K1 polysaccharide capsule (E. coli K1-) and demonstrated different degrees of susceptibility to the bactericidal action of normal human serum. Presented results showed that there were differences between E. coli K1- strains in OMPs expressing capacity. The analysis performed on OMPs has not revealed a direct association between the different OMPs band composition and the susceptibility of these strains to the serum.

Structure and interactions in biomaterials based on membrane-biopolymer self-assembly

NASA Astrophysics Data System (ADS)

Koltover, Ilya

Physical and chemical properties of artificial pure lipid membranes have been extensively studied during the last two decades and are relatively well understood. However, most real membrane systems of biological and biotechnological importance incorporate macromolecules either embedded into the membranes or absorbed onto their surfaces. We have investigated three classes of self-assembled membrane-biopolymer biomaterials: (i) Structure, interactions and stability of the two-dimensional crystals of the integral membrane protein bacteriorhodopsin (bR). We have conducted a synchrotron x-ray diffraction study of oriented bR multilayers. The important findings were as follows: (1) the protein 2D lattice exhibited diffraction patterns characteristic of a 2D solid with power-law decay of in-plane positional correlations, which allowed to measure the elastic constants of protein crystal; (2) The crystal melting temperature was a function of the multilayer hydration, reflecting the effect of inter-membrane repulsion on the stability of protein lattice; (3) Preparation of nearly perfect (mosaicity < 0.04° ) multilayers of fused bR membranes permitted, for the first time, application of powerful interface-sensitive x-ray scattering techniques to a membrane-protein system. (ii) Interactions between the particles chemically attached or absorbed onto the surfaces of flexible giant phospholipid vesicles. Using video-enhanced light microscopy we have observed a membrane-distortion induced attraction between the particles with the interaction range of the order of particle diameter. Fluid membranes decorated with many particles exhibited: (i) a finite-sized two-dimensional closed packed aggregates and (ii) a one-dimensional ring-like aggregates. (iii) Structure, stability and interactions in the cationic lipid-DNA complexes. Cationic liposomes complexed with DNA are among the most promising synthetic non-viral carriers of DNA vectors currently used in gene therapy applications. We have established that DNA complexes with cationic lipid (DOTAP) and a neutral lipid (DOPC) have a compact multilayer liquid crystalline structure ( L ca ) with DNA intercalated between the lipid bilayers in a periodic 2D smectic phase. Furthermore, a different 2D columnar phase of complexes was found in mixtures with a transfectionen-hancing lipid DOPE. This structure ( HcII ) derived from synchrotron x-ray diffraction consists of DNA coated by cationic lipid monolayers and arranged on a two-dimensional hexagonal lattice. Optical microscopy revealed that the L ca complexes bind stably to anionic vesicles (models of cellular membranes), whereas the more transfectant HcII complexes are unstable, rapidly fusing and releasing DNA upon adhering to anionic vesicles.

Interplay between membrane elasticity and active cytoskeleton forces regulates the aggregation dynamics of the immunological synapse

NASA Astrophysics Data System (ADS)

Dharan, Nadiv; Farago, Oded

Adhesion between a T cell and an antigen presenting cell is achieved by TCR-pMHC and LFA1-ICAM1 protein complexes. These segregate to form a special pattern, known as the immunological synapse (IS), consisting of a central quasi-circular domain of TCR-pMHC bonds surrounded by a peripheral domain of LFA1-ICAM1 complexes. Insights gained from imaging studies had led to the conclusion that the formation of the central adhesion domain in the IS is driven by active (ATP-driven) mechanisms. Recent studies, however, suggested that passive (thermodynamic) mechanisms may also play an important role in this process. Here, we present a simple physical model, taking into account the membrane-mediated thermodynamic attraction between the TCR-pMHC bonds and the effective forces that they experience due to ATP-driven actin retrograde flow and transport by dynein motor proteins. Monte Carlo simulations of the model exhibit a good spatio-temporal agreement with the experimentally observed pattern evolution of the TCR-pMHC microclusters. The agreement is lost when one of the aggregation mechanisms is "muted", which helps to identify the respective roles in the process. We conclude that actin retrograde flow drives the centripetal motion of TCR-pMHC bonds, while the membrane-mediated interactions facilitate microcluster formation and growth. In the absence of dynein motors, the system evolves into a ring-shaped pattern, which highlights the role of dynein motors in the formation of the final concentric pattern. The interplay between the passive and active mechanisms regulates the rate of the accumulation process, which in the absence of one them proceeds either too quickly or slowly.

Polyunsaturated Fatty Acids Inhibit T Cell Signal Transduction by Modification of Detergent-insoluble Membrane Domains

PubMed Central

Stulnig, Thomas M.; Berger, Markus; Sigmund, Thomas; Raederstorff, Daniel; Stockinger, Hannes; Waldhäusl, Werner

1998-01-01

Polyunsaturated fatty acids (PUFAs) exert immunosuppressive effects, but the molecular alterations leading to T cell inhibition are not yet elucidated. Signal transduction seems to involve detergent-resistant membrane domains (DRMs) acting as functional rafts within the plasma membrane bilayer with Src family protein tyrosine kinases being attached to their cytoplasmic leaflet. Since DRMs include predominantly saturated fatty acyl moieties, we investigated whether PUFAs could affect T cell signaling by remodeling of DRMs. Jurkat T cells cultured in PUFA-supplemented medium showed a markedly diminished calcium response when stimulated via the transmembrane CD3 complex or glycosyl phosphatidylinositol (GPI)- anchored CD59. Immunofluorescence studies indicated that CD59 but not Src family protein tyrosine kinase Lck remained in a punctate pattern after PUFA enrichment. Analysis of DRMs revealed a marked displacement of Src family kinases (Lck, Fyn) from DRMs derived from PUFA-enriched T cells compared with controls, and the presence of Lck in DRMs strictly correlated with calcium signaling. In contrast, GPI-anchored proteins (CD59, CD48) and ganglioside GM1, both residing in the outer membrane leaflet, remained in the DRM fraction. In conclusion, PUFA enrichment selectively modifies the cytoplasmic layer of DRMs and this alteration could underlie the inhibition of T cell signal transduction by PUFAs. PMID:9813086

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rouyer-Fessard, P.; Garel, M.C.; Domenget, C.

The soluble pool of alpha hemoglobin chains present in blood or bone marrow cells was measured with a new affinity method using a specific probe, beta A hemoglobin chain labeled with ({sup 3}H)N-ethylmaleimide. This pool of soluble alpha chains was 0.067 {plus minus} 0.017% of hemoglobin in blood of normal adult, 0.11 {plus minus} 0.03% in heterozygous beta thalassemia and ranged from 0.26 to 1.30% in homozygous beta thalassemia intermedia. This elevated pool of soluble alpha chains observed in human beta thalassemia intermedia decreased 33-fold from a value of 10% of total hemoglobin in bone marrow cells to 0.3% inmore » the most dense red blood cells. The amount of insoluble alpha chains was measured by using the polyacrylamide gel electrophoresis in urea and Triton X-100. In beta thalassemia intermedia the amount of insoluble alpha chains was correlated with the decreased spectrin content of red cell membrane and was associated with a decrease in ankyrin and with other abnormalities of the electrophoretic pattern of membrane proteins. The loss and topology of the reactive thiol groups of membrane proteins was determined by using ({sup 3}H)N-ethylmaleimide added to membrane ghosts prior to urea and Triton X-100 electrophoresis. Spectrin and ankyrin were the major proteins with the most important decrease of thiol groups.« less

Two Complementary Mechanisms Underpin Cell Wall Patterning during Xylem Vessel Development[OPEN

PubMed Central

Tang, Lu; Barkwill, Sarah; Lathe, Rahul; McFarlane, Heather E.

2017-01-01

The evolution of the plant vasculature was essential for the emergence of terrestrial life. Xylem vessels are solute-transporting elements in the vasculature that possess secondary wall thickenings deposited in intricate patterns. Evenly dispersed microtubule (MT) bands support the formation of these wall thickenings, but how the MTs direct cell wall synthesis during this process remains largely unknown. Cellulose is the major secondary wall constituent and is synthesized by plasma membrane-localized cellulose synthases (CesAs) whose catalytic activity propels them through the membrane. We show that the protein CELLULOSE SYNTHASE INTERACTING1 (CSI1)/POM2 is necessary to align the secondary wall CesAs and MTs during the initial phase of xylem vessel development in Arabidopsis thaliana and rice (Oryza sativa). Surprisingly, these MT-driven patterns successively become imprinted and sufficient to sustain the continued progression of wall thickening in the absence of MTs and CSI1/POM2 function. Hence, two complementary principles underpin wall patterning during xylem vessel development. PMID:28947492

Carbonic anhydrase activity in Arabidopsis thaliana thylakoid membrane and fragments enriched with PSI or PSII.

PubMed

Ignatova, Lyudmila K; Rudenko, Natalia N; Mudrik, Vilen A; Fedorchuk, Tat'yana P; Ivanov, Boris N

2011-12-01

The procedure of isolating the thylakoids and the thylakoid membrane fragments enriched with either photosystem I or photosystem II (PSI- and PSII-membranes) from Arabidopsis thaliana leaves was developed. It differed from the one used with pea and spinach in durations of detergent treatment and centrifugation, and in concentrations of detergent and Mg(2+) in the media. Both the thylakoid and the fragments preserved carbonic anhydrase (CA) activities. Using nondenaturing electrophoresis followed by detection of CA activity in the gel stained with bromo thymol blue, one low molecular mass carrier of CA activity was found in the PSI-membranes, and two carriers, a low molecular mass one and a high molecular mass one, were found in the PSII-membranes. The proteins in the PSII-membranes differed in their sensitivity to acetazolamide (AA), a specific CA inhibitor. AA at 5 × 10(-7) M inhibited the CA activity of the high molecular mass protein but stimulated the activity of the low molecular mass carrier in the PSII-membranes. At the same concentration, AA moderately inhibited, by 30%, the CA activity of PSI-membranes. CA activity of the PSII-membranes was almost completely suppressed by the lipophilic CA inhibitor, ethoxyzolamide at 10(-9) M, whereas CA activity of the PSI-membranes was inhibited by this inhibitor even at 5 × 10(-7) M just the same as for AA. The observed distribution of CA activity in the thylakoid membranes from A. thaliana was close to the one found in the membranes of pea, evidencing the general pattern of CA activity in the thylakoid membranes of C3-plants. © Springer Science+Business Media B.V. 2011

Structural changes in lymphocytes membrane of Chernobyl clean-up workers from Latvia.

PubMed

Kalnina, Inta; Zvagule, Tija; Gabruseva, Natalija; Kirilova, Jelena; Kurjane, Natalja; Bruvere, Ruta; Kesters, Andris; Kizane, Gunta; Kirilovs, Georgijs; Meirovics, Imants

2007-11-01

ABM (3-aminobenzanthrrone derivative) developed at the Riga Technical University, Riga, Latvia) has been previously shown as a potential probe for determination of the immune state of patients with different pathologies . The fist study (using probe ABM) of peripheral blood mononuclear cells (PBMC) membranes of 97 Chernobyl clean-up workers from Latvia was conducted in 1997. Now we repeatedly examine the same (n = 54) individuals in dynamics. ABM spectral parameters in PBMC suspension, fluorescence anisotropy and blood plasma albumin characteristics were recorded. In 1997 screening showed 5 different patterns of fluorescence spectra, from which in 2007 we obtained only two. These patterns of spectra had never been previously seen in healthy individuals or patients with tuberculosis, multiple sclerosis, rheumatoid arthritis, etc., examined by us. Patterns of ABM fluorescence spectra are associated with membrane anisotropy and conformational changes of blood plasma albumin. We observed that in dynamics 1997-2007 the lipid compartment of the membrane became more fluid while the lipid-protein interface became more rigid. The use of probe ANS and albumin auto-fluorescence allowed show conformational alterations in Chernobyl clean-up workers blood plasma. It is necessary to note that all investigated parameters significantly differ in observed groups of patients. These findings reinforce our understanding that that the cell membrane is a significant biological target of radiation. The role of the membrane in the expression and course of cell damage after radiation exposure must be considered. So ten years dynamic of PBMC membrane characteristics by ABM (spectral shift and anisotropy indexes) in Chernobyl clean-up workers reveal progressive trend toward certain resemblance with those of chronic B-cell lymphoid leukemia.

The Green Gut: Chlorophyll Degradation in the Gut of Spodoptera littoralis.

PubMed

Badgaa, Amarsanaa; Büchler, Rita; Wielsch, Natalie; Walde, Marie; Heintzmann, Rainer; Pauchet, Yannik; Svatos, Ales; Ploss, Kerstin; Boland, Wilhelm

2015-11-01

Chlorophylls, the most prominent natural pigments, are part of the daily diet of herbivorous insects. The spectrum of ingested and digested chlorophyll metabolites compares well to the pattern of early chlorophyll-degradation products in senescent plants. Intact chlorophyll is rapidly degraded by proteins in the front- and midgut. Unlike plants, insects convert both chlorophyll a and b into the corresponding catabolites. MALDI-TOF/MS imaging allowed monitoring the distribution of the chlorophyll catabolites along the gut of Spodoptera littoralis larvae. The chlorophyll degradation in the fore- and mid-gut is strongly pH dependent, and requires alkaline conditions. Using LC-MS/MS analysis we identified a lipocalin-type protein in the intestinal fluid of S. littoralis homolog to the chlorophyllide a binding protein from Bombyx mori. Widefield and high-resolution autofluorescence microscopy revealed that the brush border membranes are covered with the chlorophyllide binding protein tightly bound via its GPI-anchor to the gut membrane. A function in defense against gut microbes is discussed.

Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liberton, Michelle; Saha, Rajib; Jacobs, Jon M.

2016-04-07

Cyanobacteria are photosynthetic microbes with highly differentiated membrane systems. These organisms contain an outer membrane, plasma membrane, and an internal system of thylakoid membranes where the photosynthetic and respiratory machinery are found. This existence of compartmentalization and differentiation of membrane systems poses a number of challenges for cyanobacterial cells in terms of organization and distribution of proteins to the correct membrane system. Proteomics studies have long sought to identify the components of the different membrane systems, and to date about 450 different proteins have been attributed to either the plasma membrane or thylakoid membrane. Given the complexity of these membranes,more » many more proteins remain to be identified in these membrane systems, and a comprehensive catalog of plasma membrane and thylakoid membrane proteins is needed. Here we describe the identification of 635 proteins in Synechocystis sp. PCC 6803 by quantitative iTRAQ isobaric labeling; of these, 459 proteins were localized to the plasma membrane and 176 were localized to the thylakoid membrane. Surprisingly, we found over 2.5 times the number of unique proteins identified in the plasma membrane compared to the thylakoid membrane. This suggests that the protein composition of the thylakoid membrane is more homogeneous than the plasma membrane, consistent with the role of the plasma membrane in diverse cellular processes including protein trafficking and nutrient import, compared to a more specialized role for the thylakoid membrane in cellular energetics. Overall, the protein composition of the Synechocystis 6803 plasma membrane and thylakoid membrane is quite similar to the E.coli plasma membrane and Arabidopsis thylakoid membrane, respectively. Synechocystis 6803 can therefore be described as a gram-negative bacterium that has an additional internal membrane system that fulfils the energetic requirements of the cell.« less

[Adsorption characteristics of proteins on membrane surface and effect of protein solution environment on permeation behavior of berberine].

PubMed

Li, Yi-Qun; Xu, Li; Zhu, Hua-Xu; Tang, Zhi-Shu; Li, Bo; Pan, Yong-Lan; Yao, Wei-Wei; Fu, Ting-Ming; Guo, Li-Wei

2017-10-01

In order to explore the adsorption characteristics of proteins on the membrane surface and the effect of protein solution environment on the permeation behavior of berberine, berberine and proteins were used as the research object to prepare simulated solution. Low field NMR, static adsorption experiment and membrane separation experiment were used to study the interaction between the proteins and ceramic membrane or between the proteins and berberine. The static adsorption capacity of proteins, membrane relative flux, rejection rate of proteins, transmittance rate of berberine and the adsorption rate of proteins and berberine were used as the evaluation index. Meanwhile, the membrane resistance distribution, the particle size distribution and the scanning electron microscope (SEM) were determined to investigate the adsorption characteristics of proteins on ceramic membrane and the effect on membrane separation process of berberine. The results showed that the ceramic membrane could adsorb the proteins and the adsorption model was consistent with Langmuir adsorption model. In simulating the membrane separation process, proteins were the main factor to cause membrane fouling. However, when the concentration of proteins was 1 g•L⁻¹, the proteins had no significant effect on membrane separation process of berberine. Copyright© by the Chinese Pharmaceutical Association.

[Better performance of Western blotting: quick vs slow protein transfer, blotting membranes and the visualization methods].

PubMed

Kong, Ling-Quan; Pu, Ying-Hui; Ma, Shi-Kun

2008-01-01

To study how the choices of the quick vs slow protein transfer, the blotting membranes and the visualization methods influence the performance of Western blotting. The cellular proteins were abstracted from human breast cell line MDA-MB-231 for analysis with Western blotting using quick (2 h) and slow (overnight) protein transfer, different blotting membranes (nitrocellulose, PVDF and nylon membranes) and different visualization methods (ECL and DAB). In Western blotting with slow and quick protein transfer, the prestained marker presented more distinct bands on nitrocellulose membrane than on the nylon and PVDF membranes, and the latter also showed clear bands on the back of the membrane to very likely cause confusion, which did not occur with nitrocellulose membrane. PVDF membrane allowed slightly clearer visualization of the proteins with DAB method as compared with nitrocellulose and nylon membranes, and on the latter two membranes, quick protein transfer was likely to result in somehow irregular bands in comparison with slow protein transfer. With slow protein transfer and chemiluminescence for visualization, all the 3 membranes showed clear background, while with quick protein transfer, nylon membrane gave rise to obvious background noise but the other two membranes did not. Different membranes should be selected for immunoblotting according to the actual needs of the experiment. Slow transfer of the proteins onto the membranes often has better effect than quick transfer, and enhanced chemiluminescence is superior to DAB for protein visualization and allows highly specific and sensitive analysis of the protein expressions.

Membrane preparation and solubilization.

PubMed

Roy, Ankita

2015-01-01

Membrane proteins play an essential role in several biological processes like ion transport, signal transduction, and electron transfer to name a few. For structural and functional studies of integral membrane proteins, it is critically important to isolate proteins from the membrane using biological detergents. Detergents disrupt the native lipid components of the native membrane and encase the membrane protein in an unnatural environment in aqueous solution. However, a particular membrane protein is best solubilized in a specific detergent; therefore, screening for the optimal detergent is essential. Apart from keeping the membrane protein monodispered in solution, the detergent has to be compatible with downstream processes to isolate and characterize a membrane protein. Over the past several years, a number of membrane proteins have been successfully isolated for structural and functional studies that allowed an outline of general strategies for isolating a novel membrane protein of interest. © 2015 Elsevier Inc. All rights reserved.

Patterning of supported lipid bilayers and proteins using material selective nitrodopamine-mPEG.

PubMed

Spycher, Philipp R; Hall, Heike; Vogel, Viola; Reimhult, Erik

2015-01-01

We present a generic patterning process by which biomolecules in a passivated background are patterned directly from physiological buffer to microfabricated surfaces without the need for further processing. First, nitrodopamine-mPEG is self-assembled to selectively render TiO2 patterns non-fouling to biomolecule adsorption on hydrophilic and adhesive glass surfaces. After the controlled TiO2 passivation, the biomolecules can be directly adsorbed from solution in a single step creating large scale micropatterned and highly homogeneous arrays of biomolecules with very high pattern definition. We demonstrate the formation of fluid supported lipid bilayers (SLBs) down to the single μm-level limited only by the photolithographic process. Non-specific adsorption of lipid vesicles to the TiO2 background was found to be almost completely suppressed. The SLB patterns can be further selectively functionalized with retained mobility, which we demonstrate through biotin-streptavidin coupling. We envision this single step patterning approach to be very beneficial for membrane-based biosensors and for pattering of cells on a passivated background with complex, sub-cellular geometries; in each application the adherent areas have a tunable mobility of interaction sites controlled by the fluidity of the membrane.

The interactions of peripheral membrane proteins with biological membranes

DOE PAGES

Johs, Alexander; Whited, A. M.

2015-07-29

The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approachesmore » continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.« less

Fluorescence fluctuation analysis of BACE1-GFP fusion protein in cultured HEK293 cells

NASA Astrophysics Data System (ADS)

Gardeen, Spencer; Johnson, Joseph L.; Heikal, Ahmed A.

2016-10-01

Beta-site APP cleaving enzyme 1 (BACE1) is a type I transmembrane aspartyl protease. In the amyloidogenic pathway, BACE1 provides β-secretase activity that cleaves the amyloid precursor protein (APP) that leads to amyloid beta (Aβ) peptides. The aggregation of these Aβ will ultimately results in amyloid plaque formation, a hallmark of Alzheimer's disease (AD). Amyloid aggregation leads to progressive memory impairment and neural loss. Recent detergent protein extraction studies suggest that the untreated BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. Here, we examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using fluorescence correlation spectroscopy (FCS). Cells were transfected with a BACE1-EGFP fusion protein construct and imaged using confocal and DIC microscopy to monitor labeled BACE1 localization and distribution within the cell. Our one-photon fluorescence fluctuation autocorrelation of BACE1- EGFP on the plasma membrane of HEK cells is modeled using two diffusing species on the plasma membrane with estimated diffusion coefficients of 1.39 x 10-7 cm2/sec and 2.8 x 10-8 cm2/sec under resting conditions and STA-200 inhibition, respectively. Anomalous diffusion model also provided adequate description of the observed autocorrelation function of BACE1- EGFP on the plasma membrane with an estimate exponent (α) of 0.8 and 0.5 for resting and STA-200 treated cells, respectively. The corresponding hydrodynamic radius of this transmembrane fusion protein was estimated using the measured diffusion coefficients assuming both Stokes-Einstein and Saffman-Delbruck models. Our results suggest a complex diffusion pattern of BACE1-EGFP on the plasma membrane of HEK cells with the possibility for dimer formation, especially under STA-200 inhibition.

Evolution of the α-Subunit of Na/K-ATPase from Paramecium to Homo sapiens: Invariance of Transmembrane Helix Topology.

PubMed

Morrill, Gene A; Kostellow, Adele B; Liu, Lijun; Gupta, Raj K; Askari, Amir

2016-05-01

Na/K-ATPase is a key plasma membrane enzyme involved in cell signaling, volume regulation, and maintenance of electrochemical gradients. The α-subunit, central to these functions, belongs to a large family of P-type ATPases. Differences in transmembrane (TM) helix topology, sequence homology, helix-helix contacts, cell signaling, and protein domains of Na/K-ATPase α-subunit were compared in fungi (Beauveria), unicellular organisms (Paramecia), primitive multicellular organisms (Hydra), and vertebrates (Xenopus, Homo sapiens), and correlated with evolution of physiological functions in the α-subunit. All α-subunits are of similar length, with groupings of four and six helices in the N- and C-terminal regions, respectively. Minimal homology was seen for protein domain patterns in Paramecium and Hydra, with high correlation between Hydra and vertebrates. Paramecium α-subunits display extensive disorder, with minimal helix contacts. Increases in helix contacts in Hydra approached vertebrates. Protein motifs known to be associated with membrane lipid rafts and cell signaling reveal significant positional shifts between Paramecium and Hydra vulgaris, indicating that regional membrane fluidity changes occur during evolution. Putative steroid binding sites overlapping TM-3 occurred in all species. Sites associated with G-protein-receptor stimulation occur both in vertebrates and amphibia but not in Hydra or Paramecia. The C-terminus moiety "KETYY," necessary for the Na(+) activation of pump phosphorylation, is not present in unicellular species indicating the absence of classical Na(+)/K(+)-pumps. The basic protein topology evolved earliest, followed by increases in protein domains and ordered helical arrays, correlated with appearance of α-subunit regions known to involve cell signaling, membrane recycling, and ion channel formation.

Differential Protein Composition and Gene Expression in Leaf Mesophyll Cells and Bundle Sheath Cells of the C(4) Plant Digitaria sanguinalis (L.) Scop.

PubMed

Potter, J W; Black, C C

1982-08-01

The distribution and molecular weights of cellular proteins in soluble and membrane-associated locations were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining of leaf (Digitaria sanguinalis L. Scop.) extracts and isolated cell extracts. Leaf polypeptides also were pulse-labeled, followed by isolation of the labeled leaf cell types and analysis of the newly synthesized polypeptides in each cell type by electrophoresis and fluorography.Comparison of the electrophoretic patterns of crabgrass whole leaf polypeptides with isolated cell-type polypeptides indicated a difference in protein distribution patterns for the two cell types. The mesophyll cells exhibited a greater allocation of total cellular protein into membrane-associated proteins relative to soluble proteins. In contrast, the bundle sheath cells exhibited a higher percentage of total cellular protein in soluble proteins. Phosphoenolpyruvate carboxylase was the major soluble protein in the mesophyll cell and ribulose bisphosphate carboxylase was the major soluble protein in the bundle sheath cell. The majority of in vivo(35)S-pulse-labeled proteins synthesized by the two crabgrass cell types corresponded in molecular weight to the proteins present in the cell types which were detected by conventional staining techniques. The bundle sheath cell and mesophyll cell fluorograph profiles each had 15 major (35)S-labeled proteins. The major incorporation of (35)S by bundle sheath cells was into products which co-electrophoresed with the large and small subunits of ribulose bisphosphate carboxylase. In contrast, a major (35)S-labeled product in mesophyll cell extracts co-electrophoresed with the subunit of phosphoenolpyruvate carboxylase. Both cell types exhibited equivalent in vivo labeling of a polypeptide with one- and two-dimensional electrophoretic behavior similar to the major apoprotein of the light-harvesting chlorophyll a/b protein. Results from the use of protein synthesis inhibitors during pulse-labeling experiments indicated intercellular differences in both organelle and cytoplasmic protein synthesis. A majority of the (35)S incorporation by crabgrass mesophyll cell 70S ribosomes was associated with a pair of membrane-associated polypeptides of molecular weight 32,000 and 34,500; a comparison of fluorograph and stained gel profiles suggests these products resemble the precursor and mature forms of the maize chloroplast 32,000 dalton protein reported by Grebanier et al. (1978 J. Cell Biol. 28:734-746). In contrast, crabgrass bundle sheath cell organelle translation was directed predominantly into a product which co-electrophoresed with the large subunit of ribulose bisphosphate carboxylase.

Rupturing Giant Plasma Membrane Vesicles to Form Micron-sized Supported Cell Plasma Membranes with Native Transmembrane Proteins.

PubMed

Chiang, Po-Chieh; Tanady, Kevin; Huang, Ling-Ting; Chao, Ling

2017-11-09

Being able to directly obtain micron-sized cell blebs, giant plasma membrane vesicles (GPMVs), with native membrane proteins and deposit them on a planar support to form supported plasma membranes could allow the membrane proteins to be studied by various surface analytical tools in native-like bilayer environments. However, GPMVs do not easily rupture on conventional supports because of their high protein and cholesterol contents. Here, we demonstrate the possibility of using compression generated by the air-water interface to efficiently rupture GPMVs to form micron-sized supported membranes with native plasma membrane proteins. We demonstrated that not only lipid but also a native transmembrane protein in HeLa cells, Aquaporin 3 (AQP3), is mobile in the supported membrane platform. This convenient method for generating micron-sized supported membrane patches with mobile native transmembrane proteins could not only facilitate the study of membrane proteins by surface analytical tools, but could also enable us to use native membrane proteins for bio-sensing applications.

How Lipid Membranes Affect Pore Forming Toxin Activity.

PubMed

Rojko, Nejc; Anderluh, Gregor

2015-12-15

Pore forming toxins (PFTs) evolved to permeate the plasma membrane of target cells. This is achieved in a multistep mechanism that usually involves binding of soluble protein monomer to the lipid membrane, oligomerization at the plane of the membrane, and insertion of part of the polypeptide chain across the lipid membrane to form a conductive channel. Introduced pores allow uncontrolled transport of solutes across the membrane, inflicting damage to the target cell. PFTs are usually studied from the perspective of structure-function relationships, often neglecting the important role of the bulk membrane properties on the PFT mechanism of action. In this Account, we discuss how membrane lateral heterogeneity, thickness, and fluidity influence the pore forming process of PFTs. In general, lipid molecules are more accessible for binding in fluid membranes due to steric reasons. When PFT specifically binds ordered domains, it usually recognizes a specific lipid distribution pattern, like sphingomyelin (SM) clusters or SM/cholesterol complexes, and not individual lipid species. Lipid domains were also suggested to act as an additional concentration platform facilitating PFT oligomerization, but this is yet to be shown. The last stage in PFT action is the insertion of the transmembrane segment across the membranes to build the transmembrane pore walls. Conformational changes are a spontaneous process, and sufficient free energy has to be available for efficient membrane penetration. Therefore, fluid bilayers are permeabilized more readily in comparison to highly ordered and thicker liquid ordered lipid phase (Lo). Energetically more costly insertion into the Lo phase can be driven by the hydrophobic mismatch between the thinner liquid disordered phase (Ld) and large protein complexes, which are unable to tilt like single transmembrane segments. In the case of proteolipid pores, membrane properties can directly modulate pore size, stability, and even selectivity. Finally, events associated with pore formation can modulate properties of the lipid membrane and affect its organization. Model membranes do not necessarily reproduce the physicochemical properties of the native cellular membrane, and caution is needed when transferring results from model to native lipid membranes. In this context, the utilization of novel approaches that enable studying PFTs on living cells at a single molecule level should reveal complex protein-lipid membrane interactions in greater detail.

A novel lipoprotein nanoparticle system for membrane proteins

PubMed Central

Frauenfeld, Jens; Löving, Robin; Armache, Jean-Paul; Sonnen, Andreas; Guettou, Fatma; Moberg, Per; Zhu, Lin; Jegerschöld, Caroline; Flayhan, Ali; Briggs, John A.G.; Garoff, Henrik; Löw, Christian; Cheng, Yifan; Nordlund, Pär

2016-01-01

Membrane proteins are of outstanding importance in biology, drug discovery and vaccination. A common limiting factor in research and applications involving membrane proteins is the ability to solubilize and stabilize membrane proteins. Although detergents represent the major means for solubilizing membrane proteins, they are often associated with protein instability and poor applicability in structural and biophysical studies. Here, we present a novel lipoprotein nanoparticle system that allows for the reconstitution of membrane proteins into a lipid environment that is stabilized by a scaffold of Saposin proteins. We showcase the applicability of the method on two purified membrane protein complexes as well as the direct solubilization and nanoparticle-incorporation of a viral membrane protein complex from the virus membrane. We also demonstrate that this lipid nanoparticle methodology facilitates high-resolution structural studies of membrane proteins in a lipid environment by single-particle electron cryo-microscopy (cryo-EM) and allows for the stabilization of the HIV-envelope glycoprotein in a functional state. PMID:26950744

Epidemiology of virulence-associated plasmids and outer membrane protein patterns within seven common Salmonella serotypes.

PubMed

Helmuth, R; Stephan, R; Bunge, C; Hoog, B; Steinbeck, A; Bulling, E

1985-04-01

Antibiotic-sensitive Salmonella isolates belonging to seven common serotypes and originating from 29 different countries from all continents were investigated for their plasmid DNA content (337 isolates) and their outer membrane protein profiles (216 isolates). Of the S. typhimurium, S. enteritidis, S. dublin, and S. choleraesuis isolates, 90% or more carried a serotype-specific plasmid. The molecular sizes of the plasmids were 60 megadaltons (Md) for S. typhimurium, 37 Md for S. enteritidis, 56 Md for S. dublin, and 30 Md for S. choleraesuis. The outer membrane protein profiles were homogeneous within each of the seven serotypes, except that a minority of S. enteritidis and S. dublin strains were lacking one major outer membrane protein. Virulence studies were performed with 39 representative strains by measuring the 50% lethal doses (LD50S) after oral infection of mice. The LD50 values obtained for plasmid-positive strains of S. typhimurium, S. enteritidis, and S. dublin were up to 10(6)-fold lower than the values obtained for the plasmid-free strains of the same serotype. Only the plasmid-positive strains could invade the livers of orally infected mice, and only they were resistant to the bactericidal activity of 90% guinea pig serum. Strains of S. infantis were generally plasmid free, whereas S. panama and S. heidelberg isolates carried heterogeneous plasmid populations. The virulence properties of the latter three serotypes could not be correlated with the predominant plasmids found in these strains.

Epidemiology of virulence-associated plasmids and outer membrane protein patterns within seven common Salmonella serotypes.

PubMed Central

Helmuth, R; Stephan, R; Bunge, C; Hoog, B; Steinbeck, A; Bulling, E

1985-01-01

Antibiotic-sensitive Salmonella isolates belonging to seven common serotypes and originating from 29 different countries from all continents were investigated for their plasmid DNA content (337 isolates) and their outer membrane protein profiles (216 isolates). Of the S. typhimurium, S. enteritidis, S. dublin, and S. choleraesuis isolates, 90% or more carried a serotype-specific plasmid. The molecular sizes of the plasmids were 60 megadaltons (Md) for S. typhimurium, 37 Md for S. enteritidis, 56 Md for S. dublin, and 30 Md for S. choleraesuis. The outer membrane protein profiles were homogeneous within each of the seven serotypes, except that a minority of S. enteritidis and S. dublin strains were lacking one major outer membrane protein. Virulence studies were performed with 39 representative strains by measuring the 50% lethal doses (LD50S) after oral infection of mice. The LD50 values obtained for plasmid-positive strains of S. typhimurium, S. enteritidis, and S. dublin were up to 10(6)-fold lower than the values obtained for the plasmid-free strains of the same serotype. Only the plasmid-positive strains could invade the livers of orally infected mice, and only they were resistant to the bactericidal activity of 90% guinea pig serum. Strains of S. infantis were generally plasmid free, whereas S. panama and S. heidelberg isolates carried heterogeneous plasmid populations. The virulence properties of the latter three serotypes could not be correlated with the predominant plasmids found in these strains. Images PMID:3980081

Translation, modification and cellular distribution of two AC4 variants of African cassava mosaic virus in yeast and their pathogenic potential in plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hipp, Katharina, E-mail: katharina.hipp@bio.uni-st

Plant infecting geminiviruses encode a small (A)C4 protein within the open reading frame of the replication-initiator protein. In African cassava mosaic virus, two in-frame start codons may be used for the translation of a longer and a shorter AC4 variant. Both were fused to green fluorescent protein or glutathione-S-transferase genes and expressed in fission yeast. The longer variant accumulated in discrete spots in the cytoplasm, whereas the shorter variant localized to the plasma membrane. A similar expression pattern was found in plants. A myristoylation motif may promote a targeting of the shorter variant to the plasma membrane. Mass spectrometry analysismore » of the yeast-expressed shorter variant detected the corresponding myristoylation. The biological relevance of the second start codon was confirmed using mutated infectious clones. Whereas mutating the first start codon had no effect on the infectivity in Nicotiana benthamiana plants, the second start codon proved to be essential. -- Highlights: •The ACMV AC4 may be translated from one or the other in-frame start codon. •Both AC4 variants are translated in fission yeast. •The long AC4 protein localizes to the cytoplasm, the short to the plasma membrane. •The short variant is myristoylated in yeast and may promote membrane localization. •Only the shorter AC4 variant has an impact on viral infections in plants.« less

Nonenzymatic chemiluminescent detection and quantitation of total protein on Western and slot blots allowing subsequent immunodetection and sequencing.

PubMed

Alba, F J; Daban, J R

1997-10-01

We have studied the light emission efficiency of proteins labeled with different fluorescent dyes chemically excited by the bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H2O2 reaction. Using this peroxyoxalate chemiluminescence system, the best results were obtained with proteins covalently labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF). Blotted proteins on polyvinylidene difluoride (PVDF) membranes can be labeled rapidly with MDPF. Our results demonstrate that energy from the excited intermediate produced in the TCPO-H2O2 reaction can be efficiently transferred to MDPF-labeled proteins in solution and on PVDF membranes. Although this nonenzymatic chemiluminescent system produces a background emission that reduces the sensitivity, the method developed in this work allows detection of 5 ng of protein in blots after 5 min exposure to X-ray film. Chemiluminescence of MDPF-labeled proteins on Western and slot blots may also be detected and quantified using a charge-coupled device (CCD) camera or a storage phosphor imaging system. This chemiluminescent method allows the staining of the total electrophoretic pattern but does not preclude further N-terminal sequencing and immunodetection of specific bands.

Milk proteins interact with goat Binder of SPerm (BSP) proteins and decrease their binding to sperm.

PubMed

de Menezes, Erika Bezerra; van Tilburg, Mauricio; Plante, Geneviève; de Oliveira, Rodrigo V; Moura, Arlindo A; Manjunath, Puttaswamy

2016-11-01

Seminal plasma Binder of SPerm (BSP) proteins bind to sperm at ejaculation and promote capacitation. When in excess, however, BSP proteins damage the sperm membrane. It has been suggested that milk components of semen extenders associate with BSP proteins, potentially protecting sperm. Thus, this study was conducted to investigate if milk proteins interact with BSP proteins and reduce BSP binding to goat sperm. Using gel filtration chromatography, milk was incubated with goat seminal plasma proteins and loaded onto columns with and without calcium. Milk was also fractionated into parts containing mostly whey proteins or mostly caseins, incubated with seminal plasma proteins and subjected to gel filtration. Eluted fractions were evaluated by immunoblot using anti-goat BSP antibodies, confirming milk protein-BSP protein interactions. As determined by ELISA, milk proteins coated on polystyrene wells bound to increasing of goat BSP proteins. Far-western dot blots confirmed that BSP proteins bound to caseins and β-lactoglobulin in a concentration-dependent manner. Then, cauda epididymal sperm from five goats was incubated with seminal plasma; seminal plasma followed by milk; and milk followed by seminal plasma. Sperm membrane proteins were extracted and evaluated by immunoblotting. The pattern of BSP binding to sperm membrane proteins was reduced by 59.3 % when epididymal sperm were incubated with seminal plasma and then with skimmed milk (p < 0.05). When epididymal sperm were treated with milk followed by seminal plasma, coating of sperm with BSP proteins was not significantly reduced (57.6 %; p > 0.05). In conclusion, goat BSP proteins have an affinity for caseins and whey proteins. Milk reduces BSP binding to goat sperm, depending whether or not sperm had been previously exposed to seminal plasma. Such events may explain the protective effect of milk during goat sperm preservation.

Pharmacoproteomics of a metalloproteinase hydroxamate inhibitor in breast cancer cells: dynamics of membrane type 1 matrix metalloproteinase-mediated membrane protein shedding.

PubMed

Butler, Georgina S; Dean, Richard A; Tam, Eric M; Overall, Christopher M

2008-08-01

Broad-spectrum matrix metalloproteinase (MMP) inhibitors (MMPI) were unsuccessful in cancer clinical trials, partly due to side effects resulting from limited knowledge of the full repertoire of MMP substrates, termed the substrate degradome, and hence the in vivo functions of MMPs. To gain further insight into the degradome of MMP-14 (membrane type 1 MMP) an MMPI, prinomastat (drug code AG3340), was used to reduce proteolytic processing and ectodomain shedding in human MDA-MB-231 breast cancer cells transfected with MMP-14. We report a quantitative proteomic evaluation of the targets and effects of the inhibitor in this cell-based system. Proteins in cell-conditioned medium (the secretome) and membrane fractions with levels that were modulated by the MMPI were identified by isotope-coded affinity tag (ICAT) labeling and tandem mass spectrometry. Comparisons of the expression of MMP-14 with that of a vector control resulted in increased MMP-14/vector ICAT ratios for many proteins in conditioned medium, indicating MMP-14-mediated ectodomain shedding. Following MMPI treatment, the MMPI/vehicle ICAT ratio was reversed, suggesting that MMP-14-mediated shedding of these proteins was blocked by the inhibitor. The reduction in shedding or the release of substrates from pericellular sites in the presence of the MMPI was frequently accompanied by the accumulation of the protein in the plasma membrane, as indicated by high MMPI/vehicle ICAT ratios. Considered together, this is a strong predictor of biologically relevant substrates cleaved in the cellular context that led to the identification of many undescribed MMP-14 substrates, 20 of which we validated biochemically, including DJ-1, galectin-1, Hsp90alpha, pentraxin 3, progranulin, Cyr61, peptidyl-prolyl cis-trans isomerase A, and dickkopf-1. Other proteins with altered levels, such as Kunitz-type protease inhibitor 1 and beta-2-microglobulin, were not substrates in biochemical assays, suggesting an indirect affect of the MMPI, which might be important in drug development as biomarkers or, in preclinical phases, to predict systemic drug actions and adverse side effects. Hence, this approach describes the dynamic pattern of cell membrane ectodomain shedding and its perturbation upon metalloproteinase drug treatment.

Membrane Topology and Insertion of Membrane Proteins: Search for Topogenic Signals

PubMed Central

van Geest, Marleen; Lolkema, Juke S.

2000-01-01

Integral membrane proteins are found in all cellular membranes and carry out many of the functions that are essential to life. The membrane-embedded domains of integral membrane proteins are structurally quite simple, allowing the use of various prediction methods and biochemical methods to obtain structural information about membrane proteins. A critical step in the biosynthetic pathway leading to the folded protein in the membrane is its insertion into the lipid bilayer. Understanding of the fundamentals of the insertion and folding processes will significantly improve the methods used to predict the three-dimensional membrane protein structure from the amino acid sequence. In the first part of this review, biochemical approaches to elucidate membrane protein topology are reviewed and evaluated, and in the second part, the use of similar techniques to study membrane protein insertion is discussed. The latter studies search for signals in the polypeptide chain that direct the insertion process. Knowledge of the topogenic signals in the nascent chain of a membrane protein is essential for the evaluation of membrane topology studies. PMID:10704472

Membrane Compartmentalization Reducing the Mobility of Lipids and Proteins within a Model Plasma Membrane.

PubMed

Koldsø, Heidi; Reddy, Tyler; Fowler, Philip W; Duncan, Anna L; Sansom, Mark S P

2016-09-01

The cytoskeleton underlying cell membranes may influence the dynamic organization of proteins and lipids within the bilayer by immobilizing certain transmembrane (TM) proteins and forming corrals within the membrane. Here, we present coarse-grained resolution simulations of a biologically realistic membrane model of asymmetrically organized lipids and TM proteins. We determine the effects of a model of cytoskeletal immobilization of selected membrane proteins using long time scale coarse-grained molecular dynamics simulations. By introducing compartments with varying degrees of restraints within the membrane models, we are able to reveal how compartmentalization caused by cytoskeletal immobilization leads to reduced and anomalous diffusional mobility of both proteins and lipids. This in turn results in a reduced rate of protein dimerization within the membrane and of hopping of membrane proteins between compartments. These simulations provide a molecular realization of hierarchical models often invoked to explain single-molecule imaging studies of membrane proteins.

P2X7 receptors regulate multiple types of membrane trafficking responses and non-classical secretion pathways.

PubMed

Qu, Yan; Dubyak, George R

2009-06-01

Activation of the P2X7 receptor (P2X7R) triggers a remarkably diverse array of membrane trafficking responses in leukocytes and epithelial cells. These responses result in altered profiles of cell surface lipid and protein composition that can modulate the direct interactions of P2X7R-expressing cells with other cell types in the circulation, in blood vessels, at epithelial barriers, or within sites of immune and inflammatory activation. Additionally, these responses can result in the release of bioactive proteins, lipids, and large membrane complexes into extracellular compartments for remote communication between P2X7R-expressing cells and other cells that amplify or modulate inflammation, immunity, and responses to tissue damages. This review will discuss P2X7R-mediated effects on membrane composition and trafficking in the plasma membrane (PM) and intracellular organelles, as well as actions of P2X7R in controlling various modes of non-classical secretion. It will review P2X7R regulation of: (1) phosphatidylserine distribution in the PM outer leaflet; (2) shedding of PM surface proteins; (3) release of PM-derived microvesicles or microparticles; (4) PM blebbing; (5) cell-cell fusion resulting in formation of multinucleate cells; (6) phagosome maturation and fusion with lysosomes; (7) permeability of endosomes with internalized pathogen-associated molecular patterns; (8) permeability/integrity of mitochondria; (9) exocytosis of secretory lysosomes; and (10) release of exosomes from multivesicular bodies.

Immunoblot detection of class-specific humoral immune response to outer membrane proteins isolated from Salmonella typhi in humans with typhoid fever.

PubMed Central

Ortiz, V; Isibasi, A; García-Ortigoza, E; Kumate, J

1989-01-01

The studies reported here were undertaken to assess the ability of the outer membrane proteins (OMPs) of Salmonella typhi to induce a humoral immune response in humans with typhoid fever. OMPs were isolated with the nonionic detergent Triton X-100 and were found to be contaminated with approximately 4% lipopolysaccharide. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns showed protein bands with molecular size ranges from 17 to 70 kilodaltons; the major groups of proteins were those that correspond to the porins and OmpA of gram-negative bacteria. Rabbit antiserum to OMPs or to S. typhi recognized OMPs after absorption with lipopolysaccharide. Sera from patients with typhoid fever contained immunoglobulin M antibodies which reacted with a protein of 28 kilodaltons and immunoglobulin G antibodies which reacted mainly with the porins, as determined by immunoblotting. These results indicate that the porins are the major immunogenic OMPs from S. typhi and that the immune response induced in the infection could be related to the protective status. Images PMID:2768450

An Ankyrin Repeat-Containing Protein, Characterized as a Ubiquitin Ligase, Is Closely Associated with Membrane-Enclosed Organelles and Required for Pollen Germination and Pollen Tube Growth in Lily1[W

PubMed Central

Huang, Jian; Chen, Feng; Del Casino, Cecilia; Autino, Antonella; Shen, Mouhua; Yuan, Shuai; Peng, Jia; Shi, Hexin; Wang, Chen; Cresti, Mauro; Li, Yiqin

2006-01-01

Exhibiting rapid polarized growth, the pollen tube delivers the male gametes into the ovule for fertilization in higher plants. To get an overall picture of gene expression during pollen germination and pollen tube growth, we profiled the transcription patterns of 1,536 pollen cDNAs from lily (Lilium longiflorum) by microarray. Among those that exhibited significant differential expression, a cDNA named lily ankyrin repeat-containing protein (LlANK) was thoroughly studied. The full-length LlANK cDNA sequence predicts a protein containing five tandem ankyrin repeats and a RING zinc-finger domain. The LlANK protein possesses ubiquitin ligase activity in vitro. RNA blots demonstrated that LlANK transcript is present in mature pollen and its level, interestingly contrary to most pollen mRNAs, up-regulated significantly during pollen germination and pollen tube growth. When fused with green fluorescent protein and transiently expressed in pollen, LlANK was found dominantly associated with membrane-enclosed organelles as well as the generative cell. Overexpression of LlANK, however, led to abnormal growth of the pollen tube. On the other hand, transient silencing of LlANK impaired pollen germination and tube growth. Taken together, these results showed that LlANK is a ubiquitin ligase associated with membrane-enclosed organelles and required for polarized pollen tube growth. PMID:16461387

Mixing, diffusion, and percolation in binary supported membranes containing mixtures of lipids and amphiphilic block copolymers.

PubMed

Gettel, Douglas L; Sanborn, Jeremy; Patel, Mira A; de Hoog, Hans-Peter; Liedberg, Bo; Nallani, Madhavan; Parikh, Atul N

2014-07-23

Substrate-mediated fusion of small polymersomes, derived from mixtures of lipids and amphiphilic block copolymers, produces hybrid, supported planar bilayers at hydrophilic surfaces, monolayers at hydrophobic surfaces, and binary monolayer/bilayer patterns at amphiphilic surfaces, directly responding to local measures of (and variations in) surface free energy. Despite the large thickness mismatch in their hydrophobic cores, the hybrid membranes do not exhibit microscopic phase separation, reflecting irreversible adsorption and limited lateral reorganization of the polymer component. With increasing fluid-phase lipid fraction, these hybrid, supported membranes undergo a fluidity transition, producing a fully percolating fluid lipid phase beyond a critical area fraction, which matches the percolation threshold for the immobile point obstacles. This then suggests that polymer-lipid hybrid membranes might be useful models for studying obstructed diffusion, such as occurs in lipid membranes containing proteins.

Random heteropolymers preserve protein function in foreign environments

NASA Astrophysics Data System (ADS)

Panganiban, Brian; Qiao, Baofu; Jiang, Tao; DelRe, Christopher; Obadia, Mona M.; Nguyen, Trung Dac; Smith, Anton A. A.; Hall, Aaron; Sit, Izaac; Crosby, Marquise G.; Dennis, Patrick B.; Drockenmuller, Eric; Olvera de la Cruz, Monica; Xu, Ting

2018-03-01

The successful incorporation of active proteins into synthetic polymers could lead to a new class of materials with functions found only in living systems. However, proteins rarely function under the conditions suitable for polymer processing. On the basis of an analysis of trends in protein sequences and characteristic chemical patterns on protein surfaces, we designed four-monomer random heteropolymers to mimic intrinsically disordered proteins for protein solubilization and stabilization in non-native environments. The heteropolymers, with optimized composition and statistical monomer distribution, enable cell-free synthesis of membrane proteins with proper protein folding for transport and enzyme-containing plastics for toxin bioremediation. Controlling the statistical monomer distribution in a heteropolymer, rather than the specific monomer sequence, affords a new strategy to interface with biological systems for protein-based biomaterials.

Protein targeting and integration signal for the chloroplastic outer envelope membrane.

PubMed Central

Li, H M; Chen, L J

1996-01-01

Most proteins in chloroplasts are encoded by the nuclear genome and synthesized in the cytosol. With the exception of most quter envelope membrane proteins, nuclear-encoded chloroplastic proteins are synthesized with N-terminal extensions that contain the chloroplast targeting information of these proteins. Most outer membrane proteins, however, are synthesized without extensions in the cytosol. Therefore, it is not clear where the chloroplastic outer membrane targeting information resides within these polypeptides. We have analyzed a chloroplastic outer membrane protein, OEP14 (outer envelope membrane protein of 14 kD, previously named OM14), and localized its outer membrane targeting and integration signal to the first 30 amino acids of the protein. This signal consists of a positively charged N-terminal portion followed by a hydrophobic core, bearing resemblance to the signal peptides of proteins targeted to the endoplasmic reticulum. However, a chimeric protein containing this signal fused to a passenger protein did not integrate into the endoplasmic reticulum membrane. Furthermore, membrane topology analysis indicated that the signal inserts into the chloroplastic outer membrane in an orientation opposite to that predicted by the "positive inside" rule. PMID:8953775

Plasmodium knowlesi Skeleton-Binding Protein 1 Localizes to the ‘Sinton and Mulligan’ Stipplings in the Cytoplasm of Monkey and Human Erythrocytes

PubMed Central

Lucky, Amuza Byaruhanga; Sakaguchi, Miako; Katakai, Yuko; Kawai, Satoru; Yahata, Kazuhide; Templeton, Thomas J.

2016-01-01

The malaria parasite, Plasmodium, exports protein products to the infected erythrocyte to introduce modifications necessary for the establishment of nutrient acquisition and surface display of host interaction ligands. Erythrocyte remodeling impacts parasite virulence and disease pathology and is well documented for the human malaria parasite Plasmodium falciparum, but has been less described for other Plasmodium species. For P. falciparum, the exported protein skeleton-binding protein 1 (PfSBP1) is involved in the trafficking of erythrocyte surface ligands and localized to membranous structures within the infected erythrocyte, termed Maurer's clefts. In this study, we analyzed SBP1 orthologs across the Plasmodium genus by BLAST analysis and conserved gene synteny, which were also recently described by de Niz et al. (2016). To evaluate the localization of an SBP1 ortholog, we utilized the zoonotic malaria parasite, Plasmodium knowlesi. Immunofluorescence assay of transgenic P. knowlesi parasites expressing epitope-tagged recombinant PkSBP1 revealed a punctate staining pattern reminiscent of Maurer's clefts, following infection of either monkey or human erythrocytes. The recombinant PkSBP1-positive puncta co-localized with Giemsa-stained structures, known as ‘Sinton and Mulligan’ stipplings. Immunoelectron microscopy also showed that recombinant PkSBP1 localizes within or on the membranous structures akin to the Maurer's clefts. The recombinant PkSBP1 expressed in P. falciparum-infected erythrocytes co-localized with PfSBP1 at the Maurer's clefts, indicating an analogous trafficking pattern. A member of the P. knowlesi 2TM protein family was also expressed and localized to membranous structures in infected monkey erythrocytes. These results suggest that the trafficking machinery and induced erythrocyte cellular structures of P. knowlesi are similar following infection of both monkey and human erythrocytes, and are conserved with P. falciparum. PMID:27732628

Nanoscale domain formation of phosphatidylinositol 4-phosphate in the plasma and vacuolar membranes of living yeast cells.

PubMed

Tomioku, Kan-Na; Shigekuni, Mikiko; Hayashi, Hiroki; Yoshida, Akane; Futagami, Taiki; Tamaki, Hisanori; Tanabe, Kenji; Fujita, Akikazu

2018-05-01

In budding yeast Saccharomyces cerevisiae, PtdIns(4)P serves as an essential signalling molecule in the Golgi complex, endosomal system, and plasma membrane, where it is involved in the control of multiple cellular functions via direct interactions with PtdIns(4)P-binding proteins. To analyse the distribution of PtdIns(4)P in yeast cells at a nanoscale level, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the yeast membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilised in situ. We observed that PtdIns(4)P is localised on the cytoplasmic leaflet, but not the exoplasmic leaflet, of the plasma membrane, Golgi body, vacuole, and vesicular structure membranes. PtdIns(4)P labelling was not observed in the membrane of the endoplasmic reticulum, and in the outer and inner membranes of the nuclear envelope or mitochondria. PtdIns(4)P forms clusters of <100 nm in diameter in the plasma membrane and vacuolar membrane according to point pattern analysis of immunogold labelling. There are three kinds of compartments in the cytoplasmic leaflet of the plasma membrane. In the present study, we showed that PtdIns(4)P is specifically localised in the flat undifferentiated plasma membrane compartment. In the vacuolar membrane, PtdIns(4)P was concentrated in intramembrane particle (IMP)-deficient raft-like domains, which are tightly bound to lipid droplets, but not surrounding IMP-rich non-raft domains in geometrical IMP-distributed patterns in the stationary phase. This is the first report showing microdomain formations of PtdIns(4)P in the plasma membrane and vacuolar membrane of budding yeast cells at a nanoscale level, which will illuminate the functionality of PtdIns(4)P in each membrane. Copyright © 2018 Elsevier GmbH. All rights reserved.

Observing conformations of single FoF1-ATP synthases in a fast anti-Brownian electrokinetic trap

NASA Astrophysics Data System (ADS)

Su, Bertram; Düser, Monika G.; Zarrabi, Nawid; Heitkamp, Thomas; Starke, Ilka; Börsch, Michael

2015-03-01

To monitor conformational changes of individual membrane transporters in liposomes in real time, we attach two fluorophores to selected domains of a protein. Sequential distance changes between the dyes are recorded and analyzed by Förster resonance energy transfer (FRET). Using freely diffusing membrane proteins reconstituted in liposomes, observation times are limited by Brownian motion through the confocal detection volume. A. E. Cohen and W. E. Moerner have invented and built microfluidic devices to actively counteract Brownian motion of single nanoparticles in electrokinetic traps (ABELtrap). Here we present a version of an ABELtrap with a laser focus pattern generated by electro-optical beam deflectors and controlled by a programmable FPGA. This ABELtrap could hold single fluorescent nanobeads for more than 100 seconds, increasing the observation times of a single particle more than 1000-fold. Conformational changes of single FRET-labeled membrane enzymes FoF1-ATP synthase can be detected in the ABELtrap.

Boltzmann Energy-based Image Analysis Demonstrates that Extracellular Domain Size Differences Explain Protein Segregation at Immune Synapses

PubMed Central

Burroughs, Nigel J.; Köhler, Karsten; Miloserdov, Vladimir; Dustin, Michael L.; van der Merwe, P. Anton; Davis, Daniel M.

2011-01-01

Immune synapses formed by T and NK cells both show segregation of the integrin ICAM1 from other proteins such as CD2 (T cell) or KIR (NK cell). However, the mechanism by which these proteins segregate remains unclear; one key hypothesis is a redistribution based on protein size. Simulations of this mechanism qualitatively reproduce observed segregation patterns, but only in certain parameter regimes. Verifying that these parameter constraints in fact hold has not been possible to date, this requiring a quantitative coupling of theory to experimental data. Here, we address this challenge, developing a new methodology for analysing and quantifying image data and its integration with biophysical models. Specifically we fit a binding kinetics model to 2 colour fluorescence data for cytoskeleton independent synapses (2 and 3D) and test whether the observed inverse correlation between fluorophores conforms to size dependent exclusion, and further, whether patterned states are predicted when model parameters are estimated on individual synapses. All synapses analysed satisfy these conditions demonstrating that the mechanisms of protein redistribution have identifiable signatures in their spatial patterns. We conclude that energy processes implicit in protein size based segregation can drive the patternation observed in individual synapses, at least for the specific examples tested, such that no additional processes need to be invoked. This implies that biophysical processes within the membrane interface have a crucial impact on cell∶cell communication and cell signalling, governing protein interactions and protein aggregation. PMID:21829338

Determinants for membrane association and permeabilization of the coxsackievirus 2B protein and the identification of the Golgi complex as the target organelle.

PubMed

de Jong, Arjan S; Wessels, Els; Dijkman, Henri B P M; Galama, Jochem M D; Melchers, Willem J G; Willems, Peter H G M; van Kuppeveld, Frank J M

2003-01-10

The 2B protein of enterovirus is responsible for the alterations in the permeability of secretory membranes and the plasma membrane in infected cells. The structural requirements for the membrane association and the subcellular localization of this essential virus protein, however, have not been defined. Here, we provide evidence that the 2B protein is an integral membrane protein in vivo that is predominantly localized at the Golgi complex upon individual expression. Addition of organelle-specific targeting signals to the 2B protein revealed that the Golgi localization is an absolute prerequisite for the ability of the protein to modify plasma membrane permeability. Expression of deletion mutants and heterologous proteins containing specific domains of the 2B protein demonstrated that each of the two hydrophobic regions could mediate membrane binding individually. However, the presence of both hydrophobic regions was required for the correct membrane association, efficient Golgi targeting, and the membrane-permeabilizing activity of the 2B protein, suggesting that the two hydrophobic regions are cooperatively involved in the formation of a membrane-integral complex. The formation of membrane-integral pores by the 2B protein in the Golgi complex and the possible mechanism by which a Golgi-localized virus protein modifies plasma membrane permeability are discussed.

Biochemical characterization of detergent-resistant membranes: a systematic approach

PubMed Central

Babiychuk, Eduard B.; Draeger, Annette

2006-01-01

Lateral segregation of cholesterol- and sphingomyelin-rich rafts and glycerophospholipid-containing non-raft microdomains has been proposed to play a role in a variety of biological processes. The most compelling evidence for membrane segregation is based on the observation that extraction with non-ionic detergents leads to solubilization of a subset of membrane components only. However, one decade later, a large body of inconsistent detergent-extraction data is threatening the very concept of membrane segregation. We have assessed the validity of the existing paradigms and we show the following. (i) The localization of a membrane component within a particular fraction of a sucrose gradient cannot be taken as a yardstick for its solubility: a variable localization of the DRMs (detergent-resistant membranes) in sucrose gradients is the result of complex associations between the membrane skeleton and the lipid bilayer. (ii) DRMs of variable composition can be generated by using a single detergent, the increasing concentration of which gradually extracts one protein/lipid after another. Therefore any extraction pattern obtained by a single concentration experiment is bound to be ‘investigator-specific’. It follows that comparison of DRMs obtained by different detergents in a single concentration experiment is prone to misinterpretations. (iii) Depletion of cholesterol has a graded effect on membrane solubility. (iv) Differences in detergent solubility of the members of the annexin protein family arise from their association with chemically different membrane compartments; however, these cannot be attributed to the ‘brick-like’ raft-building blocks of fixed size and chemical composition. Our findings demonstrate a need for critical re-evaluation of the accumulated detergent-extraction data. PMID:16608442

Biochemical characterization of detergent-resistant membranes: a systematic approach.

PubMed

Babiychuk, Eduard B; Draeger, Annette

2006-08-01

Lateral segregation of cholesterol- and sphingomyelin-rich rafts and glycerophospholipid-containing non-raft microdomains has been proposed to play a role in a variety of biological processes. The most compelling evidence for membrane segregation is based on the observation that extraction with non-ionic detergents leads to solubilization of a subset of membrane components only. However, one decade later, a large body of inconsistent detergent-extraction data is threatening the very concept of membrane segregation. We have assessed the validity of the existing paradigms and we show the following. (i) The localization of a membrane component within a particular fraction of a sucrose gradient cannot be taken as a yardstick for its solubility: a variable localization of the DRMs (detergent-resistant membranes) in sucrose gradients is the result of complex associations between the membrane skeleton and the lipid bilayer. (ii) DRMs of variable composition can be generated by using a single detergent, the increasing concentration of which gradually extracts one protein/lipid after another. Therefore any extraction pattern obtained by a single concentration experiment is bound to be 'investigator-specific'. It follows that comparison of DRMs obtained by different detergents in a single concentration experiment is prone to misinterpretations. (iii) Depletion of cholesterol has a graded effect on membrane solubility. (iv) Differences in detergent solubility of the members of the annexin protein family arise from their association with chemically different membrane compartments; however, these cannot be attributed to the 'brick-like' raft-building blocks of fixed size and chemical composition. Our findings demonstrate a need for critical re-evaluation of the accumulated detergent-extraction data.

Release of Membrane-Bound Vesicles and Inhibition of Tumor Cell Adhesion by the Peptide Neopetrosiamide A

PubMed Central

Austin, Pamela; Heller, Markus; Williams, David E.; McIntosh, Lawrence P.; Vogl, A. Wayne; Foster, Leonard J.; Andersen, Raymond J.; Roberge, Michel; Roskelley, Calvin D.

2010-01-01

Background Neopetrosiamide A (NeoA) is a 28-amino acid tricyclic peptide originally isolated from a marine sponge as a tumor cell invasion inhibitor whose mechanism of action is unknown. Methodology/Principal Findings We show that NeoA reversibly inhibits tumor cell adhesion, disassembles focal adhesions in pre-attached cells, and decreases the level of β1 integrin subunits on the cell surface. NeoA also induces the formation of dynamic, membrane-bound protrusions on the surface of treated cells and the release of membrane-bound vesicles into the culture medium. Proteomic analysis indicates that the vesicles contain EGF and transferrin receptors as well as a number of proteins involved in adhesion and migration including: β1 integrin and numerous α integrin subunits; actin and actin-binding proteins such as cofilin, moesin and myosin 1C; and membrane modulating eps15 homology domain (EHD) proteins. Surface labeling, trafficking inhibition, and real-time imaging experiments all suggest that β1 integrin-containing vesicles are released directly from NeoA-induced cell surface protrusions rather than from vesicles generated intracellularly. The biological activity of NeoA is dependent on its disulfide bond pattern and NMR spectroscopy indicates that the peptide is globular with a continuous ridge of hydrophobic groups flanked by charged amino acid residues that could facilitate a simultaneous interaction with lipids and proteins in the membrane. Conclusions/Significance NeoA is an anti-adhesive peptide that decreases cell surface integrin levels through a novel, yet to be elucidated, mechanism that involves the release of adhesion molecule-containing vesicles from the cell surface. PMID:20520768

KCNE Regulation of K+ Channel Trafficking – a Sisyphean Task?

PubMed Central

Kanda, Vikram A.; Abbott, Geoffrey W.

2012-01-01

Voltage-gated potassium (Kv) channels shape the action potentials of excitable cells and regulate membrane potential and ion homeostasis in excitable and non-excitable cells. With 40 known members in the human genome and a variety of homomeric and heteromeric pore-forming α subunit interactions, post-translational modifications, cellular locations, and expression patterns, the functional repertoire of the Kv α subunit family is monumental. This versatility is amplified by a host of interacting proteins, including the single membrane-spanning KCNE ancillary subunits. Here, examining both the secretory and the endocytic pathways, we review recent findings illustrating the surprising virtuosity of the KCNE proteins in orchestrating not just the function, but also the composition, diaspora and retrieval of channels formed by their Kv α subunit partners. PMID:22754540

Using a biomimetic membrane surface experiment to investigate the activity of the magnetite biomineralisation protein Mms6† †Electronic supplementary information (ESI) available: Including Mms6 protein and peptide sequences, additional QCM-D and SEM data and protein modelling. See DOI: 10.1039/c5ra16469a Click here for additional data file.

PubMed Central

Bird, Scott M.; Rawlings, Andrea E.; Galloway, Johanna M.

2016-01-01

Magnetotactic bacteria are able to synthesise precise nanoparticles of the iron oxide magnetite within their cells. These particles are formed in dedicated organelles termed magnetosomes. These lipid membrane compartments use a range of biomineralisation proteins to nucleate and regulate the magnetite crystallisation process. A key component is the membrane protein Mms6, which binds to iron ions and helps to control the formation of the inorganic core. We have previously used Mms6 on gold surfaces patterned with a self-assembled monolayer to successfully produce arrays of magnetic nanoparticles. Here we use this surface system as a mimic of the interior face of the magnetosome membrane to study differences between intact Mms6 and the acid-rich C-terminal peptide subregion of the Mms6 protein. When immobilised on surfaces, the peptide is unable to reproduce the particle size or homogeneity control exhibited by the full Mms6 protein in our experimental setup. Moreover, the peptide is unable to support anchoring of a dense array of nanoparticles to the surface. This system also allows us to deconvolute particle binding from particle nucleation, and shows that Mms6 particle binding is less efficient when supplied with preformed magnetite nanoparticles when compared to particles precipitated from solution in the presence of the surface immobilised Mms6. This suggests that Mms6 binds to iron ions rather than to magnetite surfaces in our system, and is perhaps a nucleating agent rather than a controller of magnetite crystal growth. The comparison between the peptide and the protein under identical experimental conditions indicates that the full length sequence is required to support the full function of Mms6 on surfaces. PMID:27019707

Biochemical characterization of domain-specific glycoproteins of the rat hepatocyte plasma membrane.

PubMed

Bartles, J R; Braiterman, L T; Hubbard, A L

1985-10-15

Seven integral proteins (CE 9, HA 21, HA 116, HA 16, HA 4, HA 201, and HA 301) were isolated from rat hepatocyte plasma membranes by immunoaffinity chromatography on monoclonal antibody-Sepharose. Six of the proteins (all but HA 16) exhibit domain-specific localizations (either bile canalicular or sinusoidal/lateral) about the hepatocyte surface. We identified three of these protein antigens as leucine aminopeptidase (HA 201), dipeptidyl peptidase IV (HA 301), and the asialoglycoprotein receptor (HA 116). We also developed 125I-lectin blotting procedures that, when used in conjunction with chemical and glycosidase treatments, permitted a comparison of the types of oligosaccharides present on the seven proteins. All seven are sialoglycoproteins, based upon the effects of prior neuraminidase and periodate-aniline-cyanoborohydride treatments of blots on labeling by 125I-wheat germ agglutinin. 125I-labeled Ricinus communis agglutinin I and 125I-peanut agglutinin blotting of the desialylated proteins revealed few if any conventional O-linked oligosaccharides, suggesting that the sialyl residues represent termini of N-linked complex-type oligosaccharides. Depending upon the protein, we estimated the presence of 2-26 N-linked oligosaccharides/polypeptide chain from the Mr reductions accompanying chemical or enzymatic deglycosylation. Three of these mature plasma membrane proteins (HA 21, HA 116, and HA 4) have both high mannose-type and complex-type oligosaccharides on every copy of their polypeptide chains. The labeling of these three proteins by 125I-concanavalin A was sensitive to treatment with endoglycosidase H, and each exhibited a quantitative reduction in Mr after the treatment, as assessed independently by 125I-wheat germ agglutinin blotting. At this level of analysis, we were unable to discern differences in the types of oligosaccharides present on these seven glycoproteins that correlate with their patterns of expression within the plasma membrane domains of this polarized epithelial cell.

Profiling of sperm proteins and association of sperm PDC-109 with bull fertility.

PubMed

Somashekar, Lakshminarayana; Selvaraju, Sellappan; Parthipan, Sivashanmugam; Ravindra, Janivara Parameswaraiah

2015-01-01

The composition of sperm proteins influences the fertilizing ability of sperm and hence the present study was conducted (i) to profile sperm proteins expression patterns in bulls of differing fertility index and (ii) to identify and relate the abundant sperm proteins with bull fertility. The semen samples were collected from Holstein-Friesian bulls (n = 12) varying in conception rate (CR) (high/low). The frozen semen straws (three ejaculates, from each bull) were used to study (a) sperm kinetic parameters, (b) plasmalemma integrity, (c) mitochondrial membrane potential, and (d) chromatin distribution. Three bulls were randomly selected from each group (n = 3) and the neat sperm pellets were subjected to percoll purification, followed by protein isolation using 0.1% Triton X100. The sperm kinetic parameters, plasmalemma integrity, mitochondrial membrane potential, and the chromatin distribution did not differ significantly between groups. The number of acidic (pI; 3.1-5.6, 37%) and basic (pI; 7.9-10.0, 27%) proteins and their pattern of expression varied significantly (p < 0.05) between high and low fertile bulls. The abundant sperm protein spots in 2D-gel electrophoresis (2DE) were identified as seminal plasma protein PDC-109 (i.e., protein with N-terminus aspartic acid, D and carboxy terminus cystine, having 109 amino acids) and its isoform and spermadhesin-1 (SPADH1). The western blot analysis confirmed the presence of PDC-109 isoform proteins at 15.4 kDa (pI 5.3 and 5.5). The seminal plasma protein PDC-109 was abundant in the low fertile when compared to the high fertile group (p < 0.05). This study suggests that the imbalance in acidic and basic sperm proteins may influence sperm fertility and sperm PDC-109 levels above a certain threshold affects bull fertility.

Cuscuta europaea plastid apparatus in various developmental stages: localization of THF1 protein.

PubMed

Švubová, Renáta; Ovečka, Miroslav; Pavlovič, Andrej; Slováková, L'udmila; Blehová, Alžbeta

2013-05-01

It was generally accepted that Cuscuta europaea is mostly adapted to a parasitic lifestyle with no detectable levels of chlorophylls. We found out relatively high level of chlorophylls (Chls a+b) in young developmental stages of dodder. Significant lowering of Chls (a+b) content and increase of carotenoid concentration was typical only for ontogenetically more developed stages. Lower content of photosynthesis-related proteins involved in Chls biosynthesis and in photosystem formation as well as low photochemical activity of PSII indicate that photosynthesis is not the main activity of C. europaea plastids. Previously, it has been shown in other species that the Thylakoid Formation Protein 1 (THF1) is involved in thylakoid membrane differentiation, plant-fungal and plant-bacterial interactions and in sugar signaling with its preferential localization to plastids. Our immunofluorescence localization studies and analyses of haustorial plasma membrane fractions revealed that in addition to plastids, the THF1 protein localizes also to the plasma membrane and plasmodesmata in developing C. europaea haustorium, most abundantly in the digitate cells of the endophyte primordium. These results are supported by western blot analysis, documenting the highest levels of the THF1 protein in "get together" tissues of dodder and tobacco. Based on the fact that photosynthesis is not a typical process in the C. europaea haustorium and on the extra-plastidial localization pattern of the THF1, our data support rather other functions of this protein in the complex relationship between C. europaea and its host.

Basolateral junctions are sufficient to suppress epithelial invasion during Drosophila oogenesis.

PubMed

Szafranski, Przemyslaw; Goode, Scott

2007-02-01

Epithelial junctions play crucial roles during metazoan evolution and development by facilitating tissue formation, maintenance, and function. Little is known about the role of distinct types of junctions in controlling epithelial transformations leading to invasion of neighboring tissues. Discovering the key junction complexes that control these processes and how they function may also provide mechanistic insight into carcinoma cell invasion. Here, using the Drosophila ovary as a model, we show that four proteins of the basolateral junction (BLJ), Fasciclin-2, Neuroglian, Discs-large, and Lethal-giant-larvae, but not proteins of other epithelial junctions, directly suppress epithelial tumorigenesis and invasion. Remarkably, the expression pattern of Fasciclin-2 predicts which cells will invade. We compared the apicobasal polarity of BLJ tumor cells to border cells (BCs), an epithelium-derived cluster that normally migrates during mid-oogenesis. Both tumor cells and BCs differentiate a lateralized membrane pattern that is necessary but not sufficient for invasion. Independent of lateralization, derepression of motility pathways is also necessary, as indicated by a strong linear correlation between faster BC migration and an increased incidence of tumor invasion. However, without membrane lateralization, derepression of motility pathways is also not sufficient for invasion. Our results demonstrate that spatiotemporal patterns of basolateral junction activity directly suppress epithelial invasion by organizing the cooperative activity of distinct polarity and motility pathways.

Enhancing Membrane Protein Identification Using a Simplified Centrifugation and Detergent-Based Membrane Extraction Approach.

PubMed

Zhou, Yanting; Gao, Jing; Zhu, Hongwen; Xu, Jingjing; He, Han; Gu, Lei; Wang, Hui; Chen, Jie; Ma, Danjun; Zhou, Hu; Zheng, Jing

2018-02-20

Membrane proteins may act as transporters, receptors, enzymes, and adhesion-anchors, accounting for nearly 70% of pharmaceutical drug targets. Difficulties in efficient enrichment, extraction, and solubilization still exist because of their relatively low abundance and poor solubility. A simplified membrane protein extraction approach with advantages of user-friendly sample processing procedures, good repeatability and significant effectiveness was developed in the current research for enhancing enrichment and identification of membrane proteins. This approach combining centrifugation and detergent along with LC-MS/MS successfully identified higher proportion of membrane proteins, integral proteins and transmembrane proteins in membrane fraction (76.6%, 48.1%, and 40.6%) than in total cell lysate (41.6%, 16.4%, and 13.5%), respectively. Moreover, our method tended to capture membrane proteins with high degree of hydrophobicity and number of transmembrane domains as 486 out of 2106 (23.0%) had GRAVY > 0 in membrane fraction, 488 out of 2106 (23.1%) had TMs ≥ 2. It also provided for improved identification of membrane proteins as more than 60.6% of the commonly identified membrane proteins in two cell samples were better identified in membrane fraction with higher sequence coverage. Data are available via ProteomeXchange with identifier PXD008456.

Characterization of membrane association of Rinderpest virus matrix protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Subhashri, R.; Shaila, M.S.

2007-04-20

Paramyxovirus matrix protein is believed to play a crucial role in the assembly and maturation of the virus particle by bringing the major viral components together at the budding site in the host cell. The membrane association capability of many enveloped virus matrix proteins has been characterized to be their intrinsic property. In this work, we have characterized the membrane association of Rinderpest virus matrix (M) protein. The M protein of Rinderpest virus when expressed in the absence of other viral proteins is present both in the cytoplasm and plasma membrane. When expressed as GFP fusion protein, the M proteinmore » gets localized into plasma membrane protrusions. High salt and alkaline conditions resulted in partial dissociation of M protein from cell membrane. Thus, M protein behaves like an integral membrane protein although its primary structure suggests it to be a peripheral membrane protein.« less

Selective affinity labeling of a 27-kDa integral membrane protein in rat liver and kidney with N-bromoacetyl derivatives of L-thyroxine and 3,5,3'-triiodo-L-thyronine.

PubMed

Köhrle, J; Rasmussen, U B; Rokos, H; Leonard, J L; Hesch, R D

1990-04-15

125I-Labeled N-bromoacetyl derivatives of L-thyroxine and L-triiodothyronine were used as alkylating affinity labels to identify rat liver and kidney microsomal membrane proteins which specifically bind thyroid hormones. Affinity label incorporation was analyzed by ethanol precipitation and individual affinity labeled proteins were identified by autoradiography after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Six to eight membrane proteins ranging in size from 17 to 84 kDa were affinity labeled by both bromoacetyl-L-thyroxine (BrAcT4) and bromoacetyl-L-triiodothyronine (BrAcT3). Affinity labeling was time- and temperature-dependent, and both reduced dithiols and detergents increased affinity labeling, predominantly in a 27-kDa protein(s). Up to 80% of the affinity label was associated with a 27-kDa protein (p27) under optimal conditions. Affinity labeling of p27 by 0.4 nM BrAc[125I]L-T4 was blocked by 0.1 microM of the alkylating ligands BrAcT4, BrAcT3, or 100 microM iodoacetate, by 10 microM concentrations of the non-alkylating, reversible ligands N-acetyl-L-thyroxine, 3,3',5'-triiodothyronine, 3,5-diiodosalicylate, and EMD 21388, a T4-antagonistic flavonoid. Neither 10 microM L-T4, nor 10 microM N-acetyltriiodothyronine or 10 microM L-triiodothyronine blocked affinity labeling of p27 or other affinity labeled bands. Affinity labeling of a 17-kDa band was partially inhibited by excess of the alkylating ligands BrAcT4, BrAcT3, and iodoacetate, but labeling of other minor bands was not blocked by excess of the competitors. BrAc[125I]T4 yielded higher affinity label incorporation than BrAc[125I]T3, although similar banding patterns were observed, except that BrAcT3 affinity labeled more intensely a 58,000-Da band in liver and a 53,000-55,000-Da band in kidney. The pattern of other affinity labeled proteins with p27 as the predominant band was similar in liver and kidney. Peptide mapping of affinity labeled p27 and p55 bands by chemical cleavage and protease fragmentation revealed no common bands excluding that p27 is a degradation product of p55. These data indicate that N-bromoacetyl derivatives of T4 and T3 affinity label a limited but similar constellation of membrane proteins with BrAcT4 incorporation greater than that of BrAcT3. One membrane protein (p27) of low abundance (2-5 pmol/mg microsomal protein) with a reactive sulfhydryl group is selectively labeled under conditions identical to those used to measure thyroid hormone 5'-deiodination. Only p27 showed differential affinity labeling in the presence of noncovalently bound inhibitors or substrates on 5'-deiodinase suggesting that p27 is likely to be a component of type I 5'-deiodinase in rat liver and kidney.

Membrane protein synthesis in cell-free systems: from bio-mimetic systems to bio-membranes.

PubMed

Sachse, Rita; Dondapati, Srujan K; Fenz, Susanne F; Schmidt, Thomas; Kubick, Stefan

2014-08-25

When taking up the gauntlet of studying membrane protein functionality, scientists are provided with a plethora of advantages, which can be exploited for the synthesis of these difficult-to-express proteins by utilizing cell-free protein synthesis systems. Due to their hydrophobicity, membrane proteins have exceptional demands regarding their environment to ensure correct functionality. Thus, the challenge is to find the appropriate hydrophobic support that facilitates proper membrane protein folding. So far, various modes of membrane protein synthesis have been presented. Here, we summarize current state-of-the-art methodologies of membrane protein synthesis in biomimetic-supported systems. The correct folding and functionality of membrane proteins depend in many cases on their integration into a lipid bilayer and subsequent posttranslational modification. We highlight cell-free systems utilizing the advantages of biological membranes. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

A versatile palindromic amphipathic repeat coding sequence horizontally distributed among diverse bacterial and eucaryotic microbes

PubMed Central

2010-01-01

Background Intragenic tandem repeats occur throughout all domains of life and impart functional and structural variability to diverse translation products. Repeat proteins confer distinctive surface phenotypes to many unicellular organisms, including those with minimal genomes such as the wall-less bacterial monoderms, Mollicutes. One such repeat pattern in this clade is distributed in a manner suggesting its exchange by horizontal gene transfer (HGT). Expanding genome sequence databases reveal the pattern in a widening range of bacteria, and recently among eucaryotic microbes. We examined the genomic flux and consequences of the motif by determining its distribution, predicted structural features and association with membrane-targeted proteins. Results Using a refined hidden Markov model, we document a 25-residue protein sequence motif tandemly arrayed in variable-number repeats in ORFs lacking assigned functions. It appears sporadically in unicellular microbes from disparate bacterial and eucaryotic clades, representing diverse lifestyles and ecological niches that include host parasitic, marine and extreme environments. Tracts of the repeats predict a malleable configuration of recurring domains, with conserved hydrophobic residues forming an amphipathic secondary structure in which hydrophilic residues endow extensive sequence variation. Many ORFs with these domains also have membrane-targeting sequences that predict assorted topologies; others may comprise reservoirs of sequence variants. We demonstrate expressed variants among surface lipoproteins that distinguish closely related animal pathogens belonging to a subgroup of the Mollicutes. DNA sequences encoding the tandem domains display dyad symmetry. Moreover, in some taxa the domains occur in ORFs selectively associated with mobile elements. These features, a punctate phylogenetic distribution, and different patterns of dispersal in genomes of related taxa, suggest that the repeat may be disseminated by HGT and intra-genomic shuffling. Conclusions We describe novel features of PARCELs (Palindromic Amphipathic Repeat Coding ELements), a set of widely distributed repeat protein domains and coding sequences that were likely acquired through HGT by diverse unicellular microbes, further mobilized and diversified within genomes, and co-opted for expression in the membrane proteome of some taxa. Disseminated by multiple gene-centric vehicles, ORFs harboring these elements enhance accessory gene pools as part of the "mobilome" connecting genomes of various clades, in taxa sharing common niches. PMID:20626840

Epstein-Barr virus latent membrane protein expression in Hodgkin and Reed-Sternberg cells.

PubMed Central

Herbst, H; Dallenbach, F; Hummel, M; Niedobitek, G; Pileri, S; Müller-Lantzsch, N; Stein, H

1991-01-01

Cryostat sections from lymph nodes of 47 Hodgkin disease patients were examined by immunohistology for the Epstein-Barr virus (EBV)-encoded latent membrane protein (LMP), nuclear antigen 2, and late viral glycoprotein gp350/250. A distinct LMP-specific membrane and cytoplasmic staining was detected exclusively in Hodgkin and Reed-Sternberg cells in 18 patients (38%); EBV nuclear antigen 2 and gp350/250 immunoreactivity was absent in all instances. Thirty-two of 47 (68%) cases contained EBV-specific DNA sequences as detected by PCR, all LMP-positive cases being in this category. Our results confirm previous studies establishing the presence of EBV genomes in Hodgkin and Reed-Sternberg cells by demonstrating expression of an EBV-encoded protein in the tumor-cell population. The finding of LMP expression in the absence of EBV nuclear antigen 2 suggests a pattern of EBV gene expression different from that of B-lymphoblastoid cell lines and Burkitt lymphoma, whereas this finding shows similarities with that seen in undifferentiated nasopharyngeal carcinoma. Because the LMP gene has transforming potential, our findings support the concept of a pathoetiological role of EBV in many cases of Hodgkin disease. Images PMID:1647016

Development of the Cellular Immune System of Drosophila Requires the Membrane Attack Complex/Perforin-Like Protein Torso-Like.

PubMed

Forbes-Beadle, Lauren; Crossman, Tova; Johnson, Travis K; Burke, Richard; Warr, Coral G; Whisstock, James C

2016-10-01

Pore-forming members of the membrane attack complex/perforin-like (MACPF) protein superfamily perform well-characterized roles as mammalian immune effectors. For example, complement component 9 and perforin function to directly form pores in the membrane of Gram-negative pathogens or virally infected/transformed cells, respectively. In contrast, the only known MACPF protein in Drosophila melanogaster, Torso-like, plays crucial roles during development in embryo patterning and larval growth. Here, we report that in addition to these functions, Torso-like plays an important role in Drosophila immunity. However, in contrast to a hypothesized effector function in, for example, elimination of Gram-negative pathogens, we find that torso-like null mutants instead show increased susceptibility to certain Gram-positive pathogens such as Staphylococcus aureus and Enterococcus faecalis We further show that this deficit is due to a severely reduced number of circulating immune cells and, as a consequence, an impaired ability to phagocytose bacterial particles. Together these data suggest that Torso-like plays an important role in controlling the development of the Drosophila cellular immune system. Copyright © 2016 by the Genetics Society of America.

Mammalian plasma membrane proteins as potential biomarkers and drug targets.

PubMed

Rucevic, Marijana; Hixson, Douglas; Josic, Djuro

2011-06-01

Defining the plasma membrane proteome is crucial to understand the role of plasma membrane in fundamental biological processes. Change in membrane proteins is one of the first events that take place under pathological conditions, making plasma membrane proteins a likely source of potential disease biomarkers with prognostic or diagnostic potential. Membrane proteins are also potential targets for monoclonal antibodies and other drugs that block receptors or inhibit enzymes essential to the disease progress. Despite several advanced methods recently developed for the analysis of hydrophobic proteins and proteins with posttranslational modifications, integral membrane proteins are still under-represented in plasma membrane proteome. Recent advances in proteomic investigation of plasma membrane proteins, defining their roles as diagnostic and prognostic disease biomarkers and as target molecules in disease treatment, are presented. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Review: Post-translational cross-talk between brassinosteroid and sucrose signaling.

PubMed

Kühn, Christina

2016-07-01

A direct link has been elucidated between brassinosteroid function and perception, and sucrose partitioning and transport. Sucrose regulation and brassinosteroid signaling cross-talk at various levels, including the well-described regulation of transcriptional gene expression: BZR-like transcription factors link the signaling pathways. Since brassinosteroid responses depend on light quality and quantity, a light-dependent alternative pathway was postulated. Here, the focus is on post-translational events. Recent identification of sucrose transporter-interacting partners raises the question whether brassinosteroid and sugars jointly affect plant innate immunity and plant symbiotic interactions. Membrane permeability and sensitivity depends on the number of cell surface receptors and transporters. More than one endocytic route has been assigned to specific components, including brassinosteroid-receptors. The number of such proteins at the plasma membrane relies on endocytic recycling, internalization and/or degradation. Therefore, vesicular membrane trafficking is gaining considerable attention with regard to plant immunity. The organization of pattern recognition receptors (PRRs), other receptors or transporters in membrane microdomains participate in endocytosis and the formation of specific intracellular compartments, potentially impacting biotic interactions. This minireview focuses on post-translational events affecting the subcellular compartmentation of membrane proteins involved in signaling, transport, and defense, and on the cross-talk between brassinosteroid signals and sugar availability. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Proteopolymersomes: in vitro production of a membrane protein in polymersome membranes.

PubMed

Nallani, Madhavan; Andreasson-Ochsner, Mirjam; Tan, Cherng-Wen Darren; Sinner, Eva-Kathrin; Wisantoso, Yudi; Geifman-Shochat, Susana; Hunziker, Walter

2011-12-01

Polymersomes are stable self-assembled architectures which mimic cell membranes. For characterization, membrane proteins can be incorporated into such bio-mimetic membranes by reconstitution methods, leading to so-called proteopolymersomes. In this work, we demonstrate the direct incorporation of a membrane protein into polymersome membranes by a cell-free expression system. Firstly, we demonstrate pore formation in the preformed polymersome membrane using α-hemolysin. Secondly, we use claudin-2, a protein involved in cell-cell interactions, to demonstrate the in vitro expression of a membrane protein into these polymersomes. Surface plasmon resonance (Biacore) binding studies with the claudin-2 proteopolymersomes and claudin-2 specific antibodies are performed to show the presence of the in vitro expressed protein in polymersome membranes.

Glycoconjugate pattern of membranes in the acinar cell of the rat pancreas.

PubMed

Willemer, S; Köhler, H; Naumann, R; Kern, H F; Adler, G

1990-01-01

Lectin-binding studies were performed at the ultrastructural level to characterize glycoconjugate patterns on membrane systems in pancreatic acinar cells of the rat. Five lectins reacting with different sugar moieties were applied to ultrathin frozen sections: concanavalin A (ConA): glucose, mannose; wheat-germ agglutinin (WGA): N-acetylglucosamine, sialic acid; Ricinus communis agglutinin I (RCA I): galactose; Ulex europaeus agglutinin I (UEA I): L-fucose; soybean agglutinin (SBA): N-acetylgalactosamine). Binding sites of lectins were visualized either by direct conjugation to colloidal gold or by the use of a three-step procedure involving additional immune reactions. The rough endoplasmic reticulum and the nuclear envelope of acinar cells was selectively labelled for ConA. The membranes of the Golgi apparatus bound all lectins applied with an increasing intensity proceeding from the cis- to the trans-Golgi area for SBA, UEA I and WGA. In contrast RCA I selectively labelled the trans-Golgi cisternae. The membranes of condensing vacuoles and zymogen granules were labelled for all lectins used although the density of the label differed between the lectins. In contrast the content of zymogen granules failed to bind SBA and WGA. Lysosomal bodies (membranes and content) revealed binding sites for all lectins used. The plasma membranes were heavily labelled by all lectins except for SBA which showed only a weak binding to the lateral and the apical plasma membrane. These results are in accordance to current biochemical knowledge of the successive steps in the glycosylation of membrane proteins. It could be demonstrated, that the cryo-section technique is suitable for the fine structural localisation of surface glycoconjugates of plasma membranes and internal membranes in pancreatic acinar cells using plant lectins.

A Novel Form of Compensation in the Tg2576 Amyloid Mouse Model of Alzheimer’s Disease

PubMed Central

Somogyi, Attila; Katonai, Zoltán; Alpár, Alán; Wolf, Ervin

2016-01-01

One century after its first description, pathology of Alzheimer’s disease (AD) is still poorly understood. Amyloid-related dendritic atrophy and membrane alterations of susceptible brain neurons in AD, and in animal models of AD are widely recognized. However, little effort has been made to study the potential effects of combined morphological and membrane alterations on signal transfer and synaptic integration in neurons that build up affected neural networks in AD. In this study spatial reconstructions and electrophysiological measurements of layer II/III pyramidal neurons of the somatosensory cortex from wild-type (WT) and transgenic (TG) human amyloid precursor protein (hAPP) overexpressing Tg2576 mice were used to build faithful segmental cable models of these neurons. Local synaptic activities were simulated in various points of the dendritic arbors and properties of subthreshold dendritic impulse propagation and predictors of synaptic input pattern recognition ability were quantified and compared in modeled WT and TG neurons. Despite the widespread dendritic degeneration and membrane alterations in mutant mouse neurons, surprisingly little, or no change was detected in steady-state and 50 Hz sinusoidal voltage transfers, current transfers, and local and propagation delays of PSPs traveling along dendrites of TG neurons. Synaptic input pattern recognition ability was also predicted to be unaltered in TG neurons in two different soma-dendritic membrane models investigated. Our simulations predict the way how subthreshold dendritic signaling and pattern recognition are preserved in TG neurons: amyloid-related membrane alterations compensate for the pathological effects that dendritic atrophy has on subthreshold dendritic signal transfer and integration in layer II/III somatosensory neurons of this hAPP mouse model for AD. Since neither propagation of single PSPs nor integration of multiple PSPs (pattern recognition) changes in TG neurons, we conclude that AD-related neuronal hyperexcitability cannot be accounted for by altered subthreshold dendritic signaling in these neurons but hyperexcitability is related to changes in active membrane properties and network connectivity. PMID:27378850

A Novel Form of Compensation in the Tg2576 Amyloid Mouse Model of Alzheimer's Disease.

PubMed

Somogyi, Attila; Katonai, Zoltán; Alpár, Alán; Wolf, Ervin

2016-01-01

One century after its first description, pathology of Alzheimer's disease (AD) is still poorly understood. Amyloid-related dendritic atrophy and membrane alterations of susceptible brain neurons in AD, and in animal models of AD are widely recognized. However, little effort has been made to study the potential effects of combined morphological and membrane alterations on signal transfer and synaptic integration in neurons that build up affected neural networks in AD. In this study spatial reconstructions and electrophysiological measurements of layer II/III pyramidal neurons of the somatosensory cortex from wild-type (WT) and transgenic (TG) human amyloid precursor protein (hAPP) overexpressing Tg2576 mice were used to build faithful segmental cable models of these neurons. Local synaptic activities were simulated in various points of the dendritic arbors and properties of subthreshold dendritic impulse propagation and predictors of synaptic input pattern recognition ability were quantified and compared in modeled WT and TG neurons. Despite the widespread dendritic degeneration and membrane alterations in mutant mouse neurons, surprisingly little, or no change was detected in steady-state and 50 Hz sinusoidal voltage transfers, current transfers, and local and propagation delays of PSPs traveling along dendrites of TG neurons. Synaptic input pattern recognition ability was also predicted to be unaltered in TG neurons in two different soma-dendritic membrane models investigated. Our simulations predict the way how subthreshold dendritic signaling and pattern recognition are preserved in TG neurons: amyloid-related membrane alterations compensate for the pathological effects that dendritic atrophy has on subthreshold dendritic signal transfer and integration in layer II/III somatosensory neurons of this hAPP mouse model for AD. Since neither propagation of single PSPs nor integration of multiple PSPs (pattern recognition) changes in TG neurons, we conclude that AD-related neuronal hyperexcitability cannot be accounted for by altered subthreshold dendritic signaling in these neurons but hyperexcitability is related to changes in active membrane properties and network connectivity.

Improved Recovery and Identification of Membrane Proteins from Rat Hepatic Cells using a Centrifugal Proteomic Reactor*

PubMed Central

Zhou, Hu; Wang, Fangjun; Wang, Yuwei; Ning, Zhibin; Hou, Weimin; Wright, Theodore G.; Sundaram, Meenakshi; Zhong, Shumei; Yao, Zemin; Figeys, Daniel

2011-01-01

Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism. PMID:21749988

Biofunctional Paper via Covalent Modification of Cellulose

PubMed Central

Yu, Arthur; Shang, Jing; Cheng, Fang; Paik, Bradford A.; Kaplan, Justin M.; Andrade, Rodrigo B.; Ratner, Daniel M.

2012-01-01

Paper-based analytical devices are the subject of growing interest for the development of low-cost point-of-care diagnostics, environmental monitoring technologies and research tools for limited-resource settings. However, there are limited chemistries available for the conjugation of biomolecules to cellulose for use in biomedical applications. Herein, divinyl sulfone (DVS) chemistry was demonstrated to covalently immobilize small molecules, proteins and DNA onto the hydroxyl groups of cellulose membranes through nucleophilic addition. Assays on modified cellulose using protein-carbohydrate and protein-glycoprotein interactions as well as oligonucleotide hybridization showed that the membrane’s bioactivity was specific, dose-dependent, and stable over a long period of time. Use of an inkjet printer to form patterns of biomolecules on DVS-activated cellulose illustrates the adaptability of the DVS functionalization technique to pattern sophisticated designs, with potential applications in cellulose-based lateral flow devices. PMID:22708701

Genetic manipulation of isoprene emissions in poplar plants remodels the chloroplast proteome.

PubMed

Velikova, Violeta; Ghirardo, Andrea; Vanzo, Elisa; Merl, Juliane; Hauck, Stefanie M; Schnitzler, Jörg-Peter

2014-04-04

Biogenic isoprene (2-methyl-1,3-butadiene) improves the integrity and functionality of thylakoid membranes and scavenges reactive oxygen species (ROS) in plant tissue under stress conditions. On the basis of available physiological studies, we hypothesized that the suppression of isoprene production in the poplar plant by genetic engineering would cause changes in the chloroplast protein pattern, which in turn would compensate for changes in chloroplast functionality and overall plant performance under abiotic stress. To test this hypothesis, we used a stable isotope-coded protein-labeling technique in conjunction with polyacrylamide gel electrophoresis and liquid chromatography tandem mass spectrometry. We analyzed quantitative and qualitative changes in the chloroplast proteome of isoprene-emitting and non isoprene-emitting poplars. Here we demonstrate that suppression of isoprene synthase by RNA interference resulted in decreased levels of chloroplast proteins involved in photosynthesis and increased levels of histones, ribosomal proteins, and proteins related to metabolism. Overall, our results show that the absence of isoprene triggers a rearrangement of the chloroplast protein profile to minimize the negative stress effects resulting from the absence of isoprene. The present data strongly support the idea that isoprene improves/stabilizes thylakoid membrane structure and interferes with the production of ROS.

The impact of physiological crowding on the diffusivity of membrane bound proteins.

PubMed

Houser, Justin R; Busch, David J; Bell, David R; Li, Brian; Ren, Pengyu; Stachowiak, Jeanne C

2016-02-21

Diffusion of transmembrane and peripheral membrane-bound proteins within the crowded cellular membrane environment is essential to diverse biological processes including cellular signaling, endocytosis, and motility. Nonetheless we presently lack a detailed understanding of the influence of physiological levels of crowding on membrane protein diffusion. Utilizing quantitative in vitro measurements, here we demonstrate that the diffusivities of membrane bound proteins follow a single linearly decreasing trend with increasing membrane coverage by proteins. This trend holds for homogenous protein populations across a range of protein sizes and for heterogeneous mixtures of proteins of different sizes, such that protein diffusivity is controlled by the total coverage of the surrounding membrane. These results demonstrate that steric exclusion within the crowded membrane environment can fundamentally limit the diffusive rate of proteins, regardless of their size. In cells this "speed limit" could be modulated by changes in local membrane coverage, providing a mechanism for tuning the rate of molecular interaction and assembly.

Nanodisc-Tm: Rapid functional assessment of nanodisc reconstituted membrane proteins by CPM assay.

PubMed

Ashok, Yashwanth; Jaakola, Veli-Pekka

2016-01-01

Membrane proteins are generally unstable in detergents. Therefore, biochemical and biophysical studies of membrane proteins in lipidic environments provides a near native-like environment suitable for membrane proteins. However, manipulation of proteins embedded in lipid bilayer has remained difficult. Methods such as nanodiscs and lipid cubic phase have been developed for easy manipulation of membrane proteins and have yielded significant insights into membrane proteins. Traditionally functional reconstitution of receptors in nanodiscs has been studied with radioligands. We present a simple and faster method for studying the functionality of reconstituted membrane proteins for routine characterization of protein batches after initial optimization of suitable conditions using radioligands. The benefits of the method are •Faster and generic method to assess functional reconstitution of membrane proteins.•Adaptable in high throughput format (≥96 well format).•Stability measurement in near-native lipid environment and lipid dependent melting temperatures.

Functionalizing Microporous Membranes for Protein Purification and Protein Digestion

NASA Astrophysics Data System (ADS)

Dong, Jinlan; Bruening, Merlin L.

2015-07-01

This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO2 nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

Functionalizing Microporous Membranes for Protein Purification and Protein Digestion.

PubMed

Dong, Jinlan; Bruening, Merlin L

2015-01-01

This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO₂ nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

Shuttling of G protein subunits between the plasma membrane and intracellular membranes.

PubMed

Chisari, Mariangela; Saini, Deepak Kumar; Kalyanaraman, Vani; Gautam, Narasimhan

2007-08-17

Heterotrimeric G proteins (alphabetagamma) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Here we examined the spatiotemporal dynamics of G protein localization using fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and a photoswitchable fluorescent protein, Dronpa. Unexpectedly, G protein subunits shuttle rapidly (t1/2 < 1 min) between the plasma membrane and intracellular membranes. We show that consistent with such shuttling, G proteins constitutively reside in endomembranes. Furthermore, we show that shuttling is inhibited by 2-bromopalmitate. Thus, contrary to present thought, G proteins do not reside permanently on the plasma membrane but are constantly testing the cytoplasmic surfaces of the plasma membrane and endomembranes to maintain G protein pools in intracellular membranes to establish direct communication between receptors and endomembranes.

Deoxycholate-Based Glycosides (DCGs) for Membrane Protein Stabilisation.

PubMed

Bae, Hyoung Eun; Gotfryd, Kamil; Thomas, Jennifer; Hussain, Hazrat; Ehsan, Muhammad; Go, Juyeon; Loland, Claus J; Byrne, Bernadette; Chae, Pil Seok

2015-07-06

Detergents are an absolute requirement for studying the structure of membrane proteins. However, many conventional detergents fail to stabilise denaturation-sensitive membrane proteins, such as eukaryotic proteins and membrane protein complexes. New amphipathic agents with enhanced efficacy in stabilising membrane proteins will be helpful in overcoming the barriers to studying membrane protein structures. We have prepared a number of deoxycholate-based amphiphiles with carbohydrate head groups, designated deoxycholate-based glycosides (DCGs). These DCGs are the hydrophilic variants of previously reported deoxycholate-based N-oxides (DCAOs). Membrane proteins in these agents, particularly the branched diglucoside-bearing amphiphiles DCG-1 and DCG-2, displayed favourable behaviour compared to previously reported parent compounds (DCAOs) and conventional detergents (LDAO and DDM). Given their excellent properties, these agents should have significant potential for membrane protein studies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Detection of erythrocyte membrane proteins, sialoglycoproteins, and lipids in the same polyacrylamide gel using a double-staining technique.

PubMed Central

Dzandu, J K; Deh, M E; Barratt, D L; Wise, G E

1984-01-01

A silver/Coomassie brilliant blue R-250 staining technique that permits a color-coded differentiation of erythrocyte membrane proteins, sialoglycoproteins, and lipids in a single one-dimensional NaDodSO4/polyacrylamide gel has been described. Gels stained first with silver stain and then with Coomassie blue (CB) showed the characteristic blue staining of all conventional CB-sensitive membrane polypeptides, whereas periodic acid-Schiff reagent-sensitive sialoglycoproteins and lipids stained yellow. Several yellow Ag-stained bands corresponding to major and minor sialoglycoproteins were detected at Mr X 10(-3) of 88, 72, 65, 41, 35, 31, 28, 24, and 20. Neuraminidase treatment of intact erythrocytes caused shifts in the electrophoretic mobilities of several yellow-stained bands without affecting the CB-stained polypeptide pattern. These observations afforded evidence that the yellow-staining bands were sialoglycoproteins and lipids. The double-staining technique was used in a topological analysis of the membrane surface of the erythrocyte using protease digestion and selective solubilization. Trypsin cleaved the yellow bands at Mr 88,000 and 41,000. Membrane-associated cleavage products were noted at Mr 58,000 and 38,000. Pronase treatment of intact cells gave membrane-associated cleavage products at Mr 38,000 (yellow) and two CB-stained bands at Mr 58,000 and 60,000. These results suggested that the double-staining technique may be applicable in compositional and topological analyses of other biological membranes. Images PMID:6200882

Cytosolic proteins can exploit membrane localization to trigger functional assembly

PubMed Central

2018-01-01

Cell division, endocytosis, and viral budding would not function without the localization and assembly of protein complexes on membranes. What is poorly appreciated, however, is that by localizing to membranes, proteins search in a reduced space that effectively drives up concentration. Here we derive an accurate and practical analytical theory to quantify the significance of this dimensionality reduction in regulating protein assembly on membranes. We define a simple metric, an effective equilibrium constant, that allows for quantitative comparison of protein-protein interactions with and without membrane present. To test the importance of membrane localization for driving protein assembly, we collected the protein-protein and protein-lipid affinities, protein and lipid concentrations, and volume-to-surface-area ratios for 46 interactions between 37 membrane-targeting proteins in human and yeast cells. We find that many of the protein-protein interactions between pairs of proteins involved in clathrin-mediated endocytosis in human and yeast cells can experience enormous increases in effective protein-protein affinity (10–1000 fold) due to membrane localization. Localization of binding partners thus triggers robust protein complexation, suggesting that it can play an important role in controlling the timing of endocytic protein coat formation. Our analysis shows that several other proteins involved in membrane remodeling at various organelles have similar potential to exploit localization. The theory highlights the master role of phosphoinositide lipid concentration, the volume-to-surface-area ratio, and the ratio of 3D to 2D equilibrium constants in triggering (or preventing) constitutive assembly on membranes. Our simple model provides a novel quantitative framework for interpreting or designing in vitro experiments of protein complexation influenced by membrane binding. PMID:29505559

FtsZ Placement in Nucleoid-Free Bacteria

PubMed Central

Pazos, Manuel; Casanova, Mercedes; Palacios, Pilar; Margolin, William; Natale, Paolo; Vicente, Miguel

2014-01-01

We describe the placement of the cytoplasmic FtsZ protein, an essential component of the division septum, in nucleoid-free Escherichia coli maxicells. The absence of the nucleoid is accompanied in maxicells by degradation of the SlmA protein. This protein, together with the nucleoid, prevents the placement of the septum in the regions occupied by the chromosome by a mechanism called nucleoid occlusion (NO). A second septum placement mechanism, the MinCDE system (Min) involving a pole-to-pole oscillation of three proteins, nonetheless remains active in maxicells. Both Min and NO act on the polymerization of FtsZ, preventing its assembly into an FtsZ-ring except at midcell. Our results show that even in the total absence of NO, Min oscillations can direct placement of FtsZ in maxicells. Deletion of the FtsZ carboxyl terminal domain (FtsZ*), a central hub that receives signals from a variety of proteins including MinC, FtsA and ZipA, produces a Min-insensitive form of FtsZ unable to interact with the membrane-anchoring FtsA and ZipA proteins. This protein produces a totally disorganized pattern of FtsZ localization inside the maxicell cytoplasm. In contrast, FtsZ*-VM, an artificially cytoplasmic membrane-anchored variant of FtsZ*, forms helical or repetitive ring structures distributed along the entire length of maxicells even in the absence of NO. These results show that membrane anchoring is needed to organize FtsZ into rings and underscore the role of the C-terminal hub of FtsZ for their correct placement. PMID:24638110

FtsZ placement in nucleoid-free bacteria.

PubMed

Pazos, Manuel; Casanova, Mercedes; Palacios, Pilar; Margolin, William; Natale, Paolo; Vicente, Miguel

2014-01-01

We describe the placement of the cytoplasmic FtsZ protein, an essential component of the division septum, in nucleoid-free Escherichia coli maxicells. The absence of the nucleoid is accompanied in maxicells by degradation of the SlmA protein. This protein, together with the nucleoid, prevents the placement of the septum in the regions occupied by the chromosome by a mechanism called nucleoid occlusion (NO). A second septum placement mechanism, the MinCDE system (Min) involving a pole-to-pole oscillation of three proteins, nonetheless remains active in maxicells. Both Min and NO act on the polymerization of FtsZ, preventing its assembly into an FtsZ-ring except at midcell. Our results show that even in the total absence of NO, Min oscillations can direct placement of FtsZ in maxicells. Deletion of the FtsZ carboxyl terminal domain (FtsZ*), a central hub that receives signals from a variety of proteins including MinC, FtsA and ZipA, produces a Min-insensitive form of FtsZ unable to interact with the membrane-anchoring FtsA and ZipA proteins. This protein produces a totally disorganized pattern of FtsZ localization inside the maxicell cytoplasm. In contrast, FtsZ*-VM, an artificially cytoplasmic membrane-anchored variant of FtsZ*, forms helical or repetitive ring structures distributed along the entire length of maxicells even in the absence of NO. These results show that membrane anchoring is needed to organize FtsZ into rings and underscore the role of the C-terminal hub of FtsZ for their correct placement.

Membrane curvature and its generation by BAR proteins

PubMed Central

Mim, Carsten; Unger, Vinzenz M

2012-01-01

Membranes are flexible barriers that surround the cell and its compartments. To execute vital functions such as locomotion or receptor turnover, cells need to control the shapes of their membranes. In part, this control is achieved through membrane-bending proteins, such as the bin/amphiphysin/rvs domain (BAR) proteins. Many open questions remain about the mechanisms by which membrane-bending proteins function. Addressing this shortfall, recent structures of BAR protein:membrane complexes support existing mechanistic models, but also produced novel insights into how BAR-domain proteins sense, stabilize and generate curvature. Here we review these recent findings, focusing on how BAR proteins interact with the membrane, and how the resulting scaffold structures might aid the recruitment of other proteins to the sites where membranes are bent. PMID:23058040

Analysis of Membrane Protein Topology in the Plant Secretory Pathway.

PubMed

Guo, Jinya; Miao, Yansong; Cai, Yi

2017-01-01

Topology of membrane proteins provides important information for the understanding of protein function and intermolecular associations. Integrate membrane proteins are generally transported from endoplasmic reticulum (ER) to Golgi and downstream compartments in the plant secretory pathway. Here, we describe a simple method to study membrane protein topology along the plant secretory pathway by transiently coexpressing a fluorescent protein (XFP)-tagged membrane protein and an ER export inhibitor protein, ARF1 (T31N), in tobacco BY-2 protoplast. By fractionation, microsome isolation, and trypsin digestion, membrane protein topology could be easily detected by either direct confocal microscopy imaging or western-blot analysis using specific XFP antibodies. A similar strategy in determining membrane protein topology could be widely adopted and applied to protein analysis in a broad range of eukaryotic systems, including yeast cells and mammalian cells.

The styrene-maleic acid copolymer: a versatile tool in membrane research.

PubMed

Dörr, Jonas M; Scheidelaar, Stefan; Koorengevel, Martijn C; Dominguez, Juan J; Schäfer, Marre; van Walree, Cornelis A; Killian, J Antoinette

2016-01-01

A new and promising tool in membrane research is the detergent-free solubilization of membrane proteins by styrene-maleic acid copolymers (SMAs). These amphipathic molecules are able to solubilize lipid bilayers in the form of nanodiscs that are bounded by the polymer. Thus, membrane proteins can be directly extracted from cells in a water-soluble form while conserving a patch of native membrane around them. In this review article, we briefly discuss current methods of membrane protein solubilization and stabilization. We then zoom in on SMAs, describe their physico-chemical properties, and discuss their membrane-solubilizing effect. This is followed by an overview of studies in which SMA has been used to isolate and investigate membrane proteins. Finally, potential future applications of the methodology are discussed for structural and functional studies on membrane proteins in a near-native environment and for characterizing protein-lipid and protein-protein interactions.

A family of cellular proteins related to snake venom disintegrins.

PubMed

Weskamp, G; Blobel, C P

1994-03-29

Disintegrins are short soluble integrin ligands that were initially identified in snake venom. A previously recognized cellular protein with a disintegrin domain was the guinea pig sperm protein PH-30, a protein implicated in sperm-egg membrane binding and fusion. Here we present peptide sequences that are characteristic for several cellular disintegrin-domain proteins. These peptide sequences were deduced from cDNA sequence tags that were generated by polymerase chain reaction from various mouse tissue and a mouse muscle cell line. Northern blot analysis with four sequence tags revealed distinct mRNA expression patterns. Evidently, cellular proteins containing a disintegrin domain define a superfamily of potential integrin ligands that are likely to function in important cell-cell and cell-matrix interactions.

A bacterial view of the periodic table: genes and proteins for toxic inorganic ions.

PubMed

Silver, Simon; Phung, Le T

2005-12-01

Essentially all bacteria have genes for toxic metal ion resistances and these include those for Ag+, AsO2-, AsO4(3-), Cd2+ Co2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, TeO3(2-), Tl+ and Zn2+. The largest group of resistance systems functions by energy-dependent efflux of toxic ions. Fewer involve enzymatic transformations (oxidation, reduction, methylation, and demethylation) or metal-binding proteins (for example, metallothionein SmtA, chaperone CopZ and periplasmic silver binding protein SilE). Some of the efflux resistance systems are ATPases and others are chemiosmotic ion/proton exchangers. For example, Cd2+-efflux pumps of bacteria are either inner membrane P-type ATPases or three polypeptide RND chemiosmotic complexes consisting of an inner membrane pump, a periplasmic-bridging protein and an outer membrane channel. In addition to the best studied three-polypeptide chemiosmotic system, Czc (Cd2+, Zn2+, and Co2), others are known that efflux Ag+, Cu+, Ni2+, and Zn2+. Resistance to inorganic mercury, Hg2+ (and to organomercurials, such as CH3Hg+ and phenylmercury) involve a series of metal-binding and membrane transport proteins as well as the enzymes mercuric reductase and organomercurial lyase, which overall convert more toxic to less toxic forms. Arsenic resistance and metabolizing systems occur in three patterns, the widely-found ars operon that is present in most bacterial genomes and many plasmids, the more recently recognized arr genes for the periplasmic arsenate reductase that functions in anaerobic respiration as a terminal electron acceptor, and the aso genes for the periplasmic arsenite oxidase that functions as an initial electron donor in aerobic resistance to arsenite.

On the molecular basis of the receptor mosaic hypothesis of the engram.

PubMed

Agnati, Luigi F; Ferré, Sergi; Leo, Giuseppina; Lluis, Carme; Canela, Enric I; Franco, Rafael; Fuxe, Kjell

2004-08-01

1. This paper revisits the so-called "receptor mosaic hypothesis" for memory trace formation in the light of recent findings in "functional (or interaction) proteomics." The receptor mosaic hypothesis maintains that receptors may form molecular aggregates at the plasma membrane level representing part of the computational molecular networks. 2. Specific interactions between receptors occur as a consequence of the pattern of transmitter release from the source neurons, which release the chemical code impinging on the receptor mosaics of the target neuron. Thus, the decoding of the chemical message depends on the receptors forming the receptor mosaics and on the type of interactions among receptors and other proteins in the molecular network with novel long-term mosaics formed by their stabilization via adapter proteins formed in target neurons through the incoming neurotransmitter code. The internalized receptor heteromeric complexes or parts of them may act as transcription factors for the formation of such adapter proteins. 3. Receptor mosaics are formed both at the pre- and postsynaptic level of the plasma membranes and this phenomenon can play a role in the Hebbian behavior of some synaptic contacts. The appropriate "matching" of the pre- with the postsynaptic receptor mosaic can be thought of as the "clamping of the synapse to the external teaching signal." According to our hypothesis the behavior of the molecular networks at plasma membrane level to which the receptor mosaics belong can be set in a "frozen" conformation (i.e. in a frozen functional state) and this may represent a mechanism to maintain constant the input to a neuron. 4. Thus, we are suggesting that molecular networks at plasma membrane level may display multiple "attractors" each of which stores the memory of a specific neurotransmitter code due to a unique firing pattern. Hence, this mechanism may play a role in learning processes where the input to a neuron is likely to remain constant for a while.

Membrane skeletal proteins and their integral membrane protein anchors are targets for tyrosine and threonine kinases in Euglena.

PubMed

Fazio, M J; Da Silva, A C; Rosiere, T K; Bouck, G B

1995-01-01

Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [32P]-orthophosphate or gamma-[32P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.

Pannexin2 oligomers localize in the membranes of endosomal vesicles in mammalian cells while Pannexin1 channels traffic to the plasma membrane.

PubMed

Boassa, Daniela; Nguyen, Phuong; Hu, Junru; Ellisman, Mark H; Sosinsky, Gina E

2014-01-01

Pannexin2 (Panx2) is the largest of three members of the pannexin proteins. Pannexins are topologically related to connexins and innexins, but serve different functional roles than forming gap junctions. We previously showed that pannexins form oligomeric channels but unlike connexins and innexins, they form only single membrane channels. High levels of Panx2 mRNA and protein in the Central Nervous System (CNS) have been documented. Whereas Pannexin1 (Panx1) is fairly ubiquitous and Pannexin3 (Panx3) is found in skin and connective tissue, both are fully glycosylated, traffic to the plasma membrane and have functions correlated with extracellular ATP release. Here, we describe trafficking and subcellular localizations of exogenous Panx2 and Panx1 protein expression in MDCK, HeLa, and HEK 293T cells as well as endogenous Panx1 and Panx2 patterns in the CNS. Panx2 was found in intracellular localizations, was partially N-glycosylated, and localizations were non-overlapping with Panx1. Confocal images of hippocampal sections immunolabeled for the astrocytic protein GFAP, Panx1 and Panx2 demonstrated that the two isoforms, Panx1 and Panx2, localized at different subcellular compartments in both astrocytes and neurons. Using recombinant fusions of Panx2 with appended genetic tags developed for correlated light and electron microscopy and then expressed in different cell lines, we determined that Panx2 is localized in the membrane of intracellular vesicles and not in the endoplasmic reticulum as initially indicated by calnexin colocalization experiments. Dual immunofluorescence imaging with protein markers for specific vesicle compartments showed that Panx2 vesicles are early endosomal in origin. In electron tomographic volumes, cross-sections of these vesicles displayed fine structural details and close proximity to actin filaments. Thus, pannexins expressed at different subcellular compartments likely exert distinct functional roles, particularly in the nervous system.

Membrane fusion and exocytosis.

PubMed

Jahn, R; Südhof, T C

1999-01-01

Membrane fusion involves the merger of two phospholipid bilayers in an aqueous environment. In artificial lipid bilayers, fusion proceeds by means of defined transition states, including hourglass-shaped intermediates in which the proximal leaflets of the fusing membranes are merged whereas the distal leaflets are separate (fusion stalk), followed by the reversible opening of small aqueous fusion pores. Fusion of biological membranes requires the action of specific fusion proteins. Best understood are the viral fusion proteins that are responsible for merging the viral with the host cell membrane during infection. These proteins undergo spontaneous and dramatic conformational changes upon activation. In the case of the paradigmatic fusion proteins of the influenza virus and of the human immunodeficiency virus, an amphiphilic fusion peptide is inserted into the target membrane. The protein then reorients itself, thus forcing the fusing membranes together and inducing lipid mixing. Fusion of intracellular membranes in eukaryotic cells involves several protein families including SNAREs, Rab proteins, and Sec1/Munc-18 related proteins (SM-proteins). SNAREs form a novel superfamily of small and mostly membrane-anchored proteins that share a common motif of about 60 amino acids (SNARE motif). SNAREs reversibly assemble into tightly packed helical bundles, the core complexes. Assembly is thought to pull the fusing membranes closely together, thus inducing fusion. SM-proteins comprise a family of soluble proteins that bind to certain types of SNAREs and prevent the formation of core complexes. Rab proteins are GTPases that undergo highly regulated GTP-GDP cycles. In their GTP form, they interact with specific proteins, the effector proteins. Recent evidence suggests that Rab proteins function in the initial membrane contact connecting the fusing membranes but are not involved in the fusion reaction itself.

Cloning of the Pseudomonas aeruginosa outer membrane porin protein P gene: evidence for a linked region of DNA homology.

PubMed Central

Siehnel, R J; Worobec, E A; Hancock, R E

1988-01-01

The gene encoding the outer membrane phosphate-selective porin protein P from Pseudomonas aeruginosa was cloned into Escherichia coli. The protein product was expressed and transported to the outer membrane of an E. coli phoE mutant and assembled into functional trimers. Expression of a product of the correct molecular weight was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, using polyclonal antibodies to protein P monomer and trimer forms. Protein P trimers were partially purified from the E. coli clone and shown to form channels with the same conductance as those formed by protein P from P. aeruginosa. The location and orientation of the protein P-encoding (oprP) gene on the cloned DNA was identified by three methods: (i) mapping the insertion point of transposon Tn501 in a previously isolated P. aeruginosa protein P-deficient mutant; (ii) hybridization of restriction fragments from the cloned DNA to an oligonucleotide pool synthesized on the basis of the amino-terminal protein sequence of protein P; and (iii) fusion of a PstI fragment of the cloned DNA to the amino terminus of the beta-galactosidase gene of pUC8, producing a fusion protein that contained protein P-antigenic epitopes. Structural analysis of the cloned DNA and P. aeruginosa chromosomal DNA revealed the presence of two adjacent PstI fragments which cross-hybridized, suggesting a possible gene duplication. The P-related (PR) region hybridized to the oligonucleotide pool described above. When the PstI fragment which contained the PR region was fused to the beta-galactosidase gene of pUC8, a fusion protein was produced which reacted with a protein P-specific antiserum. However, the restriction endonuclease patterns of the PR region and the oprP gene differed significantly beyond the amino-terminal one-third of the two genes. Images PMID:2834340

Two Complementary Mechanisms Underpin Cell Wall Patterning during Xylem Vessel Development.

PubMed

Schneider, Rene; Tang, Lu; Lampugnani, Edwin R; Barkwill, Sarah; Lathe, Rahul; Zhang, Yi; McFarlane, Heather E; Pesquet, Edouard; Niittyla, Totte; Mansfield, Shawn D; Zhou, Yihua; Persson, Staffan

2017-10-01

The evolution of the plant vasculature was essential for the emergence of terrestrial life. Xylem vessels are solute-transporting elements in the vasculature that possess secondary wall thickenings deposited in intricate patterns. Evenly dispersed microtubule (MT) bands support the formation of these wall thickenings, but how the MTs direct cell wall synthesis during this process remains largely unknown. Cellulose is the major secondary wall constituent and is synthesized by plasma membrane-localized cellulose synthases (CesAs) whose catalytic activity propels them through the membrane. We show that the protein CELLULOSE SYNTHASE INTERACTING1 (CSI1)/POM2 is necessary to align the secondary wall CesAs and MTs during the initial phase of xylem vessel development in Arabidopsis thaliana and rice ( Oryza sativa ). Surprisingly, these MT-driven patterns successively become imprinted and sufficient to sustain the continued progression of wall thickening in the absence of MTs and CSI1/POM2 function. Hence, two complementary principles underpin wall patterning during xylem vessel development. © 2017 American Society of Plant Biologists. All rights reserved.

Effects of short-term tocopherol (T) feeding on structure-localized protein tyrosine nitration (pTN) patterns of mitochondrial ATPase following endotoxin (LPS) challenge in beef calves.

USDA-ARS?s Scientific Manuscript database

Mitochondrial ATPase/Complex-V (MCV) is an electron transport chain (ETC) component needed for ATP synthesis. The ETC, exquisitely sensitive to proinflammatory mediators (PIM), generates oxynitrogen reactants leading to pTN formation as mitochondrial membrane leakage occurs. Immunohistochemical loca...

Coarse-Grained Simulations of Membrane Insertion and Folding of Small Helical Proteins Using the CABS Model.

PubMed

Pulawski, Wojciech; Jamroz, Michal; Kolinski, Michal; Kolinski, Andrzej; Kmiecik, Sebastian

2016-11-28

The CABS coarse-grained model is a well-established tool for modeling globular proteins (predicting their structure, dynamics, and interactions). Here we introduce an extension of the CABS representation and force field (CABS-membrane) to the modeling of the effect of the biological membrane environment on the structure of membrane proteins. We validate the CABS-membrane model in folding simulations of 10 short helical membrane proteins not using any knowledge about their structure. The simulations start from random protein conformations placed outside the membrane environment and allow for full flexibility of the modeled proteins during their spontaneous insertion into the membrane. In the resulting trajectories, we have found models close to the experimental membrane structures. We also attempted to select the correctly folded models using simple filtering followed by structural clustering combined with reconstruction to the all-atom representation and all-atom scoring. The CABS-membrane model is a promising approach for further development toward modeling of large protein-membrane systems.

Exploring the Spatiotemporal Organization of Membrane Proteins in Living Plant Cells.

PubMed

Wang, Li; Xue, Yiqun; Xing, Jingjing; Song, Kai; Lin, Jinxing

2018-04-29

Plasma membrane proteins have important roles in transport and signal transduction. Deciphering the spatiotemporal organization of these proteins provides crucial information for elucidating the links between the behaviors of different molecules. However, monitoring membrane proteins without disrupting their membrane environment remains difficult. Over the past decade, many studies have developed single-molecule techniques, opening avenues for probing the stoichiometry and interactions of membrane proteins in their native environment by providing nanometer-scale spatial information and nanosecond-scale temporal information. In this review, we assess recent progress in the development of labeling and imaging technology for membrane protein analysis. We focus in particular on several single-molecule techniques for quantifying the dynamics and assembly of membrane proteins. Finally, we provide examples of how these new techniques are advancing our understanding of the complex biological functions of membrane proteins.

Unraveling sterol-dependent membrane phenotypes by analysis of protein abundance-ratio distributions in different membrane fractions under biochemical and endogenous sterol depletion.

PubMed

Zauber, Henrik; Szymanski, Witold; Schulze, Waltraud X

2013-12-01

During the last decade, research on plasma membrane focused increasingly on the analysis of so-called microdomains. It has been shown that function of many membrane-associated proteins involved in signaling and transport depends on their conditional segregation within sterol-enriched membrane domains. High throughput proteomic analysis of sterol-protein interactions are often based on analyzing detergent resistant membrane fraction enriched in sterols and associated proteins, which also contain proteins from these microdomain structures. Most studies so far focused exclusively on the characterization of detergent resistant membrane protein composition and abundances. This approach has received some criticism because of its unspecificity and many co-purifying proteins. In this study, by a label-free quantitation approach, we extended the characterization of membrane microdomains by particularly studying distributions of each protein between detergent resistant membrane and detergent-soluble fractions (DSF). This approach allows a more stringent definition of dynamic processes between different membrane phases and provides a means of identification of co-purifying proteins. We developed a random sampling algorithm, called Unicorn, allowing for robust statistical testing of alterations in the protein distribution ratios of the two different fractions. Unicorn was validated on proteomic data from methyl-β-cyclodextrin treated plasma membranes and the sterol biosynthesis mutant smt1. Both, chemical treatment and sterol-biosynthesis mutation affected similar protein classes in their membrane phase distribution and particularly proteins with signaling and transport functions.

Unraveling Sterol-dependent Membrane Phenotypes by Analysis of Protein Abundance-ratio Distributions in Different Membrane Fractions Under Biochemical and Endogenous Sterol Depletion*

PubMed Central

Zauber, Henrik; Szymanski, Witold; Schulze, Waltraud X.

2013-01-01

During the last decade, research on plasma membrane focused increasingly on the analysis of so-called microdomains. It has been shown that function of many membrane-associated proteins involved in signaling and transport depends on their conditional segregation within sterol-enriched membrane domains. High throughput proteomic analysis of sterol-protein interactions are often based on analyzing detergent resistant membrane fraction enriched in sterols and associated proteins, which also contain proteins from these microdomain structures. Most studies so far focused exclusively on the characterization of detergent resistant membrane protein composition and abundances. This approach has received some criticism because of its unspecificity and many co-purifying proteins. In this study, by a label-free quantitation approach, we extended the characterization of membrane microdomains by particularly studying distributions of each protein between detergent resistant membrane and detergent-soluble fractions (DSF). This approach allows a more stringent definition of dynamic processes between different membrane phases and provides a means of identification of co-purifying proteins. We developed a random sampling algorithm, called Unicorn, allowing for robust statistical testing of alterations in the protein distribution ratios of the two different fractions. Unicorn was validated on proteomic data from methyl-β-cyclodextrin treated plasma membranes and the sterol biosynthesis mutant smt1. Both, chemical treatment and sterol-biosynthesis mutation affected similar protein classes in their membrane phase distribution and particularly proteins with signaling and transport functions. PMID:24030099

Biomimetic devices functionalized by membrane channel proteins

NASA Astrophysics Data System (ADS)

Schmidt, Jacob

2004-03-01

We are developing a new family of active materials which derive their functional properties from membrane proteins. These materials have two primary components: the proteins and the membranes themselves. I will discuss our recent work directed toward development of a generic platform for a "plug-and-play" philosophy of membrane protein engineering. By creating a stable biomimetic polymer membrane a single molecular monolayer thick, we will enable the exploitation of the function of any membrane protein, from pores and pumps to sensors and energy transducers. Our initial work has centered on the creation, study, and characterization of the biomimetic membranes. We are attempting to make large areas of membrane monolayers using Langmuir-Blodgett film formation as well as through arrays of microfabricated black lipid membrane-type septa. A number of techniques allow the insertion of protein into the membranes. As a benchmark, we have been employing a model system of voltage-gated pore proteins, which have electrically controllable porosities. I will report on the progress of this work, the characterization of the membranes, protein insertion processes, and the yield and functionality of the composite.

Effects of L-arginine on solubilization and purification of plant membrane proteins.

PubMed

Arakawa, Junji; Uegaki, Masamichi; Ishimizu, Takeshi

2011-11-01

Biochemical analysis of membrane proteins is problematic at the level of solubilization and/or purification because of their hydrophobic nature. Here, we developed methods for efficient solubilization and purification of membrane proteins using L-arginine. The addition of 100 mM of basic amino acids (L-arginine, L-lysine, and L-ornithine) to a detergent-containing solubilization buffer enhanced solubilization (by 2.6-4.3 fold) of a model membrane protein-polygalacturonic acid synthase. Of all the amino acids, arginine was the most effective additive for solubilization of this membrane protein. Arginine addition also resulted in the best solubilization of other plant membrane proteins. Next, we examined the effects of arginine on purification of a model membrane protein. In anion-exchange chromatography, the addition of arginine to the loading and elution buffers resulted in a greater recovery of a membrane protein. In ultrafiltration, the addition of arginine to a protein solution significantly improved the recovery of a membrane protein. These results were thought to be due to the properties of arginine that prevent aggregation of hydrophobic proteins. Taken together, the results of our study showed that arginine is useful for solubilization and purification of aggregate-prone membrane proteins. Copyright © 2011 Elsevier Inc. All rights reserved.

Membrane tension controls the assembly of curvature-generating proteins

PubMed Central

Simunovic, Mijo; Voth, Gregory A.

2015-01-01

Proteins containing a Bin/Amphiphysin/Rvs (BAR) domain regulate membrane curvature in the cell. Recent simulations have revealed that BAR proteins assemble into linear aggregates, strongly affecting membrane curvature and its in-plane stress profile. Here, we explore the opposite question: do mechanical properties of the membrane impact protein association? By using coarse-grained molecular dynamics simulations, we show that increased surface tension significantly impacts the dynamics of protein assembly. While tensionless membranes promote a rapid formation of long-living linear aggregates of N-BAR proteins, increase in tension alters the geometry of protein association. At high tension, protein interactions are strongly inhibited. Increasing surface density of proteins leads to a wider range of protein association geometries, promoting the formation of meshes, which can be broken apart with membrane tension. Our work indicates that surface tension may play a key role in recruiting proteins to membrane-remodelling sites in the cell. PMID:26008710

Membrane Protein Structure, Function, and Dynamics: a Perspective from Experiments and Theory

DOE PAGES

Cournia, Zoe; Allen, Toby W.; Andricioaei, Ioan; ...

2015-06-11

It is fundamental for the flourishing biological cells that membrane proteins mediate the process. Membrane-embedded transporters move ions and larger solutes across membranes; receptors mediate communication between the cell and its environment and membrane-embedded enzymes catalyze chemical reactions. Understanding these mechanisms of action requires knowledge of how the proteins couple to their fluid, hydrated lipid membrane environment. Here, we present here current studies in computational and experimental membrane protein biophysics, and show how they address outstanding challenges in understanding the complex environmental effects on the structure, function, and dynamics of membrane proteins.

G protein-membrane interactions II: Effect of G protein-linked lipids on membrane structure and G protein-membrane interactions.

PubMed

Casas, Jesús; Ibarguren, Maitane; Álvarez, Rafael; Terés, Silvia; Lladó, Victoria; Piotto, Stefano P; Concilio, Simona; Busquets, Xavier; López, David J; Escribá, Pablo V

2017-09-01

G proteins often bear myristoyl, palmitoyl and isoprenyl moieties, which favor their association with the membrane and their accumulation in G Protein Coupled Receptor-rich microdomains. These lipids influence the biophysical properties of membranes and thereby modulate G protein binding to bilayers. In this context, we showed here that geranylgeraniol, but neither myristate nor palmitate, increased the inverted hexagonal (H II ) phase propensity of phosphatidylethanolamine-containing membranes. While myristate and palmitate preferentially associated with phosphatidylcholine membranes, geranylgeraniol favored nonlamellar-prone membranes. In addition, Gαi 1 monomers had a higher affinity for lamellar phases, while Gβγ and Gαβγ showed a marked preference for nonlamellar prone membranes. Moreover, geranylgeraniol enhanced the binding of G protein dimers and trimers to phosphatidylethanolamine-containing membranes, yet it decreased that of monomers. By contrast, both myristate and palmitate increased the Gαi 1 preference for lamellar membranes. Palmitoylation reinforced the binding of the monomer to PC membranes and myristoylation decreased its binding to PE-enriched bilayer. Finally, binding of dimers and trimers to lamellar-prone membranes was decreased by palmitate and myristate, but it was increased in nonlamellar-prone bilayers. These results demonstrate that co/post-translational G protein lipid modifications regulate the membrane lipid structure and that they influence the physico-chemical properties of membranes, which in part explains why G protein subunits sort to different plasma membrane domains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 Elsevier B.V. All rights reserved.

Membrane re-modelling by BAR domain superfamily proteins via molecular and non-molecular factors.

PubMed

Nishimura, Tamako; Morone, Nobuhiro; Suetsugu, Shiro

2018-04-17

Lipid membranes are structural components of cell surfaces and intracellular organelles. Alterations in lipid membrane shape are accompanied by numerous cellular functions, including endocytosis, intracellular transport, and cell migration. Proteins containing Bin-Amphiphysin-Rvs (BAR) domains (BAR proteins) are unique, because their structures correspond to the membrane curvature, that is, the shape of the lipid membrane. BAR proteins present at high concentration determine the shape of the membrane, because BAR domain oligomers function as scaffolds that mould the membrane. BAR proteins co-operate with various molecular and non-molecular factors. The molecular factors include cytoskeletal proteins such as the regulators of actin filaments and the membrane scission protein dynamin. Lipid composition, including saturated or unsaturated fatty acid tails of phospholipids, also affects the ability of BAR proteins to mould the membrane. Non-molecular factors include the external physical forces applied to the membrane, such as tension and friction. In this mini-review, we will discuss how the BAR proteins orchestrate membrane dynamics together with various molecular and non-molecular factors. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Biophysics of α-synuclein membrane interactions.

PubMed

Pfefferkorn, Candace M; Jiang, Zhiping; Lee, Jennifer C

2012-02-01

Membrane proteins participate in nearly all cellular processes; however, because of experimental limitations, their characterization lags far behind that of soluble proteins. Peripheral membrane proteins are particularly challenging to study because of their inherent propensity to adopt multiple and/or transient conformations in solution and upon membrane association. In this review, we summarize useful biophysical techniques for the study of peripheral membrane proteins and their application in the characterization of the membrane interactions of the natively unfolded and Parkinson's disease (PD) related protein, α-synuclein (α-syn). We give particular focus to studies that have led to the current understanding of membrane-bound α-syn structure and the elucidation of specific membrane properties that affect α-syn-membrane binding. Finally, we discuss biophysical evidence supporting a key role for membranes and α-syn in PD pathogenesis. This article is part of a Special Issue entitled: Membrane protein structure and function. Copyright © 2011. Published by Elsevier B.V.

Proteome analysis of the triton-insoluble erythrocyte membrane skeleton.

PubMed

Basu, Avik; Harper, Sandra; Pesciotta, Esther N; Speicher, Kaye D; Chakrabarti, Abhijit; Speicher, David W

2015-10-14

Erythrocyte shape and membrane integrity is imparted by the membrane skeleton, which can be isolated as a Triton X-100 insoluble structure that retains the biconcave shape of intact erythrocytes, indicating isolation of essentially intact membrane skeletons. These erythrocyte "Triton Skeletons" have been studied morphologically and biochemically, but unbiased proteome analysis of this substructure of the membrane has not been reported. In this study, different extraction buffers and in-depth proteome analyses were used to more fully define the protein composition of this functionally critical macromolecular complex. As expected, the major, well-characterized membrane skeleton proteins and their associated membrane anchors were recovered in good yield. But surprisingly, a substantial number of additional proteins that are not considered in erythrocyte membrane skeleton models were recovered in high yields, including myosin-9, lipid raft proteins (stomatin, flotillin1 and 2), multiple chaperone proteins (HSPs, protein disulfide isomerase and calnexin), and several other proteins. These results show that the membrane skeleton is substantially more complex than previous biochemical studies indicated, and it apparently has localized regions with unique protein compositions and functions. This comprehensive catalog of the membrane skeleton should lead to new insights into erythrocyte membrane biology and pathogenic mutations that perturb membrane stability. Biological significance Current models of erythrocyte membranes describe fairly simple homogenous structures that are incomplete. Proteome analysis of the erythrocyte membrane skeleton shows that it is quite complex and includes a substantial number of proteins whose roles and locations in the membrane are not well defined. Further elucidation of interactions involving these proteins and definition of microdomains in the membrane that contain these proteins should yield novel insights into how the membrane skeleton produces the normal biconcave erythrocyte shape and how it is perturbed in pathological conditions that destabilize the membrane. Copyright © 2015 Elsevier B.V. All rights reserved.

Shotgun proteomics of plant plasma membrane and microdomain proteins using nano-LC-MS/MS.

PubMed

Takahashi, Daisuke; Li, Bin; Nakayama, Takato; Kawamura, Yukio; Uemura, Matsuo

2014-01-01

Shotgun proteomics allows the comprehensive analysis of proteins extracted from plant cells, subcellular organelles, and membranes. Previously, two-dimensional gel electrophoresis-based proteomics was used for mass spectrometric analysis of plasma membrane proteins. In order to get comprehensive proteome profiles of the plasma membrane including highly hydrophobic proteins with a number of transmembrane domains, a mass spectrometry-based shotgun proteomics method using nano-LC-MS/MS for proteins from the plasma membrane proteins and plasma membrane microdomain fraction is described. The results obtained are easily applicable to label-free protein semiquantification.

Mining Missing Membrane Proteins by High-pH Reverse-Phase StageTip Fractionation and Multiple Reaction Monitoring Mass Spectrometry.

PubMed

Kitata, Reta Birhanu; Dimayacyac-Esleta, Baby Rorielyn T; Choong, Wai-Kok; Tsai, Chia-Feng; Lin, Tai-Du; Tsou, Chih-Chiang; Weng, Shao-Hsing; Chen, Yi-Ju; Yang, Pan-Chyr; Arco, Susan D; Nesvizhskii, Alexey I; Sung, Ting-Yi; Chen, Yu-Ju

2015-09-04

Despite significant efforts in the past decade toward complete mapping of the human proteome, 3564 proteins (neXtProt, 09-2014) are still "missing proteins". Over one-third of these missing proteins are annotated as membrane proteins, owing to their relatively challenging accessibility with standard shotgun proteomics. Using nonsmall cell lung cancer (NSCLC) as a model study, we aim to mine missing proteins from disease-associated membrane proteome, which may be still largely under-represented. To increase identification coverage, we employed Hp-RP StageTip prefractionation of membrane-enriched samples from 11 NSCLC cell lines. Analysis of membrane samples from 20 pairs of tumor and adjacent normal lung tissue was incorporated to include physiologically expressed membrane proteins. Using multiple search engines (X!Tandem, Comet, and Mascot) and stringent evaluation of FDR (MAYU and PeptideShaker), we identified 7702 proteins (66% membrane proteins) and 178 missing proteins (74 membrane proteins) with PSM-, peptide-, and protein-level FDR of 1%. Through multiple reaction monitoring using synthetic peptides, we provided additional evidence of eight missing proteins including seven with transmembrane helix domains. This study demonstrates that mining missing proteins focused on cancer membrane subproteome can greatly contribute to map the whole human proteome. All data were deposited into ProteomeXchange with the identifier PXD002224.

Peripheral Protein Unfolding Drives Membrane Bending.

PubMed

Siaw, Hew Ming Helen; Raghunath, Gokul; Dyer, R Brian

2018-06-20

Dynamic modulation of lipid membrane curvature can be achieved by a number of peripheral protein binding mechanisms such as hy-drophobic insertion of amphipathic helices and membrane scaffolding. Recently, an alternative mechanism was proposed in which crowding of peripherally bound proteins induces membrane curvature through steric pressure generated by lateral collisions. This effect was enhanced using intrinsically disordered proteins that possess high hydrodynamic radii, prompting us to explore whether membrane bending can be triggered by the folding-unfolding transition of surface-bound proteins. We utilized histidine-tagged human serum albumin bound to Ni-NTA-DGS containing liposomes as our model system to test this hypothesis. We found that reduction of the disulfide bonds in the protein resulted in unfolding of HSA, which subsequently led to membrane tubule formation. The frequency of tubule formation was found to be significantly higher when the proteins were unfolded while being localized to a phase-separated domain as opposed to randomly distributed in fluid phase liposomes, indicating that the steric pressure generated from protein unfolding is directly responsible for membrane deformation. Our results are critical for the design of peripheral membrane protein-immobilization strategies and open new avenues for exploring mechanisms of membrane bending driven by conformational changes of peripheral membrane proteins.

Challenges in the Development of Functional Assays of Membrane Proteins

PubMed Central

Tiefenauer, Louis; Demarche, Sophie

2012-01-01

Lipid bilayers are natural barriers of biological cells and cellular compartments. Membrane proteins integrated in biological membranes enable vital cell functions such as signal transduction and the transport of ions or small molecules. In order to determine the activity of a protein of interest at defined conditions, the membrane protein has to be integrated into artificial lipid bilayers immobilized on a surface. For the fabrication of such biosensors expertise is required in material science, surface and analytical chemistry, molecular biology and biotechnology. Specifically, techniques are needed for structuring surfaces in the micro- and nanometer scale, chemical modification and analysis, lipid bilayer formation, protein expression, purification and solubilization, and most importantly, protein integration into engineered lipid bilayers. Electrochemical and optical methods are suitable to detect membrane activity-related signals. The importance of structural knowledge to understand membrane protein function is obvious. Presently only a few structures of membrane proteins are solved at atomic resolution. Functional assays together with known structures of individual membrane proteins will contribute to a better understanding of vital biological processes occurring at biological membranes. Such assays will be utilized in the discovery of drugs, since membrane proteins are major drug targets.

Closely related, yet unique: Distinct homo- and heterodimerization patterns of G protein coupled chemokine receptors and their fine-tuning by cholesterol

PubMed Central

Gahbauer, Stefan; Pluhackova, Kristyna

2018-01-01

Chemokine receptors, a subclass of G protein coupled receptors (GPCRs), play essential roles in the human immune system, they are involved in cancer metastasis as well as in HIV-infection. A plethora of studies show that homo- and heterodimers or even higher order oligomers of the chemokine receptors CXCR4, CCR5, and CCR2 modulate receptor function. In addition, membrane cholesterol affects chemokine receptor activity. However, structural information about homo- and heterodimers formed by chemokine receptors and their interplay with cholesterol is limited. Here, we report homo- and heterodimer configurations of the chemokine receptors CXCR4, CCR5, and CCR2 at atomistic detail, as obtained from thousands of molecular dynamics simulations. The observed homodimerization patterns were similar for the closely related CC chemokine receptors, yet they differed significantly between the CC receptors and CXCR4. Despite their high sequence identity, cholesterol modulated the CC homodimer interfaces in a subtype-specific manner. Chemokine receptor heterodimers display distinct dimerization patterns for CXCR4/CCR5 and CXCR4/CCR2. Furthermore, associations between CXCR4 and CCR5 reveal an increased cholesterol-sensitivity as compared to CXCR4/CCR2 heterodimerization patterns. This work provides a first comprehensive structural overview over the complex interaction network between chemokine receptors and indicates how heterodimerization and the interaction with the membrane environment diversifies the function of closely related GPCRs. PMID:29529028

Protein diffusion in plant cell plasma membranes: the cell-wall corral.

PubMed

Martinière, Alexandre; Runions, John

2013-01-01

Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment.

Vertebrate Membrane Proteins: Structure, Function, and Insights from Biophysical Approaches

PubMed Central

MÜLLER, DANIEL J.; WU, NAN; PALCZEWSKI, KRZYSZTOF

2008-01-01

Membrane proteins are key targets for pharmacological intervention because they are vital for cellular function. Here, we analyze recent progress made in the understanding of the structure and function of membrane proteins with a focus on rhodopsin and development of atomic force microscopy techniques to study biological membranes. Membrane proteins are compartmentalized to carry out extra- and intracellular processes. Biological membranes are densely populated with membrane proteins that occupy approximately 50% of their volume. In most cases membranes contain lipid rafts, protein patches, or paracrystalline formations that lack the higher-order symmetry that would allow them to be characterized by diffraction methods. Despite many technical difficulties, several crystal structures of membrane proteins that illustrate their internal structural organization have been determined. Moreover, high-resolution atomic force microscopy, near-field scanning optical microscopy, and other lower resolution techniques have been used to investigate these structures. Single-molecule force spectroscopy tracks interactions that stabilize membrane proteins and those that switch their functional state; this spectroscopy can be applied to locate a ligand-binding site. Recent development of this technique also reveals the energy landscape of a membrane protein, defining its folding, reaction pathways, and kinetics. Future development and application of novel approaches during the coming years should provide even greater insights to the understanding of biological membrane organization and function. PMID:18321962

Plant membrane proteomics.

PubMed

Ephritikhine, Geneviève; Ferro, Myriam; Rolland, Norbert

2004-12-01

Plant membrane proteins are involved in many different functions according to their location in the cell. For instance, the chloroplast has two membrane systems, thylakoids and envelope, with specialized membrane proteins for photosynthesis and metabolite and ion transporters, respectively. Although recent advances in sample preparation and analytical techniques have been achieved for the study of membrane proteins, the characterization of these proteins, especially the hydrophobic ones, is still challenging. The present review highlights recent advances in methodologies for identification of plant membrane proteins from purified subcellular structures. The interest of combining several complementary extraction procedures to take into account specific features of membrane proteins is discussed in the light of recent proteomics data, notably for chloroplast envelope, mitochondrial membranes and plasma membrane from Arabidopsis. These examples also illustrate how, on one hand, proteomics can feed bioinformatics for a better definition of prediction tools and, on the other hand, although prediction tools are not 100% reliable, they can give valuable information for biological investigations. In particular, membrane proteomics brings new insights over plant membrane systems, on both the membrane compartment where proteins are working and their putative cellular function.

Functional diversification and specialization of cytosolic 70-kDa heat shock proteins.

PubMed

McCallister, Chelsea; Siracusa, Matthew C; Shirazi, Farzaneh; Chalkia, Dimitra; Nikolaidis, Nikolas

2015-03-20

A fundamental question in molecular evolution is how protein functional differentiation alters the ability of cells and organisms to cope with stress and survive. To answer this question we used two paralogous Hsp70s from mouse and explored whether these highly similar cytosolic molecular chaperones, which apart their temporal expression have been considered functionally interchangeable, are differentiated with respect to their lipid-binding function. We demonstrate that the two proteins bind to diverse lipids with different affinities and therefore are functionally specialized. The observed lipid-binding patterns may be related with the ability of both Hsp70s to induce cell death by binding to a particular plasma-membrane lipid, and the potential of only one of them to promote cell survival by binding to a specific lysosomal-membrane lipid. These observations reveal that two seemingly identical proteins differentially modulate cellular adaptation and survival by having acquired specialized functions via sequence divergence. Therefore, this study provides an evolutionary paradigm, where promiscuity, specificity, sub- and neo-functionalization orchestrate one of the most conserved systems in nature, the cellular stress-response.

Shape matters in protein mobility within membranes

PubMed Central

Quemeneur, François; Sigurdsson, Jon K.; Renner, Marianne; Atzberger, Paul J.; Bassereau, Patricia; Lacoste, David

2014-01-01

The lateral mobility of proteins within cell membranes is usually thought to be dependent on their size and modulated by local heterogeneities of the membrane. Experiments using single-particle tracking on reconstituted membranes demonstrate that protein diffusion is significantly influenced by the interplay of membrane curvature, membrane tension, and protein shape. We find that the curvature-coupled voltage-gated potassium channel (KvAP) undergoes a significant increase in protein mobility under tension, whereas the mobility of the curvature-neutral water channel aquaporin 0 (AQP0) is insensitive to it. Such observations are well explained in terms of an effective friction coefficient of the protein induced by the local membrane deformation. PMID:24706877

Biopores/membrane proteins in synthetic polymer membranes.

PubMed

Garni, Martina; Thamboo, Sagana; Schoenenberger, Cora-Ann; Palivan, Cornelia G

2017-04-01

Mimicking cell membranes by simple models based on the reconstitution of membrane proteins in lipid bilayers represents a straightforward approach to understand biological function of these proteins. This biomimetic strategy has been extended to synthetic membranes that have advantages in terms of chemical and mechanical stability, thus providing more robust hybrid membranes. We present here how membrane proteins and biopores have been inserted both in the membrane of nanosized and microsized compartments, and in planar membranes under various conditions. Such bio-hybrid membranes have new properties (as for example, permeability to ions/molecules), and functionality depending on the specificity of the inserted biomolecules. Interestingly, membrane proteins can be functionally inserted in synthetic membranes provided these have appropriate properties to overcome the high hydrophobic mismatch between the size of the biomolecule and the membrane thickness. Functional insertion of membrane proteins and biopores in synthetic membranes of compartments or in planar membranes is possible by an appropriate selection of the amphiphilic copolymers, and conditions of the self-assembly process. These hybrid membranes have new properties and functionality based on the specificity of the biomolecules and the nature of the synthetic membranes. Bio-hybrid membranes represent new solutions for the development of nanoreactors, artificial organelles or active surfaces/membranes that, by further gaining in complexity and functionality, will promote translational applications. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider. Copyright © 2016. Published by Elsevier B.V.

Topologically Diverse Human Membrane Proteins Partition to Liquid-Disordered Domains in Phase-Separated Lipid Vesicles.

PubMed

Schlebach, Jonathan P; Barrett, Paul J; Day, Charles A; Kim, Ji Hun; Kenworthy, Anne K; Sanders, Charles R

2016-02-23

The integration of membrane proteins into "lipid raft" membrane domains influences many biochemical processes. The intrinsic structural properties of membrane proteins are thought to mediate their partitioning between membrane domains. However, whether membrane topology influences the targeting of proteins to rafts remains unclear. To address this question, we examined the domain preference of three putative raft-associated membrane proteins with widely different topologies: human caveolin-3, C99 (the 99 residue C-terminal domain of the amyloid precursor protein), and peripheral myelin protein 22. We find that each of these proteins are excluded from the ordered domains of giant unilamellar vesicles containing coexisting liquid-ordered and liquid-disordered phases. Thus, the intrinsic structural properties of these three topologically distinct disease-linked proteins are insufficient to confer affinity for synthetic raft-like domains.

Simulation of controllable permeation in PNIPAAm coated membranes

NASA Astrophysics Data System (ADS)

Ehrenhofer, Adrian; Wallmersperger, Thomas; Richter, Andreas

2016-04-01

Membranes separate fluid compartments and can comprise transport structures for selective permeation. In biology, channel proteins are specialized in their atomic structure to allow transport of specific compounds (selectivity). Conformational changes in protein structure allow the control of the permeation abilities by outer stimuli (gating). In polymeric membranes, the selectivity is due to electrostatic or size-exclusion. It can thus be controlled by size variation or electric charges. Controllable permeation can be useful to determine particle-size distributions in continuous flow, e.g. in microfluidics and biomedicine to gain cell diameter profiles in blood. The present approach uses patterned polyethylene terephthalate (PET) membranes with hydrogel surface coating for permeation control by size-exclusion. The thermosensitive hydrogel poly(N-isopropylacrylamide) (PNIPAAm) is structured with a cross-shaped pore geometry. A change in the temperature of the water flow through the membrane leads to a pore shape variation. The temperature dependent behavior of PNIPAAm can be numerically modeled with a temperature expansion model, where the swelling and deswelling is depicted by temperature dependent expansion coefficients. In the present study, the free swelling behavior was implemented to the Finite Element tool ABAQUS for the complex composite structure of the permeation control membrane. Experimental values of the geometry characteristics were derived from microscopy images with the tool Image J and compared to simulation results. Numerical simulations using the derived thermo-mechanical model for different pore geometries (circular, rectangle, cross and triangle) were performed. With this study, we show that the temperature expansion model with values from the free swelling behavior can be used to adequately predict the deformation behavior of the complex membrane system. The predictions can be used to optimize the behavior of the membrane pores and the overall performance of the smart membrane.

Cell-free Co-expression of Functional Membrane Proteins and Apolipoprotein, Forming Soluble Nanolipoprotein Particles*S⃞

PubMed Central

Cappuccio, Jenny A.; Blanchette, Craig D.; Sulchek, Todd A.; Arroyo, Erin S.; Kralj, Joel M.; Hinz, Angela K.; Kuhn, Edward A.; Chromy, Brett A.; Segelke, Brent W.; Rothschild, Kenneth J.; Fletcher, Julia E.; Katzen, Federico; Peterson, Todd C.; Kudlicki, Wieslaw A.; Bench, Graham; Hoeprich, Paul D.; Coleman, Matthew A.

2008-01-01

Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Δ1–49 apolipoprotein A-I fragment (Δ49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR → M transition. Importantly the functional bR was solubilized in discoidal bR·NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Δ49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies. PMID:18603642

Microfabricated Patch Clamp Electrodes for Improved Ion Channel Protein Measurements

NASA Astrophysics Data System (ADS)

Klemic, James; Klemic, Kathryn; Reed, Mark; Sigworth, Frederick

2002-03-01

Ion channels are trans-membrane proteins that underlie many cell functions including hormone and neurotransmitter release, muscle contraction and cell signaling cascades. Ion channel proteins are commonly characterized via the patch clamp method in which an extruded glass tube containing ionic solution, manipulated by an expert technician, is brought into contact with a living cell to record ionic current through the cell membrane. Microfabricated planar patch electrodes, micromolded in the silicone elastomer poly-dimethylsiloxane (PDMS) from microlithographically patterned structures, have been developed that improve on this method. Microfabrication techniques allow arrays of patch electrodes to be fabricated, increasing the throughput of the measurement technique. Planar patch electrodes readily allow the automation of cell sealing, further increasing throughput. Microfabricated electrode arrays may be readily integrated with microfluidic structures to allow fast, in situ solution exchange. Miniaturization of the electrode geometry should increase both the signal to noise and the bandwidth of the measurement. Microfabricated patch electrode arrays have been fabricated and measurements have been taken.

Membrane Protein Production in E. coli Lysates in Presence of Preassembled Nanodiscs.

PubMed

Rues, Ralf-Bernhardt; Gräwe, Alexander; Henrich, Erik; Bernhard, Frank

2017-01-01

Cell-free expression allows to synthesize membrane proteins in completely new formats that can relatively easily be customized for particular applications. Amphiphilic superstructures such as micelles, lipomicelles, or nanodiscs can be provided as nano-devices for the solubilization of membrane proteins. Defined empty bilayers in the form of nanodiscs offer native like environments for membrane proteins, supporting functional folding, proper oligomeric assembly as well as stability. Even very difficult and detergent-sensitive membrane proteins can be addressed by the combination of nanodisc technology with efficient cell-free expression systems as the direct co-translational insertion of nascent membrane proteins into supplied preassembled nanodiscs is possible. This chapter provides updated protocols for the synthesis of membrane proteins in presence of preassembled nanodiscs suitable for emerging applications such as screening of lipid effects on membrane protein function and the modulation of oligomeric complex formation.

Systematically Ranking the Tightness of Membrane Association for Peripheral Membrane Proteins (PMPs)*

PubMed Central

Gao, Liyan; Ge, Haitao; Huang, Xiahe; Liu, Kehui; Zhang, Yuanya; Xu, Wu; Wang, Yingchun

2015-01-01

Large-scale quantitative evaluation of the tightness of membrane association for nontransmembrane proteins is important for identifying true peripheral membrane proteins with functional significance. Herein, we simultaneously ranked more than 1000 proteins of the photosynthetic model organism Synechocystis sp. PCC 6803 for their relative tightness of membrane association using a proteomic approach. Using multiple precisely ranked and experimentally verified peripheral subunits of photosynthetic protein complexes as the landmarks, we found that proteins involved in two-component signal transduction systems and transporters are overall tightly associated with the membranes, whereas the associations of ribosomal proteins are much weaker. Moreover, we found that hypothetical proteins containing the same domains generally have similar tightness. This work provided a global view of the structural organization of the membrane proteome with respect to divergent functions, and built the foundation for future investigation of the dynamic membrane proteome reorganization in response to different environmental or internal stimuli. PMID:25505158

Cuscuta europaea plastid apparatus in various developmental stages

PubMed Central

Švubová, Renáta; Ovečka, Miroslav; Pavlovič, Andrej; Slováková, Ľudmila; Blehová, Alžbeta

2013-01-01

It was generally accepted that Cuscuta europaea is mostly adapted to a parasitic lifestyle with no detectable levels of chlorophylls. We found out relatively high level of chlorophylls (Chls a+b) in young developmental stages of dodder. Significant lowering of Chls (a+b) content and increase of carotenoid concentration was typical only for ontogenetically more developed stages. Lower content of photosynthesis-related proteins involved in Chls biosynthesis and in photosystem formation as well as low photochemical activity of PSII indicate that photosynthesis is not the main activity of C. europaea plastids. Previously, it has been shown in other species that the Thylakoid Formation Protein 1 (THF1) is involved in thylakoid membrane differentiation, plant-fungal and plant-bacterial interactions and in sugar signaling with its preferential localization to plastids. Our immunofluorescence localization studies and analyses of haustorial plasma membrane fractions revealed that in addition to plastids, the THF1 protein localizes also to the plasma membrane and plasmodesmata in developing C. europaea haustorium, most abundantly in the digitate cells of the endophyte primordium. These results are supported by western blot analysis, documenting the highest levels of the THF1 protein in “get together” tissues of dodder and tobacco. Based on the fact that photosynthesis is not a typical process in the C. europaea haustorium and on the extra-plastidial localization pattern of the THF1, our data support rather other functions of this protein in the complex relationship between C. europaea and its host. PMID:23438585

Crystal structural characterization reveals novel oligomeric interactions of human voltage-dependent anion channel 1.

PubMed

Hosaka, Toshiaki; Okazaki, Masateru; Kimura-Someya, Tomomi; Ishizuka-Katsura, Yoshiko; Ito, Kaori; Yokoyama, Shigeyuki; Dodo, Kosuke; Sodeoka, Mikiko; Shirouzu, Mikako

2017-09-01

Voltage-dependent anion channel 1 (VDAC1), which is located in the outer mitochondrial membrane, plays important roles in various cellular processes. For example, oligomerization of VDAC1 is involved in the release of cytochrome c to the cytoplasm, leading to apoptosis. However, it is unknown how VDAC1 oligomerization occurs in the membrane. In the present study, we determined high-resolution crystal structures of oligomeric human VDAC1 (hVDAC1) prepared by using an Escherichia coli cell-free protein synthesis system, which avoided the need for denaturation and refolding of the protein. Broad-range screening using a bicelle crystallization method produced crystals in space groups C222 and P22 1 2 1 , which diffracted to a resolution of 3.10 and 3.15 Å, respectively. Each crystal contained two hVDAC1 protomers in the asymmetric unit. Dimer within the asymmetrical unit of the crystal in space group C222 were oriented parallel, whereas those of the crystal in space group P22 1 2 1 were oriented anti-parallel. From a model of the crystal in space group C222, which we constructed by using crystal symmetry operators, a heptameric structure with eight patterns of interaction between protomers, including hydrophobic interactions with β-strands, hydrophilic interactions with loop regions, and protein-lipid interactions, was observed. It is possible that by having multiple patterns of interaction, VDAC1 can form homo- or hetero-oligomers not only with other VDAC1 protomers but also with other proteins such as VDAC2, VDAC3 and apoptosis-regulating proteins in the Bcl-2 family. © 2017 The Protein Society.

Binding constants of membrane-anchored receptors and ligands depend strongly on the nanoscale roughness of membranes.

PubMed

Hu, Jinglei; Lipowsky, Reinhard; Weikl, Thomas R

2013-09-17

Cell adhesion and the adhesion of vesicles to the membranes of cells or organelles are pivotal for immune responses, tissue formation, and cell signaling. The adhesion processes depend sensitively on the binding constant of the membrane-anchored receptor and ligand proteins that mediate adhesion, but this constant is difficult to measure in experiments. We have investigated the binding of membrane-anchored receptor and ligand proteins with molecular dynamics simulations. We find that the binding constant of the anchored proteins strongly decreases with the membrane roughness caused by thermally excited membrane shape fluctuations on nanoscales. We present a theory that explains the roughness dependence of the binding constant for the anchored proteins from membrane confinement and that relates this constant to the binding constant of soluble proteins without membrane anchors. Because the binding constant of soluble proteins is readily accessible in experiments, our results provide a useful route to compute the binding constant of membrane-anchored receptor and ligand proteins.

The membrane skeleton in Paramecium: Molecular characterization of a novel epiplasmin family and preliminary GFP expression results.

PubMed

Pomel, Sébastien; Diogon, Marie; Bouchard, Philippe; Pradel, Lydie; Ravet, Viviane; Coffe, Gérard; Viguès, Bernard

2006-02-01

Previous attempts to identify the membrane skeleton of Paramecium cells have revealed a protein pattern that is both complex and specific. The most prominent structural elements, epiplasmic scales, are centered around ciliary units and are closely apposed to the cytoplasmic side of the inner alveolar membrane. We sought to characterize epiplasmic scale proteins (epiplasmins) at the molecular level. PCR approaches enabled the cloning and sequencing of two closely related genes by amplifications of sequences from a macronuclear genomic library. Using these two genes (EPI-1 and EPI-2), we have contributed to the annotation of the Paramecium tetraurelia macronuclear genome and identified 39 additional (paralogous) sequences. Two orthologous sequences were found in the Tetrahymena thermophila genome. Structural analysis of the 43 sequences indicates that the hallmark of this new multigenic family is a 79 aa domain flanked by two Q-, P- and V-rich stretches of sequence that are much more variable in amino-acid composition. Such features clearly distinguish members of the multigenic family from epiplasmic proteins previously sequenced in other ciliates. The expression of Green Fluorescent Protein (GFP)-tagged epiplasmin showed significant labeling of epiplasmic scales as well as oral structures. We expect that the GFP construct described herein will prove to be a useful tool for comparative subcellular localization of different putative epiplasmins in Paramecium.

Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT.

PubMed

Roundhill, E; Burchill, S

2013-07-09

Primary Ewing's sarcoma family of tumours (ESFTs) may respond to chemotherapy, although many patients experience subsequent disease recurrence and relapse. The survival of ESFT cells following chemotherapy has been attributed to the development of resistant disease, possibly through the expression of ABC transporter proteins. MRP-1 and Pgp mRNA and protein expression in primary ESFTs was determined by quantitative reverse-transcriptase PCR (RT-qPCR) and immunohistochemistry, respectively, and alternative splicing of MRP-1 by RT-PCR. We observed MRP-1 protein expression in 92% (43 out of 47) of primary ESFTs, and cell membrane MRP-1 was highly predictive of both overall survival (P<0.0001) and event-free survival (P<0.0001). Alternative splicing of MRP-1 was detected in primary ESFTs, although the pattern of splicing variants was not predictive of patient outcome, with the exception of loss of exon 9 in six patients, which predicted relapse (P=0.041). Pgp protein was detected in 6% (38 out of 44) of primary ESFTs and was not associated with patient survival. For the first time we have established that cell membrane expression of MRP-1 or loss of exon 9 is predictive of outcome but not the number of splicing events or expression of Pgp, and both may be valuable factors for the stratification of patients for more intensive therapy.

Dendronic trimaltoside amphiphiles (DTMs) for membrane protein study† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc03700g

PubMed Central

Sadaf, Aiman; Du, Yang; Santillan, Claudia; Mortensen, Jonas S.; Molist, Iago; Seven, Alpay B.; Hariharan, Parameswaran; Skiniotis, Georgios; Loland, Claus J.; Kobilka, Brian K.; Guan, Lan; Byrne, Bernadette

2017-01-01

The critical contribution of membrane proteins in normal cellular function makes their detailed structure and functional analysis essential. Detergents, amphipathic agents with the ability to maintain membrane proteins in a soluble state in aqueous solution, have key roles in membrane protein manipulation. Structural and functional stability is a prerequisite for biophysical characterization. However, many conventional detergents are limited in their ability to stabilize membrane proteins, making development of novel detergents for membrane protein manipulation an important research area. The architecture of a detergent hydrophobic group, that directly interacts with the hydrophobic segment of membrane proteins, is a key factor in dictating their efficacy for both membrane protein solubilization and stabilization. In the current study, we developed two sets of maltoside-based detergents with four alkyl chains by introducing dendronic hydrophobic groups connected to a trimaltoside head group, designated dendronic trimaltosides (DTMs). Representative DTMs conferred enhanced stabilization to multiple membrane proteins compared to the benchmark conventional detergent, DDM. One DTM (i.e., DTM-A6) clearly outperformed DDM in stabilizing human β2 adrenergic receptor (β2AR) and its complex with Gs protein. A further evaluation of this DTM led to a clear visualization of β2AR-Gs complex via electron microscopic analysis. Thus, the current study not only provides novel detergent tools useful for membrane protein study, but also suggests that the dendronic architecture has a role in governing detergent efficacy for membrane protein stabilization. PMID:29619178

HIP12 is a non-proapoptotic member of a gene family including HIP1, an interacting protein with huntingtin.

PubMed

Chopra, V S; Metzler, M; Rasper, D M; Engqvist-Goldstein, A E; Singaraja, R; Gan, L; Fichter, K M; McCutcheon, K; Drubin, D; Nicholson, D W; Hayden, M R

2000-11-01

Huntingtin-interacting protein I (HIP1) is a membrane-associated protein that interacts with huntingtin, the protein altered in Huntington disease. HIP1 shows homology to Sla2p, a protein essential for the assembly and function of the cytoskeleton and endocytosis in Saccharomyces cerevisiae. We have determined that the HIP1 gene comprises 32 exons spanning approximately 215 kb of genomic DNA and gives rise to two alternate splice forms termed HIP1-1 and HIP1-2. Additionally, we have identified a novel protein termed HIP12 with significant sequence and biochemical similarities to HIP1 and high sequence similarity to Sla2p. HIP12 differs from HIP1 in its pattern of expression both at the mRNA and protein level. However, HIP1 and HIP12 are both found within the brain and show a similar subcellular distribution pattern. In contrast to HIP1, which is toxic in cell culture, HIP12 does not confer toxicity in the same assay systems. Interestingly, HIP12 does not interact with huntingtin but can interact with HIP1. suggesting a potential interaction in vivo that may influence the function of each respective protein.

Cytoskeletal Components Define Protein Location to Membrane Microdomains*

PubMed Central

Szymanski, Witold G.; Zauber, Henrik; Erban, Alexander; Gorka, Michal; Wu, Xu Na; Schulze, Waltraud X.

2015-01-01

The plasma membrane is an important compartment that undergoes dynamic changes in composition upon external or internal stimuli. The dynamic subcompartmentation of proteins in ordered low-density (DRM) and disordered high-density (DSM) membrane phases is hypothesized to require interactions with cytoskeletal components. Here, we systematically analyzed the effects of actin or tubulin disruption on the distribution of proteins between membrane density phases. We used a proteomic screen to identify candidate proteins with altered submembrane location, followed by biochemical or cell biological characterization in Arabidopsis thaliana. We found that several proteins, such as plasma membrane ATPases, receptor kinases, or remorins resulted in a differential distribution between membrane density phases upon cytoskeletal disruption. Moreover, in most cases, contrasting effects were observed: Disruption of actin filaments largely led to a redistribution of proteins from DRM to DSM membrane fractions while disruption of tubulins resulted in general depletion of proteins from the membranes. We conclude that actin filaments are necessary for dynamic movement of proteins between different membrane phases and that microtubules are not necessarily important for formation of microdomains as such, but rather they may control the protein amount present in the membrane phases. PMID:26091700

Stability of milk fat globule membrane proteins toward human enzymatic gastrointestinal digestion.

PubMed

Le, T T; Van de Wiele, T; Do, T N H; Debyser, G; Struijs, K; Devreese, B; Dewettinck, K; Van Camp, J

2012-05-01

The milk fat globule membrane (MFGM) fraction refers to the thin film of polar lipids and membrane proteins that surrounds fat globules in milk. It is its unique biochemical composition that renders MFGM with some beneficial biological activities, such as anti-adhesive effects toward pathogens. However, a prerequisite for the putative bioactivity of MFGM is its stability during gastrointestinal digestion. We, therefore, subjected MFGM material, isolated from raw milk, to an in vitro enzymatic gastrointestinal digestion. Sodium dodecyl sulfate PAGE, in combination with 2 staining methods, Coomassie Blue and periodic acid Schiff staining, was used to evaluate polypeptide patterns of the digest, whereas mass spectrometry was used to confirm the presence of specific MFGM proteins. Generally, it was observed that glycoproteins showed higher resistance to endogenous proteases compared with non-glycosylated proteins. Mucin 1 displayed the highest resistance to digestion and a considerable part of this protein was still detected at its original molecular weight after gastric and small intestine digestion. Cluster of differentiation 36 was also quite resistant to pepsin. A significant part of periodic acid Schiff 6/7 survived the gastric digestion, provided that the lipid moiety was not removed from the MFGM material. Overall, MFGM glycoproteins are generally more resistant to gastrointestinal digestion than serum milk proteins and the presence of lipids, besides glycosylation, may protect MFGM glycoproteins from gastrointestinal digestion. This gastrointestinal stability makes MFGM glycoproteins amenable to further studies in which their putative health-promoting effects can be explored. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Applications of solid-state NMR to membrane proteins.

PubMed

Ladizhansky, Vladimir

2017-11-01

Membrane proteins mediate flow of molecules, signals, and energy between cells and intracellular compartments. Understanding membrane protein function requires a detailed understanding of the structural and dynamic properties involved. Lipid bilayers provide a native-like environment for structure-function investigations of membrane proteins. In this review we give a general discourse on the recent progress in the field of solid-state NMR of membrane proteins. Solid-state NMR is a variation of NMR spectroscopy that is applicable to molecular systems with restricted mobility, such as high molecular weight proteins and protein complexes, supramolecular assemblies, or membrane proteins in a phospholipid environment. We highlight recent advances in applications of solid-state NMR to membrane proteins, specifically focusing on the recent developments in the field of Dynamic Nuclear Polarization, proton detection, and solid-state NMR applications in situ (in cell membranes). This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

The membrane separation mechanism in protein concentration from the extract of waste press cake in biofuel manufacturing process of Jatropha seeds

NASA Astrophysics Data System (ADS)

Chung, T. W.; Chen, C. K.; Hsu, S. H.

2017-11-01

Protein concentration process using filter membrane has a significant advantage on energy saving compared to the traditional drying processes. However, fouling on large membrane area and frequent membrane cleaning will increase the energy consumption and operation cost for the protein concentration process with filter membrane. In this study, the membrane filtration for protein concentration will be conducted and compared with the recent protein concentration technology. The analysis of operating factors for protein concentration process using filter membrane was discussed. The separation mechanism of membrane filtration was developed according to the size difference between the pore of membrane and the particle of filter material. The Darcy’s Law was applied to discuss the interaction on flux, TMP (transmembrane pressure) and resistance in this study. The effect of membrane pore size, pH value and TMP on the steady-state flux (Jst) and protein rejection (R) were studied. It is observed that the Jst increases with decreasing membrane pore size, the Jst increases with increasing TMP, and R increased with decreasing solution pH value. Compare to other variables, the pH value is the most significant variable for separation between protein and water.

Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs.

PubMed

Richards, Mark J; Hsia, Chih-Yun; Singh, Rohit R; Haider, Huma; Kumpf, Julia; Kawate, Toshimitsu; Daniel, Susan

2016-03-29

Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions on protein activity and the roles of membrane proteins in disease pathways.

Interaction of murine macrophage-membrane proteins with components of the pathogenic fungus Histoplasma capsulatum

PubMed Central

Taylor, M L; Duarte-Escalante, E; Reyes-Montes, M R; Elizondo, N; Maldonado, G; Zenteno, E

1998-01-01

The interaction of macrophage-membrane proteins and histoplasmin, a crude antigen of the pathogenic fungus Histoplasma capsulatum, was studied using murine peritoneal macrophages. Membrane proteins were purified via membrane attachment to polycationic beads and solubilized in Tris–HCl/SDS/DTT/glycerol for protein extraction; afterwards they were adsorbed or not with H. capsulatum yeast or lectin binding-enriched by affinity chromatography. Membrane proteins and histoplasmin interactions were detected by ELISA and immunoblotting assays using anti-H. capsulatum human or mouse serum and biotinylated goat anti-human or anti-mouse IgG/streptavidin-peroxidase system to reveal the interaction. Results indicate that macrophage-membrane proteins and histoplasmin components interact in a dose-dependent reaction, and adsorption of macrophage-membrane proteins by yeast cells induces a critical decrease in the interaction. Macrophage-membrane glycoproteins with terminal d-galactosyl residues, purified by chromatography with Abrus precatorius lectin, bound to histoplasmin; and two bands of 68 kD and 180 kD of transferred membrane protein samples interacted with histoplasmin components, as revealed by immunoblot assays. Specificity for β-galactoside residues on the macrophage-membrane was confirmed by galactose inhibition of the interaction between macrophage-membrane proteins and histoplasmin components, in competitive ELISA using sugars, as well as by enzymatic cleavage of the galactoside residues. PMID:9737672

Detection of Proteins on Blot Membranes

PubMed Central

Goldman, Aaron; Harper, Sandra; Speicher, David W.

2017-01-01

Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. PMID:27801518

Detection of Proteins on Blot Membranes.

PubMed

Goldman, Aaron; Harper, Sandra; Speicher, David W

2016-11-01

Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Spatial distributions of AQP5 and AQP0 in embryonic and postnatal mouse lens development

PubMed Central

Petrova, Rosica S.; Schey, Kevin L.; Donaldson, Paul J.; Grey, Angus C.

2015-01-01

The expression of the water channel protein aquaporin (AQP)-5 in adult rodent and human lenses was recently reported using immunohistochemistry, molecular biology, and mass spectrometry techniques, confirming a second transmembrane water channel that is present in lens fibre cells in addition to the abundant AQP0 protein. Interestingly, the sub-cellular distribution and level of post-translational modification of both proteins changes with fibre cell differentiation and location in the adult rodent lens. This study compares the sub-cellular distribution of AQP0 and AQP5 during embryonic and postnatal fibre cell development in the mouse lens to understand how the immunolabelling patterns for both AQPs observed in adult lens are first established. Immunohistochemistry was used to map the cellular and sub-cellular distribution of AQP5 and AQP0 throughout the lens in cryosections from adult (6 weeks to 8 months) and postnatal (0-2 weeks) mouse lenses and in sections from paraffin embedded mouse embryos (E10-E19). All sections were imaged by fluorescence confocal microscopy. Using antibodies directed against the C-terminus of each AQP, AQP5 was abundantly expressed early in development, being found in the cytoplasm of cells of the lens vesicle and surrounding tissues (E10), while AQP0 was detected later (E11), and only in the membranes of elongating primary fibre cells. During the course of subsequent embryonic and postnatal development the pattern of cytoplasmic AQP5 and membranous AQP0 labelling was maintained until postnatal day 6 (P6). From P6 AQP5 labelling became progressively more membranous initially in the lens nucleus and then later in all regions of the lens, while AQP0 labelling was abruptly lost in the lens nucleus due to C-terminal truncation. Our results show that the spatial distribution patterns of AQP0 and AQP5 observed in the adult lens are established during a narrow window of post natal development (P6-P15) that precedes eye opening and coincides with regression of the hyaloid vascular system. Our results support the hypothesis that, in the older fibre cells, insertion of AQP5 into the fibre cell membrane may compensate for any change in the functionality of AQP0 induced by truncation of its C-terminal tail. PMID:25595964

Profiling the outer membrane proteome during growth and development of the social bacterium Myxococcus xanthus by selective biotinylation and analyses of outer membrane vesicles.

PubMed

Kahnt, Jörg; Aguiluz, Kryssia; Koch, Jürgen; Treuner-Lange, Anke; Konovalova, Anna; Huntley, Stuart; Hoppert, Michael; Søgaard-Andersen, Lotte; Hedderich, Reiner

2010-10-01

Social behavior in the bacterium Myxococcus xanthus relies on contact-dependent activities involving cell-cell and cell-substratum interactions. To identify outer membrane proteins that have a role in these activities, we profiled the outer membrane proteome of growing and starving cells using two strategies. First, outer membrane proteins were enriched by biotinylation of intact cells using the reagent NHS (N-hydroxysuccinimide)-PEO(12) (polyethylene oxide)-biotin with subsequent membrane solubilization and affinity chromatography. Second, the proteome of outer membrane vesicles (OMV) was determined. Comparisons of detected proteins show that these methods have different detection profiles and together provide a comprehensive view of the outer membrane proteome. From 362 proteins identified, 274 (76%) were cell envelope proteins including 64 integral outer membrane proteins and 85 lipoproteins. The majority of these proteins were of unknown function. Among integral outer membrane proteins with homologues of known function, TonB-dependent transporters comprise the largest group. Our data suggest novel functions for these transporters. Among lipoproteins with homologues of known function, proteins with hydrolytic functions comprise the largest group. The luminal load of OMV was enriched for proteins with hydrolytic functions. Our data suggest that OMV have functions in predation and possibly in transfer of intercellular signaling molecules between cells.

Topographical analysis of the plasma membrane-associated sucrose binding protein from soybean.

PubMed

Overvoorde, P J; Grimes, H D

1994-05-27

Plasma membranes of soybean cells actively engaged in sucrose transport have a sucrose binding protein (SBP) that does not appear to be an integral membrane protein. Experiments were undertaken to analyze the topographical association of this protein with the membrane. Treatment of purified plasma membrane vesicles with either 1 M KCl or KI released less than 35% of the sucrose binding protein from the membrane whereas treatment with either 4 M urea or 0.1 M Na2CO3, pH 11.5, disassociated between 50 and 70%, respectively, of this protein from the membrane. SDS, at either 0.5x, 1x, or 10x of its critical micelle concentration, effectively solubilized the sucrose binding protein. The nonionic detergents Triton X-100 and CHAPS, at either 0.5x, 1x, or 10x of their critical micelle concentration, solubilized between 65 and 75% of this protein. When either native plasma membrane-associated or in vitro-transcribed and -translated SBP were subjected to Triton X-114 phase separation, 80% partitioned into the detergent-poor aqueous phase. These results indicate that the SBP is a peripheral membrane protein but also suggest that there is a population of this protein that is tethered to the membrane.

An Integrated Framework Advancing Membrane Protein Modeling and Design

PubMed Central

Weitzner, Brian D.; Duran, Amanda M.; Tilley, Drew C.; Elazar, Assaf; Gray, Jeffrey J.

2015-01-01

Membrane proteins are critical functional molecules in the human body, constituting more than 30% of open reading frames in the human genome. Unfortunately, a myriad of difficulties in overexpression and reconstitution into membrane mimetics severely limit our ability to determine their structures. Computational tools are therefore instrumental to membrane protein structure prediction, consequently increasing our understanding of membrane protein function and their role in disease. Here, we describe a general framework facilitating membrane protein modeling and design that combines the scientific principles for membrane protein modeling with the flexible software architecture of Rosetta3. This new framework, called RosettaMP, provides a general membrane representation that interfaces with scoring, conformational sampling, and mutation routines that can be easily combined to create new protocols. To demonstrate the capabilities of this implementation, we developed four proof-of-concept applications for (1) prediction of free energy changes upon mutation; (2) high-resolution structural refinement; (3) protein-protein docking; and (4) assembly of symmetric protein complexes, all in the membrane environment. Preliminary data show that these algorithms can produce meaningful scores and structures. The data also suggest needed improvements to both sampling routines and score functions. Importantly, the applications collectively demonstrate the potential of combining the flexible nature of RosettaMP with the power of Rosetta algorithms to facilitate membrane protein modeling and design. PMID:26325167

Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork.

PubMed

Lenne, Pierre-François; Wawrezinieck, Laure; Conchonaud, Fabien; Wurtz, Olivier; Boned, Annie; Guo, Xiao-Jun; Rigneault, Hervé; He, Hai-Tao; Marguet, Didier

2006-07-26

It is by now widely recognized that cell membranes show complex patterns of lateral organization. Two mechanisms involving either a lipid-dependent (microdomain model) or cytoskeleton-based (meshwork model) process are thought to be responsible for these plasma membrane organizations. In the present study, fluorescence correlation spectroscopy measurements on various spatial scales were performed in order to directly identify and characterize these two processes in live cells with a high temporal resolution, without any loss of spatial information. Putative raft markers were found to be dynamically compartmented within tens of milliseconds into small microdomains (Ø <120 nm) that are sensitive to the cholesterol and sphingomyelin levels, whereas actin-based cytoskeleton barriers are responsible for the confinement of the transferrin receptor protein. A free-like diffusion was observed when both the lipid-dependent and cytoskeleton-based organizations were disrupted, which suggests that these are two main compartmentalizing forces at work in the plasma membrane.

Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots.

PubMed

Abeyrathne, Priyanka D; Lam, Joseph S

2007-04-01

A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.

A protein interaction network analysis for yeast integral membrane protein.

PubMed

Shi, Ming-Guang; Huang, De-Shuang; Li, Xue-Ling

2008-01-01

Although the yeast Saccharomyces cerevisiae is the best exemplified single-celled eukaryote, the vast number of protein-protein interactions of integral membrane proteins of Saccharomyces cerevisiae have not been characterized by experiments. Here, based on the kernel method of Greedy Kernel Principal Component analysis plus Linear Discriminant Analysis, we identify 300 protein-protein interactions involving 189 membrane proteins and get the outcome of a highly connected protein-protein interactions network. Furthermore, we study the global topological features of integral membrane proteins network of Saccharomyces cerevisiae. These results give the comprehensive description of protein-protein interactions of integral membrane proteins and reveal global topological and robustness of the interactome network at a system level. This work represents an important step towards a comprehensive understanding of yeast protein interactions.

Identification of Genes Involved in Chemoreception in Plutella xyllostella by Antennal Transcriptome Analysis.

PubMed

Yang, Shiyong; Cao, Depan; Wang, Guirong; Liu, Yang

2017-09-20

Perception of environmental and habitat cues is of significance for insect survival and reproduction. Odor detection in insects is mediated by a number of proteins in antennae such as odorant receptors (ORs), ionotropic receptors (IRs), odorant binding proteins (OBPs), chemosensory proteins (CSPs), sensory neuron membrane proteins (SNMPs) and odorant degrading enzymes. In this study, we sequenced and assembled the adult male and female antennal transcriptomes of a destructive agricultural pest, the diamondback moth Plutella xyllostella. In these transcriptomes, we identified transcripts belonging to 6 chemoreception gene families related to ordor detection, including 54 ORs, 16 IRs, 7 gustatory receptors (GRs), 15 CSPs, 24 OBPs and 2 SNMPs. Semi-quantitative reverse transcription PCR analysis of expression patterns indicated that some of these ORs and IRs have clear sex-biased and tissue-specific expression patterns. Our results lay the foundation for future characterization of the functions of these P. xyllostella chemosensory receptors at the molecular level and development of novel semiochemicals for integrated control of this agricultural pest.

Production of membrane proteins without cells or detergents.

PubMed

Rajesh, Sundaresan; Knowles, Timothy; Overduin, Michael

2011-04-30

The production of membrane proteins in cellular systems is besieged by several problems due to their hydrophobic nature which often causes misfolding, protein aggregation and cytotoxicity, resulting in poor yields of stable proteins. Cell-free expression has emerged as one of the most versatile alternatives for circumventing these obstacles by producing membrane proteins directly into designed hydrophobic environments. Efficient optimisation of expression and solubilisation conditions using a variety of detergents, membrane mimetics and lipids has yielded structurally and functionally intact membrane proteins, with yields several fold above the levels possible from cell-based systems. Here we review recently developed techniques available to produce functional membrane proteins, and discuss amphipols, nanodisc and styrene maleic acid lipid particle (SMALP) technologies that can be exploited alongside cell-free expression of membrane proteins. Copyright © 2010 Elsevier B.V. All rights reserved.

Role of membrane contact sites in protein import into mitochondria

PubMed Central

Horvath, Susanne E; Rampelt, Heike; Oeljeklaus, Silke; Warscheid, Bettina; van der Laan, Martin; Pfanner, Nikolaus

2015-01-01

Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long-standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence-carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture. PMID:25514890

A Continuum Poisson-Boltzmann Model for Membrane Channel Proteins

PubMed Central

Xiao, Li; Diao, Jianxiong; Greene, D'Artagnan; Wang, Junmei; Luo, Ray

2017-01-01

Membrane proteins constitute a large portion of the human proteome and perform a variety of important functions as membrane receptors, transport proteins, enzymes, signaling proteins, and more. Computational studies of membrane proteins are usually much more complicated than those of globular proteins. Here we propose a new continuum model for Poisson-Boltzmann calculations of membrane channel proteins. Major improvements over the existing continuum slab model are as follows:1) The location and thickness of the slab model are fine-tuned based on explicit-solvent MD simulations. 2) The highly different accessibility in the membrane and water regions are addressed with a two-step, two-probe grid labeling procedure, and 3) The water pores/channels are automatically identified. The new continuum membrane model is optimized (by adjusting the membrane probe, as well as the slab thickness and center) to best reproduce the distributions of buried water molecules in the membrane region as sampled in explicit water simulations. Our optimization also shows that the widely adopted water probe of 1.4 Å for globular proteins is a very reasonable default value for membrane protein simulations. It gives the best compromise in reproducing the explicit water distributions in membrane channel proteins, at least in the water accessible pore/channel regions that we focus on. Finally, we validate the new membrane model by carrying out binding affinity calculations for a potassium channel, and we observe a good agreement with experiment results. PMID:28564540

Generation of wavy structure on lipid membrane by peripheral proteins: a linear elastic analysis.

PubMed

Mahata, Paritosh; Das, Sovan Lal

2017-05-01

We carry out a linear elastic analysis to study wavy structure generation on lipid membrane by peripheral membrane proteins. We model the lipid membrane as linearly elastic and anisotropic material. The hydrophobic insertion by proteins into the lipid membrane has been idealized as penetration of rigid rod-like inclusions into the membrane and the electrostatic interaction between protein and membrane has been modeled by a distributed surface traction acting on the membrane surface. With the proposed model we study curvature generation by several binding domains of peripheral membrane proteins containing BAR domains and amphipathic alpha-helices. It is observed that electrostatic interaction is essential for curvature generation by the BAR domains. © 2017 Federation of European Biochemical Societies.

Pharmacoproteomics of a Metalloproteinase Hydroxamate Inhibitor in Breast Cancer Cells: Dynamics of Membrane Type 1 Matrix Metalloproteinase-Mediated Membrane Protein Shedding ▿ ‡

PubMed Central

Butler, Georgina S.; Dean, Richard A.; Tam, Eric M.; Overall, Christopher M.

2008-01-01

Broad-spectrum matrix metalloproteinase (MMP) inhibitors (MMPI) were unsuccessful in cancer clinical trials, partly due to side effects resulting from limited knowledge of the full repertoire of MMP substrates, termed the substrate degradome, and hence the in vivo functions of MMPs. To gain further insight into the degradome of MMP-14 (membrane type 1 MMP) an MMPI, prinomastat (drug code AG3340), was used to reduce proteolytic processing and ectodomain shedding in human MDA-MB-231 breast cancer cells transfected with MMP-14. We report a quantitative proteomic evaluation of the targets and effects of the inhibitor in this cell-based system. Proteins in cell-conditioned medium (the secretome) and membrane fractions with levels that were modulated by the MMPI were identified by isotope-coded affinity tag (ICAT) labeling and tandem mass spectrometry. Comparisons of the expression of MMP-14 with that of a vector control resulted in increased MMP-14/vector ICAT ratios for many proteins in conditioned medium, indicating MMP-14-mediated ectodomain shedding. Following MMPI treatment, the MMPI/vehicle ICAT ratio was reversed, suggesting that MMP-14-mediated shedding of these proteins was blocked by the inhibitor. The reduction in shedding or the release of substrates from pericellular sites in the presence of the MMPI was frequently accompanied by the accumulation of the protein in the plasma membrane, as indicated by high MMPI/vehicle ICAT ratios. Considered together, this is a strong predictor of biologically relevant substrates cleaved in the cellular context that led to the identification of many undescribed MMP-14 substrates, 20 of which we validated biochemically, including DJ-1, galectin-1, Hsp90α, pentraxin 3, progranulin, Cyr61, peptidyl-prolyl cis-trans isomerase A, and dickkopf-1. Other proteins with altered levels, such as Kunitz-type protease inhibitor 1 and beta-2-microglobulin, were not substrates in biochemical assays, suggesting an indirect affect of the MMPI, which might be important in drug development as biomarkers or, in preclinical phases, to predict systemic drug actions and adverse side effects. Hence, this approach describes the dynamic pattern of cell membrane ectodomain shedding and its perturbation upon metalloproteinase drug treatment. PMID:18505826

Virus-Mimetic Fusogenic Exosomes for Direct Delivery of Integral Membrane Proteins to Target Cell Membranes.

PubMed

Yang, Yoosoo; Hong, Yeonsun; Nam, Gi-Hoon; Chung, Jin Hwa; Koh, Eunee; Kim, In-San

2017-04-01

An efficient system for direct delivery of integral membrane proteins is successfully developed using a new biocompatible exosome-based platform. Fusogenic exosomes harboring viral fusogen, vascular stomatitis virus (VSV)-G protein, can fuse with and modify plasma membranes in a process called "membrane editing." This can facilitate the transfer of biologically active membrane proteins into the target cell membranes both in vitro and in vivo. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cournia, Zoe; Allen, Toby W.; Andricioaei, Ioan

It is fundamental for the flourishing biological cells that membrane proteins mediate the process. Membrane-embedded transporters move ions and larger solutes across membranes; receptors mediate communication between the cell and its environment and membrane-embedded enzymes catalyze chemical reactions. Understanding these mechanisms of action requires knowledge of how the proteins couple to their fluid, hydrated lipid membrane environment. Here, we present here current studies in computational and experimental membrane protein biophysics, and show how they address outstanding challenges in understanding the complex environmental effects on the structure, function, and dynamics of membrane proteins.

Cluster Formation of Anchored Proteins Induced by Membrane-Mediated Interaction

PubMed Central

Li, Shuangyang; Zhang, Xianren; Wang, Wenchuan

2010-01-01

Abstract Computer simulations were used to study the cluster formation of anchored proteins in a membrane. The rate and extent of clustering was found to be dependent upon the hydrophobic length of the anchored proteins embedded in the membrane. The cluster formation mechanism of anchored proteins in our work was ascribed to the different local perturbations on the upper and lower monolayers of the membrane and the intermonolayer coupling. Simulation results demonstrated that only when the penetration depth of anchored proteins was larger than half the membrane thickness, could the structure of the lower monolayer be significantly deformed. Additionally, studies on the local structures of membranes indicated weak perturbation of bilayer thickness for a shallowly inserted protein, while there was significant perturbation for a more deeply inserted protein. The origin of membrane-mediated protein-protein interaction is therefore due to the local perturbation of the membrane thickness, and the entropy loss—both of which are caused by the conformation restriction on the lipid chains and the enhanced intermonolayer coupling for a deeply inserted protein. Finally, in this study we addressed the difference of cluster formation mechanisms between anchored proteins and transmembrane proteins. PMID:20513399

Improving membrane protein expression and function using genomic edits

DOE PAGES

Jensen, Heather M.; Eng, Thomas; Chubukov, Victor; ...

2017-10-12

Expression of membrane proteins often leads to growth inhibition and perturbs central metabolism and this burden varies with the protein being overexpressed. There are also known strain backgrounds that allow greater expression of membrane proteins but that differ in efficacy across proteins. Here, we hypothesized that for any membrane protein, it may be possible to identify a modified strain background where its expression can be accommodated with less burden. To directly test this hypothesis, we used a bar-coded transposon insertion library in tandem with cell sorting to assess genome-wide impact of gene deletions on membrane protein expression. The expression ofmore » five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST), each fused to GFP, was examined. We identified Escherichia coli mutants that demonstrated increased membrane protein expression relative to that in wild type. For two of the proteins (CyoB and CydB), we conducted functional assays to confirm that the increase in protein expression also led to phenotypic improvement in function. This study represents a systematic approach to broadly identify genetic loci that can be used to improve membrane protein expression, and our method can be used to improve expression of any protein that poses a cellular burden.« less

Improving membrane protein expression and function using genomic edits

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jensen, Heather M.; Eng, Thomas; Chubukov, Victor

Expression of membrane proteins often leads to growth inhibition and perturbs central metabolism and this burden varies with the protein being overexpressed. There are also known strain backgrounds that allow greater expression of membrane proteins but that differ in efficacy across proteins. Here, we hypothesized that for any membrane protein, it may be possible to identify a modified strain background where its expression can be accommodated with less burden. To directly test this hypothesis, we used a bar-coded transposon insertion library in tandem with cell sorting to assess genome-wide impact of gene deletions on membrane protein expression. The expression ofmore » five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST), each fused to GFP, was examined. We identified Escherichia coli mutants that demonstrated increased membrane protein expression relative to that in wild type. For two of the proteins (CyoB and CydB), we conducted functional assays to confirm that the increase in protein expression also led to phenotypic improvement in function. This study represents a systematic approach to broadly identify genetic loci that can be used to improve membrane protein expression, and our method can be used to improve expression of any protein that poses a cellular burden.« less

Mining Missing Membrane Proteins by High-pH Reverse Phase StageTip Fractionation and Multiple Reaction Monitoring Mass Spectrometry

PubMed Central

Kitata, Reta Birhanu; Dimayacyac-Esleta, Baby Rorielyn T.; Choong, Wai-Kok; Tsai, Chia-Feng; Lin, Tai-Du; Tsou, Chih-Chiang; Weng, Shao-Hsing; Chen, Yi-Ju; Yang, Pan-Chyr; Arco, Susan D.; Nesvizhskii, Alexey I.; Sung, Ting-Yi; Chen, Yu-Ju

2016-01-01

Despite significant efforts in the past decade towards complete mapping of the human proteome, 3564 proteins (neXtProt, 09-2014) are still “missing proteins”. Over one-third of these missing proteins are annotated as membrane proteins, owing to their relatively challenging accessibility with standard shotgun proteomics. Using non-small cell lung cancer (NSCLC) as a model study, we aim to mine missing proteins from disease-associated membrane proteome, which may be still largely under-represented. To increase identification coverage, we employed Hp-RP StageTip pre-fractionation of membrane-enriched samples from 11 NSCLC cell lines. Analysis of membrane samples from 20 pairs of tumor and adjacent normal lung tissue were incorporated to include physiologically expressed membrane proteins. Using multiple search engines (X!Tandem, Comet and Mascot) and stringent evaluation of FDR (MAYU and PeptideShaker), we identified 7702 proteins (66% membrane proteins) and 178 missing proteins (74 membrane proteins) with PSM-, peptide-, and protein-level FDR of 1%. Through multiple reaction monitoring (MRM) using synthetic peptides, we provided additional evidences for 8 missing proteins including 7 with transmembrane helix domains (TMH). This study demonstrates that mining missing proteins focused on cancer membrane sub-proteome can greatly contribute to map the whole human proteome. All data were deposited into ProteomeXchange with the identifier PXD002224. PMID:26202522

SCARFACE encodes an ARF-GAP that is required for normal auxin efflux and vein patterning in Arabidopsis.

PubMed

Sieburth, Leslie E; Muday, Gloria K; King, Edward J; Benton, Geoff; Kim, Sun; Metcalf, Kasee E; Meyers, Lindsay; Seamen, Emylie; Van Norman, Jaimie M

2006-06-01

To identify molecular mechanisms controlling vein patterns, we analyzed scarface (sfc) mutants. sfc cotyledon and leaf veins are largely fragmented, unlike the interconnected networks in wild-type plants. SFC encodes an ADP ribosylation factor GTPase activating protein (ARF-GAP), a class with well-established roles in vesicle trafficking regulation. Quadruple mutants of SCF and three homologs (ARF-GAP DOMAIN1, 2, and 4) showed a modestly enhanced vascular phenotype. Genetic interactions between sfc and pinoid and between sfc and gnom suggest a possible function for SFC in trafficking of auxin efflux regulators. Genetic analyses also revealed interaction with cotyledon vascular pattern2, suggesting that lipid-based signals may underlie some SFC ARF-GAP functions. To assess possible roles for SFC in auxin transport, we analyzed sfc roots, which showed exaggerated responses to exogenous auxin and higher auxin transport capacity. To determine whether PIN1 intracellular trafficking was affected, we analyzed PIN1:green fluorescent protein (GFP) dynamics using confocal microscopy in sfc roots. We found normal PIN1:GFP localization at the apical membrane of root cells, but treatment with brefeldin A resulted in PIN1 accumulating in smaller and more numerous compartments than in the wild type. These data suggest that SFC is required for normal intracellular transport of PIN1 from the plasma membrane to the endosome.

Influence of cell shape, inhomogeneities and diffusion barriers in cell polarization models

NASA Astrophysics Data System (ADS)

Giese, Wolfgang; Eigel, Martin; Westerheide, Sebastian; Engwer, Christian; Klipp, Edda

2015-12-01

In silico experiments bear the potential for further understanding of biological transport processes by allowing a systematic modification of any spatial property and providing immediate simulation results. Cell polarization and spatial reorganization of membrane proteins are fundamental for cell division, chemotaxis and morphogenesis. We chose the yeast Saccharomyces cerevisiae as an exemplary model system which entails the shuttling of small Rho GTPases such as Cdc42 and Rho, between an active membrane-bound form and an inactive cytosolic form. We used partial differential equations to describe the membrane-cytosol shuttling of proteins. In this study, a consistent extension of a class of 1D reaction-diffusion systems into higher space dimensions is suggested. The membrane is modeled as a thin layer to allow for lateral diffusion and the cytosol is modeled as an enclosed volume. Two well-known polarization mechanisms were considered. One shows the classical Turing-instability patterns, the other exhibits wave-pinning dynamics. For both models, we investigated how cell shape and diffusion barriers like septin structures or bud scars influence the formation of signaling molecule clusters and subsequent polarization. An extensive set of in silico experiments with different modeling hypotheses illustrated the dependence of cell polarization models on local membrane curvature, cell size and inhomogeneities on the membrane and in the cytosol. In particular, the results of our computer simulations suggested that for both mechanisms, local diffusion barriers on the membrane facilitate Rho GTPase aggregation, while diffusion barriers in the cytosol and cell protrusions limit spontaneous molecule aggregations of active Rho GTPase locally.

Ionic protein-lipid interaction at the plasma membrane: what can the charge do?

PubMed

Li, Lunyi; Shi, Xiaoshan; Guo, Xingdong; Li, Hua; Xu, Chenqi

2014-03-01

Phospholipids are the major components of cell membranes, but they have functional roles beyond forming lipid bilayers. In particular, acidic phospholipids form microdomains in the plasma membrane and can ionically interact with proteins via polybasic sequences, which can have functional consequences for the protein. The list of proteins regulated by ionic protein-lipid interaction has been quickly expanding, and now includes membrane proteins, cytoplasmic soluble proteins, and viral proteins. Here we review how acidic phospholipids in the plasma membrane regulate protein structure and function via ionic interactions, and how Ca(2+) regulates ionic protein-lipid interactions via direct and indirect mechanisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

Prostate cancer targeting motifs: expression of αν β3, neurotensin receptor 1, prostate specific membrane antigen, and prostate stem cell antigen in human prostate cancer cell lines and xenografts.

PubMed

Taylor, Robert M; Severns, Virginia; Brown, David C; Bisoffi, Marco; Sillerud, Laurel O

2012-04-01

Membrane receptors are frequent targets of cancer therapeutic and imaging agents. However, promising in vitro results often do not translate to in vivo clinical applications. To better understand this obstacle, we measured the expression differences in receptor signatures among several human prostate cancer cell lines and xenografts as a function of tumorigenicity. Messenger RNA and protein expression levels for integrin α(ν) β(3), neurotensin receptor 1 (NTSR1), prostate specific membrane antigen (PSMA), and prostate stem cell antigen (PSCA) were measured in LNCaP, C4-2, and PC-3 human prostate cancer cell lines and in murine xenografts using quantitative reverse transcriptase polymerase chain reaction, flow cytometry, and immunohistochemistry. Stable expression patterns were observed for integrin α(ν) and PSMA in all cells and corresponding xenografts. Integrin β(3) mRNA expression was greatly reduced in C4-2 xenografts and greatly elevated in PC-3 xenografts compared with the corresponding cultured cells. NTSR1 mRNA expression was greatly elevated in LNCaP and PC-3 xenografts. PSCA mRNA expression was elevated in C4-2 xenografts when compared with C4-2 cells cultured in vitro. Furthermore, at the protein level, PSCA was re-expressed in all xenografts compared with cells in culture. The regulation of mRNA and protein expression of the cell-surface target proteins α(ν) β(3), NTSR1, PSMA, and PSCA, in prostate cancer cells with different tumorigenic potential, was influenced by factors of the microenvironment, differing between cell cultures and murine xenotransplants. Integrin α(ν) β(3), NTRS1 and PSCA mRNA expression increased with tumorigenic potential, but mRNA expression levels for these proteins do not translate directly to equivalent expression levels of membrane bound protein. Copyright © 2011 Wiley Periodicals, Inc.

Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

PubMed Central

Barros, Marilia; Nanda, Hirsh

2016-01-01

ABSTRACT By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals—electrostatic, hydrophobic, and lipid-specific—to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCE Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane-bounded protein lattice that recruits genomic RNA into the virus and forms the shell of a budding immature viral capsid. In binding studies of HIV-1 Gag MA to model membranes with well-controlled lipid composition, we dissect the multiple interactions of the MA domain with its target membrane. This results in a detailed understanding of the thermodynamic aspects that determine membrane association, preferential lipid recruitment to the viral shell, and those aspects of Gag assembly into the membrane-bound protein lattice that are determined by MA. PMID:26912608

Backbone dynamics of a model membrane protein: assignment of the carbonyl carbon /sup 13/C NMR resonances in detergent-solubilized M13 coat protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Henry, G.D.; Weiner, J.H.; Sykes, B.D.

The major coat protein of the filamentous bacteriophage M13 is a 50-residue amphiphilic polypeptide which is inserted, as an integral membrane-spanning protein, in the inner membrane of the Escherichia coli host during infection. /sup 13/C was incorporated biosynthetically into a total of 23 of the peptide carbonyls using labeled amino acids (alanine, glycine, lysine, phenylalanine, and proline). The structure and dynamics of carbonyl-labeled M13 coat protein were monitored by /sup 13/C nuclear magnetic resonance (NMR) spectroscopy. Assignment of many resonances was achieved by using protease digestion, pH titration, or labeling of the peptide bond with both /sup 13/C and /supmore » 15/N. The carbonyl region of the natural-abundance /sup 13/C NMR spectrum of M13 coat protein in sodium dodecyl sulfate solution shows approximately eight backbone carbonyl resonances with line widths much narrower than the rest. Three of these more mobile residues correspond to assigned peaks (glycine-3, lysine-48, and alanine-49) in the individual amino acid spectra, and another almost certainly arises from glutamic acid-2. A ninth residue, alanine-1, also gives rise to a very narrow carbonyl resonance if the pH is well above or below the pK/sub a/ of the terminal amino group. These data suggest that only about four residues at either end of the protein experience large-amplitude spatial fluctuations; the rest of the molecule is essentially rigid on the time scale of the overall rotational tumbling of the protein-detergent complex. The relative exposure of different regions of detergent-bound protein was monitored by limited digestion with proteinase K. Comparable spectra and digestion patterns were obtained when the protein was solubilized in sodium deoxycholate, suggesting that the coat protein binds both amphiphiles in a similar fashion.« less

Expression of nestin, mesothelin and epithelial membrane antigen (EMA) in developing and adult human meninges and meningiomas.

PubMed

Petricevic, Josko; Forempoher, Gea; Ostojic, Ljerka; Mardesic-Brakus, Snjezana; Andjelinovic, Simun; Vukojevic, Katarina; Saraga-Babic, Mirna

2011-11-01

The spatial and temporal pattern of appearance of nestin, epithelial membrane antigen (EMA) and mesothelin proteins was immunohistochemically determined in the cells of normal developing and adult human meninges and meningiomas. Human meninges developed as two mesenchymal condensations in the head region. The simple squamous epithelium on the surface of leptomeninges developed during mesenchymal to epithelial transformation. Nestin appeared for the first time in week 7, EMA in week 8, while mesothelin appeared in week 22 of development. In the late fetal period and after birth, nestin expression decreased, whereas expression of EMA and mesothelin increased. EMA appeared in all surface epithelial cells and nodules, while mesothelin was found only in some of them. In adult meninges, all three proteins were predominantly localized in the surface epithelium and meningeal nodules. In meningothelial meningiomas (WHO grade I), EMA was detected in all tumor cells except in the endothelial cells, mesothelin characterized nests of tumor cells, while nestin was found predominantly in the walls of blood vessels. The distribution pattern of those proteins in normal meningeal and tumor cells indicates that nestin might characterize immature cells, while EMA and mesothelin appeared in maturing epithelial cells. Neoplastic transformation of these specific cell lineages contributes to the cell population in meningiomas. Copyright © 2010 Elsevier GmbH. All rights reserved.

Noise reduction in the intracellular pom1p gradient by a dynamic clustering mechanism.

PubMed

Saunders, Timothy E; Pan, Kally Z; Angel, Andrew; Guan, Yinghua; Shah, Jagesh V; Howard, Martin; Chang, Fred

2012-03-13

Chemical gradients can generate pattern formation in biological systems. In the fission yeast Schizosaccharomyces pombe, a cortical gradient of pom1p (a DYRK-type protein kinase) functions to position sites of cytokinesis and cell polarity and to control cell length. Here, using quantitative imaging, fluorescence correlation spectroscopy, and mathematical modeling, we study how its gradient distribution is formed. Pom1p gradients exhibit large cell-to-cell variability, as well as dynamic fluctuations in each individual gradient. Our data lead to a two-state model for gradient formation in which pom1p molecules associate with the plasma membrane at cell tips and then diffuse on the membrane while aggregating into and fragmenting from clusters, before disassociating from the membrane. In contrast to a classical one-component gradient, this two-state gradient buffers against cell-to-cell variations in protein concentration. This buffering mechanism, together with time averaging to reduce intrinsic noise, allows the pom1p gradient to specify positional information in a robust manner. Copyright © 2012 Elsevier Inc. All rights reserved.

Dynamic nuclear polarization methods in solids and solutions to explore membrane proteins and membrane systems.

PubMed

Cheng, Chi-Yuan; Han, Songi

2013-01-01

Membrane proteins regulate vital cellular processes, including signaling, ion transport, and vesicular trafficking. Obtaining experimental access to their structures, conformational fluctuations, orientations, locations, and hydration in membrane environments, as well as the lipid membrane properties, is critical to understanding their functions. Dynamic nuclear polarization (DNP) of frozen solids can dramatically boost the sensitivity of current solid-state nuclear magnetic resonance tools to enhance access to membrane protein structures in native membrane environments. Overhauser DNP in the solution state can map out the local and site-specific hydration dynamics landscape of membrane proteins and lipid membranes, critically complementing the structural and dynamics information obtained by electron paramagnetic resonance spectroscopy. Here, we provide an overview of how DNP methods in solids and solutions can significantly increase our understanding of membrane protein structures, dynamics, functions, and hydration in complex biological membrane environments.

Biophysics of α-Synuclein Membrane Interactions

PubMed Central

Pfefferkorn, Candace M.; Jiang, Zhiping; Lee, Jennifer C.

2011-01-01

Membrane proteins participate in nearly all cellular processes; however, because of experimental limitations, their characterization lags far behind that of soluble proteins. Peripheral membrane proteins are particularly challenging to study because of their inherent propensity to adopt multiple and/or transient conformations in solution and upon membrane association. In this review, we summarize useful biophysical techniques for the study of peripheral membrane proteins and their application in the characterization of the membrane interactions of the natively unfolded and Parkinson’s disease (PD) related protein, α-synuclein (α-syn). We give particular focus to studies that have led to the current understanding of membrane-bound α-syn structure and the elucidation of specific membrane properties that affect α-syn-membrane binding. Finally, we discuss biophysical evidence supporting a key role for membranes and α-syn in PD pathogenesis. PMID:21819966

The Pleiotropic Antibacterial Mechanisms of Ursolic Acid against Methicillin-Resistant Staphylococcus aureus (MRSA).

PubMed

Wang, Chao-Min; Jhan, Yun-Lian; Tsai, Shang-Jie; Chou, Chang-Hung

2016-07-07

(1) BACKGROUND: Several triterpenoids were found to act synergistically with classes of antibiotic, indicating that plant-derived chemicals have potential to be used as therapeutics to enhance the activity of antibiotics against multidrug-resistant pathogens. However, the mode of action of triterpenoids against bacterial pathogens remains unclear. The objective of this study is to evaluate the interaction between ursolic acid against methicillin-resistant Staphylococcus aureus (MRSA); (2) METHODS: The ability of ursolic acid to damage mammalian and bacterial membranes was examined. The proteomic response of methicillin-resistant S. aureus in ursolic acid treatment was investigated using two-dimensional (2D) proteomic analysis; (3) RESULTS: Ursolic acid caused the loss of staphylococcal membrane integrity without hemolytic activity. The comparison of the protein pattern of ursolic acid-treated and normal MRSA cells revealed that ursolic acid affected a variety of proteins involved in the translation process with translational accuracy, ribonuclease and chaperon subunits, glycolysis and oxidative responses; (4) CONCLUSION: The mode of action of ursolic acid appears to be the influence on the integrity of the bacterial membrane initially, followed by inhibition of protein synthesis and the metabolic pathway. These findings reflect that the pleiotropic effects of ursolic acid against MRSA make it a promising antibacterial agent in pharmaceutical research.

The G-protein coupled estrogen receptor, GPER: The inside and inside-out story.

PubMed

Gaudet, H M; Cheng, S B; Christensen, E M; Filardo, E J

2015-12-15

GPER possesses structural and functional characteristics shared by members of the G-protein-coupled receptor (GPCR) superfamily, the largest class of plasma membrane receptors. This newly appreciated estrogen receptor is localized predominately within intracellular membranes in most, but not all, cell types and its surface expression is modulated by steroid hormones and during tissue injury. An intracellular staining pattern is not unique among GPCRs, which employ a diverse array of molecular mechanisms that restrict cell surface expression and effectively regulating receptor binding and activation. The finding that GPER displays an intracellular predisposition has created some confusion as the estrogen-inducible transcription factors, ERα and ERβ, also reside intracellularly, and has led to complex suggestions of receptor interaction. GPER undergoes constitutive retrograde trafficking from the plasma membrane to the endoplasmic reticulum and recent studies indicate its interaction with PDZ binding proteins that sort transmembrane receptors to synaptosomes and endosomes. Genetic targeting and selective ligand approaches as well as cell models that express GPER in the absence of ERs clearly supports GPER as a bonafide "stand alone" receptor. Here, the molecular details that regulate GPER action, its cell biological activities and its implicated roles in physiological and pathological processes are reviewed. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Campylobacter jejuni Colonization in Wild Birds: Results from an Infection Experiment

PubMed Central

Waldenström, Jonas; Axelsson-Olsson, Diana; Olsen, Björn; Hasselquist, Dennis; Griekspoor, Petra; Jansson, Lena; Teneberg, Susann; Svensson, Lovisa; Ellström, Patrik

2010-01-01

Campylobacter jejuni is a common cause of bacterial gastroenteritis in most parts of the world. The bacterium has a broad host range and has been isolated from many animals and environments. To investigate shedding patterns and putative effects on an avian host, we developed a colonization model in which a wild bird species, the European Robin Erithacus rubecula, was inoculated orally with C. jejuni from either a human patient or from another wild bird species, the Song Thrush Turdus philomelos. These two isolates were genetically distinct from each other and provoked very different host responses. The Song Thrush isolate colonized all challenged birds and colonization lasted 6.8 days on average. Birds infected with this isolate also showed a transient but significant decrease in body mass. The human isolate did not colonize the birds and could be detected only in the feces of the birds shortly after inoculation. European Robins infected with the wild bird isolate generated a specific antibody response to C. jejuni membrane proteins from the avian isolate, which also was cross-reactive to membrane proteins of the human isolate. In contrast, European Robins infected with the human isolate did not mount a significant response to bacterial membrane proteins from either of the two isolates. The difference in colonization ability could indicate host adaptations. PMID:20140204

Disturbed vesicular trafficking of membrane proteins in prion disease.

PubMed

Uchiyama, Keiji; Miyata, Hironori; Sakaguchi, Suehiro

2013-01-01

The pathogenic mechanism of prion diseases remains unknown. We recently reported that prion infection disturbs post-Golgi trafficking of certain types of membrane proteins to the cell surface, resulting in reduced surface expression of membrane proteins and abrogating the signal from the proteins. The surface expression of the membrane proteins was reduced in the brains of mice inoculated with prions, well before abnormal symptoms became evident. Prions or pathogenic prion proteins were mainly detected in endosomal compartments, being particularly abundant in recycling endosomes. Some newly synthesized membrane proteins are delivered to the surface from the Golgi apparatus through recycling endosomes, and some endocytosed membrane proteins are delivered back to the surface through recycling endosomes. These results suggest that prions might cause neuronal dysfunctions and cell loss by disturbing post-Golgi trafficking of membrane proteins via accumulation in recycling endosomes. Interestingly, it was recently shown that delivery of a calcium channel protein to the cell surface was impaired and its function was abrogated in a mouse model of hereditary prion disease. Taken together, these results suggest that impaired delivery of membrane proteins to the cell surface is a common pathogenic event in acquired and hereditary prion diseases.

Evaluation of detergents for the soluble expression of alpha-helical and beta-barrel-type integral membrane proteins by a preparative scale individual cell-free expression system.

PubMed

Klammt, Christian; Schwarz, Daniel; Fendler, Klaus; Haase, Winfried; Dötsch, Volker; Bernhard, Frank

2005-12-01

Cell-free expression has become a highly promising tool for the fast and efficient production of integral membrane proteins. The proteins can be produced as precipitates that solubilize in mild detergents usually without any prior denaturation steps. Alternatively, membrane proteins can be synthesized in a soluble form by adding detergents to the cell-free system. However, the effects of a representative variety of detergents on the production, solubility and activity of a wider range of membrane proteins upon cell-free expression are currently unknown. We therefore analyzed the cell-free expression of three structurally very different membrane proteins, namely the bacterial alpha-helical multidrug transporter, EmrE, the beta-barrel nucleoside transporter, Tsx, and the porcine vasopressin receptor of the eukaryotic superfamily of G-protein coupled receptors. All three membrane proteins could be produced in amounts of several mg per one ml of reaction mixture. In general, the detergent 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] was found to be most effective for the resolubilization of membrane protein precipitates, while long chain polyoxyethylene-alkyl-ethers proved to be most suitable for the soluble expression of all three types of membrane proteins. The yield of soluble expressed membrane protein remained relatively stable above a certain threshold concentration of the detergents. We report, for the first time, the high-level cell-free expression of a beta-barrel type membrane protein in a functional form. Structural and functional variations of the analyzed membrane proteins are evident that correspond with the mode of expression and that depend on the supplied detergent.

The Fluid-Mosaic Model of Membrane Structure: still relevant to understanding the structure, function and dynamics of biological membranes after more than 40 years.

PubMed

Nicolson, Garth L

2014-06-01

In 1972 the Fluid-Mosaic Membrane Model of membrane structure was proposed based on thermodynamic principals of organization of membrane lipids and proteins and available evidence of asymmetry and lateral mobility within the membrane matrix [S. J. Singer and G. L. Nicolson, Science 175 (1972) 720-731]. After over 40years, this basic model of the cell membrane remains relevant for describing the basic nano-structures of a variety of intracellular and cellular membranes of plant and animal cells and lower forms of life. In the intervening years, however, new information has documented the importance and roles of specialized membrane domains, such as lipid rafts and protein/glycoprotein complexes, in describing the macrostructure, dynamics and functions of cellular membranes as well as the roles of membrane-associated cytoskeletal fences and extracellular matrix structures in limiting the lateral diffusion and range of motion of membrane components. These newer data build on the foundation of the original model and add new layers of complexity and hierarchy, but the concepts described in the original model are still applicable today. In updated versions of the model more emphasis has been placed on the mosaic nature of the macrostructure of cellular membranes where many protein and lipid components are limited in their rotational and lateral motilities in the membrane plane, especially in their natural states where lipid-lipid, protein-protein and lipid-protein interactions as well as cell-matrix, cell-cell and intracellular membrane-associated protein and cytoskeletal interactions are important in restraining the lateral motility and range of motion of particular membrane components. The formation of specialized membrane domains and the presence of tightly packed integral membrane protein complexes due to membrane-associated fences, fenceposts and other structures are considered very important in describing membrane dynamics and architecture. These structures along with membrane-associated cytoskeletal and extracellular structures maintain the long-range, non-random mosaic macro-organization of membranes, while smaller membrane nano- and submicro-sized domains, such as lipid rafts and protein complexes, are important in maintaining specialized membrane structures that are in cooperative dynamic flux in a crowded membrane plane. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy. © 2013.

Structural elucidation of the interaction between neurodegenerative disease-related tau protein with model lipid membranes

NASA Astrophysics Data System (ADS)

Jones, Emmalee M.

A protein's sequence of amino acids determines how it folds. That folded structure is linked to protein function, and misfolding to dysfunction. Protein misfolding and aggregation into beta-sheet rich fibrillar aggregates is connected with over 20 neurodegenerative diseases, including Alzheimer's disease (AD). AD is characterized in part by misfolding, aggregation and deposition of the microtubule associated tau protein into neurofibrillary tangles (NFTs). However, two questions remain: What is tau's fibrillization mechanism, and what is tau's cytotoxicity mechanism? Tau is prone to heterogeneous interactions, including with lipid membranes. Lipids have been found in NFTs, anionic lipid vesicles induced aggregation of the microtubule binding domain of tau, and other protein aggregates induced ion permeability in cells. This evidence prompted our investigation of tau's interaction with model lipid membranes to elucidate the structural perturbations those interactions induced in tau protein and in the membrane. We show that although tau is highly charged and soluble, it is highly surface active and preferentially interacts with anionic membranes. To resolve molecular-scale structural details of tau and model membranes, we utilized X-ray and neutron scattering techniques. X-ray reflectivity indicated tau aggregated at air/water and anionic lipid membrane interfaces and penetrated into membranes. More significantly, membrane interfaces induced tau protein to partially adopt a more compact conformation with density similar to folded protein and ordered structure characteristic of beta-sheet formation. This suggests possible membrane-based mechanisms of tau aggregation. Membrane morphological changes were seen using fluorescence microscopy, and X-ray scattering techniques showed tau completely disrupts anionic membranes, suggesting an aggregate-based cytotoxicity mechanism. Further investigation of protein constructs and a "hyperphosphorylation" disease mimic helped clarify the role of the microtubule binding domain in anionic lipid affinity and demonstrated even "hyperphosphorylation" did not prevent interaction with anionic membranes. Additional studies investigated more complex membrane models to increase physiological relevance. These insights revealed structural changes in tau protein and lipid membranes after interaction. We observed tau's affinity for interfaces, and aggregation and compaction once tau partitions to interfaces. We observed the beginnings of beta-sheet formation in tau at anionic lipid membranes. We also examined disruption to the membrane on a molecular scale.

High-throughput Isolation and Characterization of Untagged Membrane Protein Complexes: Outer Membrane Complexes of Desulfovibrio vulgaris

PubMed Central

2012-01-01

Cell membranes represent the “front line” of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a “tagless” process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein–protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms. PMID:23098413

Perturbations of Native Membrane Protein Structure in Alkyl Phosphocholine Detergents: A Critical Assessment of NMR and Biophysical Studies

PubMed Central

2018-01-01

Membrane proteins perform a host of vital cellular functions. Deciphering the molecular mechanisms whereby they fulfill these functions requires detailed biophysical and structural investigations. Detergents have proven pivotal to extract the protein from its native surroundings. Yet, they provide a milieu that departs significantly from that of the biological membrane, to the extent that the structure, the dynamics, and the interactions of membrane proteins in detergents may considerably vary, as compared to the native environment. Understanding the impact of detergents on membrane proteins is, therefore, crucial to assess the biological relevance of results obtained in detergents. Here, we review the strengths and weaknesses of alkyl phosphocholines (or foscholines), the most widely used detergent in solution-NMR studies of membrane proteins. While this class of detergents is often successful for membrane protein solubilization, a growing list of examples points to destabilizing and denaturing properties, in particular for α-helical membrane proteins. Our comprehensive analysis stresses the importance of stringent controls when working with this class of detergents and when analyzing the structure and dynamics of membrane proteins in alkyl phosphocholine detergents. PMID:29488756

Protein-centric N-glycoproteomics analysis of membrane and plasma membrane proteins.

PubMed

Sun, Bingyun; Hood, Leroy

2014-06-06

The advent of proteomics technology has transformed our understanding of biological membranes. The challenges for studying membrane proteins have inspired the development of many analytical and bioanalytical tools, and the techniques of glycoproteomics have emerged as an effective means to enrich and characterize membrane and plasma-membrane proteomes. This Review summarizes the development of various glycoproteomics techniques to overcome the hurdles formed by the unique structures and behaviors of membrane proteins with a focus on N-glycoproteomics. Example contributions of N-glycoproteomics to the understanding of membrane biology are provided, and the areas that require future technical breakthroughs are discussed.

Distance Measurement on an Endogenous Membrane Transporter in E. coli Cells and Native Membranes Using EPR Spectroscopy.

PubMed

Joseph, Benesh; Sikora, Arthur; Bordignon, Enrica; Jeschke, Gunnar; Cafiso, David S; Prisner, Thomas F

2015-05-18

Membrane proteins may be influenced by the environment, and they may be unstable in detergents or fail to crystallize. As a result, approaches to characterize structures in a native environment are highly desirable. Here, we report a novel general strategy for precise distance measurements on outer membrane proteins in whole Escherichia coli cells and isolated outer membranes. The cobalamin transporter BtuB was overexpressed and spin-labeled in whole cells and outer membranes and interspin distances were measured to a spin-labeled cobalamin using pulse EPR spectroscopy. A comparative analysis of the data reveals a similar interspin distance between whole cells, outer membranes, and synthetic vesicles. This approach provides an elegant way to study conformational changes or protein-protein/ligand interactions at surface-exposed sites of membrane protein complexes in whole cells and native membranes, and provides a method to validate outer membrane protein structures in their native environment. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Membrane shape modulates transmembrane protein distribution.

PubMed

Aimon, Sophie; Callan-Jones, Andrew; Berthaud, Alice; Pinot, Mathieu; Toombes, Gilman E S; Bassereau, Patricia

2014-01-27

Although membrane shape varies greatly throughout the cell, the contribution of membrane curvature to transmembrane protein targeting is unknown because of the numerous sorting mechanisms that take place concurrently in cells. To isolate the effect of membrane shape, we used cell-sized giant unilamellar vesicles (GUVs) containing either the potassium channel KvAP or the water channel AQP0 to form membrane nanotubes with controlled radii. Whereas the AQP0 concentrations in flat and curved membranes were indistinguishable, KvAP was enriched in the tubes, with greater enrichment in more highly curved membranes. Fluorescence recovery after photobleaching measurements showed that both proteins could freely diffuse through the neck between the tube and GUV, and the effect of each protein on membrane shape and stiffness was characterized using a thermodynamic sorting model. This study establishes the importance of membrane shape for targeting transmembrane proteins and provides a method for determining the effective shape and flexibility of membrane proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

Interactions between Membranes and "Metaphilic" Polypeptide Architectures with Diverse Side-Chain Populations.

PubMed

Lee, Michelle W; Han, Ming; Bossa, Guilherme Volpe; Snell, Carly; Song, Ziyuan; Tang, Haoyu; Yin, Lichen; Cheng, Jianjun; May, Sylvio; Luijten, Erik; Wong, Gerard C L

2017-03-28

At physiological conditions, most proteins or peptides can fold into relatively stable structures that present on their molecular surfaces specific chemical patterns partially smeared out by thermal fluctuations. These nanoscopically defined patterns of charge, hydrogen bonding, and/or hydrophobicity, along with their elasticity and shape stability (folded proteins have Young's moduli of ∼1 × 10 8 Pa), largely determine and limit the interactions of these molecules, such as molecular recognition and allosteric regulation. In this work, we show that the membrane-permeating activity of antimicrobial peptides (AMPs) and cell-penetrating peptides (CPPs) can be significantly enhanced using prototypical peptides with "molten" surfaces: metaphilic peptides with quasi-liquid surfaces and adaptable shapes. These metaphilic peptides have a bottlebrush-like architecture consisting of a rigid helical core decorated with mobile side chains that are terminated by cationic or hydrophobic groups. Computer simulations show that these flexible side chains can undergo significant rearrangement in response to different environments, giving rise to adaptable surface chemistry of the peptide. This quality makes it possible to control their hydrophobicity over a broad range while maintaining water solubility, unlike many AMPs and CPPs. Thus, we are able to show how the activity of these peptides is amplified by hydrophobicity and cationic charge, and rationalize these results using a quantitative mean-field theory. Computer simulations show that the shape-changing properties of the peptides and the resultant adaptive presentation of chemistry play a key enabling role in their interactions with membranes.

The cell-based L-glutathione protection assays to study endocytosis and recycling of plasma membrane proteins.

PubMed

Cihil, Kristine M; Swiatecka-Urban, Agnieszka

2013-12-13

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane Stored Curvature Elastic Stress Modulates Recruitment of Maintenance Proteins PspA and Vipp1.

PubMed

McDonald, Christopher; Jovanovic, Goran; Ces, Oscar; Buck, Martin

2015-09-01

Phage shock protein A (PspA), which is responsible for maintaining inner membrane integrity under stress in enterobacteria, and vesicle-inducting protein in plastids 1 (Vipp1), which functions for membrane maintenance and thylakoid biogenesis in cyanobacteria and plants, are similar peripheral membrane-binding proteins. Their homologous N-terminal amphipathic helices are required for membrane binding; however, the membrane features recognized and required for expressing their functionalities have remained largely uncharacterized. Rigorously controlled, in vitro methodologies with lipid vesicles and purified proteins were used in this study and provided the first biochemical and biophysical characterizations of membrane binding by PspA and Vipp1. Both proteins are found to sense stored curvature elastic (SCE) stress and anionic lipids within the membrane. PspA has an enhanced sensitivity for SCE stress and a higher affinity for the membrane than Vipp1. These variations in binding may be crucial for some of the proteins' differing roles in vivo. Assays probing the transcriptional regulatory function of PspA in the presence of vesicles showed that a relief of transcription inhibition occurs in an SCE stress-specific manner. This in vitro recapitulation of membrane stress-dependent transcription control suggests that the Psp response may be mounted in vivo when a cell's inner membrane experiences increased SCE stress. All cell types maintain the integrity of their membrane systems. One widely distributed membrane stress response system in bacteria is the phage shock protein (Psp) system. The central component, peripheral membrane protein PspA, which mitigates inner membrane stress in bacteria, has a counterpart, Vipp1, which functions for membrane maintenance and thylakoid biogenesis in plants and photosynthetic bacteria. Membrane association of both these proteins is accepted as playing a pivotal role in their functions. Here we show that direct membrane binding by PspA and Vipp1 is driven by two physio-chemical signals, one of which is membrane stress specific. Our work points to alleviation of membrane stored curvature elastic stress by amphipathic helix insertions as an attractive mechanism for membrane maintenance by PspA and Vipp1. Furthermore, the identification of a physical, stress-related membrane signal suggests a unilateral mechanism that promotes both binding of PspA and induction of the Psp response. Copyright © 2015 McDonald et al.

Isomeric Detergent Comparison for Membrane Protein Stability: Importance of Inter-Alkyl-Chain Distance and Alkyl Chain Length

PubMed Central

Cho, Kyung Ho; Hariharan, Parameswaran; Mortensen, Jonas S.; Du, Yang; Nielsen, Anne K.; Byrne, Bernadette; Kobilka, Brian K.; Loland, Claus J.; Guan, Lan

2017-01-01

Membrane proteins encapsulated by detergent micelles are widely used for structural study. Because of their amphipathic property, detergents have the ability to maintain protein solubility and stability in an aqueous medium. However, conventional detergents have serious limitations in their scope and utility, particularly for eukaryotic membrane proteins and membrane protein complexes. Thus, a number of new agents have been devised; some have made significant contributions to membrane protein structural studies. However, few detergent design principles are available. In this study, we prepared meta and ortho isomers of the previously reported para-substituted xylene-linked maltoside amphiphiles (XMAs), along with alkyl chain-length variation. The isomeric XMAs were assessed with three membrane proteins, and the meta isomer with a C12 alkyl chain was most effective at maintaining solubility/stability of the membrane proteins. We propose that interplay between the hydrophile–lipophile balance (HLB) and alkyl chain length is of central importance for high detergent efficacy. In addition, differences in inter-alkyl-chain distance between the isomers influence the ability of the detergents to stabilise membrane proteins. PMID:27981750

Antigenic Protein In Microgravity-Grown Human Mixed Mullerian Tumor (LN1) Cells Preserved In RNA Stabilizing Agent

NASA Technical Reports Server (NTRS)

Hammond, Dianne K.; Becker, Jeanne; Elliott, T. F.; Holubec, K.; Baker, T. L.; Love, J. E.

2004-01-01

Cells treated with RNAlater(TradeMark) have previously been shown to contain antigenic proteins that can be visualized using Western blot analysis. These proteins seem to be stable for several months when stored in RNA stabilizer at 4 C. Antigenic protein can be recovered from cells that have been processed using an Ambion RNAqueous(Registered TradeMark) kit to remove RNA. In this set of experiments, human mixed Mullerian tumor (LNI) cells grown on the International Space Station during Expedition 3 were examined for antigenic stability after removal of RNA. The cells were stored for three months in RNAlater(TradeMark) and RNA was extracted. The RNA filtrate containing the protein was precipitated, washed, and suspended in buffer containing sodium dodecyl sulfate (SDS). Samples containing equal concentrations of protein were loaded onto SDS-polyacrylamide gels. Proteins were separated by electrophoresis and transferred by Western blot to polyvinylidene fluoride (PVDF) membrane. The Western blots were stained with an enhanced chemiluminescent ECL(Registered Trademark) Plus detection kit (Amersham) and scanned using a Storm 840 gel image analyzer (Amersham, Molecular Dynamics). ImageQuant(Registered TradeMark) software was used to quantify the densities of the protein bands. The ground control and flight LN1 cell samples showed a similar staining pattern over time with antibodies to vimentin, glyceraldehyde-3-phosphate dehydrogenase, and epithelial membrane antigens.

Antigenic Protein In Microgravity-Grown Human Mixed Mullerian Tumor (LN1) Cells Preserved In RNA Stabilizing Agent

NASA Technical Reports Server (NTRS)

Hammond, Dianne K.; Becker, Jeanne; Holubec, K.; Baker, T. L.; Love, J. E.

2004-01-01

Cells treated with RNAlater(TradeMark) have previously been shown to contain antigenic proteins that can be visualized using Western blot analysis. These proteins seem to be stable for several months when stored in RNA stabilizer at 4 C. Antigenic protein can be recovered from cells that have been processed using an Ambion RNAqueous(Registered TradeMark) kit to remove RNA. In this set of experiments, human mixed Mullerian tumor (LN1) cells grown on the International Space Station during Expedition 3 were examined for antigenic stability after removal of RNA. The cells were stored for three months in RNAlater(TradeMark) and RNA was extracted. The RNA filtrate Containing the protein was precipitated, washed, and suspended in buffer containing sodium dodecyl sulfate (SDS). Samples containing equal concentrations of protein were loaded onto SDS-polyacrylamide gels. Proteins were separated by electrophoresis and transferred by Western blot to polyvinylidene fluoride (PVDF) membrane. The Western blots were stained with an enhanced chemiluminescent ECL(Registered TradeMark)Plus detection kit (Amersham) and scanned using a Storm 840 gel image analyzer (Amersham, Molecular Dynamics). ImageQuant(Registered TradeMark)a software was used to quantify the densities of the protein bands. The ground control and flight LN1 cell samples showed a similar staining pattern over time with antibodies to vimentin, glyceraldehyde-3-phosphate dehydrogenase, and epithelial membrane antigens.

Application of Fragment Based Drug Discovery to Membrane Proteins: Biophysical Identification of Ligands of the Integral Membrane Enzyme DsbB

PubMed Central

Früh, Virginie; Zhou, Yunpeng; Chen, Dan; Loch, Caroline; Eiso, AB; Grinkova, Yelena N.; Verheij, Herman; Sligar, Stephen G; Bushweller, John H.; Siegal, Gregg

2014-01-01

Summary Membrane proteins are important pharmaceutical targets, but they pose significant challenges for fragment based drug discovery approaches. Here we present the first successful use of biophysical methods to screen for fragment ligands to an integral membrane protein. The E. coli inner membrane protein DsbB was solubilized in detergent micelles and lipid bilayer nanodiscs. The solubilized protein was immobilized with retention of functionality and used to screen 1,071 drug fragments for binding using Target Immobilized NMR Screening. Biochemical and biophysical validation of the 8 most potent hits revealed an IC50 range of 7 to 200 μM. The ability to insert a broad array of membrane proteins into nanodiscs, combined with the efficiency of TINS, demonstrates the feasibility of finding fragments targeting membrane proteins. PMID:20797617

Life at the border: Adaptation of proteins to anisotropic membrane environment

PubMed Central

Pogozheva, Irina D; Mosberg, Henry I; Lomize, Andrei L

2014-01-01

This review discusses main features of transmembrane (TM) proteins which distinguish them from water-soluble proteins and allow their adaptation to the anisotropic membrane environment. We overview the structural limitations on membrane protein architecture, spatial arrangement of proteins in membranes and their intrinsic hydrophobic thickness, co-translational and post-translational folding and insertion into lipid bilayers, topogenesis, high propensity to form oligomers, and large-scale conformational transitions during membrane insertion and transport function. Special attention is paid to the polarity of TM protein surfaces described by profiles of dipolarity/polarizability and hydrogen-bonding capacity parameters that match polarity of the lipid environment. Analysis of distributions of Trp resides on surfaces of TM proteins from different biological membranes indicates that interfacial membrane regions with preferential accumulation of Trp indole rings correspond to the outer part of the lipid acyl chain region—between double bonds and carbonyl groups of lipids. These “midpolar” regions are not always symmetric in proteins from natural membranes. We also examined the hydrophobic effect that drives insertion of proteins into lipid bilayer and different free energy contributions to TM protein stability, including attractive van der Waals forces and hydrogen bonds, side-chain conformational entropy, the hydrophobic mismatch, membrane deformations, and specific protein–lipid binding. PMID:24947665

Overcoming bottlenecks in the membrane protein structural biology pipeline.

PubMed

Hardy, David; Bill, Roslyn M; Jawhari, Anass; Rothnie, Alice J

2016-06-15

Membrane proteins account for a third of the eukaryotic proteome, but are greatly under-represented in the Protein Data Bank. Unfortunately, recent technological advances in X-ray crystallography and EM cannot account for the poor solubility and stability of membrane protein samples. A limitation of conventional detergent-based methods is that detergent molecules destabilize membrane proteins, leading to their aggregation. The use of orthologues, mutants and fusion tags has helped improve protein stability, but at the expense of not working with the sequence of interest. Novel detergents such as glucose neopentyl glycol (GNG), maltose neopentyl glycol (MNG) and calixarene-based detergents can improve protein stability without compromising their solubilizing properties. Styrene maleic acid lipid particles (SMALPs) focus on retaining the native lipid bilayer of a membrane protein during purification and biophysical analysis. Overcoming bottlenecks in the membrane protein structural biology pipeline, primarily by maintaining protein stability, will facilitate the elucidation of many more membrane protein structures in the near future. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

A moving view: subcellular trafficking processes in pattern recognition receptor-triggered plant immunity.

PubMed

Ben Khaled, Sara; Postma, Jelle; Robatzek, Silke

2015-01-01

A significant challenge for plants is to induce localized defense responses at sites of pathogen attack. Therefore, host subcellular trafficking processes enable accumulation and exchange of defense compounds, which contributes to the plant on-site defenses in response to pathogen perception. This review summarizes our current understanding of the transport processes that facilitate immunity, the significance of which is highlighted by pathogens reprogramming membrane trafficking through host cell translocated effectors. Prominent immune-related cargos of plant trafficking pathways are the pattern recognition receptors (PRRs), which must be present at the plasma membrane to sense microbes in the apoplast. We focus on the dynamic localization of the FLS2 receptor and discuss the pathways that regulate receptor transport within the cell and their link to FLS2-mediated immunity. One emerging theme is that ligand-induced late endocytic trafficking is conserved across different PRR protein families as well as across different plant species.

Membrane remodeling by amyloidogenic and non-amyloidogenic proteins studied by EPR

NASA Astrophysics Data System (ADS)

Varkey, Jobin; Langen, Ralf

2017-07-01

The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single protein can generate different type of membrane curvatures using specific conformations for specific membrane structures and how EPR is a versatile tool well-suited to analyze subtle alterations in structures under such modifying conditions which otherwise would have been difficult using other biophysical tools.

Improving membrane protein expression by optimizing integration efficiency

PubMed Central

2017-01-01

The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were 4-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effects of double mutations on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies. PMID:28918393

Mutants in three novel complementation groups inhibit membrane protein insertion into and soluble protein translocation across the endoplasmic reticulum membrane of Saccharomyces cerevisiae

PubMed Central

1992-01-01

We have isolated mutants that inhibit membrane protein insertion into the ER membrane of Saccharomyces cerevisiae. The mutants were contained in three complementation groups, which we have named SEC70, SEC71, and SEC72. The mutants also inhibited the translocation of soluble proteins into the lumen of the ER, indicating that they pleiotropically affect protein transport across and insertion into the ER membrane. Surprisingly, the mutants inhibited the translocation and insertion of different proteins to drastically different degrees. We have also shown that mutations in SEC61 and SEC63, which were previously isolated as mutants inhibiting the translocation of soluble proteins, also affect the insertion of membrane proteins into the ER. Taken together our data indicate that the process of protein translocation across the ER membrane involves a much larger number of gene products than previously appreciated. Moreover, different translocation substrates appear to have different requirements for components of the cellular targeting and translocation apparatus. PMID:1730771

Organization and Dynamics of Receptor Proteins in a Plasma Membrane.

PubMed

Koldsø, Heidi; Sansom, Mark S P

2015-11-25

The interactions of membrane proteins are influenced by their lipid environment, with key lipid species able to regulate membrane protein function. Advances in high-resolution microscopy can reveal the organization and dynamics of proteins and lipids within living cells at resolutions <200 nm. Parallel advances in molecular simulations provide near-atomic-resolution models of the dynamics of the organization of membranes of in vivo-like complexity. We explore the dynamics of proteins and lipids in crowded and complex plasma membrane models, thereby closing the gap in length and complexity between computations and experiments. Our simulations provide insights into the mutual interplay between lipids and proteins in determining mesoscale (20-100 nm) fluctuations of the bilayer, and in enabling oligomerization and clustering of membrane proteins.

A Finite Element Solution of Lateral Periodic Poisson–Boltzmann Model for Membrane Channel Proteins

PubMed Central

Xu, Jingjie; Lu, Benzhuo

2018-01-01

Membrane channel proteins control the diffusion of ions across biological membranes. They are closely related to the processes of various organizational mechanisms, such as: cardiac impulse, muscle contraction and hormone secretion. Introducing a membrane region into implicit solvation models extends the ability of the Poisson–Boltzmann (PB) equation to handle membrane proteins. The use of lateral periodic boundary conditions can properly simulate the discrete distribution of membrane proteins on the membrane plane and avoid boundary effects, which are caused by the finite box size in the traditional PB calculations. In this work, we: (1) develop a first finite element solver (FEPB) to solve the PB equation with a two-dimensional periodicity for membrane channel proteins, with different numerical treatments of the singular charges distributions in the channel protein; (2) add the membrane as a dielectric slab in the PB model, and use an improved mesh construction method to automatically identify the membrane channel/pore region even with a tilt angle relative to the z-axis; and (3) add a non-polar solvation energy term to complete the estimation of the total solvation energy of a membrane protein. A mesh resolution of about 0.25 Å (cubic grid space)/0.36 Å (tetrahedron edge length) is found to be most accurate in linear finite element calculation of the PB solvation energy. Computational studies are performed on a few exemplary molecules. The results indicate that all factors, the membrane thickness, the length of periodic box, membrane dielectric constant, pore region dielectric constant, and ionic strength, have individually considerable influence on the solvation energy of a channel protein. This demonstrates the necessity to treat all of those effects in the PB model for membrane protein simulations. PMID:29495644

A Finite Element Solution of Lateral Periodic Poisson-Boltzmann Model for Membrane Channel Proteins.

PubMed

Ji, Nan; Liu, Tiantian; Xu, Jingjie; Shen, Longzhu Q; Lu, Benzhuo

2018-02-28

Membrane channel proteins control the diffusion of ions across biological membranes. They are closely related to the processes of various organizational mechanisms, such as: cardiac impulse, muscle contraction and hormone secretion. Introducing a membrane region into implicit solvation models extends the ability of the Poisson-Boltzmann (PB) equation to handle membrane proteins. The use of lateral periodic boundary conditions can properly simulate the discrete distribution of membrane proteins on the membrane plane and avoid boundary effects, which are caused by the finite box size in the traditional PB calculations. In this work, we: (1) develop a first finite element solver (FEPB) to solve the PB equation with a two-dimensional periodicity for membrane channel proteins, with different numerical treatments of the singular charges distributions in the channel protein; (2) add the membrane as a dielectric slab in the PB model, and use an improved mesh construction method to automatically identify the membrane channel/pore region even with a tilt angle relative to the z -axis; and (3) add a non-polar solvation energy term to complete the estimation of the total solvation energy of a membrane protein. A mesh resolution of about 0.25 Å (cubic grid space)/0.36 Å (tetrahedron edge length) is found to be most accurate in linear finite element calculation of the PB solvation energy. Computational studies are performed on a few exemplary molecules. The results indicate that all factors, the membrane thickness, the length of periodic box, membrane dielectric constant, pore region dielectric constant, and ionic strength, have individually considerable influence on the solvation energy of a channel protein. This demonstrates the necessity to treat all of those effects in the PB model for membrane protein simulations.

Cell shape can mediate the spatial organization of the bacterial cytoskeleton

NASA Astrophysics Data System (ADS)

Wang, Siyuan; Wingreen, Ned

2013-03-01

The bacterial cytoskeleton guides the synthesis of cell wall and thus regulates cell shape. Since spatial patterning of the bacterial cytoskeleton is critical to the proper control of cell shape, it is important to ask how the cytoskeleton spatially self-organizes in the first place. In this work, we develop a quantitative model to account for the various spatial patterns adopted by bacterial cytoskeletal proteins, especially the orientation and length of cytoskeletal filaments such as FtsZ and MreB in rod-shaped cells. We show that the combined mechanical energy of membrane bending, membrane pinning, and filament bending of a membrane-attached cytoskeletal filament can be sufficient to prescribe orientation, e.g. circumferential for FtsZ or helical for MreB, with the accuracy of orientation increasing with the length of the cytoskeletal filament. Moreover, the mechanical energy can compete with the chemical energy of cytoskeletal polymerization to regulate filament length. Notably, we predict a conformational transition with increasing polymer length from smoothly curved to end-bent polymers. Finally, the mechanical energy also results in a mutual attraction among polymers on the same membrane, which could facilitate tight polymer spacing or bundling. The predictions of the model can be verified through genetic, microscopic, and microfluidic approaches.

A New Poly(Methyl Methacrylate) Membrane Dialyzer, NF, with Adsorptive and Antithrombotic Properties.

PubMed

Oshihara, Wataru; Fujieda, Hiroaki; Ueno, Yoshiyuki

2017-01-01

Poly(methyl methacrylate) (PMMA) membranes adsorb several kinds of proteins and can remove high-molecular-weight proteins, including uremic toxins, which are not removed efficiently by hemodialysis or hemodiafiltration. However, the antithrombogenicity of PMMA membranes is insufficient due to their adsorptive properties. Coagulation during hemodialysis occurs because proteins that are adsorbed to the PMMA membrane undergo structural changes and are recognized by platelets, which are then activated by adhesion to the membrane surface. In developing a new PMMA membrane dialyzer, NF, we intended to inhibit platelet adhesion to the membrane surface by suppressing the structural change in the proteins adsorbed on the membrane. In addition, we give examples of clinical trials of the NF in Japan and describe its advantages. Key Message: PMMA membrane dialyzers have been used for 40 years. The PMMA dialyzer NF can suppress the adhesion of platelets to the membrane while maintaining protein adsorption. © 2017 S. Karger AG, Basel.

Proteomics of plasma membranes from poplar trees reveals tissue distribution of transporters, receptors, and proteins in cell wall formation.

PubMed

Nilsson, Robert; Bernfur, Katja; Gustavsson, Niklas; Bygdell, Joakim; Wingsle, Gunnar; Larsson, Christer

2010-02-01

By exploiting the abundant tissues available from Populus trees, 3-4 m high, we have been able to isolate plasma membranes of high purity from leaves, xylem, and cambium/phloem at a time (4 weeks after bud break) when photosynthesis in the leaves and wood formation in the xylem should have reached a steady state. More than 40% of the 956 proteins identified were found in the plasma membranes of all three tissues and may be classified as "housekeeping" proteins, a typical example being P-type H(+)-ATPases. Among the 213 proteins predicted to be integral membrane proteins, transporters constitute the largest class (41%) followed by receptors (14%) and proteins involved in cell wall and carbohydrate metabolism (8%) and membrane trafficking (8%). ATP-binding cassette transporters (all members of subfamilies B, C, and G) and receptor-like kinases (four subfamilies) were two of the largest protein families found, and the members of these two families showed pronounced tissue distribution. Leaf plasma membranes were characterized by a very high proportion of transporters, constituting almost half of the integral proteins. Proteins involved in cell wall synthesis (such as cellulose and sucrose synthases) and membrane trafficking were most abundant in xylem plasma membranes in agreement with the role of the xylem in wood formation. Twenty-five integral proteins and 83 soluble proteins were exclusively found in xylem plasma membranes, which identifies new candidates associated with cell wall synthesis and wood formation. Among the proteins uniquely found in xylem plasma membranes were most of the enzymes involved in lignin biosynthesis, which suggests that they may exist as a complex linked to the plasma membrane.

X-ray Diffraction from Membrane Protein Nanocrystals

PubMed Central

Hunter, M.S.; DePonte, D.P.; Shapiro, D.A.; Kirian, R.A.; Wang, X.; Starodub, D.; Marchesini, S.; Weierstall, U.; Doak, R.B.; Spence, J.C.H.; Fromme, P.

2011-01-01

Membrane proteins constitute >30% of the proteins in an average cell, and yet the number of currently known structures of unique membrane proteins is <300. To develop new concepts for membrane protein structure determination, we have explored the serial nanocrystallography method, in which fully hydrated protein nanocrystals are delivered to an x-ray beam within a liquid jet at room temperature. As a model system, we have collected x-ray powder diffraction data from the integral membrane protein Photosystem I, which consists of 36 subunits and 381 cofactors. Data were collected from crystals ranging in size from 100 nm to 2 μm. The results demonstrate that there are membrane protein crystals that contain <100 unit cells (200 total molecules) and that 3D crystals of membrane proteins, which contain <200 molecules, may be suitable for structural investigation. Serial nanocrystallography overcomes the problem of x-ray damage, which is currently one of the major limitations for x-ray structure determination of small crystals. By combining serial nanocrystallography with x-ray free-electron laser sources in the future, it may be possible to produce molecular-resolution electron-density maps using membrane protein crystals that contain only a few hundred or thousand unit cells. PMID:21190672

Comparison of membrane ATPases from extreme halophiles isolated from ancient salt deposits

NASA Technical Reports Server (NTRS)

Stan-Lotter, Helga; Sulzner, Michael; Egelseer, Eva; Norton, Cynthia F.; Hochstein, Lawrence I.

1993-01-01

Halophilic microorganisms were isolated from Triassic and Permian salt deposits. Two were rods and grew as red colonies; another was a coccus and produced pink colonies. The rods lysed in solutions that lacked added sodium chloride. Growth of all isolates was inhibited by aphidicolin and their bulk-proteins were acidic as judged from isoelectric focusing. Therefore, these organisms were tentatively identified as extreme halophiles. Whole cell proteins patterns of the isolates following gel electrophoresis were distinct and differed from those of representative type strains of halophilic bacteria. The membrane ATPases from the rods were similar to the enzyme from Halobacterium saccharovorum with respect to subunit composition, enzymatic properties and immunological cross-reaction, but differed slightly in amino acid composition. If the age of the microbial isolated is similar to that of the salt deposits, they can be considered repositories of molecular information of great evolutionary interest.

Comparison of Membrane ATPases from Extreme Halophiles Isolated from Ancient Salt Deposits

NASA Technical Reports Server (NTRS)

Stan-Lotter, Helga; Sulzner, Michael; Egelseer, Eva; Norton, Cynthia F.; Hochstein, Lawrence I.

1993-01-01

Halophilic microorganisms were isolated from Triassic and Permian salt deposits. Two were rods and grew as red colonies; another was a coccus and produced pink colonies. The rods lysed in solutions that lacked added sodium chloride. Growth of all isolates was inhibited by aphidicolin and their bulk proteins were acidic as judged from isoelectric focusing. Therefore, these organisms were tentatively identified as extreme halophiles. Whole cell proteins patterns of the isolates following gel electrophoresis were distinct and differed from those of representative type strains of halophilic bacteria. The membrane ATPases from the rods were similar to the enzyme from Halobacterium saccharovorum with respect to sub unit composition. enzymatic properties and immunological cross-reaction, but differed slightly in amino acid composition. If the age of the microbial isolated is similar to that of the salt deposits, they can be considered repositories of molecular information of great evolutionary interest.

Voltage-dependent-anion-channels (VDACs) in Arabidopsis have a dual localization in the cell but show a distinct role in mitochondria.

PubMed

Robert, Nadia; d'Erfurth, Isabelle; Marmagne, Anne; Erhardt, Mathieu; Allot, Michèle; Boivin, Karine; Gissot, Lionel; Monachello, Dario; Michaud, Morgane; Duchêne, Anne-Marie; Barbier-Brygoo, Hélène; Maréchal-Drouard, Laurence; Ephritikhine, Geneviève; Filleur, Sophie

2012-03-01

In mammals, the Voltage-dependent anion channels (VDACs) are predominant proteins of the outer mitochondrial membrane (OMM) where they contribute to the exchange of small metabolites essential for respiration. They were shown to be as well associated with the plasma membrane (PM) and act as redox enzyme or are involved in ATP release for example. In Arabidopsis, we show that four out of six genomic sequences encode AtVDAC proteins. All four AtVDACs are ubiquitously expressed in the plant but each of them displays a specific expression pattern in root cell types. Using two complementary approaches, we demonstrate conclusively that the four expressed AtVDACs are targeted to both mitochondria and plasma membrane but in differential abundance, AtVDAC3 being the most abundant in PM, and conversely, AtVDAC4 almost exclusively associated with mitochondria. These are the first plant proteins to be shown to reside in both these two membranes. To investigate a putative function of AtVDACs, we analyzed T-DNA insertion lines in each of the corresponding genes. Knock-out mutants for AtVDAC1, AtVDAC2 and AtVDAC4 present slow growth, reduced fertility and yellow spots in leaves when atvdac3 does not show any visible difference compared to wildtype plants. Analyses of atvdac1 and atvdac4 reveal that yellow areas correspond to necrosis and the mitochondria are swollen in these two mutants. All these results suggest that, in spite of a localization in plasma membrane for three of them, AtVDAC1, AtVDAC2 and AtVDAC4 have a main function in mitochondria.

Structure and biochemical composition of desmosomes and tonofilaments isolated from calf muzzle epidermis

PubMed Central

1978-01-01

Complexes of plasma membrane segments with desmosomes and attached tonofilaments were separated from the stratum spinosum cells of calf muzzle by means of moderately alkaline buffers of low ionic strength and mechanical homogenization. These structures were further fractionated by the use of various treatments including sonication, sucrose gradient centrifugation, and extraction with buffers containing high concentrations of salt, urea, citric acid, or detergents. Subfractions enriched in desmosome-tonofilament-complexes and tonofilament fragments were studied in detail. The desmosome structures such as the midline, the trilaminar membrane profile, and the desmosomal plaque appeared well preserved and were notably resistant to the various treatments employed. Fractions containing desmosome- tonofilament complexes were invariably dominated by the nonmembranous proteins of the tonofilaments which appeared as five major polypeptide bands (apparent molecular weights: 48,000; 51,000; 58,000; 60,000; 68,000) present in molar ratios of approx. 2:1:1:2:2. Four of these polypeptide bands showed electrophoretic mobilities similar to those of prekeratin polypeptides from bovine hoof. However, the largest polypeptide (68,000 mol wt) migrated significantly less in polyacrylamide gels than the largest component of the hoof prekeratin (approximately 63,000 mol wt). In addition, a series of minor bands, including carbohydrate-containing proteins, were identified and concluded to represent constituents of the desmosomal membrane. The analysis of protein-bound carbohydrates (total 270 microgram/mg phospholipid in desmosome-enriched subfractions) showed the presence of relatively high amounts of glucosamine, mannose, galactose, and sialic acids. These data as well as the lipid composition (e.g., high ratio of cholesterol to phospholipids, relatively high contents of sphingomyelin and gangliosides, and fatty acid pattern) indicate that the desmosomal membrane is complex in protein and lipid composition and has a typical plasma membrane character. The similarity of the desmosome-associated tonofilaments to prekeratin filaments and other forms of intermediate- sized filaments is discussed. PMID:569157

Structure and biochemical composition of desmosomes and tonofilaments isolated from calf muzzle epidermis.

PubMed

Drochmans, P; Freudenstein, C; Wanson, J C; Laurent, L; Keenan, T W; Stadler, J; Leloup, R; Franke, W W

1978-11-01

Complexes of plasma membrane segments with desmosomes and attached tonofilaments were separated from the stratum spinosum cells of calf muzzle by means of moderately alkaline buffers of low ionic strength and mechanical homogenization. These structures were further fractionated by the use of various treatments including sonication, sucrose gradient centrifugation, and extraction with buffers containing high concentrations of salt, urea, citric acid, or detergents. Subfractions enriched in desmosome-tonofilament-complexes and tonofilament fragments were studied in detail. The desmosome structures such as the midline, the trilaminar membrane profile, and the desmosomal plaque appeared well preserved and were notably resistant to the various treatments employed. Fractions containing desmosome-tonofilament complexes were invariably dominated by the nonmembranous proteins of the tonofilaments which appeared as five major polypeptide bands (apparent molecular weights: 48,000; 51,000; 58,000; 60,000; 68,000) present in molar ratios of approx. 2:1:1:2:2. Four of these polypeptide bands showed electrophoretic mobilities similar to those of prekeratin polypeptides from bovine hoof. However, the largest polypeptide (68,000 mol wt) migrated significantly less in polyacrylamide gels than the largest component of the hoof prekeratin (approximately 63,000 mol wt). In addition, a series of minor bands, including carbohydrate-containing proteins, were identified and concluded to represent constituents of the desmosomal membrane. The analysis of protein-bound carbohydrates (total 270 microgram/mg phospholipid in desmosome-enriched subfractions) showed the presence of relatively high amounts of glucosamine, mannose, galactose, and sialic acids. These data as well as the lipid composition (e.g., high ratio of cholesterol to phospholipids, relatively high contents of sphingomyelin and gangliosides, and fatty acid pattern) indicate that the desmosomal membrane is complex in protein and lipid composition and has a typical plasma membrane character. The similarity of the desmosome-associated tonofilaments to prekeratin filaments and other forms of intermediate-sized filaments is discussed.

Current strategies for protein production and purification enabling membrane protein structural biology.

PubMed

Pandey, Aditya; Shin, Kyungsoo; Patterson, Robin E; Liu, Xiang-Qin; Rainey, Jan K

2016-12-01

Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles, owing to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and (or) amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through the introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10-15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM).

Current strategies for protein production and purification enabling membrane protein structural biology

PubMed Central

Pandey, Aditya; Shin, Kyungsoo; Patterson, Robin E.; Liu, Xiang-Qin; Rainey, Jan K.

2017-01-01

Membrane proteins are still heavily underrepresented in the protein data bank (PDB) due to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles due to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and/or amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10–15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM). PMID:27010607

Correlation Study of PVDF Membrane Morphology with Protein Adsorption: Quantitative Analysis by FTIR/ATR Technique

NASA Astrophysics Data System (ADS)

Ideris, N.; Ahmad, A. L.; Ooi, B. S.; Low, S. C.

2018-05-01

Microporous PVDF membranes were used as protein capture matrices in immunoassays. Because the most common labels in immunoassays were detected based on the colour change, an understanding of how protein concentration varies on different PVDF surfaces was needed. Herein, the correlation between the membrane pore size and protein adsorption was systematically investigated. Five different PVDF membrane morphologies were prepared and FTIR/ATR was employed to accurately quantify the surface protein concentration on membranes with small pore sizes. SigmaPlot® was used to find a suitable curve fit for protein adsorption and membrane pore size, with a high correlation coefficient, R2, of 0.9971.

Durable vesicles for reconstitution of membrane proteins in biotechnology.

PubMed

Beales, Paul A; Khan, Sanobar; Muench, Stephen P; Jeuken, Lars J C

2017-02-08

The application of membrane proteins in biotechnology requires robust, durable reconstitution systems that enhance their stability and support their functionality in a range of working environments. Vesicular architectures are highly desirable to provide the compartmentalisation to utilise the functional transmembrane transport and signalling properties of membrane proteins. Proteoliposomes provide a native-like membrane environment to support membrane protein function, but can lack the required chemical and physical stability. Amphiphilic block copolymers can also self-assemble into polymersomes: tough vesicles with improved stability compared with liposomes. This review discusses the reconstitution of membrane proteins into polymersomes and the more recent development of hybrid vesicles, which blend the robust nature of block copolymers with the biofunctionality of lipids. These novel synthetic vesicles hold great promise for enabling membrane proteins within biotechnologies by supporting their enhanced in vitro performance and could also contribute to fundamental biochemical and biophysical research by improving the stability of membrane proteins that are challenging to work with. © 2017 The Author(s).

Liver plasma membranes: an effective method to analyze membrane proteome.

PubMed

Cao, Rui; Liang, Songping

2012-01-01

Plasma membrane proteins are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of plasma membrane proteins make them difficult to analyze. The protocols given here are the efficient isolation/digestion procedures for liver plasma membrane proteomic analysis. Both protocol for the isolation of plasma membranes and protocol for the in-gel digestion of gel-embedded plasma membrane proteins are presented. The later method allows the use of a high detergent concentration to achieve efficient solubilization of hydrophobic plasma membrane proteins while avoiding interference with the subsequent LC-MS/MS analysis.

Controlling the shape of membrane protein polyhedra

NASA Astrophysics Data System (ADS)

Li, Di; Kahraman, Osman; Haselwandter, Christoph A.

2017-03-01

Membrane proteins and lipids can self-assemble into membrane protein polyhedral nanoparticles (MPPNs). MPPNs have a closed spherical surface and a polyhedral protein arrangement, and may offer a new route for structure determination of membrane proteins and targeted drug delivery. We develop here a general analytic model of how MPPN self-assembly depends on bilayer-protein interactions and lipid bilayer mechanical properties. We find that the bilayer-protein hydrophobic thickness mismatch is a key molecular control parameter for MPPN shape that can be used to bias MPPN self-assembly towards highly symmetric and uniform MPPN shapes. Our results suggest strategies for optimizing MPPN shape for structural studies of membrane proteins and targeted drug delivery.

A brief review of other notable protein blotting methods.

PubMed

Kurien, Biji T; Scofield, R Hal

2009-01-01

A plethora of methods have been used for transferring proteins from the gel to the membrane. These include centrifuge blotting, electroblotting of proteins to Teflon tape and membranes for N- and C-terminal sequence analysis, multiple tissue blotting, a two-step transfer of low and high molecular weight proteins, blotting of Coomassie Brilliant Blue (CBB)-stained proteins from polyacrylamide gels to transparencies, acid electroblotting onto activated glass, membrane-array method for the detection of human intestinal bacteria in fecal samples, protein microarray using a new black cellulose nitrate support, electrotransfer using square wave alternating voltage for enhanced protein recovery, polyethylene glycol-mediated significant enhancement of the immunoblotting transfer, parallel protein chemical processing before and during western blot and the molecular scanner concept, electronic western blot of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-identified polypeptides from parallel processed gel-separated proteins, semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels, transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride (PVDF) membranes, and the display of K(+) channel proteins on a solid nitrocellulose support for assaying toxin binding. The quantification of proteins bound to PVDF membranes by elution of CBB, clarification of immunoblots on PVDF for transmission densitometry, gold coating of nonconductive membranes before MALDI tandem mass spectrometric analysis to prevent charging effect for analysis of peptides from PVDF membranes, and a simple method for coating native polysaccharides onto nitrocellulose are some of the methods involving either the manipulation of membranes with transferred proteins or just a passive transfer of antigens to membranes. All these methods are briefly reviewed in this chapter.

Plasma membrane lipid–protein interactions affect signaling processes in sterol-biosynthesis mutants in Arabidopsis thaliana

PubMed Central

Zauber, Henrik; Burgos, Asdrubal; Garapati, Prashanth; Schulze, Waltraud X.

2014-01-01

The plasma membrane is an important organelle providing structure, signaling and transport as major biological functions. Being composed of lipids and proteins with different physicochemical properties, the biological functions of membranes depend on specific protein–protein and protein–lipid interactions. Interactions of proteins with their specific sterol and lipid environment were shown to be important factors for protein recruitment into sub-compartmental structures of the plasma membrane. System-wide implications of altered endogenous sterol levels for membrane functions in living cells were not studied in higher plant cells. In particular, little is known how alterations in membrane sterol composition affect protein and lipid organization and interaction within membranes. Here, we conducted a comparative analysis of the plasma membrane protein and lipid composition in Arabidopsis sterol-biosynthesis mutants smt1 and ugt80A2;B1. smt1 shows general alterations in sterol composition while ugt80A2;B1 is significantly impaired in sterol glycosylation. By systematically analyzing different cellular fractions and combining proteomic with lipidomic data we were able to reveal contrasting alterations in lipid–protein interactions in both mutants, with resulting differential changes in plasma membrane signaling status. PMID:24672530

In vitro membrane protein synthesis inside Sec translocon-reconstituted cell-sized liposomes

PubMed Central

Ohta, Naoki; Kato, Yasuhiko; Watanabe, Hajime; Mori, Hirotada; Matsuura, Tomoaki

2016-01-01

Protein synthesis using an in vitro transcription-translation system (IVTT) inside cell-sized liposomes has become a valuable tool to study the properties of biological systems under cell-mimicking conditions. However, previous liposome systems lacked the machinery for membrane protein translocation. Here, we reconstituted the translocon consisting of SecYEG from Escherichia coli inside cell-sized liposomes. The cell-sized liposomes also carry the reconstituted IVTT, thereby providing a cell-mimicking environment for membrane protein synthesis. By using EmrE, a multidrug transporter from E. coli, as a model membrane protein, we found that both the amount and activity of EmrE synthesized inside the liposome is increased approximately three-fold by incorporating the Sec translocon. The topological change of EmrE induced by the translocon was also identified. The membrane integration of 6 out of 9 E. coli inner membrane proteins that was tested was increased by incorporation of the translocon. By introducing the Sec translocon, the membrane integration efficiency of the membrane protein of interest was increased, and enabled the integration of membrane proteins that otherwise cannot be inserted. In addition, this work represents an essential step toward the construction of an artificial cell through a bottom-up approach. PMID:27808179

Protein Composition of Trypanosoma brucei Mitochondrial Membranes

PubMed Central

Acestor, Nathalie; Panigrahi, Aswini K.; Ogata, Yuko; Anupama, Atashi; Stuart, Kenneth D.

2010-01-01

Mitochondria consist of four compartments, outer membrane, intermembrane space, inner membrane and matrix; each harboring specific functions and structures. In this study, we used mass spectrometry (LC-MS/MS) to characterize the protein composition of Trypanosoma brucei mitochondrial membranes, which were enriched by different biochemical fractionation techniques. The analyses identified 202 proteins that contain one or more transmembrane domain(s) and/or positive GRAVY scores. Of these, various criteria were used to assign 72 proteins to mitochondrial membranes with high confidence, and 106 with moderate to low confidence. The sub-cellular localization of a selected subset of 13 membrane assigned proteins was confirmed by tagging and immunofluorescence analysis. While most proteins assigned to mitochondrial membrane have putative roles in metabolic, energy generating, and transport processes, ~50% have no known function. These studies result in a comprehensive profile of the composition and sub-organellar location of proteins in the T. brucei mitochondrion thus, providing useful information on mitochondrial functions. PMID:19834910

Structural study of the membrane protein MscL using cell-free expression and solid-state NMR

NASA Astrophysics Data System (ADS)

Abdine, Alaa; Verhoeven, Michiel A.; Park, Kyu-Ho; Ghazi, Alexandre; Guittet, Eric; Berrier, Catherine; Van Heijenoort, Carine; Warschawski, Dror E.

2010-05-01

High-resolution structures of membrane proteins have so far been obtained mostly by X-ray crystallography, on samples where the protein is surrounded by detergent. Recent developments of solid-state NMR have opened the way to a new approach for the study of integral membrane proteins inside a membrane. At the same time, the extension of cell-free expression to the production of membrane proteins allows for the production of proteins tailor made for NMR. We present here an in situ solid-state NMR study of a membrane protein selectively labeled through the use of cell-free expression. The sample consists of MscL (mechano-sensitive channel of large conductance), a 75 kDa pentameric α-helical ion channel from Escherichia coli, reconstituted in a hydrated lipid bilayer. Compared to a uniformly labeled protein sample, the spectral crowding is greatly reduced in the cell-free expressed protein sample. This approach may be a decisive step required for spectral assignment and structure determination of membrane proteins by solid-state NMR.

Mapping the yeast host cell response to recombinant membrane protein production: relieving the biological bottlenecks.

PubMed

Ashe, Mark P; Bill, Roslyn M

2011-06-01

Understanding the structures and functions of membrane proteins is an active area of research within bioscience. Membrane proteins are key players in essential cellular processes such as the uptake of nutrients, the export of waste products, and the way in which cells communicate with their environment. It is therefore not surprising that membrane proteins are targeted by over half of all prescription drugs. Since most membrane proteins are not abundant in their native membranes, it is necessary to produce them in recombinant host cells to enable further structural and functional studies. Unfortunately, achieving the required yields of functional recombinant membrane proteins is still a bottleneck in contemporary bioscience. This has highlighted the need for defined and rational optimization strategies based upon experimental observation rather than relying on trial and error. We have published a transcriptome and subsequent genetic analysis that has identified genes implicated in high-yielding yeast cells. These results have highlighted a role for alterations to a cell's protein synthetic capacity in the production of high yields of recombinant membrane protein: paradoxically, reduced protein synthesis favors higher yields. These results highlight a potential bottleneck at the protein folding or translocation stage of protein production. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Metaproteomic analysis of biocake proteins to understand membrane fouling in a submerged membrane bioreactor.

PubMed

Zhou, Zhongbo; Meng, Fangang; He, Xiang; Chae, So-Ryong; An, Yujia; Jia, Xiaoshan

2015-01-20

Metaproteomic analyses, including two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separation and matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)/TOF mass spectrometer (MS) detection, were used to trace and identify biocake proteins on membranes in a bench-scale submerged membrane bioreactor (MBR). 2D-PAGE images showed that proteins in the biocake (S3) at a low transmembrane pressure (TMP) level (i.e., before the TMP jump) had larger gray intensities in the pH 5.5–7.0 region regardless of the membrane flux, similar to soluble microbial product (SMP) proteins. However, the biocake (S2 and S4) at a high TMP level (i.e., after the TMP jump) had many more proteins in the pH range of 4.0–5.5, similar to extracellular polymeric substance (EPS) proteins. Such similarities between biocake proteins and SMP or EPS proteins can be useful for tracing the sources of proteins resulting in membrane fouling. In total, 183 differentially abundant protein spots were marked in the three biocakes (S2, S3, and S4). However, only 32 protein spots co-occurred in the 2D gels of the three biocakes, indicating that membrane fluxes and TMP evolution levels had significant effects on the abundance of biocake proteins. On the basis of the MS and MS/MS data, 23 of 71 protein spots were successfully identified. Of the 23 proteins, outer membrane proteins (Omp) were a major contributor (60.87%). These Omps were mainly from potential surface colonizers such as Aeromonas, Enterobacter, Pseudomonas, and Thauera. Generally, the metaproteomic analysis is a useful alternative to trace the sources and compositions of biocake proteins on the levels of molecules and bacteria species that can provide new insight into membrane fouling.

Microfluidic platform for efficient Nanodisc assembly, membrane protein incorporation, and purification.

PubMed

Wade, James H; Jones, Joshua D; Lenov, Ivan L; Riordan, Colleen M; Sligar, Stephen G; Bailey, Ryan C

2017-08-22

The characterization of integral membrane proteins presents numerous analytical challenges on account of their poor activity under non-native conditions, limited solubility in aqueous solutions, and low expression in most cell culture systems. Nanodiscs are synthetic model membrane constructs that offer many advantages for studying membrane protein function by offering a native-like phospholipid bilayer environment. The successful incorporation of membrane proteins within Nanodiscs requires experimental optimization of conditions. Standard protocols for Nanodisc formation can require large amounts of time and input material, limiting the facile screening of formation conditions. Capitalizing on the miniaturization and efficient mass transport inherent to microfluidics, we have developed a microfluidic platform for efficient Nanodisc assembly and purification, and demonstrated the ability to incorporate functional membrane proteins into the resulting Nanodiscs. In addition to working with reduced sample volumes, this platform simplifies membrane protein incorporation from a multi-stage protocol requiring several hours or days into a single platform that outputs purified Nanodiscs in less than one hour. To demonstrate the utility of this platform, we incorporated Cytochrome P450 into Nanodiscs of variable size and lipid composition, and present spectroscopic evidence for the functional active site of the membrane protein. This platform is a promising new tool for membrane protein biology and biochemistry that enables tremendous versatility for optimizing the incorporation of membrane proteins using microfluidic gradients to screen across diverse formation conditions.

A Class of Rigid Linker-bearing Glucosides for Membrane Protein Structural Study.

PubMed

Sadaf, Aiman; Mortensen, Jonas S; Capaldi, Stefano; Tikhonova, Elena; Hariharan, Parameswaran; de Castro Ribeiro, Orquidea; Loland, Claus J; Guan, Lan; Byrne, Bernadette; Chae, Pil Seok

2016-03-01

Membrane proteins are amphipathic bio-macromolecules incompatible with the polar environments of aqueous media. Conventional detergents encapsulate the hydrophobic surfaces of membrane proteins allowing them to exist in aqueous solution. Membrane proteins stabilized by detergent micelles are used for structural and functional analysis. Despite the availability of a large number of detergents, only a few agents are sufficiently effective at maintaining the integrity of membrane proteins to allow successful crystallization. In the present study, we describe a novel class of synthetic amphiphiles with a branched tail group and a triglucoside head group. These head and tail groups were connected via an amide or ether linkage by using a tris(hydroxylmethyl)aminomethane (TRIS) or neopentyl glycol (NPG) linker to produce TRIS-derived triglucosides (TDTs) and NPG-derived triglucosides (NDTs), respectively. Members of this class conferred enhanced stability on target membrane proteins compared to conventional detergents. Because of straightforward synthesis of the novel agents and their favourable effects on a range of membrane proteins, these agents should be of wide applicability to membrane protein science.

A Class of Rigid Linker-bearing Glucosides for Membrane Protein Structural Study

PubMed Central

Sadaf, Aiman; Mortensen, Jonas S.; Capaldi, Stefano; Tikhonova, Elena; Hariharan, Parameswaran; de Castro Ribeiro, Orquidea; Loland, Claus J; Guan, Lan; Byrne, Bernadette

2015-01-01

Membrane proteins are amphipathic bio-macromolecules incompatible with the polar environments of aqueous media. Conventional detergents encapsulate the hydrophobic surfaces of membrane proteins allowing them to exist in aqueous solution. Membrane proteins stabilized by detergent micelles are used for structural and functional analysis. Despite the availability of a large number of detergents, only a few agents are sufficiently effective at maintaining the integrity of membrane proteins to allow successful crystallization. In the present study, we describe a novel class of synthetic amphiphiles with a branched tail group and a triglucoside head group. These head and tail groups were connected via an amide or ether linkage by using a tris(hydroxylmethyl)aminomethane (TRIS) or neopentyl glycol (NPG) linker to produce TRIS-derived triglucosides (TDTs) and NPG-derived triglucosides (NDTs), respectively. Members of this class conferred enhanced stability on target membrane proteins compared to conventional detergents. Because of straightforward synthesis of the novel agents and their favourable effects on a range of membrane proteins, these agents should be of wide applicability to membrane protein science. PMID:27110345

UL31 and UL34 Proteins of Herpes Simplex Virus Type 1 Form a Complex That Accumulates at the Nuclear Rim and Is Required for Envelopment of Nucleocapsids

PubMed Central

Reynolds, Ashley E.; Ryckman, Brent J.; Baines, Joel D.; Zhou, Yuping; Liang, Li; Roller, Richard J.

2001-01-01

The herpes simplex virus type 1 (HSV-1) UL34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the UL31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with UL34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing UL31 protein fused to glutathione-S-transferase (UL31-GST) and UL34 protein fused to GST (UL34-GST) were demonstrated to specifically recognize the UL31 and UL34 proteins of approximately 34,000 and 30,000 Da, respectively. The UL31 and UL34 gene products colocalized in a smooth pattern throughout the nuclear rim of infected cells by 10 h postinfection. UL34 protein also accumulated in pleiomorphic cytoplasmic structures at early times and associated with an altered nuclear envelope late in infection. Localization of UL31 protein at the nuclear rim required the presence of UL34 protein, inasmuch as cells infected with a UL34 null mutant virus contained UL31 protein primarily in central intranuclear domains separate from the nuclear rim, and to a lesser extent in the cytoplasm. Conversely, localization of UL34 protein exclusively at the nuclear rim required the presence of the UL31 gene product, inasmuch as UL34 protein was detectable at the nuclear rim, in replication compartments, and in the cytoplasm of cells infected with a UL31 null virus. When transiently expressed in the absence of other viral factors, UL31 protein localized diffusely in the nucleoplasm, whereas UL34 protein localized primarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the UL31 and UL34 proteins was sufficient to target both proteins exclusively to the nuclear rim. The proteins were also shown to directly interact in vitro in the absence of other viral proteins. In cells infected with a virus lacking the US3-encoded protein kinase, previously shown to phosphorylate the UL34 gene product, UL31 and UL34 proteins colocalized in small punctate areas that accumulated on the nuclear rim. Thus, US3 kinase is required for even distribution of UL31 and UL34 proteins throughout the nuclear rim. Taken together with the similar phenotypes of the UL31 and UL34 deletion mutants, these data strongly suggest that the UL31 and UL34 proteins form a complex that accumulates at the nuclear membrane and plays an important role in nucleocapsid envelopment at the inner nuclear membrane. PMID:11507225

The Escherichia coli Peripheral Inner Membrane Proteome*

PubMed Central

Papanastasiou, Malvina; Orfanoudaki, Georgia; Koukaki, Marina; Kountourakis, Nikos; Sardis, Marios Frantzeskos; Aivaliotis, Michalis; Karamanou, Spyridoula; Economou, Anastassios

2013-01-01

Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected ∼19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies ∼25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with biological membrane surfaces that we are only beginning to decipher. PMID:23230279

The Charcot Marie Tooth disease protein LITAF is a zinc-binding monotopic membrane protein

PubMed Central

Qin, Wenxia; Wunderley, Lydia; Barrett, Anne L.; High, Stephen; Woodman, Philip G.

2016-01-01

LITAF (LPS-induced TNF-activating factor) is an endosome-associated integral membrane protein important for multivesicular body sorting. Several mutations in LITAF cause autosomal-dominant Charcot Marie Tooth disease type 1C. These mutations map to a highly conserved C-terminal region, termed the LITAF domain, which includes a 22 residue hydrophobic sequence and flanking cysteine-rich regions that contain peptide motifs found in zinc fingers. Although the LITAF domain is thought to be responsible for membrane integration, the membrane topology of LITAF has not been established. Here, we have investigated whether LITAF is a tail-anchored (TA) membrane-spanning protein or monotopic membrane protein. When translated in vitro, LITAF integrates poorly into ER-derived microsomes compared with Sec61β, a bona fide TA protein. Furthermore, introduction of N-linked glycosylation reporters shows that neither the N-terminal nor C-terminal domains of LITAF translocate into the ER lumen. Expression in cells of an LITAF construct containing C-terminal glycosylation sites confirms that LITAF is not a TA protein in cells. Finally, an immunofluorescence-based latency assay showed that both the N- and C-termini of LITAF are exposed to the cytoplasm. Recombinant LITAF contains 1 mol/mol zinc, while mutation of predicted zinc-binding residues disrupts LITAF membrane association. Hence, we conclude that LITAF is a monotopic membrane protein whose membrane integration is stabilised by a zinc finger. The related human protein, CDIP1 (cell death involved p53 target 1), displays identical membrane topology, suggesting that this mode of membrane integration is conserved in LITAF family proteins. PMID:27582497

Interaction between La(III) and proteins on the plasma membrane of horseradish

NASA Astrophysics Data System (ADS)

Yang, Guang-Mei; Chu, Yun-Xia; Lv, Xiao-Fen; Zhou, Qing; Huang, Xiao-Hua

2012-06-01

Lanthanum (La) is an important rare earth element in the ecological environment of plant. The proteins on the plasma membrane control the transport of molecules into and out of cell. It is very important to investigate the effect of La(III) on the proteins on the plasma membrane in the plant cell. In the present work, the interaction between La(III) and proteins on the plasma membrane of horseradish was investigated using optimization of the fluorescence microscopy and fluorescence spectroscopy. It is found that the fluorescence of the complex system of protoplasts and 1-aniline Kenai-8-sulfonic acid in horseradish treated with the low concentration of La(III) is increased compared with that of the control horseradish. The opposite effect is observed in horseradish treated with the high concentration of La(III). These results indicated that the low concentration of La(III) can interact with the proteins on the plasma membrane of horseradish, causing the improvement in the structure of proteins on the plasma membrane. The high concentration of La(III) can also interact with the proteins on the plasma membrane of horseradish, leading to the destruction of the structure of proteins on the plasma membrane. We demonstrate that the proteins on the plasma membrane are the targets of La(III) action on plant cell.

In silico local structure approach: a case study on outer membrane proteins.

PubMed

Martin, Juliette; de Brevern, Alexandre G; Camproux, Anne-Claude

2008-04-01

The detection of Outer Membrane Proteins (OMP) in whole genomes is an actual question, their sequence characteristics have thus been intensively studied. This class of protein displays a common beta-barrel architecture, formed by adjacent antiparallel strands. However, due to the lack of available structures, few structural studies have been made on this class of proteins. Here we propose a novel OMP local structure investigation, based on a structural alphabet approach, i.e., the decomposition of 3D structures using a library of four-residue protein fragments. The optimal decomposition of structures using hidden Markov model results in a specific structural alphabet of 20 fragments, six of them dedicated to the decomposition of beta-strands. This optimal alphabet, called SA20-OMP, is analyzed in details, in terms of local structures and transitions between fragments. It highlights a particular and strong organization of beta-strands as series of regular canonical structural fragments. The comparison with alphabets learned on globular structures indicates that the internal organization of OMP structures is more constrained than in globular structures. The analysis of OMP structures using SA20-OMP reveals some recurrent structural patterns. The preferred location of fragments in the distinct regions of the membrane is investigated. The study of pairwise specificity of fragments reveals that some contacts between structural fragments in beta-sheets are clearly favored whereas others are avoided. This contact specificity is stronger in OMP than in globular structures. Moreover, SA20-OMP also captured sequential information. This can be integrated in a scoring function for structural model ranking with very promising results. (c) 2007 Wiley-Liss, Inc.

A method for detergent-free isolation of membrane proteins in their local lipid environment.

PubMed

Lee, Sarah C; Knowles, Tim J; Postis, Vincent L G; Jamshad, Mohammed; Parslow, Rosemary A; Lin, Yu-Pin; Goldman, Adrian; Sridhar, Pooja; Overduin, Michael; Muench, Stephen P; Dafforn, Timothy R

2016-07-01

Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins.

Fold-Unfold Transitions in the Selectivity and Mechanism of Action of the N-Terminal Fragment of the Bactericidal/Permeability-Increasing Protein (rBPI21)

PubMed Central

Domingues, Marco M.; Lopes, Sílvia C.D.N.; Santos, Nuno C.; Quintas, Alexandre; Castanho, Miguel A.R.B.

2009-01-01

Septic or endotoxic shock is a common cause of death in hospital intensive care units. In the last decade numerous antimicrobial peptides and proteins have been tested in the search for an efficient drug to treat this lethal disease. Now in phase III clinical trials, rBPI21, a recombinant N-terminal fragment of the bactericidal/permeability-increasing protein (BPI), is a promising drug to reduce lesions caused by meningococcal sepsis. We correlated structural and stability data with functional information of rBPI21 bound to both model systems of eukaryotic and bacterial membranes. On interaction with membranes, rBPI21 loses its conformational stability, as studied by circular dichroism. This interaction of rBPI21 at membrane level was higher in the presence of negatively charged phospholipid relatively to neutral ones, with higher partition coefficients (Kp), suggesting a preference for bacterial membranes over mammalian membranes. rBPI21 binding to membranes is reinforced when its disulfide bond is broken due to conformational changes of the protein. This interaction is followed by liposome aggregation due to unfolding, which ensures protein aggregation, and interfacial localization of rBPI21 in membranes, as studied by extensive quenching by acrylamide and 5-deoxylstearic acid and not by 16-deoxylstearic acid. An uncommon model of the selectivity and mechanism of action is proposed, where membrane induces unfolding of the antimicrobial protein, rBPI21. The unfolding ensures protein aggregation, established by protein-protein interaction at membrane surface or between adjacent membranes covered by the unfolded protein. This protein aggregation step may lead to membrane perturbation. PMID:19186136

Direct Capture of Functional Proteins from Mammalian Plasma Membranes into Nanodiscs.

PubMed

Roy, Jahnabi; Pondenis, Holly; Fan, Timothy M; Das, Aditi

2015-10-20

Mammalian plasma membrane proteins make up the largest class of drug targets yet are difficult to study in a cell free system because of their intransigent nature. Herein, we perform direct encapsulation of plasma membrane proteins derived from mammalian cells into a functional nanodisc library. Peptide fingerprinting was used to analyze the proteome of the incorporated proteins in nanodiscs and to further demonstrate that the lipid composition of the nanodiscs directly affects the class of protein that is incorporated. Furthermore, the functionality of the incorporated membrane proteome was evaluated by measuring the activity of membrane proteins: Na(+)/K(+)-ATPase and receptor tyrosine kinases. This work is the first report of the successful establishment and characterization of a cell free functional library of mammalian membrane proteins into nanodiscs.

Membrane bending: the power of protein imbalance.

PubMed

Derganc, Jure; Antonny, Bruno; Copič, Alenka

2013-11-01

Many cellular processes require membrane deformation, which is driven by specialized protein machinery and can often be recapitulated using pure lipid bilayers. However, biological membranes contain a large amount of embedded proteins. Recent research suggests that membrane-bound proteins with asymmetric distribution of mass across the bilayer can influence membrane bending in a nonspecific manner due to molecular crowding. This mechanism is physical in nature and arises from collisions between such 'mushroom-shaped' proteins. It can either facilitate or impede the action of protein coats, for example COPII, during vesicle budding. We describe the physics of how molecular crowding can influence membrane bending and discuss the implications for other cellular processes, such as sorting of glycosylphosphatidylinositol-anchored proteins (GPI-APs) and production of intraluminal vesicles. Copyright © 2013 Elsevier Ltd. All rights reserved.

The p14 fusion-associated small transmembrane (FAST) protein effects membrane fusion from a subset of membrane microdomains.

PubMed

Corcoran, Jennifer A; Salsman, Jayme; de Antueno, Roberto; Touhami, Ahmed; Jericho, Manfred H; Clancy, Eileen K; Duncan, Roy

2006-10-20

The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WX- and Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)-resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipid-anchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process.

Specific Adhesion of Lipid Membranes Can Simultaneously Produce Two Types of Lipid and Protein Heterogeneities

NASA Astrophysics Data System (ADS)

Shindell, Orrin; Micah, Natalie; Ritzer, Max; Gordon, Vernita

2015-03-01

Living cells adhere to one another and their environment. Adhesion is associated with re-organization of the lipid and protein components of the cell membrane. The resulting heterogeneities are functional structures involved in biological processes. We use artificial lipid membranes that contain a single type of binding protein. Before adhesion, the lipid, protein, and dye components in the membrane are well-mixed and constitute a single disordered-liquid phase (Ld) . After adhesion, two distinct types of heterogeneities coexist in the adhesion zone: a central domain of ordered lipid phase that excludes both binding proteins and membrane dye, and a peripheral domain of disordered lipid phase that is densely packed with adhesion proteins and enriched in membrane dye relative to the non-adhered portion of the vesicle. Thus, we show that adhesion that is mediated by only one type of protein can organize the lipid and protein components of the membranes into heterogeneities that resemble those found in biology, for example the immune synapse.

Functions of intrinsic disorder in transmembrane proteins.

PubMed

Kjaergaard, Magnus; Kragelund, Birthe B

2017-09-01

Intrinsic disorder is common in integral membrane proteins, particularly in the intracellular domains. Despite this observation, these domains are not always recognized as being disordered. In this review, we will discuss the biological functions of intrinsically disordered regions of membrane proteins, and address why the flexibility afforded by disorder is mechanistically important. Intrinsically disordered regions are present in many common classes of membrane proteins including ion channels and transporters; G-protein coupled receptors (GPCRs), receptor tyrosine kinases and cytokine receptors. The functions of the disordered regions are many and varied. We will discuss selected examples including: (1) Organization of receptors, kinases, phosphatases and second messenger sources into signaling complexes. (2) Modulation of the membrane-embedded domain function by ball-and-chain like mechanisms. (3) Trafficking of membrane proteins. (4) Transient membrane associations. (5) Post-translational modifications most notably phosphorylation and (6) disorder-linked isoform dependent function. We finish the review by discussing the future challenges facing the membrane protein community regarding protein disorder.

Using Haloarcula marismortui Bacteriorhodopsin as a Fusion Tag for Enhancing and Visible Expression of Integral Membrane Proteins in Escherichia coli

PubMed Central

Hsu, Min-Feng; Yu, Tsung-Fu; Chou, Chia-Cheng; Fu, Hsu-Yuan; Yang, Chii-Shen; Wang, Andrew H. J.

2013-01-01

Membrane proteins are key targets for pharmacological intervention because of their vital functions. Structural and functional studies of membrane proteins have been severely hampered because of the difficulties in producing sufficient quantities of properly folded and biologically active proteins. Here we generate a high-level expression system of integral membrane proteins in Escherichia coli by using a mutated bacteriorhodopsin (BR) from Haloarcula marismortui (HmBRI/D94N) as a fusion partner. A purification strategy was designed by incorporating a His-tag on the target membrane protein for affinity purification and an appropriate protease cleavage site to generate the final products. The fusion system can be used to detect the intended target membrane proteins during overexpression and purification either with the naked eye or by directly monitoring their characteristic optical absorption. In this study, we applied this approach to produce two functional integral membrane proteins, undecaprenyl pyrophosphate phosphatase and carnitine/butyrobetaine antiporter with significant yield enhancement. This technology could facilitate the development of a high-throughput strategy to screen for conditions that improve the yield of correctly folded target membrane proteins. Other robust BRs can also be incorporated in this system. PMID:23457558

Isomeric Detergent Comparison for Membrane Protein Stability: Importance of Inter-Alkyl-Chain Distance and Alkyl Chain Length.

PubMed

Cho, Kyung Ho; Hariharan, Parameswaran; Mortensen, Jonas S; Du, Yang; Nielsen, Anne K; Byrne, Bernadette; Kobilka, Brian K; Loland, Claus J; Guan, Lan; Chae, Pil Seok

2016-12-14

Membrane proteins encapsulated by detergent micelles are widely used for structural study. Because of their amphipathic property, detergents have the ability to maintain protein solubility and stability in an aqueous medium. However, conventional detergents have serious limitations in their scope and utility, particularly for eukaryotic membrane proteins and membrane protein complexes. Thus, a number of new agents have been devised; some have made significant contributions to membrane protein structural studies. However, few detergent design principles are available. In this study, we prepared meta and ortho isomers of the previously reported para-substituted xylene-linked maltoside amphiphiles (XMAs), along with alkyl chain-length variation. The isomeric XMAs were assessed with three membrane proteins, and the meta isomer with a C 12 alkyl chain was most effective at maintaining solubility/stability of the membrane proteins. We propose that interplay between the hydrophile-lipophile balance (HLB) and alkyl chain length is of central importance for high detergent efficacy. In addition, differences in inter-alkyl-chain distance between the isomers influence the ability of the detergents to stabilise membrane proteins. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane.

PubMed

Leser, George P; Lamb, Robert A

2017-05-01

Influenza virus assembles and buds at the plasma membrane of virus-infected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some proteins, like hemagglutinin (HA), NA, and M2, are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains, whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immunogold staining. The distribution of these proteins was examined individually and pairwise by using the Ripley K function, a type of nearest-neighbor analysis. Individually, HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly coclustered in the plasma membrane; however, in the case of NA and M2, clustering depends upon the expression system used. Despite both proteins being raft resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly cocluster, but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the coexpression of other viral proteins. Similarly, M2 and NP occupy separate compartments, but an association can be bridged by the coexpression of M1. IMPORTANCE The complement of influenza virus proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft-like domains, whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships between viral proteins in the plasma membrane. Some proteins, such as HA and M2, inherently cocluster within the membrane, although M2 is found mostly at the periphery of regions of HA, consistent with the proposed role of M2 in scission at the end of budding. The association between some pairs of influenza virus proteins, such as M2 and NP, appears to be brokered by additional influenza virus proteins, in this case M1. HA and NA, while raft associated, reside in distinct domains, reflecting their distributions in the viral membrane. Copyright © 2017 American Society for Microbiology.

Profiling of kidney vascular endothelial cell plasma membrane proteins by liquid chromatography-tandem mass spectrometry.

PubMed

Liu, Zan; Xu, Bo; Nameta, Masaaki; Zhang, Ying; Magdeldin, Sameh; Yoshida, Yutaka; Yamamoto, Keiko; Fujinaka, Hidehiko; Yaoita, Eishin; Tasaki, Masayuki; Nakagawa, Yuki; Saito, Kazuhide; Takahashi, Kota; Yamamoto, Tadashi

2013-06-01

Vascular endothelial cells (VECs) play crucial roles in physiological and pathologic conditions in tissues and organs. Most of these roles are related to VEC plasma membrane proteins. In the kidney, VECs are closely associated with structures and functions; however, plasma membrane proteins in kidney VECs remain to be fully elucidated. Rat kidneys were perfused with cationic colloidal silica nanoparticles (CCSN) to label the VEC plasma membrane. The CCSN-labeled plasma membrane fraction was collected by gradient ultracentrifugation. The VEC plasma membrane or whole-kidney lysate proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and digested with trypsin in gels for liquid chromatography-tandem mass spectrometry. Enrichment analysis was then performed. The VEC plasma membrane proteins were purified by the CCSN method with high yield (approximately 20 μg from 1 g of rat kidney). By Mascot search, 582 proteins were identified in the VEC plasma membrane fraction, and 1,205 proteins were identified in the kidney lysate. In addition to 16 VEC marker proteins such as integrin beta-1 and intercellular adhesion molecule-2 (ICAM-2), 8 novel proteins such as Deltex 3-like protein and phosphatidylinositol binding clathrin assembly protein (PICALM) were identified. As expected, many key functions of plasma membranes in general and of endothelial cells in particular (i.e., leukocyte adhesion) were significantly overrepresented in the proteome of CCSN-labeled kidney VEC fraction. The CCSN method is a reliable technique for isolation of VEC plasma membrane from the kidney, and proteomic analysis followed by bioinformatics revealed the characteristics of in vivo VECs in the kidney.

The JNK-like MAPK KGB-1 of Caenorhabditis elegans promotes reproduction, lifespan, and gene expressions for protein biosynthesis and germline homeostasis but interferes with hyperosmotic stress tolerance.

PubMed

Gerke, Peter; Keshet, Alex; Mertenskötter, Ansgar; Paul, Rüdiger J

2014-01-01

This study focused on the role of the JNK-like MAPK (mitogen-activated protein kinase) KGB-1 (kinase, GLH-binding 1) for osmoprotection and other vital functions. We mapped KGB-1 expression patterns and determined lifespan, reproduction and survival rates as well as changes in body volume, motility, and GPDH (glycerol-3-phosphate dehydrogenase) activity for glycerol production in wildtype (WT), different signaling mutants (including a kgb-1 deletion mutant, kgb-1∆) and RNAi-treated worms under control and hyperosmotic conditions. KGB-1-mediated gene expressions were studied, for instance, by RNA Sequencing, with the resulting transcriptome data analyzed using orthology-based approaches. Surprisingly, mutation/RNAi of kgb-1 and fos-1 (gene for an AP-1, activator protein 1, element) significantly promoted hyperosmotic resistance, even though hyperosmotic GPDH activity was higher in WT than in kgb-1∆. KGB-1 and moderate hyperosmolarity promoted and severe hyperosmolarity repressed kgb-1, fos-1, and jun-1 (gene for another AP-1 element) expression. Transcriptome profiling revealed, for instance, down-regulated genes for protein biosynthesis and up-regulated genes for membrane transporters in kgb-1∆ and up-regulated genes for GPDH-1 or detoxification in WT, with the latter indicating cellular damage and less effective osmoprotection in WT. KGB-1 promotes reproduction and lifespan and fosters gene expressions for AP-1 elements, protein biosynthesis, and balanced gametogenesis, but inhibits expressions for membrane transporters perhaps in order to control energy consumption. Reduced protein biosyntheses and enhanced membrane transports in kgb-1∆ most likely contribute to the high hyperosmotic tolerance of the mutant by easing the burden of the existing chaperone machinery and promoting regulatory volume increases upon hyperosmotic stress.

The ENU-3 protein family members function in the Wnt pathway parallel to UNC-6/Netrin to promote motor neuron axon outgrowth in C. elegans.

PubMed

Florica, Roxana Oriana; Hipolito, Victoria; Bautista, Stephen; Anvari, Homa; Rapp, Chloe; El-Rass, Suzan; Asgharian, Alimohammad; Antonescu, Costin N; Killeen, Marie T

2017-10-01

The axons of the DA and DB classes of motor neurons fail to reach the dorsal cord in the absence of the guidance cue UNC-6/Netrin or its receptor UNC-5 in C. elegans. However, the axonal processes usually exit their cell bodies in the ventral cord in the absence of both molecules. Strains lacking functional versions of UNC-6 or UNC-5 have a low level of DA and DB motor neuron axon outgrowth defects. We found that mutations in the genes for all six of the ENU-3 proteins function to enhance the outgrowth defects of the DA and DB axons in strains lacking either UNC-6 or UNC-5. A mutation in the gene for the MIG-14/Wntless protein also enhances defects in a strain lacking either UNC-5 or UNC-6, suggesting that the ENU-3 and Wnt pathways function parallel to the Netrin pathway in directing motor neuron axon outgrowth. Our evidence suggests that the ENU-3 proteins are novel members of the Wnt pathway in nematodes. Five of the six members of the ENU-3 family are predicted to be single-pass trans-membrane proteins. The expression pattern of ENU-3.1 was consistent with plasma membrane localization. One family member, ENU-3.6, lacks the predicted signal peptide and the membrane-spanning domain. In HeLa cells ENU-3.6 had a cytoplasmic localization and caused actin dependent processes to appear. We conclude that the ENU-3 family proteins function in a pathway parallel to the UNC-6/Netrin pathway for motor neuron axon outgrowth, most likely in the Wnt pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

LDRD final report on imaging self-organization of proteins in membranes by photocatalytic nano-tagging.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zavadil, Kevin Robert; Shelnutt, John Allen; Sasaki, Darryl Yoshio

We have developed a new nanotagging technology for detecting and imaging the self-organization of proteins and other components of membranes at nanometer resolution for the purpose of investigating cell signaling and other membrane-mediated biological processes. We used protein-, lipid-, or drug-bound porphyrin photocatalysts to grow in-situ nanometer-sized metal particles, which reveal the location of the porphyrin-labeled molecules by electron microscopy. We initially used photocatalytic nanotagging to image assembled multi-component proteins and to monitor the distribution of lipids and porphyrin labels in liposomes. For example, by exchanging the heme molecules in hemoproteins with a photocatalytic tin porphyrin, a nanoparticle was grownmore » at each heme site of the protein. The result obtained from electron microscopy for a tagged multi-subunit protein such as hemoglobin is a symmetric constellation of a specific number of nanoparticle tags, four in the case of the hemoglobin tetramer. Methods for covalently linking photocatalytic porphyrin labels to lipids and proteins were also developed to detect and image the self-organization of lipids, protein-protein supercomplexes, and membrane-protein complexes. Procedures for making photocatalytic porphyrin-drug, porphyrin-lipid, and porphyrin-protein hybrids for non-porphyrin-binding proteins and membrane components were pursued and the first porphyrin-labeled lipids was investigated in liposomal membrane models. Our photocatalytic nanotagging technique may ultimately allow membrane self-organization and cell signaling processes to be imaged in living cells. Fluorescence and plasmonic spectra of the tagged proteins might also provide additional information about protein association and membrane organization. In addition, a porphyrin-aspirin or other NSAID hybrid may be used to grow metal nanotags for the pharmacologically important COX enzymes in membranes so that the distribution of the protein can be imaged at the nanometer scale.« less

Other notable protein blotting methods: a brief review.

PubMed

Kurien, Biji T; Scofield, R Hal

2015-01-01

Proteins have been transferred from the gel to the membrane by a variety of methods. These include vacuum blotting, centrifuge blotting, electroblotting of proteins to Teflon tape and membranes for N- and C-terminal sequence analysis, multiple tissue blotting, a two-step transfer of low- and high-molecular-weight proteins, acid electroblotting onto activated glass, membrane-array method for the detection of human intestinal bacteria in fecal samples, protein microarray using a new black cellulose nitrate support, electrotransfer using square wave alternating voltage for enhanced protein recovery, polyethylene glycol-mediated significant enhancement of the immunoblotting transfer, parallel protein chemical processing before and during western blot and the molecular scanner concept, electronic western blot of matrix-assisted laser desorption/ionization mass spectrometric-identified polypeptides from parallel processed gel-separated proteins, semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels, transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride (PVDF) membranes, and the display of K(+) channel proteins on a solid nitrocellulose support for assaying toxin binding. The quantification of proteins bound to PVDF membranes by elution of CBB, clarification of immunoblots on PVDF for transmission densitometry, gold coating of nonconductive membranes before matrix-assisted laser desorption/ionization tandem mass spectrometric analysis to prevent charging effect for analysis of peptides from PVDF membranes, and a simple method for coating native polysaccharides onto nitrocellulose are some of the methods involving either the manipulation of membranes with transferred proteins or just a passive transfer of antigens to membranes. All these methods are briefly reviewed in this chapter.

The presequence pathway is involved in protein sorting to the mitochondrial outer membrane.

PubMed

Wenz, Lena-Sophie; Opaliński, Lukasz; Schuler, Max-Hinderk; Ellenrieder, Lars; Ieva, Raffaele; Böttinger, Lena; Qiu, Jian; van der Laan, Martin; Wiedemann, Nils; Guiard, Bernard; Pfanner, Nikolaus; Becker, Thomas

2014-06-01

The mitochondrial outer membrane contains integral α-helical and β-barrel proteins that are imported from the cytosol. The machineries importing β-barrel proteins have been identified, however, different views exist on the import of α-helical proteins. It has been reported that the biogenesis of Om45, the most abundant signal-anchored protein, does not depend on proteinaceous components, but involves direct insertion into the outer membrane. We show that import of Om45 occurs via the translocase of the outer membrane and the presequence translocase of the inner membrane. Assembly of Om45 in the outer membrane involves the MIM machinery. Om45 thus follows a new mitochondrial biogenesis pathway that uses elements of the presequence import pathway to direct a protein to the outer membrane. © 2014 The Authors.

Rapid Preparation of a Plasma Membrane Fraction: Western Blot Detection of Translocated Glucose Transporter 4 from Plasma Membrane of Muscle and Adipose Cells and Tissues.

PubMed

Yamamoto, Norio; Yamashita, Yoko; Yoshioka, Yasukiyo; Nishiumi, Shin; Ashida, Hitoshi

2016-08-01

Membrane proteins account for 70% to 80% of all pharmaceutical targets, indicating their clinical relevance and underscoring the importance of identifying differentially expressed membrane proteins that reflect distinct disease properties. The translocation of proteins from the bulk of the cytosol to the plasma membrane is a critical step in the transfer of information from membrane-embedded receptors or transporters to the cell interior. To understand how membrane proteins work, it is important to separate the membrane fraction of cells. This unit provides a protocol for rapidly obtaining plasma membrane fractions for western blot analysis. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Membrane Contact Sites: Complex Zones for Membrane Association and Lipid Exchange

PubMed Central

Quon, Evan; Beh, Christopher T.

2015-01-01

Lipid transport between membranes within cells involves vesicle and protein carriers, but as agents of nonvesicular lipid transfer, the role of membrane contact sites has received increasing attention. As zones for lipid metabolism and exchange, various membrane contact sites mediate direct associations between different organelles. In particular, membrane contact sites linking the plasma membrane (PM) and the endoplasmic reticulum (ER) represent important regulators of lipid and ion transfer. In yeast, cortical ER is stapled to the PM through membrane-tethering proteins, which establish a direct connection between the membranes. In this review, we consider passive and facilitated models for lipid transfer at PM–ER contact sites. Besides the tethering proteins, we examine the roles of an additional repertoire of lipid and protein regulators that prime and propagate PM–ER membrane association. We conclude that instead of being simple mediators of membrane association, regulatory components of membrane contact sites have complex and multilayered functions. PMID:26949334

Caveolin-1 interacts with the Gag precursor of murine leukaemia virus and modulates virus production

PubMed Central

Yu, Zheng; Beer, Christiane; Koester, Mario; Wirth, Manfred

2006-01-01

Background Retroviral Gag determines virus assembly at the plasma membrane and the formation of virus-like particles in intracellular multivesicular bodies. Thereby, retroviruses exploit by interaction with cellular partners the cellular machineries for vesicular transport in various ways. Results The retroviral Gag precursor protein drives assembly of murine leukaemia viruses (MLV) at the plasma membrane (PM) and the formation of virus like particles in multivesicular bodies (MVBs). In our study we show that caveolin-1 (Cav-1), a multifunctional membrane-associated protein, co-localizes with Gag in a punctate pattern at the PM of infected NIH 3T3 cells. We provide evidence that Cav-1 interacts with the matrix protein (MA) of the Gag precursor. This interaction is mediated by a Cav-1 binding domain (CBD) within the N-terminus of MA. Interestingly, the CBD motif identified within MA is highly conserved among most other γ-retroviruses. Furthermore, Cav-1 is incorporated into MLV released from NIH 3T3 cells. Overexpression of a GFP fusion protein containing the putative CBD of the retroviral MA resulted in a considerable decrease in production of infectious retrovirus. Moreover, expression of a dominant-negative Cav-1 mutant affected retroviral titres significantly. Conclusion This study demonstrates that Cav-1 interacts with MLV Gag, co-localizes with Gag at the PM and affects the production of infectious virus. The results strongly suggest a role for Cav-1 in the process of virus assembly. PMID:16956408

Sorting of Pmel17 to melanosomes through the plasma membrane by AP1 and AP2: evidence for the polarized nature of melanocytes

PubMed Central

Valencia, Julio C.; Watabe, Hidenori; Chi, An; Rouzaud, Francois; Chen, Kevin G.; Vieira, Wilfred D.; Takahashi, Kaoruko; Yamaguchi, Yuji; Berens, Werner; Nagashima, Kunio; Shabanowitz, Jeffrey; Hunt, Donald F.; Appella, Ettore; Hearing, Vincent J.

2015-01-01

Summary Adaptor proteins (AP) play important roles in the sorting of proteins from the trans-Golgi network, but how they function in the sorting of various melanosome-specific proteins such as Pmel17, an essential structural component of melanosomes, in melanocytes is unknown. We characterized the processing and trafficking of Pmel17 via adaptor protein complexes within melanocytic cells. Proteomics analysis detected Pmel17, AP1 and AP2, but not AP3 or AP4 in early melanosomes. Real-time PCR, immunolabeling and tissue in-situ hybridization confirmed the coexpression of AP1 isoforms μ1A and μ1B (expressed only in polarized cells) in melanocytes and keratinocytes, but expression of μ1B is missing in some melanoma cell lines. Transfection with AP1 isoforms (μ1A or μ1B) showed two distinct distribution patterns that involved Pmel17, and only μ1B was able to restore the sorting of Pmel17 to the plasma membrane in cells lacking μ1B expression. Finally, we established that expression of μ1B is regulated physiologically in melanocytes by UV radiation or DKK1. These results show that Pmel17 is sorted to melanosomes by various intracellular routes, directly or indirectly through the plasma membrane, and the presence of basolateral elements in melanocytes suggests their polarized nature. PMID:16492709

Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT

PubMed Central

Roundhill, E; Burchill, S

2013-01-01

Background: Primary Ewing's sarcoma family of tumours (ESFTs) may respond to chemotherapy, although many patients experience subsequent disease recurrence and relapse. The survival of ESFT cells following chemotherapy has been attributed to the development of resistant disease, possibly through the expression of ABC transporter proteins. Methods: MRP-1 and Pgp mRNA and protein expression in primary ESFTs was determined by quantitative reverse-transcriptase PCR (RT-qPCR) and immunohistochemistry, respectively, and alternative splicing of MRP-1 by RT-PCR. Results: We observed MRP-1 protein expression in 92% (43 out of 47) of primary ESFTs, and cell membrane MRP-1 was highly predictive of both overall survival (P<0.0001) and event-free survival (P<0.0001). Alternative splicing of MRP-1 was detected in primary ESFTs, although the pattern of splicing variants was not predictive of patient outcome, with the exception of loss of exon 9 in six patients, which predicted relapse (P=0.041). Pgp protein was detected in 6% (38 out of 44) of primary ESFTs and was not associated with patient survival. Conclusion: For the first time we have established that cell membrane expression of MRP-1 or loss of exon 9 is predictive of outcome but not the number of splicing events or expression of Pgp, and both may be valuable factors for the stratification of patients for more intensive therapy. PMID:23799853

Nitrate and ammonium lead to distinct global dynamic phosphorylation patterns when resupplied to nitrogen-starved Arabidopsis seedlings.

PubMed

Engelsberger, Wolfgang R; Schulze, Waltraud X

2012-03-01

Nitrogen is an essential macronutrient for plant growth and development. Inorganic nitrogen and its assimilation products control various metabolic, physiological and developmental processes. Although the transcriptional responses induced by nitrogen have been extensively studied in the past, our work here focused on the discovery of candidate proteins for regulatory events that are complementary to transcriptional changes. Most signaling pathways involve modulation of protein abundance and/or activity by protein phosphorylation. Therefore, we analyzed the dynamic changes in protein phosphorylation in membrane and soluble proteins from plants exposed to rapid changes in nutrient availability over a time course of 30 min. Plants were starved of nitrogen and subsequently resupplied with nitrogen in the form of nitrate or ammonium. Proteins with maximum change in their phosphorylation level at up to 5 min after nitrogen resupply (fast responses) included GPI-anchored proteins, receptor kinases and transcription factors, while proteins with maximum change in their phosphorylation level after 10 min of nitrogen resupply (late responses) included proteins involved in protein synthesis and degradation, as well as proteins with functions in central metabolism and hormone metabolism. Resupply of nitrogen in the form of nitrate or ammonium resulted in distinct phosphorylation patterns, mainly of proteins with signaling functions, transcription factors and transporters. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Differences in the protein composition of bovine retinal rod outer segment disk and plasma membranes isolated by a ricin-gold-dextran density perturbation method

PubMed Central

1987-01-01

The plasma membrane and disk membranes of bovine retinal rod outer segments (ROS) have been purified by a novel density-gradient perturbation method for analysis of their protein compositions. Purified ROS were treated with neuraminidase to expose galactose residues on plasma membrane-specific glycoproteins and labeled with ricin-gold-dextran particles. After the ROS were lysed in hypotonic buffer, the plasma membrane was dissociated from the disks by either mild trypsin digestion or prolonged exposure to low ionic strength buffer. The dense ricin-gold-dextran-labeled plasma membrane was separated from disks by sucrose gradient centrifugation. Electron microscopy was used to follow this fractionation procedure. The dense red pellet primarily consisted of inverted plasma membrane vesicles containing gold particles; the membrane fraction of density 1.13 g/cc consisted of unlabeled intact disks and vesicles. Ricin-binding studies indicated that the plasma membrane from trypsin-treated ROS was purified between 10-15-fold. The protein composition of plasma membranes and disks was significantly different as analyzed by SDS gels and Western blots labeled with lectins and monoclonal antibodies. ROS plasma membrane exhibited three major proteins of 36 (rhodopsin), 38, and 52 kD, three ricin-binding glycoproteins of 230, 160, and 110 kD, and numerous minor proteins in the range of 14-270 kD. In disk membranes rhodopsin appeared as the only major protein. A 220-kD concanavalin A-binding glycoprotein and peripherin, a rim-specific protein, were also present along with minor proteins of 43 and 57-63 kD. Radioimmune assays indicated that the ROS plasma membrane contained about half as much rhodopsin as disk membranes. PMID:2447095

Folding DNA into a Lipid-Conjugated Nanobarrel for Controlled Reconstitution of Membrane Proteins.

PubMed

Dong, Yuanchen; Chen, Shuobing; Zhang, Shijian; Sodroski, Joseph; Yang, Zhongqiang; Liu, Dongsheng; Mao, Youdong

2018-02-19

Building upon DNA origami technology, we introduce a method to reconstitute a single membrane protein into a self-assembled DNA nanobarrel that scaffolds a nanodisc-like lipid environment. Compared with the membrane-scaffolding-protein nanodisc technique, our approach gives rise to defined stoichiometry, controlled sizes, as well as enhanced stability and homogeneity in membrane protein reconstitution. We further demonstrate potential applications of the DNA nanobarrels in the structural analysis of membrane proteins. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Production of okara and soy protein concentrates using membrane technology.

PubMed

Vishwanathan, K H; Govindaraju, K; Singh, Vasudeva; Subramanian, R

2011-01-01

Microfiltration (MF) membranes with pore sizes of 200 and 450 nm and ultrafiltration (UF) membranes with molecular weight cut off of 50, 100, and 500 kDa were assessed for their ability to eliminate nonprotein substances from okara protein extract in a laboratory cross-flow membrane system. Both MF and UF improved the protein content of okara extract to a similar extent from approximately 68% to approximately 81% owing to the presence of protein in the feed leading to the formation of dynamic layer controlling the performance rather than the actual pore size of membranes. Although normalized flux in MF-450 (117 LMH/MPa) was close to UF-500 (118 LMH/MPa), the latter was selected based on higher average flux (47 LMH) offering the advantage of reduced processing time. Membrane processing of soy extract improved the protein content from 62% to 85% much closer to the target value. However, the final protein content in okara (approximately 80%) did not reach the target value (90%) owing to the greater presence of soluble fibers that were retained by the membrane. Solubility curve of membrane okara protein concentrate (MOPC) showed lower solubility than soy protein concentrate and a commercial isolate in the entire pH range. However, water absorption and fat-binding capacities of MOPC were either superior or comparable while emulsifying properties were in accordance with its solubility. The results of this study showed that okara protein concentrate (80%) could be produced using membrane technology without loss of any true proteins, thus offering value addition to okara, hitherto underutilized. Practical Application: Okara, a byproduct obtained during processing soybean for soymilk, is either underutilized or unutilized in spite of the fact that its protein quality is as good as that of soy milk and tofu. Membrane-processed protein products have been shown to possess superior functional properties compared to conventionally produced protein products. However, the potential of membrane technology has not been exploited for the recovery of okara protein. Our study showed that protein content of okara extract could be improved from approximately 68% to approximately 81% without losing any true proteins in the process.

Systematic Identification and Characterization of Novel Human Skin-Associated Genes Encoding Membrane and Secreted Proteins

PubMed Central

Buhren, Bettina Alexandra; Martinez, Cynthia; Schrumpf, Holger; Gasis, Marcia; Grether-Beck, Susanne; Krutmann, Jean

2013-01-01

Through bioinformatics analyses of a human gene expression database representing 105 different tissues and cell types, we identified 687 skin-associated genes that are selectively and highly expressed in human skin. Over 50 of these represent uncharacterized genes not previously associated with skin and include a subset that encode novel secreted and plasma membrane proteins. The high levels of skin-associated expression for eight of these novel therapeutic target genes were confirmed by semi-quantitative real time PCR, western blot and immunohistochemical analyses of normal skin and skin-derived cell lines. Four of these are expressed specifically by epidermal keratinocytes; two that encode G-protein-coupled receptors (GPR87 and GPR115), and two that encode secreted proteins (WFDC5 and SERPINB7). Further analyses using cytokine-activated and terminally differentiated human primary keratinocytes or a panel of common inflammatory, autoimmune or malignant skin diseases revealed distinct patterns of regulation as well as disease associations that point to important roles in cutaneous homeostasis and disease. Some of these novel uncharacterized skin genes may represent potential biomarkers or drug targets for the development of future diagnostics or therapeutics. PMID:23840300

Outer Membrane Targeting of Passenger Proteins by the Vacuolating Cytotoxin Autotransporter of Helicobacter pylori

PubMed Central

Fischer, Wolfgang; Buhrdorf, Renate; Gerland, Elke; Haas, Rainer

2001-01-01

Helicobacter pylori produces a number of proteins associated with the outer membrane, including adhesins and the vacuolating cytotoxin. These proteins are supposed to integrate into the outer membrane by β-barrel structures, characteristic of the family of autotransporter proteins. By using the SOMPES (shuttle vector-based outer membrane protein expression) system for outer membrane protein production, we were able to functionally express in H. pylori the cholera toxin B subunit genetically fused to the C-terminal VacA domain. We demonstrate that the fusion protein is translocated to the H. pylori outer membrane and that the CtxB domain is exposed on the H. pylori surface. Thus, we provide the first experimental evidence that the C-terminal β-domain of VacA can transport a foreign passenger protein to the H. pylori surface and hence acts as a functional autotransporter. PMID:11598049

Retention mechanisms for ER and Golgi membrane proteins.

PubMed

Gao, Caiji; Cai, Yi; Wang, Yejun; Kang, Byung-Ho; Aniento, Fernando; Robinson, David G; Jiang, Liwen

2014-08-01

Unless there are mechanisms to selectively retain membrane proteins in the endoplasmic reticulum (ER) or in the Golgi apparatus, they automatically proceed downstream to the plasma or vacuole membranes. Two types of coat protein complex I (COPI)-interacting motifs in the cytosolic tails of membrane proteins seem to facilitate membrane retention in the early secretory pathway of plants: a dilysine (KKXX) motif (which is typical of p24 proteins) for the ER and a KXE/D motif (which occurs in the Arabidopsis endomembrane protein EMP12) for the Golgi apparatus. The KXE/D motif is highly conserved in all eukaryotic EMPs and is additionally present in hundreds of other proteins of unknown subcellular localization and function. This novel signal may represent a new general mechanism for Golgi targeting and the retention of polytopic integral membrane proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

The fine art of integral membrane protein crystallisation.

PubMed

Birch, James; Axford, Danny; Foadi, James; Meyer, Arne; Eckhardt, Annette; Thielmann, Yvonne; Moraes, Isabel

2018-05-18

Integral membrane proteins are among the most fascinating and important biomolecules as they play a vital role in many biological functions. Knowledge of their atomic structures is fundamental to the understanding of their biochemical function and key in many drug discovery programs. However, over the years, structure determination of integral membrane proteins has proven to be far from trivial, hence they are underrepresented in the protein data bank. Low expression levels, insolubility and instability are just a few of the many hurdles one faces when studying these proteins. X-ray crystallography has been the most used method to determine atomic structures of membrane proteins. However, the production of high quality membrane protein crystals is always very challenging, often seen more as art than a rational experiment. Here we review valuable approaches, methods and techniques to successful membrane protein crystallisation. Copyright © 2018 Diamond Light Source LTD. Published by Elsevier Inc. All rights reserved.

Endocytosis of GPI-anchored proteins in human lymphocytes: role of glycolipid-based domains, actin cytoskeleton, and protein kinases

PubMed Central

1996-01-01

GPI-anchored surface proteins mediate many important functions, including transport, signal transduction, adhesion, and protection against complement. They cluster into glycolipid-based membrane domains and caveolae, plasmalemmal vesicles involved in the transcytosis and endocytosis of these surface proteins. However, in lymphocytes, neither the characteristic flask shaped caveolae nor caveolin, a transmembrane protein typical of caveolae, have been observed. Here, we show that the GPI-anchored CD59 molecule on Jurkat T cells is internalized after cross-linking, a process inhibited by nystatin, a sterol chelating agent. Clustered CD59 molecules mostly accumulate in non-coated invaginations of the lymphocyte membrane before endocytosis, in marked contrast with the pattern of CD3-TCR internalization. Cytochalasin H blocked CD59 internalization in lymphocytes, but neither CD3 internalization nor transferrin uptake. Confocal microscopy analysis of F-actin distribution within lymphocytes showed that CD59 clusters were associated with patches of polymerized actin. Also, we found that internalization of CD59 was prevented by the protein kinase C inhibitor staurosporine and by the protein kinase A activator forskolin. Thus, in lymphocytes, as in other cell types, glycolipid-based domains provide sites of integration of signaling pathways involved in GPI-anchored protein endocytosis. This process, which is regulated by both protein kinase C and A activity, is tightly controlled by the dynamic organization of actin cytoskeleton, and may be critical for polarized contacts of circulating cells. PMID:8666664

Membrane Protein Crystallization Using Laser Irradiation

NASA Astrophysics Data System (ADS)

Adachi, Hiroaki; Murakami, Satoshi; Niino, Ai; Matsumura, Hiroyoshi; Takano, Kazufumi; Inoue, Tsuyoshi; Mori, Yusuke; Yamaguchi, Akihito; Sasaki, Takatomo

2004-10-01

We demonstrate the crystallization of a membrane protein using femtosecond laser irradiation. This method, which we call the laser irradiated growth technique (LIGHT), is useful for producing AcrB crystals in a solution of low supersaturation range. LIGHT is characterized by reduced nucleation times. This feature is important for crystallizing membrane proteins because of their labile properties when solubilized as protein-detergent micelles. Using LIGHT, high-quality crystals of a membrane transporter protein, AcrB, were obtained. The resulting crystals were found to be of sufficiently high resolution for X-ray diffraction. The results reported here indicate that LIGHT is a powerful tool for membrane protein crystallization, as well as for the growth of soluble proteins.

Investigation of Surface Enhanced Coherent Raman Scattering on Nano-patterned Insect Wings

NASA Astrophysics Data System (ADS)

Ujj, Laszlo; Lawhead, Carlos

2015-03-01

Many insect wings (cicadas, butterflies, mosquitos) poses nano-patterned surface structure. Characterization of surface morphology and chemical composition of insect wings is important to understand the extreme mechanical properties and the biophysical functionalities of the wings. We have measured the image of the membrane of a cicada's wing with the help of Scanning Electron Microscopy (SEM). The results confirm the existing periodic structure of the wing measured previously. In order to identify the chemical composition of the wing, we have deposited silver nanoparticles on it and applied Coherent anti-Stokes Raman Spectroscopy to measure the vibrational spectra of the molecules comprising the wing for the first time. The measured spectra are consistent with the original assumption that the wing membrane is composed of protein, wax, and chitin. The results of these studies can be used to measure other nano-patterned surfaces and to make artificial materials in the future. Authors grateful for financial support from the Department of Physics of the College of Sciences Engineering and Health of UWF and the Pall Corporation for SEM imaging.

Predicting membrane protein types by fusing composite protein sequence features into pseudo amino acid composition.

PubMed

Hayat, Maqsood; Khan, Asifullah

2011-02-21

Membrane proteins are vital type of proteins that serve as channels, receptors, and energy transducers in a cell. Prediction of membrane protein types is an important research area in bioinformatics. Knowledge of membrane protein types provides some valuable information for predicting novel example of the membrane protein types. However, classification of membrane protein types can be both time consuming and susceptible to errors due to the inherent similarity of membrane protein types. In this paper, neural networks based membrane protein type prediction system is proposed. Composite protein sequence representation (CPSR) is used to extract the features of a protein sequence, which includes seven feature sets; amino acid composition, sequence length, 2 gram exchange group frequency, hydrophobic group, electronic group, sum of hydrophobicity, and R-group. Principal component analysis is then employed to reduce the dimensionality of the feature vector. The probabilistic neural network (PNN), generalized regression neural network, and support vector machine (SVM) are used as classifiers. A high success rate of 86.01% is obtained using SVM for the jackknife test. In case of independent dataset test, PNN yields the highest accuracy of 95.73%. These classifiers exhibit improved performance using other performance measures such as sensitivity, specificity, Mathew's correlation coefficient, and F-measure. The experimental results show that the prediction performance of the proposed scheme for classifying membrane protein types is the best reported, so far. This performance improvement may largely be credited to the learning capabilities of neural networks and the composite feature extraction strategy, which exploits seven different properties of protein sequences. The proposed Mem-Predictor can be accessed at http://111.68.99.218/Mem-Predictor. Copyright © 2010 Elsevier Ltd. All rights reserved.

Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens.

PubMed

Raguz, Marija; Mainali, Laxman; O'Brien, William J; Subczynski, Witold K

2014-03-01

The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O'Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

Lipid-Protein Interactions in Plasma Membranes of Fiber Cells Isolated from the Human Eye Lens

PubMed Central

Raguz, Marija; Mainali, Laxman; O’Brien, William J.; Subczynski, Witold K.

2014-01-01

The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali,L., Raguz, M., O’Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed. PMID:24486794

The synthesis of recombinant membrane proteins in yeast for structural studies.

PubMed

Routledge, Sarah J; Mikaliunaite, Lina; Patel, Anjana; Clare, Michelle; Cartwright, Stephanie P; Bawa, Zharain; Wilks, Martin D B; Low, Floren; Hardy, David; Rothnie, Alice J; Bill, Roslyn M

2016-02-15

Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Membrane remodeling by amyloidogenic and non-amyloidogenic proteins studied by EPR.

PubMed

Varkey, Jobin; Langen, Ralf

2017-07-01

The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single protein can generate different type of membrane curvatures using specific conformations for specific membrane structures and how EPR is a versatile tool well-suited to analyze subtle alterations in structures under such modifying conditions which otherwise would have been difficult using other biophysical tools. Copyright © 2017 Elsevier Inc. All rights reserved.

Examining hemodialyzer membrane performance using proteomic technologies

PubMed Central

Bonomini, Mario; Pieroni, Luisa; Di Liberato, Lorenzo; Sirolli, Vittorio; Urbani, Andrea

2018-01-01

The success and the quality of hemodialysis therapy are mainly related to both clearance and biocompatibility properties of the artificial membrane packed in the hemodialyzer. Performance of a membrane is strongly influenced by its interaction with the plasma protein repertoire during the extracorporeal procedure. Recognition that a number of medium–high molecular weight solutes, including proteins and protein-bound molecules, are potentially toxic has prompted the development of more permeable membranes. Such membrane engineering, however, may cause loss of vital proteins, with membrane removal being nonspecific. In addition, plasma proteins can be adsorbed onto the membrane surface upon blood contact during dialysis. Adsorption can contribute to the removal of toxic compounds and governs the biocompatibility of a membrane, since surface-adsorbed proteins may trigger a variety of biologic blood pathways with pathophysiologic consequences. Over the last years, use of proteomic approaches has allowed polypeptide spectrum involved in the process of hemodialysis, a key issue previously hampered by lack of suitable technology, to be assessed in an unbiased manner and in its full complexity. Proteomics has been successfully applied to identify and quantify proteins in complex mixtures such as dialysis outflow fluid and fluid desorbed from dialysis membrane containing adsorbed proteins. The identified proteins can also be characterized by their involvement in metabolic and signaling pathways, molecular networks, and biologic processes through application of bioinformatics tools. Proteomics may thus provide an actual functional definition as to the effect of a membrane material on plasma proteins during hemodialysis. Here, we review the results of proteomic studies on the performance of hemodialysis membranes, as evaluated in terms of solute removal efficiency and blood–membrane interactions. The evidence collected indicates that the information provided by proteomic investigations yields improved molecular and functional knowledge and may lead to the development of more efficient membranes for the potential benefit of the patient. PMID:29296087

Examining hemodialyzer membrane performance using proteomic technologies.

PubMed

Bonomini, Mario; Pieroni, Luisa; Di Liberato, Lorenzo; Sirolli, Vittorio; Urbani, Andrea

2018-01-01

The success and the quality of hemodialysis therapy are mainly related to both clearance and biocompatibility properties of the artificial membrane packed in the hemodialyzer. Performance of a membrane is strongly influenced by its interaction with the plasma protein repertoire during the extracorporeal procedure. Recognition that a number of medium-high molecular weight solutes, including proteins and protein-bound molecules, are potentially toxic has prompted the development of more permeable membranes. Such membrane engineering, however, may cause loss of vital proteins, with membrane removal being nonspecific. In addition, plasma proteins can be adsorbed onto the membrane surface upon blood contact during dialysis. Adsorption can contribute to the removal of toxic compounds and governs the biocompatibility of a membrane, since surface-adsorbed proteins may trigger a variety of biologic blood pathways with pathophysiologic consequences. Over the last years, use of proteomic approaches has allowed polypeptide spectrum involved in the process of hemodialysis, a key issue previously hampered by lack of suitable technology, to be assessed in an unbiased manner and in its full complexity. Proteomics has been successfully applied to identify and quantify proteins in complex mixtures such as dialysis outflow fluid and fluid desorbed from dialysis membrane containing adsorbed proteins. The identified proteins can also be characterized by their involvement in metabolic and signaling pathways, molecular networks, and biologic processes through application of bioinformatics tools. Proteomics may thus provide an actual functional definition as to the effect of a membrane material on plasma proteins during hemodialysis. Here, we review the results of proteomic studies on the performance of hemodialysis membranes, as evaluated in terms of solute removal efficiency and blood-membrane interactions. The evidence collected indicates that the information provided by proteomic investigations yields improved molecular and functional knowledge and may lead to the development of more efficient membranes for the potential benefit of the patient.

Membrane protein stoichiometry studied in intact mammalian cells using liquid-phase electron microscopy.

PubMed

DE Jonge, N

2018-02-01

Receptor membrane proteins in the plasma membranes of cells respond to extracellular chemical signals by conformational changes, spatial redistribution, and (re-)assembly into protein complexes, for example, into homodimers (pairs of the same protein type). The functional state of the proteins can be determined from information about how subunits are assembled into protein complexes. Stoichiometric information about the protein complex subunits, however, is generally not obtained from intact cells but from pooled material extracted from many cells, resulting in a lack of fundamental knowledge about the functioning of membrane proteins. First, functional states may dramatically differ from cell to cell on account of cell heterogeneity. Second, extracting the membrane proteins from the plasma membrane may lead to many artefacts. Liquid-phase scanning transmission electron microscopy (STEM), in short liquid STEM, is a new technique capable of determining the locations of individual membrane proteins within the intact plasma membranes of cells in liquid. Many tens of whole cells can readily be imaged. It is possible to analyse the stoichiometry of membrane proteins in single cells while accounting for heterogenic cell populations. Liquid STEM was used to image epidermal growth factor receptors in whole COS7 cells. A study of the dimerisation of the HER2 protein in breast cancer cells revealed the presence of rare cancer cells in which HER2 was in a different functional state than in the bulk cells. Stoichiometric information about receptors is essential not only for basic science but also for biomedical application because they present many important pharmaceutical targets. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.

PubMed

Dormeyer, Wilma; van Hoof, Dennis; Mummery, Christine L; Krijgsveld, Jeroen; Heck, Albert J R

2008-10-01

The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological mechanisms that regulate proliferation and differentiation. The comparison of their membrane proteomes will help unravel the biological principles of pluripotency, and the identification of biomarker proteins in their plasma membranes is considered a crucial step to fully exploit pluripotent cells for therapeutic purposes. For these tasks, membrane proteomics is the method of choice, but as indicated by the scarce identification of membrane and plasma membrane proteins in global proteomic surveys it is not an easy task. In this minireview, we first describe the general challenges of membrane proteomics. We then review current sample preparation steps and discuss protocols that we found particularly beneficial for the identification of large numbers of (plasma) membrane proteins in human tumour- and embryo-derived stem cells. Our optimized assembled protocol led to the identification of a large number of membrane proteins. However, as the composition of cells and membranes is highly variable we still recommend adapting the sample preparation protocol for each individual system.

Protein-membrane interaction and fatty acid transfer from intestinal fatty acid-binding protein to membranes. Support for a multistep process.

PubMed

Falomir-Lockhart, Lisandro J; Laborde, Lisandro; Kahn, Peter C; Storch, Judith; Córsico, Betina

2006-05-19

Fatty acid transfer from intestinal fatty acid-binding protein (IFABP) to phospholipid membranes occurs during protein-membrane collisions. Electrostatic interactions involving the alpha-helical "portal" region of the protein have been shown to be of great importance. In the present study, the role of specific lysine residues in the alpha-helical region of IFABP was directly examined. A series of point mutants in rat IFABP was engineered in which the lysine positive charges in this domain were eliminated or reversed. Using a fluorescence resonance energy transfer assay, we analyzed the rates and mechanism of fatty acid transfer from wild type and mutant proteins to acceptor membranes. Most of the alpha-helical domain mutants showed slower absolute fatty acid transfer rates to zwitterionic membranes, with substitution of one of the lysines of the alpha2 helix, Lys27, resulting in a particularly dramatic decrease in the fatty acid transfer rate. Sensitivity to negatively charged phospholipid membranes was also reduced, with charge reversal mutants in the alpha2 helix the most affected. The results support the hypothesis that the portal region undergoes a conformational change during protein-membrane interaction, which leads to release of the bound fatty acid to the membrane and that the alpha2 segment is of particular importance in the establishment of charge-charge interactions between IFABP and membranes. Cross-linking experiments with a phospholipid-photoactivable reagent underscored the importance of charge-charge interactions, showing that the physical interaction between wild-type intestinal fatty acid-binding protein and phospholipid membranes is enhanced by electrostatic interactions. Protein-membrane interactions were also found to be enhanced by the presence of ligand, suggesting different collisional complex structures for holo- and apo-IFABP.

Interplay between membrane curvature and protein conformational equilibrium investigated by solid-state NMR.

PubMed

Liao, Shu Y; Lee, Myungwoon; Hong, Mei

2018-03-01

Many membrane proteins sense and induce membrane curvature for function, but structural information about how proteins modulate their structures to cause membrane curvature is sparse. We review our recent solid-state NMR studies of two virus membrane proteins whose conformational equilibrium is tightly coupled to membrane curvature. The influenza M2 proton channel has a drug-binding site in the transmembrane (TM) pore. Previous chemical shift data indicated that this pore-binding site is lost in an M2 construct that contains the TM domain and a curvature-inducing amphipathic helix. We have now obtained chemical shift perturbation, protein-drug proximity, and drug orientation data that indicate that the pore-binding site is restored when the full cytoplasmic domain is present. This finding indicates that the curvature-inducing amphipathic helix distorts the TM structure to interfere with drug binding, while the cytoplasmic tail attenuates this effect. In the second example, we review our studies of a parainfluenza virus fusion protein that merges the cell membrane and the virus envelope during virus entry. Chemical shifts of two hydrophobic domains of the protein indicate that both domains have membrane-dependent backbone conformations, with the β-strand structure dominating in negative-curvature phosphatidylethanolamine (PE) membranes. 31 P NMR spectra and 1 H- 31 P correlation spectra indicate that the β-strand-rich conformation induces saddle-splay curvature to PE membranes and dehydrates them, thus stabilizing the hemifusion state. These results highlight the indispensable role of solid-state NMR to simultaneously determine membrane protein structures and characterize the membrane curvature in which these protein structures exist. Copyright © 2018 Elsevier Inc. All rights reserved.

Pannexin-1 channels show distinct morphology and no gap junction characteristics in mammalian cells.

PubMed

Beckmann, Anja; Grissmer, Alexander; Krause, Elmar; Tschernig, Thomas; Meier, Carola

2016-03-01

Pannexins (Panx) are proteins with a similar membrane topology to connexins, the integral membrane protein of gap junctions. Panx1 channels are generally of major importance in a large number of system and cellular processes and their function has been thoroughly characterized. In contrast, little is known about channel structure and subcellular distribution. We therefore determine the subcellular localization of Panx1 channels in cultured cells and aim at the identification of channel morphology in vitro. Using freeze-fracture replica immunolabeling on EYFP-Panx1-overexpressing HEK 293 cells, large particles were identified in plasma membranes, which were immunogold-labeled using either GFP or Panx1 antibodies. There was no labeling or particles in the nuclear membranes of these cells, pointing to plasma membrane localization of Panx1-EYFP channels. The assembly of particles was irregular, this being in contrast to the regular pattern of gap junctions. The fact that no counterparts were identified on apposing cells, which would have been indicative of intercellular signaling, supported the idea of Panx1 channels within one membrane. Control cells (transfected with EYFP only, non-transfected) were devoid of both particles and immunogold labeling. Altogether, this study provides the first demonstration of Panx1 channel morphology and assembly in intact cells. The identification of Panx1 channels as large particles within the plasma membrane provides the knowledge required to enable recognition of Panx1 channels in tissues in future studies. Thus, these results open up new avenues for the detailed analysis of the subcellular localization of Panx1 and of its nearest neighbors such as purinergic receptors in vivo.

Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

NASA Technical Reports Server (NTRS)

Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

2000-01-01

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography.

PubMed

Hu, S; Brady, S R; Kovar, D R; Staiger, C J; Clark, G B; Roux, S J; Muday, G K

2000-10-01

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

Inhibition of phosphatidylinositol 3,5-bisphosphate production has pleiotropic effects on various membrane trafficking routes in Arabidopsis.

PubMed

Hirano, Tomoko; Munnik, Teun; Sato, Masa H

2017-01-01

Phosphoinositides play an important role in various membrane trafficking events in eukaryotes. One of them, however, phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2], has not been studied widely in plants. Using a combination of fluorescent reporter proteins and the PI(3,5)P2-specific inhibitor YM202636, here we demonstrated that in Arabidopsis thaliana, PI(3,5)P2 affects various membrane trafficking events, mostly in the post-Golgi routes. We found that YM201636 treatment effectively reduced PI(3,5)P2 concentration not only in the wild type but also in FAB1A-overexpressing Arabidopsis plants. In particular, reduced PI(3,5)P2 levels caused abnormal membrane dynamics of plasma membrane proteins, AUX1 and BOR1, with different trafficking patterns. Secretion and morphological characteristics of late endosomes and vacuoles were also affected by the decreased PI(3,5)P2 production. These pleiotropic defects in the post-Golgi trafficking events were caused by the inhibition of PI(3,5)P2 production. This effect is probably mediated by the inhibition of maturation of FAB1-positive late endosomes, thereby impairing late endosome function. In conclusion, our results imply that in Arabidopsis, late endosomes are involved in multiple post-Golgi membrane trafficking routes including not only vacuolar trafficking and endocytosis but also secretion. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Affinity reagent technology development and application to rapid immunochromatographic pathogen detection

NASA Astrophysics Data System (ADS)

Sooter, Letha J.; Stratis-Cullum, Dimitra N.; Zhang, Yanting; Daugherty, Patrick S.; Soh, H. Tom; Pellegrino, Paul; Stagliano, Nancy

2007-09-01

Immunochromatography is a rapid, reliable, and cost effective method of detecting biowarfare agents. The format is similar to that of an over-the-counter pregnancy test. A sample is applied to one end of a cassette and then a control line, and possibly a sample line, are visualized at the other end of the cassette. The test is based upon a sandwich assay. For the control, a line of Protein A is immobilized on the membrane. Gold nanoparticle bound IgG flows through the membrane and binds the Protein A, creating a visible line on the membrane. For the sample, one epitope is immobilized on the membrane and another epitope is attached to gold nanoparticles. The sample binds gold bound epitope, travels through the membrane, and binds membrane bound epitope. The two epitopes are not cross-reactive, therefore a sample line is only visible if the sample is present. In order to efficiently screen for binders to a sample target, a novel, Continuous Magnetic Activated Cell Sorter (CMACS) has been developed on a disposable, microfluidic platform. The CMACS chip quickly sorts E. coli peptide libraries for target binders with high affinity. Peptide libraries, are composed of approximately ten million bacteria, each displaying a different peptide on their surface. The target of interest is conjugated to a micrometer sized magnetic particle. After the library and the target are incubated together to allow binding, the mixture is applied to the CMACS chip. In the presence of patterned nickel and an external magnet, separation occurs of the bead-bound bacteria from the bulk material. The bead fraction is added to bacterial growth media where any attached E. coli grow and divide. These cells are cloned, sequenced, and the peptides are assayed for target binding affinity. As a proof-of-principle, assays were developed for human C-reactive protein. More defense relevant targets are currently being pursued.

The interaction between Pseudomonas aeruginosa cells and cationic PC:Chol:DOTAP liposomal vesicles versus outer-membrane structure and envelope properties of bacterial cell.

PubMed

Drulis-Kawa, Zuzanna; Dorotkiewicz-Jach, Agata; Gubernator, Jerzy; Gula, Grzegorz; Bocer, Tomasz; Doroszkiewicz, Wlodzimierz

2009-02-09

The interactions between cationic liposomal formulations (PC:Chol:DOTAP 3:4:3) and 23 Pseudomonas aeruginosa strains were tested. The study was undertaken because different antimicrobial results had been obtained by the authors for Pseudomonas aeruginosa strains and liposomal antibiotics (Drulis-Kawa, Z., Gubernator, J., Dorotkiewicz-Jach, A., Doroszkiewicz, W., Kozubek, A., 2006. The comparison of in vitro antimicrobial activity of liposomes containing meropenem and gentamicin. Cell. Mol. Biol. Lett., 11, 360-375; Drulis-Kawa, Z., Gubernator, J., Dorotkiewicz-Jach, A., Doroszkiewicz W., Kozubek, A., 2006. In vitro antimicrobial activity of liposomal meropenem against Pseudomonas aeruginosa strains. Int. J. Pharm., 315, 59-66). The experiments evaluate the roles of the bacterial outer-membrane structure, especially outer-membrane proteins and LPS, and envelope properties (hydrophobicity and electrostatic potential) in the interactions/fusion process between cells and lipid vesicles. The interactions were examined by fluorescent microscopy using PE-rhodamine-labelled liposomes. Some of the strains exhibited red-light emission (fusion with vesicles or vesicles surrounding the cell) and some showed negative reaction (no red-light emission). The main aim of the study was to determine what kinds of bacterial structure or envelope properties have a major influence on the fusion process. Negatively charged cells and hydrophobic properties promote interaction with cationic lipid vesicles, but no specific correlation was noted for the tested strains. A similar situation concerned LPS structure, where parent strains and their mutants possessing identical ladder-like band patterns in SDS-PAGE analysis exhibited totally different results with fluorescent microscopy. Outer-membrane protein analysis showed that an 18-kDA protein occurred in the isolates showing fusion with rhodamine-labelled vesicles and, conversely, strains lacking the 18-kDA protein exhibited no positive reaction (red emission). This suggests that even one protein may be responsible for favouring stronger interactions between Pseudomonas aeruginosa cells and cationic liposomal formulations (PC:Chol:DOTAP 3:4:3).

Palm is expressed in both developing and adult mouse lens and retina

PubMed Central

Castellini, Meryl; Wolf, Louise V; Chauhan, Bharesh K; Galileo, Deni S; Kilimann, Manfred W; Cvekl, Ales; Duncan, Melinda K

2005-01-01

Background Paralemmin (Palm) is a prenyl-palmitoyl anchored membrane protein that can drive membrane and process formation in neurons. Earlier studies have shown brain preferred Palm expression, although this protein is a major water insoluble protein in chicken lens fiber cells and the Palm gene may be regulated by Pax6. Methods The expression profile of Palm protein in the embryonic, newborn and adult mouse eye as well as dissociated retinal neurons was determined by confocal immunofluorescence. The relative mRNA levels of Palm, Palmdelphin (PalmD) and paralemmin2 (Palm2) in the lens and retina were determined by real time rt-PCR. Results In the lens, Palm is already expressed at 9.5 dpc in the lens placode, and this expression is maintained in the lens vesicle throughout the formation of the adult lens. Palm is largely absent from the optic vesicle but is detectable at 10.5 dpc in the optic cup. In the developing retina, Palm expression transiently upregulates during the formation of optic nerve as well as in the formation of both the inner and outer plexiform layers. In short term dissociated chick retinal cultures, Palm protein is easily detectable, but the levels appear to reduce sharply as the cultures age. Palm mRNA was found at much higher levels relative to Palm2 or PalmD in both the retina and lens. Conclusion Palm is the major paralemmin family member expressed in the retina and lens and its expression in the retina transiently upregulates during active neurite outgrowth. The expression pattern of Palm in the eye is consistent with it being a Pax6 responsive gene. Since Palm is known to be able to drive membrane formation in brain neurons, it is possible that this molecule is crucial for the increase in membrane formation during lens fiber cell differentiation. PMID:15969763

Computational and theoretical approaches for studies of a lipid recognition protein on biological membranes

PubMed Central

Yamamoto, Eiji

2017-01-01

Many cellular functions, including cell signaling and related events, are regulated by the association of peripheral membrane proteins (PMPs) with biological membranes containing anionic lipids, e.g., phosphatidylinositol phosphate (PIP). This association is often mediated by lipid recognition modules present in many PMPs. Here, I summarize computational and theoretical approaches to investigate the molecular details of the interactions and dynamics of a lipid recognition module, the pleckstrin homology (PH) domain, on biological membranes. Multiscale molecular dynamics simulations using combinations of atomistic and coarse-grained models yielded results comparable to those of actual experiments and could be used to elucidate the molecular mechanisms of the formation of protein/lipid complexes on membrane surfaces, which are often difficult to obtain using experimental techniques. Simulations revealed some modes of membrane localization and interactions of PH domains with membranes in addition to the canonical binding mode. In the last part of this review, I address the dynamics of PH domains on the membrane surface. Local PIP clusters formed around the proteins exhibit anomalous fluctuations. This dynamic change in protein-lipid interactions cause temporally fluctuating diffusivity of proteins, i.e., the short-term diffusivity of the bound protein changes substantially with time, and may in turn contribute to the formation/dissolution of protein complexes in membranes. PMID:29159013

Mitochondrial Protein Synthesis, Import, and Assembly

PubMed Central

Fox, Thomas D.

2012-01-01

The mitochondrion is arguably the most complex organelle in the budding yeast cell cytoplasm. It is essential for viability as well as respiratory growth. Its innermost aqueous compartment, the matrix, is bounded by the highly structured inner membrane, which in turn is bounded by the intermembrane space and the outer membrane. Approximately 1000 proteins are present in these organelles, of which eight major constituents are coded and synthesized in the matrix. The import of mitochondrial proteins synthesized in the cytoplasm, and their direction to the correct soluble compartments, correct membranes, and correct membrane surfaces/topologies, involves multiple pathways and macromolecular machines. The targeting of some, but not all, cytoplasmically synthesized mitochondrial proteins begins with translation of messenger RNAs localized to the organelle. Most proteins then pass through the translocase of the outer membrane to the intermembrane space, where divergent pathways sort them to the outer membrane, inner membrane, and matrix or trap them in the intermembrane space. Roughly 25% of mitochondrial proteins participate in maintenance or expression of the organellar genome at the inner surface of the inner membrane, providing 7 membrane proteins whose synthesis nucleates the assembly of three respiratory complexes. PMID:23212899

Identification of the triazine receptor protein as a chloroplast gene product

PubMed Central

Steinback, Katherine E.; McIntosh, Lee; Bogorad, Lawrence; Arntzen, Charles J.

1981-01-01

The triazine herbicides inhibit photosynthesis by blocking electron transport at the second stable electron acceptor of photosystem II. This electron transport component of chloroplast thylakoid membranes is a protein-plastoquinone complex termed “B.” The polypeptide that is believed to be a component of the B complex has recently been identified as a 32- to 34-kilo-dalton polypeptide by using a photoaffinity labeling probe, azido-[14C]atrazine. A 34-kilodalton polypeptide of pea chloroplasts rapidly incorporates [35S]methionine in vivo and is also a rapidly labeled product of chloroplast-directed protein synthesis. Trypsin treatment of membranes tagged with azido-[14C]atrazine, [35S]methionine in vivo, or [35S]methionine in isolated intact chloroplasts results in identical, sequential alterations of the 34-kilo-dalton polypeptide to species of 32, then 18 and 16 kilodaltons. From the identical pattern of susceptibility to trypsin we conclude that the rapidly synthesized 34-kilodalton polypeptide that is a product of chloroplast-directed protein synthesis is identical to the triazine herbicide-binding protein of photosystem II. Chloroplasts of both triazine-susceptible and triazine-resistant biotypes of Amaranthus hybridus synthesize the 34-kilodalton polypeptide, but that of the resistant biotype does not bind the herbicide. Images PMID:16593133

Functional binding interaction identified between the axonal CAM L1 and members of the ERM family.

PubMed

Dickson, Tracey C; Mintz, C David; Benson, Deanna L; Salton, Stephen R J

2002-06-24

A yeast two-hybrid library was screened using the cytoplasmic domain of the axonal cell adhesion molecule L1 to identify binding partners that may be involved in the regulation of L1 function. The intracellular domain of L1 bound to ezrin, a member of the ezrin, radixin, and moesin (ERM) family of membrane-cytoskeleton linking proteins, at a site overlapping that for AP2, a clathrin adaptor. Binding of bacterial fusion proteins confirmed this interaction. To determine whether ERM proteins interact with L1 in vivo, extracellular antibodies to L1 were used to force cluster the protein on cultured hippocampal neurons and PC12 cells, which were then immunolabeled for ERM proteins. Confocal analysis revealed a precise pattern of codistribution between ERMs and L1 clusters in axons and PC12 neurites, whereas ERMs in dendrites and spectrin labeling remained evenly distributed. Transfection of hippocampal neurons grown on an L1 substrate with a dominant negative ERM construct resulted in extensive and abnormal elaboration of membrane protrusions and an increase in axon branching, highlighting the importance of the ERM-actin interaction in axon development. Together, our data indicate that L1 binds directly to members of the ERM family and suggest this association may coordinate aspects of axonal morphogenesis.

Reciprocal Regulation of Endocytosis and Metabolism

PubMed Central

Antonescu, Costin N.; McGraw, Timothy E.; Klip, Amira

2014-01-01

The cellular uptake of many nutrients and micronutrients governs both their cellular availability and their systemic homeostasis. The cellular rate of nutrient or ion uptake (e.g., glucose, Fe3+, K+) or efflux (e.g., Na+) is governed by a complement of membrane transporters and receptors that show dynamic localization at both the plasma membrane and defined intracellular membrane compartments. Regulation of the rate and mechanism of endocytosis controls the amounts of these proteins on the cell surface, which in many cases determines nutrient uptake or secretion. Moreover, the metabolic action of diverse hormones is initiated upon binding to surface receptors that then undergo regulated endocytosis and show distinct signaling patterns once internalized. Here, we examine how the endocytosis of nutrient transporters and carriers as well as signaling receptors governs cellular metabolism and thereby systemic (whole-body) metabolite homeostasis. PMID:24984778

Role of Transbilayer Distribution of Lipid Molecules on the Structure and Protein-Lipid Interaction of an Amyloidogenic Protein on the Membrane Surface

NASA Astrophysics Data System (ADS)

Cheng, Kwan; Cheng, Sara

We used molecular dynamics simulations to examine the effects of transbilayer distribution of lipid molecules, particularly anionic lipids with negatively charged headgroups, on the structure and binding kinetics of an amyloidogenic protein on the membrane surface and subsequent protein-induced structural disruption of the membrane. Our systems consisted of a model beta-sheet rich dimeric protein absorbed on asymmetric bilayers with neutral and anionic lipids and symmetric bilayers with neutral lipids. We observed larger folding, domain aggregation, and tilt angle of the absorbed protein on the asymmetric bilayer surfaces. We also detected more focused bilayer thinning in the asymmetric bilayer due to weak lipid-protein interactions. Our results support the mechanism that the higher lipid packing in the protein-contacting lipid leaflet promotes stronger protein-protein but weaker protein-lipid interactions of an amyloidogenic protein on the membrane surface. We speculate that the observed surface-induced structural and protein-lipid interaction of our model amyloidogenic protein may play a role in the early membrane-associated amyloid cascade pathway that leads to membrane structural damage of neurons in Alzheimer's disease. NSF ACI-1531594.

Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins

NASA Astrophysics Data System (ADS)

Smith, Elizabeth Myhra

The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.

Sensing Membrane Stresses by Protein Insertions

PubMed Central

Campelo, Felix; Kozlov, Michael M.

2014-01-01

Protein domains shallowly inserting into the membrane matrix are ubiquitous in peripheral membrane proteins involved in various processes of intracellular membrane shaping and remodeling. It has been suggested that these domains sense membrane curvature through their preferable binding to strongly curved membranes, the binding mechanism being mediated by lipid packing defects. Here we make an alternative statement that shallow protein insertions are universal sensors of the intra-membrane stresses existing in the region of the insertion embedding rather than sensors of the curvature per se. We substantiate this proposal computationally by considering different independent ways of the membrane stress generation among which some include changes of the membrane curvature whereas others do not alter the membrane shape. Our computations show that the membrane-binding coefficient of shallow protein insertions is determined by the resultant stress independently of the way this stress has been produced. By contrast, consideration of the correlation between the insertion binding and the membrane curvature demonstrates that the binding coefficient either increases or decreases with curvature depending on the factors leading to the curvature generation. To validate our computational model, we treat quantitatively the experimental results on membrane binding by ALPS1 and ALPS2 motifs of ArfGAP1. PMID:24722359

Rastering strategy for screening and centring of microcrystal samples of human membrane proteins with a sub-10 µm size X-ray synchrotron beam

PubMed Central

Cherezov, Vadim; Hanson, Michael A.; Griffith, Mark T.; Hilgart, Mark C.; Sanishvili, Ruslan; Nagarajan, Venugopalan; Stepanov, Sergey; Fischetti, Robert F.; Kuhn, Peter; Stevens, Raymond C.

2009-01-01

Crystallization of human membrane proteins in lipidic cubic phase often results in very small but highly ordered crystals. Advent of the sub-10 µm minibeam at the APS GM/CA CAT has enabled the collection of high quality diffraction data from such microcrystals. Herein we describe the challenges and solutions related to growing, manipulating and collecting data from optically invisible microcrystals embedded in an opaque frozen in meso material. Of critical importance is the use of the intense and small synchrotron beam to raster through and locate the crystal sample in an efficient and reliable manner. The resulting diffraction patterns have a significant reduction in background, with strong intensity and improvement in diffraction resolution compared with larger beam sizes. Three high-resolution structures of human G protein-coupled receptors serve as evidence of the utility of these techniques that will likely be useful for future structural determination efforts. We anticipate that further innovations of the technologies applied to microcrystallography will enable the solving of structures of ever more challenging targets. PMID:19535414

Lateral release of proteins from the TOM complex into the outer membrane of mitochondria.

PubMed

Harner, Max; Neupert, Walter; Deponte, Marcel

2011-07-15

The TOM complex of the outer membrane of mitochondria is the entry gate for the vast majority of precursor proteins that are imported into the mitochondria. It is made up by receptors and a protein conducting channel. Although precursor proteins of all subcompartments of mitochondria use the TOM complex, it is not known whether its channel can only mediate passage across the outer membrane or also lateral release into the outer membrane. To study this, we have generated fusion proteins of GFP and Tim23 which are inserted into the inner membrane and, at the same time, are spanning either the TOM complex or are integrated into the outer membrane. Our results demonstrate that the TOM complex, depending on sequence determinants in the precursors, can act both as a protein conducting pore and as an insertase mediating lateral release into the outer membrane.

Tandem neopentyl glycol maltosides (TNMs) for membrane protein stabilisation†

PubMed Central

Bae, Hyoung Eun; Mortensen, Jonas S.; Ribeiro, Orquidea; Du, Yang; Ehsan, Muhammad; Kobilka, Brian K.; Loland, Claus J.; Byrne, Bernadette

2017-01-01

A novel class of detergents, designated tandem neopentyl glycol maltosides (TNMs), were evaluated with four target membrane proteins. The best detergent varied depending on the target, but TNM-C12L and TNM-C11S were notable for their ability to confer increased membrane protein stability compared to DDM. These agents have potential for use in membrane protein research. PMID:27711401

Tandem neopentyl glycol maltosides (TNMs) for membrane protein stabilisation.

PubMed

Bae, Hyoung Eun; Mortensen, Jonas S; Ribeiro, Orquidea; Du, Yang; Ehsan, Muhammad; Kobilka, Brian K; Loland, Claus J; Byrne, Bernadette; Chae, Pil Seok

2016-10-04

A novel class of detergents, designated tandem neopentyl glycol maltosides (TNMs), were evaluated with four target membrane proteins. The best detergent varied depending on the target, but TNM-C12L and TNM-C11S were notable for their ability to confer increased membrane protein stability compared to DDM. These agents have potential for use in membrane protein research.

Domain Formation Induced by the Adsorption of Charged Proteins on Mixed Lipid Membranes

PubMed Central

Mbamala, Emmanuel C.; Ben-Shaul, Avinoam; May, Sylvio

2005-01-01

Peripheral proteins can trigger the formation of domains in mixed fluid-like lipid membranes. We analyze the mechanism underlying this process for proteins that bind electrostatically onto a flat two-component membrane, composed of charged and neutral lipid species. Of particular interest are membranes in which the hydrocarbon lipid tails tend to segregate owing to nonideal chain mixing, but the (protein-free) lipid membrane is nevertheless stable due to the electrostatic repulsion between the charged lipid headgroups. The adsorption of charged, say basic, proteins onto a membrane containing anionic lipids induces local lipid demixing, whereby charged lipids migrate toward (or away from) the adsorption site, so as to minimize the electrostatic binding free energy. Apart from reducing lipid headgroup repulsion, this process creates a gradient in lipid composition around the adsorption zone, and hence a line energy whose magnitude depends on the protein's size and charge and the extent of lipid chain nonideality. Above a certain critical lipid nonideality, the line energy is large enough to induce domain formation, i.e., protein aggregation and, concomitantly, macroscopic lipid phase separation. We quantitatively analyze the thermodynamic stability of the dressed membrane based on nonlinear Poisson-Boltzmann theory, accounting for both the microscopic characteristics of the proteins and lipid composition modulations at and around the adsorption zone. Spinodal surfaces and critical points of the dressed membranes are calculated for several different model proteins of spherical and disk-like shapes. Among the models studied we find the most substantial protein-induced membrane destabilization for disk-like proteins whose charges are concentrated in the membrane-facing surface. If additional charges reside on the side faces of the proteins, direct protein-protein repulsion diminishes considerably the propensity for domain formation. Generally, a highly charged flat face of a macroion appears most efficient in inducing large compositional gradients, hence a large and unfavorable line energy and consequently lateral macroion aggregation and, concomitantly, macroscopic lipid phase separation. PMID:15626713

The eisosome core is composed of BAR domain proteins

PubMed Central

Olivera-Couto, Agustina; Graña, Martin; Harispe, Laura; Aguilar, Pablo S.

2011-01-01

Eisosomes define sites of plasma membrane organization. In Saccharomyces cerevisiae, eisosomes delimit furrow-like plasma membrane invaginations that concentrate sterols, transporters, and signaling molecules. Eisosomes are static macromolecular assemblies composed of cytoplasmic proteins, most of which have no known function. In this study, we used a bioinformatics approach to analyze a set of 20 eisosome proteins. We found that the core components of eisosomes, paralogue proteins Pil1 and Lsp1, are distant homologues of membrane-sculpting Bin/amphiphysin/Rvs (BAR) proteins. Consistent with this finding, purified recombinant Pil1 and Lsp1 tubulated liposomes and formed tubules when the proteins were overexpressed in mammalian cells. Structural homology modeling and site-directed mutagenesis indicate that Pil1 positively charged surface patches are needed for membrane binding and liposome tubulation. Pil1 BAR domain mutants were defective in both eisosome assembly and plasma membrane domain organization. In addition, we found that eisosome-associated proteins Slm1 and Slm2 have F-BAR domains and that these domains are needed for targeting to furrow-like plasma membrane invaginations. Our results support a model in which BAR domain protein–mediated membrane bending leads to clustering of lipids and proteins within the plasma membrane. PMID:21593205

Effects of oxygen-augmented atmosphere on the immune response.

NASA Technical Reports Server (NTRS)

Guttman, H. N.

1973-01-01

Antibovine serum albumin antibody and nonspecific protein production was evaluated in female rabbits (11-14.5 kg) housed in special cages ventilated with 20% or 40% oxygen at normal barometric pressure. Animals exposed to 40% oxygen do not show normal steady increase of serum antibody. Instead, their titers show a pattern of undershoot, overshoot, undershoot, and finally equilibration at a subnormal level; they have a depressed popliteal node polysome level and have an abnormally low proportion of membrane-bound polysomes. They also show reduced capability of popliteal nodes to synthesize protein (as expected from the reduced number of polysomes). However, the ratio of newly-synthesized specific antibody: nonspecific protein remains normal.

A high-throughput differential filtration assay to screen and select detergents for membrane proteins

PubMed Central

Vergis, James M.; Purdy, Michael D.; Wiener, Michael C.

2015-01-01

Structural studies on integral membrane proteins are routinely performed on protein–detergent complexes (PDCs) consisting of purified protein solubilized in a particular detergent. Of all the membrane protein crystal structures solved to date, a subset of only four detergents has been used in more than half of these structures. Unfortunately, many membrane proteins are not well behaved in these four detergents and/or fail to yield well-diffracting crystals. Identification of detergents that maintain the solubility and stability of a membrane protein is a critical step and can be a lengthy and “protein-expensive” process. We have developed an assay that characterizes the stability and size of membrane proteins exchanged into a panel of 94 commercially available and chemically diverse detergents. This differential filtration assay (DFA), using a set of filtered microplates, requires sub-milligram quantities of purified protein and small quantities of detergents and other reagents and is performed in its entirety in several hours. PMID:20667442

Effect of ceramic membrane channel diameter on limiting retentate protein concentration during skim milk microfiltration.

PubMed

Adams, Michael C; Barbano, David M

2016-01-01

Our objective was to determine the effect of retentate flow channel diameter (4 or 6mm) of nongraded permeability 100-nm pore size ceramic membranes operated in nonuniform transmembrane pressure mode on the limiting retentate protein concentration (LRPC) while microfiltering (MF) skim milk at a temperature of 50°C, a flux of 55 kg · m(-2) · h(-1), and an average cross-flow velocity of 7 m · s(-1). At the above conditions, the retentate true protein concentration was incrementally increased from 7 to 11.5%. When temperature, flux, and average cross-flow velocity were controlled, ceramic membrane retentate flow channel diameter did not affect the LRPC. This indicates that LRPC is not a function of the Reynolds number. Computational fluid dynamics data, which indicated that both membranes had similar radial velocity profiles within their retentate flow channels, supported this finding. Membranes with 6-mm flow channels can be operated at a lower pressure decrease from membrane inlet to membrane outlet (ΔP) or at a higher cross-flow velocity, depending on which is controlled, than membranes with 4-mm flow channels. This implies that 6-mm membranes could achieve a higher LRPC than 4-mm membranes at the same ΔP due to an increase in cross-flow velocity. In theory, the higher LRPC of the 6-mm membranes could facilitate 95% serum protein removal in 2 MF stages with diafiltration between stages if no serum protein were rejected by the membrane. At the same flux, retentate protein concentration, and average cross-flow velocity, 4-mm membranes require 21% more energy to remove a given amount of permeate than 6-mm membranes, despite the lower surface area of the 6-mm membranes. Equations to predict skim milk MF retentate viscosity as a function of protein concentration and temperature are provided. Retentate viscosity, retentate recirculation pump frequency required to maintain a given cross-flow velocity at a given retentate viscosity, and retentate protein determination by mid-infrared spectrophotometry were all useful tools for monitoring the retentate protein concentration to ensure a sustainable MF process. Using 6-mm membranes instead of 4-mm membranes would be advantageous for processors who wish to reduce energy costs or maximize the protein concentration of a MF retentate. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Solubilization of human cells by the styrene-maleic acid copolymer: Insights from fluorescence microscopy.

PubMed

Dörr, Jonas M; van Coevorden-Hameete, Marleen H; Hoogenraad, Casper C; Killian, J Antoinette

2017-11-01

Extracting membrane proteins from biological membranes by styrene-maleic acid copolymers (SMAs) in the form of nanodiscs has developed into a powerful tool in membrane research. However, the mode of action of membrane (protein) solubilization in a cellular context is still poorly understood and potential specificity for cellular compartments has not been investigated. Here, we use fluorescence microscopy to visualize the process of SMA solubilization of human cells, exemplified by the immortalized human HeLa cell line. Using fluorescent protein fusion constructs that mark distinct subcellular compartments, we found that SMA solubilizes membranes in a concentration-dependent multi-stage process. While all major intracellular compartments were affected without a strong preference, plasma membrane solubilization was found to be generally slower than the solubilization of organelle membranes. Interestingly, some plasma membrane-localized proteins were more resistant against solubilization than others, which might be explained by their presence in specific membrane domains with differing properties. Our results support the general applicability of SMA for the isolation of membrane proteins from different types of (sub)cellular membranes. Copyright © 2017 Elsevier B.V. All rights reserved.

A Circular Dichroism Reference Database for Membrane Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wallace,B.; Wien, F.; Stone, T.

2006-01-01

Membrane proteins are a major product of most genomes and the target of a large number of current pharmaceuticals, yet little information exists on their structures because of the difficulty of crystallising them; hence for the most part they have been excluded from structural genomics programme targets. Furthermore, even methods such as circular dichroism (CD) spectroscopy which seek to define secondary structure have not been fully exploited because of technical limitations to their interpretation for membrane embedded proteins. Empirical analyses of circular dichroism (CD) spectra are valuable for providing information on secondary structures of proteins. However, the accuracy of themore » results depends on the appropriateness of the reference databases used in the analyses. Membrane proteins have different spectral characteristics than do soluble proteins as a result of the low dielectric constants of membrane bilayers relative to those of aqueous solutions (Chen & Wallace (1997) Biophys. Chem. 65:65-74). To date, no CD reference database exists exclusively for the analysis of membrane proteins, and hence empirical analyses based on current reference databases derived from soluble proteins are not adequate for accurate analyses of membrane protein secondary structures (Wallace et al (2003) Prot. Sci. 12:875-884). We have therefore created a new reference database of CD spectra of integral membrane proteins whose crystal structures have been determined. To date it contains more than 20 proteins, and spans the range of secondary structures from mostly helical to mostly sheet proteins. This reference database should enable more accurate secondary structure determinations of membrane embedded proteins and will become one of the reference database options in the CD calculation server DICHROWEB (Whitmore & Wallace (2004) NAR 32:W668-673).« less

Molecular basis of endosomal-membrane association for the dengue virus envelope protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rogers, David M.; Kent, Michael S.; Rempe, Susan B.

Dengue virus is coated by an icosahedral shell of 90 envelope protein dimers that convert to trimers at low pH and promote fusion of its membrane with the membrane of the host endosome. We provide the first estimates for the free energy barrier and minimum for two key steps in this process: host membrane bending and protein–membrane binding. Both are studied using complementary membrane elastic, continuum electrostatics and all-atom molecular dynamics simulations. The predicted host membrane bending required to form an initial fusion stalk presents a 22–30 kcal/mol free energy barrier according to a constrained membrane elastic model. Combined continuummore » and molecular dynamics results predict a 15 kcal/mol free energy decrease on binding of each trimer of dengue envelope protein to a membrane with 30% anionic phosphatidylglycerol lipid. The bending cost depends on the preferred curvature of the lipids composing the host membrane leaflets, while the free energy gained for protein binding depends on the surface charge density of the host membrane. The fusion loop of the envelope protein inserts exactly at the level of the interface between the membrane's hydrophobic and head-group regions. As a result, the methods used in this work provide a means for further characterization of the structures and free energies of protein-assisted membrane fusion.« less

Molecular basis of endosomal-membrane association for the dengue virus envelope protein

DOE PAGES

Rogers, David M.; Kent, Michael S.; Rempe, Susan B.

2015-01-02

Dengue virus is coated by an icosahedral shell of 90 envelope protein dimers that convert to trimers at low pH and promote fusion of its membrane with the membrane of the host endosome. We provide the first estimates for the free energy barrier and minimum for two key steps in this process: host membrane bending and protein–membrane binding. Both are studied using complementary membrane elastic, continuum electrostatics and all-atom molecular dynamics simulations. The predicted host membrane bending required to form an initial fusion stalk presents a 22–30 kcal/mol free energy barrier according to a constrained membrane elastic model. Combined continuummore » and molecular dynamics results predict a 15 kcal/mol free energy decrease on binding of each trimer of dengue envelope protein to a membrane with 30% anionic phosphatidylglycerol lipid. The bending cost depends on the preferred curvature of the lipids composing the host membrane leaflets, while the free energy gained for protein binding depends on the surface charge density of the host membrane. The fusion loop of the envelope protein inserts exactly at the level of the interface between the membrane's hydrophobic and head-group regions. As a result, the methods used in this work provide a means for further characterization of the structures and free energies of protein-assisted membrane fusion.« less

Multiple Lines of Evidence Localize Signaling, Morphology, and Lipid Biosynthesis Machinery to the Mitochondrial Outer Membrane of Arabidopsis[W][OA

PubMed Central

Duncan, Owen; Taylor, Nicolas L.; Carrie, Chris; Eubel, Holger; Kubiszewski-Jakubiak, Szymon; Zhang, Botao; Narsai, Reena; Millar, A. Harvey; Whelan, James

2011-01-01

The composition of the mitochondrial outer membrane is notoriously difficult to deduce by orthology to other organisms, and biochemical enrichments are inevitably contaminated with the closely associated inner mitochondrial membrane and endoplasmic reticulum. In order to identify novel proteins of the outer mitochondrial membrane in Arabidopsis (Arabidopsis thaliana), we integrated a quantitative mass spectrometry analysis of highly enriched and prefractionated samples with a number of confirmatory biochemical and cell biology approaches. This approach identified 42 proteins, 27 of which were novel, more than doubling the number of confirmed outer membrane proteins in plant mitochondria and suggesting novel functions for the plant outer mitochondrial membrane. The novel components identified included proteins that affected mitochondrial morphology and/or segregation, a protein that suggests the presence of bacterial type lipid A in the outer membrane, highly stress-inducible proteins, as well as proteins necessary for embryo development and several of unknown function. Additionally, proteins previously inferred via orthology to be present in other compartments, such as an NADH:cytochrome B5 reductase required for hydroxyl fatty acid accumulation in developing seeds, were shown to be located in the outer membrane. These results also revealed novel proteins, which may have evolved to fulfill plant-specific requirements of the mitochondrial outer membrane, and provide a basis for the future functional characterization of these proteins in the context of mitochondrial intracellular interaction. PMID:21896887

Detergent-associated solution conformations of helical and beta-barrel membrane proteins.

PubMed

Mo, Yiming; Lee, Byung-Kwon; Ankner, John F; Becker, Jeffrey M; Heller, William T

2008-10-23

Membrane proteins present major challenges for structural biology. In particular, the production of suitable crystals for high-resolution structural determination continues to be a significant roadblock for developing an atomic-level understanding of these vital cellular systems. The use of detergents for extracting membrane proteins from the native membrane for either crystallization or reconstitution into model lipid membranes for further study is assumed to leave the protein with the proper fold with a belt of detergent encompassing the membrane-spanning segments of the structure. Small-angle X-ray scattering was used to probe the detergent-associated solution conformations of three membrane proteins, namely bacteriorhodopsin (BR), the Ste2p G-protein coupled receptor from Saccharomyces cerevisiae, and the Escherichia coli porin OmpF. The results demonstrate that, contrary to the traditional model of a detergent-associated membrane protein, the helical proteins BR and Ste2p are not in the expected, compact conformation and associated with detergent micelles, while the beta-barrel OmpF is indeed embedded in a disk-like micelle in a properly folded state. The comparison provided by the BR and Ste2p, both members of the 7TM family of helical membrane proteins, further suggests that the interhelical interactions between the transmembrane helices of the two proteins differ, such that BR, like other rhodopsins, can properly refold to crystallize, while Ste2p continues to prove resistant to crystallization from an initially detergent-associated state.

Detergent-associated Solution Conformations of Helical and Beta-barrel Membrane Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mo, Yiming; Lee, Byung-Kwon; Ankner, John Francis

2008-01-01

Membrane proteins present major challenges for structural biology. In particular, the production of suitable crystals for high-resolution structural determination continues to be a significant roadblock for developing an atomic-level understanding of these vital cellular systems. The use of detergents for extracting membrane proteins from the native membrane for either crystallization or reconstitution into model lipid membranes for further study is assumed to leave the protein with the proper fold with a belt of detergent encompassing the membrane-spanning segments of the structure. Small-angle X-ray scattering was used to probe the detergent-associated solution conformations of three membrane proteins, namely bacteriorhodopsin (BR), themore » Ste2p G-protein coupled receptor from Saccharomyces cerevisiae, and the Escherichia coli porin OmpF. The results demonstrate that, contrary to the traditional model of a detergent-associated membrane protein, the helical proteins BR and Ste2p are not in the expected, compact conformation and associated with detergent micelles, while the ?-barrel OmpF is indeed embedded in a disk-like micelle in a properly folded state. The comparison provided by the BR and Ste2p, both members of the 7TM family of helical membrane proteins, further suggests that the interhelical interactions between the transmembrane helices of the two proteins differ, such that BR, like other rhodopsins, can properly refold to crystallize, while Ste2p continues to prove resistant to crystallization from an initially detergent-associated state.« less

A fluorogenic probe for SNAP-tagged plasma membrane proteins based on the solvatochromic molecule Nile Red.

PubMed

Prifti, Efthymia; Reymond, Luc; Umebayashi, Miwa; Hovius, Ruud; Riezman, Howard; Johnsson, Kai

2014-03-21

A fluorogenic probe for plasma membrane proteins based on the dye Nile Red and SNAP-tag is introduced. It takes advantage of Nile Red, a solvatochromic molecule highly fluorescent in an apolar environment, such as cellular membranes, but almost dark in a polar aqueous environment. The probe possesses a tuned affinity for membranes allowing its Nile Red moiety to insert into the lipid bilayer of the plasma membrane, becoming fluorescent, only after its conjugation to a SNAP-tagged plasma membrane protein. The fluorogenic character of the probe was demonstrated for different SNAP-tag fusion proteins, including the human insulin receptor. This work introduces a new approach for generating a powerful turn-on probe for "no-wash" labeling of plasma membrane proteins with numerous applications in bioimaging.

High-resolution Structures of Protein-Membrane Complexes by Neutron Reflection and MD Simulation: Membrane Association of the PTEN Tumor Suppressor

NASA Astrophysics Data System (ADS)

Lösche, Matthias

2012-02-01

The lipid matrix of biomembranes is an in-plane fluid, thermally and compositionally disordered leaflet of 5 nm thickness and notoriously difficult to characterize in structural terms. Yet, biomembranes are ubiquitous in the cell, and membrane-bound proteins are implicated in a variety of signaling pathways and intra-cellular transport. We developed methodology to study proteins associated with model membranes using neutron reflection measurements and showed recently that this approach can resolve the penetration depth and orientation of membrane proteins with ångstrom resolution if their crystal or NMR structure is known. Here we apply this technology to determine the membrane bindung and unravel functional details of the PTEN phosphatase, a key player in the PI3K apoptosis pathway. PTEN is an important regulatory protein and tumor suppressor that performs its phosphatase activity as an interfacial enzyme at the plasma membrane-cytoplasm boundary. Acting as an antagonist to phosphoinositide-3-kinase (PI3K) in cell signaling, it is deleted in many human cancers. Despite its importance in regulating the levels of the phosphoinositoltriphosphate PI(3,4,5)P3, there is little understanding of how PTEN binds to membranes, is activated and then acts as a phosphatase. We investigated the structure and function of PTEN by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P2) act synergetically in attracting the enzyme to the membrane surface. Membrane affinities depend strongly on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P2. Neutron reflection measurements show that the PTEN phosphatase ``scoots'' along the membrane surface (penetration < 5 å) but binds the membrane tightly with its two major domains, the C2 and phosphatase domains. In the bound state, PTEN's regulatory C-terminal tail is displaced from the membrane and organized on the far side of the protein, ˜ 60 å away from the bilayer surface, in a rather compact structure. The combination of binding studies and neutron reflection allows us to distinguish between PTEN mutant proteins and ultimately may identify the structural features required for membrane binding and activation of PTEN. Molecular dynamics simulations, currently in progress, refine this structural picture further.

Binding of (/sup 3/H)forskolin to solubilized preparations of adenylate cyclase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nelson, C.A.; Seamon, K.B.

1988-01-01

The binding of (/sup 3/H)forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating (/sup 3/H)forskolin bound to protein from free (/sup 3/H)forskolin by rapid filtration. The K/sub d/ for (/sup 3/H)forskolin binding to solubilized proteins was 14 nM which was similar to that for (/sup 3/H)forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for (/sup 3/H)forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. (/sup 3/H)forskolin bound to proteins solubilized from membranes with a Bmaxmore » of 38 fmolmg protein which increased to 94 fmolmg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on (/sup 3/H)forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmolmgmin which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmolmgmin which was not stimulated by GppNHp or forskolin« less

Z-membranes: artificial organelles for overexpressing recombinant integral membrane proteins.

PubMed Central

Gong, F C; Giddings, T H; Meehl, J B; Staehelin, L A; Galbraith, D W

1996-01-01

We have expressed a fusion protein formed between the avian infectious bronchitis virus M protein and the bacterial enzyme beta-glucuronidase in transgenic tobacco cells. Electron microscope images of such cells demonstrate that overexpression of this fusion protein gives rise to a type of endoplasmic reticulum membrane domain in which adjacent membranes become zippered together apparently as a consequence of the oligomerizing action of beta-glucuronidase. These zippered (Z-) membranes lack markers of the endoplasmic reticulum (NADH cytochrome c reductase and ribosomes) and accumulate in the cells in the form of multilayered scroll-like structures (up to 2 micrometers in diameter; 20-50 per cell) without affecting plant growth. The discovery of Z-membranes has broad implications for biology and biotechnology in that they provide a means for accumulating large quantities of recombinant membrane proteins within discrete domains of native membranes. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8700911

Interaction of partially denatured insulin with a DSPC floating lipid bilayer.

PubMed

Dennison, A J C; Jones, R A L; Staniforth, R A; Parnell, A J

2016-01-21

The carefully controlled permeability of cellular membranes to biological molecules is key to life. In degenerative diseases associated with protein misfolding and aggregation, protein molecules or their aggregates are believed to permeate these barriers and threaten membrane integrity. We used neutron reflectivity to study the interaction of insulin, a model amyloidogenic protein, with a DSPC floating lipid bilayer. Structural changes consistent with protein partitioning to the membrane interior and adsorption to a gel phase model lipid bilayer were observed under conditions where the native fold of the protein is significantly destabilised. We propose that the perturbation of the membrane by misfolded proteins involves long term occupation of the membrane by these proteins, rather than transient perforation events.

Preprotein transport machineries of yeast mitochondrial outer membrane are not required for Bax-induced release of intermembrane space proteins.

PubMed

Sanjuán Szklarz, Luiza K; Kozjak-Pavlovic, Vera; Vögtle, F-Nora; Chacinska, Agnieszka; Milenkovic, Dusanka; Vogel, Sandra; Dürr, Mark; Westermann, Benedikt; Guiard, Bernard; Martinou, Jean-Claude; Borner, Christoph; Pfanner, Nikolaus; Meisinger, Chris

2007-04-20

The mitochondrial outer membrane contains protein import machineries, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been speculated that TOM or SAM are required for Bax-induced release of intermembrane space (IMS) proteins; however, experimental evidence has been scarce. We used isolated yeast mitochondria as a model system and report that Bax promoted an efficient release of soluble IMS proteins while preproteins were still imported, excluding an unspecific damage of mitochondria. Removal of import receptors by protease treatment did not inhibit the release of IMS proteins by Bax. Yeast mutants of each Tom receptor and the Tom40 channel were not impaired in Bax-induced protein release. We analyzed a large collection of mutants of mitochondrial outer membrane proteins, including SAM, fusion and fission components, but none of these components was required for Bax-induced protein release. The released proteins included complexes up to a size of 230 kDa. We conclude that Bax promotes efficient release of IMS proteins through the outer membrane of yeast mitochondria while the inner membrane remains intact. Inactivation of the known protein import and sorting machineries of the outer membrane does not impair the function of Bax at the mitochondria.

Far Western: probing membranes.

PubMed

Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

2007-08-01

INTRODUCTIONThe far-Western technique described in this protocol is fundamentally similar to Western blotting. In Western blots, an antibody is used to detect a query protein on a membrane. In contrast, in a far-Western blot (also known as an overlay assay) the antibody is replaced by a recombinant GST fusion protein (produced and purified from bacteria), and the assay detects the interaction of this protein with target proteins on a membrane. The membranes are washed and blocked, incubated with probe protein, washed again, and subjected to autoradiography. The GST fusion (probe) proteins are often labeled with (32)P; alternatively, the membrane can be probed with unlabeled GST fusion protein, followed by detection using commercially available GST antibodies. The nonradioactive approach is substantially more expensive (due to the purchase of antibody and detection reagents) than using radioactively labeled proteins. In addition, care must be taken to control for nonspecific interactions with GST alone and a signal resulting from antibody cross-reactivity. In some instances, proteins on the membrane are not able to interact after transfer. This may be due to improper folding, particularly in the case of proteins expressed from a phage expression library. This protocol describes a way to overcome this by washing the membrane in denaturation buffer, which is then serially diluted to permit slow renaturation of the proteins.

Comparative analysis of soybean plasma membrane proteins under osmotic stress using gel-based and LC MS/MS-based proteomics approaches.

PubMed

Nouri, Mohammad-Zaman; Komatsu, Setsuko

2010-05-01

To study the soybean plasma membrane proteome under osmotic stress, two methods were used: a gel-based and a LC MS/MS-based proteomics method. Two-day-old seedlings were subjected to 10% PEG for 2 days. Plasma membranes were purified from seedlings using a two-phase partitioning method and their purity was verified by measuring ATPase activity. Using the gel-based proteomics, four and eight protein spots were identified as up- and downregulated, respectively, whereas in the nanoLC MS/MS approach, 11 and 75 proteins were identified as up- and downregulated, respectively, under PEG treatment. Out of osmotic stress responsive proteins, most of the transporter proteins and all proteins with high number of transmembrane helices as well as low-abundance proteins could be identified by the LC MS/MS-based method. Three homologues of plasma membrane H(+)-ATPase, which are transporter proteins involved in ion efflux, were upregulated under osmotic stress. Gene expression of this protein was increased after 12 h of stress exposure. Among the identified proteins, seven proteins were mutual in two proteomics techniques, in which calnexin was the highly upregulated protein. Accumulation of calnexin in plasma membrane was confirmed by immunoblot analysis. These results suggest that under hyperosmotic conditions, calnexin accumulates in the plasma membrane and ion efflux accelerates by upregulation of plasma membrane H(+)-ATPase protein.

Membrane-tethered peptides patterned after the TRP domain (TRPducins) selectively inhibit TRPV1 channel activity.

PubMed

Valente, Pierluigi; Fernández-Carvajal, Asia; Camprubí-Robles, María; Gomis, Ana; Quirce, Susana; Viana, Félix; Fernández-Ballester, Gregorio; González-Ros, José M; Belmonte, Carlos; Planells-Cases, Rosa; Ferrer-Montiel, Antonio

2011-05-01

The transient receptor potential vanilloid 1 (TRPV1) channel is a thermosensory receptor implicated in diverse physiological and pathological processes. The TRP domain, a highly conserved region in the C terminus adjacent to the internal channel gate, is critical for subunit tetramerization and channel gating. Here, we show that cell-penetrating, membrane-anchored peptides patterned after this protein domain are moderate and selective TRPV1 antagonists both in vitro and in vivo, blocking receptor activity in intact rat primary sensory neurons and their peripheral axons with mean decline time of 30 min. The most potent lipopeptide, TRP-p5, blocked all modes of TRPV1 gating with micromolar efficacy (IC(50)<10 μM), without significantly affecting other thermoTRP channels. In contrast, its retrosequence or the corresponding sequences of other TRPV channels did not alter TRPV1 channel activity (IC(50)>100 μM). TRP-p5 did not affect the capsaicin sensitivity of the vanilloid receptor. Our data suggest that TRP-p5 interferes with protein-protein interactions at the level of the TRP domain that are essential for the "conformational" change that leads to gate opening. Therefore, these palmitoylated peptides, which we termed TRPducins, are noncompetitive, voltage-independent, sequence-specific TRPV1 blockers. Our findings indicate that TRPducin-like peptides may embody a novel molecular strategy that can be exploited to generate a selective pharmacological arsenal for the TRP superfamily of ion channels.

Printing-assisted surface modifications of patterned ultrafiltration membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wardrip, Nathaniel C.; Dsouza, Melissa; Urgun-Demirtas, Meltem

Understanding and restricting microbial surface attachment will enhance wastewater treatment with membranes. We report a maskless lithographic patterning technique for the generation of patterned polymer coatings on ultrafiltration membranes. Polyethylene glycol, zwitterionic, or negatively charged hydrophilic polymer compositions in parallel- or perpendicular-striped patterns with respect to feed flow were evaluated using wastewater. Membrane fouling was dependent on the orientation and chemical composition of the coatings. Modifications reduced alpha diversity in the attached microbial community (Shannon indices decreased from 2.63 to 1.89) which nevertheless increased with filtration time. Sphingomonas species, which condition membrane surfaces and facilitate cellular adhesion, were depleted inmore » all modified membranes. Microbial community structure was significantly different between control, different patterns, and different chemistries. Lastly, this study broadens the tools for surface modification of membranes with polymer coatings and for understanding and optimization of antifouling surfaces.« less

Printing-assisted surface modifications of patterned ultrafiltration membranes

DOE PAGES

Wardrip, Nathaniel C.; Dsouza, Melissa; Urgun-Demirtas, Meltem; ...

2016-10-17

Understanding and restricting microbial surface attachment will enhance wastewater treatment with membranes. We report a maskless lithographic patterning technique for the generation of patterned polymer coatings on ultrafiltration membranes. Polyethylene glycol, zwitterionic, or negatively charged hydrophilic polymer compositions in parallel- or perpendicular-striped patterns with respect to feed flow were evaluated using wastewater. Membrane fouling was dependent on the orientation and chemical composition of the coatings. Modifications reduced alpha diversity in the attached microbial community (Shannon indices decreased from 2.63 to 1.89) which nevertheless increased with filtration time. Sphingomonas species, which condition membrane surfaces and facilitate cellular adhesion, were depleted inmore » all modified membranes. Microbial community structure was significantly different between control, different patterns, and different chemistries. Lastly, this study broadens the tools for surface modification of membranes with polymer coatings and for understanding and optimization of antifouling surfaces.« less

Functional domains of the T lymphocyte plasma membrane: characterization of the polypeptide composition.

PubMed

Szamel, M; Kaever, V; Resch, K

1987-01-01

Highly purified plasma membranes from calf thymocytes were fractionated by affinity chromatography on Concanavalin A-Sepharose into two subfractions, one eluting freely from the affinity column (MF1) and a second being specifically retained (MF2). SDS-polyacrylamide-gel-electrophoresis revealed different polypeptide patterns of the two plasma membrane subfractions. Polypeptides of apparent molecular weights of 170, 150, 110, 94, 39, and 30 kDa were several-fold enriched in the adherent fraction, MF2. In contrast, several proteins in the 55-65 kDa range were preferentially recovered in the non-adherent fraction. Five Five of the six polypeptides, preferentially recovered in MF2 proved to be glycoproteins, the 39 kDa peptide was non-glycosilated. The differences in the amounts of the polypeptides specifically enriched in the adherent fraction MF2 became even more clear-cut when plasma membranes solubilized with non-ionic detergents (lysolecithin, ET-18-2H, Triton-X-100) were separated by affinity chromatography on Concanavalin A-Sepharose. The non-glycosilated peptide of apparent molecular weight of 39 kDa was recovered together with several glycoproteins in the adherent fraction, MF2, suggesting that not single glycoproteins, but plasma membrane domains were separated by Concanavalin A-Sepharose. Although the glycoproteins of the non-adherent fraction MF1 bound significant amounts of Concanavalin A, the major Concanavalin A binding glycoproteins were recovered in the adherent fraction, MF2. The plasma membrane subfractions showed also different functional properties, the specific activities [Na+ + K+]AT-Pase, Ca2+ ATPase and lysolecithin acyltransferase were several-fold enriched in the adherent fraction, MF2, as compared to MF1. The data suggest the existence of plasma membrane domains in the plasma membranes of thymocytes consisting of a different set of proteins, among others the major Concanavalin A binding glycoproteins with some membrane bound enzymes, probably implicated in the initiation of lymphocyte activation.

Physical basis of some membrane shaping mechanisms

PubMed Central

2016-01-01

In vesicular transport pathways, membrane proteins and lipids are internalized, externalized or transported within cells, not by bulk diffusion of single molecules, but embedded in the membrane of small vesicles or thin tubules. The formation of these ‘transport carriers’ follows sequential events: membrane bending, fission from the donor compartment, transport and eventually fusion with the acceptor membrane. A similar sequence is involved during the internalization of drug or gene carriers inside cells. These membrane-shaping events are generally mediated by proteins binding to membranes. The mechanisms behind these biological processes are actively studied both in the context of cell biology and biophysics. Bin/amphiphysin/Rvs (BAR) domain proteins are ideally suited for illustrating how simple soft matter principles can account for membrane deformation by proteins. We review here some experimental methods and corresponding theoretical models to measure how these proteins affect the mechanics and the shape of membranes. In more detail, we show how an experimental method employing optical tweezers to pull a tube from a giant vesicle may give important quantitative insights into the mechanism by which proteins sense and generate membrane curvature and the mechanism of membrane scission. This article is part of the themed issue ‘Soft interfacial materials: from fundamentals to formulation’. PMID:27298443

LAMBADA and InflateGRO2: efficient membrane alignment and insertion of membrane proteins for molecular dynamics simulations.

PubMed

Schmidt, Thomas H; Kandt, Christian

2012-10-22

At the beginning of each molecular dynamics membrane simulation stands the generation of a suitable starting structure which includes the working steps of aligning membrane and protein and seamlessly accommodating the protein in the membrane. Here we introduce two efficient and complementary methods based on pre-equilibrated membrane patches, automating these steps. Using a voxel-based cast of the coarse-grained protein, LAMBADA computes a hydrophilicity profile-derived scoring function based on which the optimal rotation and translation operations are determined to align protein and membrane. Employing an entirely geometrical approach, LAMBADA is independent from any precalculated data and aligns even large membrane proteins within minutes on a regular workstation. LAMBADA is the first tool performing the entire alignment process automatically while providing the user with the explicit 3D coordinates of the aligned protein and membrane. The second tool is an extension of the InflateGRO method addressing the shortcomings of its predecessor in a fully automated workflow. Determining the exact number of overlapping lipids based on the area occupied by the protein and restricting expansion, compression and energy minimization steps to a subset of relevant lipids through automatically calculated and system-optimized operation parameters, InflateGRO2 yields optimal lipid packing and reduces lipid vacuum exposure to a minimum preserving as much of the equilibrated membrane structure as possible. Applicable to atomistic and coarse grain structures in MARTINI format, InflateGRO2 offers high accuracy, fast performance, and increased application flexibility permitting the easy preparation of systems exhibiting heterogeneous lipid composition as well as embedding proteins into multiple membranes. Both tools can be used separately, in combination with other methods, or in tandem permitting a fully automated workflow while retaining a maximum level of usage control and flexibility. To assess the performance of both methods, we carried out test runs using 22 membrane proteins of different size and transmembrane structure.

Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein is PAM involved in the capacitative calcium entry?

PubMed

Kozieł, Katarzyna; Lebiedzinska, Magdalena; Szabadkai, Gyorgy; Onopiuk, Marta; Brutkowski, Wojciech; Wierzbicka, Katarzyna; Wilczyński, Grzegorz; Pinton, Paolo; Duszyński, Jerzy; Zabłocki, Krzysztof; Wieckowski, Mariusz R

2009-12-01

A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.

Myristoylation Restricts Orientation of the GRASP Domain on Membranes and Promotes Membrane Tethering*

PubMed Central

Heinrich, Frank; Nanda, Hirsh; Goh, Haw Zan; Bachert, Collin; Lösche, Mathias; Linstedt, Adam D.

2014-01-01

The mammalian Golgi reassembly stacking protein (GRASP) proteins are Golgi-localized homotypic membrane tethers that organize Golgi stacks into a long, contiguous ribbon-like structure. It is unknown how GRASPs undergo trans pairing given that cis interactions between the proteins in the plane of the membrane are intrinsically favored. To test the hypothesis that myristoylation of the self-interacting GRASP domain restricts its orientation on the membrane to favor trans pairing, we established an in vitro assay that recapitulates GRASP-dependent membrane tethering and used neutron reflection under similar conditions to determine the orientation of the GRASP domain. In vivo, the membrane association of GRASP proteins is conferred by the simultaneous insertion of an N-terminal myristic acid and binding to a Golgi-associated binding partner. In our assay, the latter contact was replaced using a C-terminal hexa-His moiety, which bound to Ni2+-conjugated lipids incorporated into a substrate-supported bilayer lipid membrane. Nonmyristoylated protein lacked a fixed orientation on the membrane and inefficiently tethered liposomes. In contrast, myristoylated GRASP promoted tethering and exhibited a unique membrane complex. Thus, myristoylation restricts the membrane orientation of the GRASP domain favoring interactions in trans for membrane tethering. PMID:24505136

Serum protein adsorption and platelet adhesion on aspartic-acid-immobilized polysulfone membranes.

PubMed

Higuchi, Akon; Hashiba, Hirokazu; Hayashi, Rika; Yoon, Boo Ok; Sakurai, Masaru; Hara, Mariko

2004-01-01

Polysulfone (PSf) membranes that covalently conjugated with aspartic acid (ASP-PSf) were prepared and analyzed for hemocompatability. Compared to PSf or other types of surface-modified PSf membranes, the ASP-PSf membranes had a reduced ability to adsorb protein from either a plasma solution or a mixed solution of albumin, globulin and fibrinogen. This appears to be due to the creation of a hydrophilic surface by the aspartic acid zwitterion immobilized on the ASP-PSf membranes. Furthermore, the analyses of membrane protein adsorption showed that a mixed protein solution recapitulates the cooperative adsorption of proteins that occurs in plasma. We also found that the number of adhering platelets was the lowest on the ASP-PSf membranes and, in general, that platelet adhesion decreased in parallel with fibrinogen adsorption. In summary, aspartic acid immobilized on the ASP-PSf membranes, which have zwitterions with a net zero charge, effectively contributes to the hydrophilic and hemocompatible sites on the surface of the hydrophobic PSf membranes.

Misregulation of membrane trafficking processes in human nonalcoholic steatohepatitis.

PubMed

Dzierlenga, Anika L; Cherrington, Nathan J

2018-03-01

Nonalcoholic steatohepatitis (NASH) remodels the expression and function of genes and proteins that are critical for drug disposition. This study sought to determine whether disruption of membrane protein trafficking pathways in human NASH contributes to altered localization of multidrug resistance-associated protein 2 (MRP2). A comprehensive immunoblot analysis assessed the phosphorylation, membrane translocation, and expression of transporter membrane insertion regulators, including several protein kinases (PK), radixin, MARCKS, and Rab11. Radixin exhibited a decreased phosphorylation and total expression, whereas Rab11 had an increased membrane localization. PKCδ, PKCα, and PKA had increased membrane activation, whereas PKCε had a decreased phosphorylation and membrane expression. Radixin dephosphorylation may activate MRP2 membrane retrieval in NASH; however, the activation of Rab11/PKCδ and PKA/PKCα suggest an activation of membrane insertion pathways as well. Overall these data suggest an altered regulation of protein trafficking in human NASH, although other processes may be involved in the regulation of MRP2 localization. © 2018 Wiley Periodicals, Inc.

Chemical Composition of Salmon Ovary Outer Membrane and Its Protein Increases Fecal Mucins Content in C57BL/6J and Type 2 Diabetic/Obese KK-Ay Mice

PubMed Central

Maeda, Hayato; Hosomi, Ryota; Chiba, Utako; Fukunaga, Kenji

2013-01-01

Salmon ovary outer membrane (SOM) is a byproduct of the salmon industry; however, the effective utilization of SOM for food materials and supplements is anticipated as the demand for fish and seafood increases worldwide. The purposes of the present study were to assess the chemical composition of SOM, the characteristics of SOM protein (SOMP), and its effects on serum and fecal biochemical parameters in mice. SOM contained high levels of crude protein (61.9 g/100 g) and crude lipid (18.9 g/100 g). The protein pattern of SOMP was different from those of fish muscle protein and roe; it was abundant in collagen, as calculated from the hydroxyproline content. In addition, SOMP exhibited lower protein digestibility during in vitro digestion analyses compared with casein. Male C57BL/6J and KK-Ay mice were fed a casein-based semi-purified diet or a diet with replacement of part of the dietary protein (50%) by SOMP for four weeks. Mice fed the diet containing SOMP showed elevated fecal nitrogen and mucins contents and reduced levels of serum liver injury markers and fecal ammonia. These results show for the first time that chemical composition of SOM, and SOMP, contain a resistant protein fraction and a large amount of collagen. Therefore, SOM is a potential source of marine collagen and functional food material for promoting the health of the liver and colon. PMID:28239126

Differential expression of syndecan isoforms during mouse incisor amelogenesis.

PubMed

Muto, Taro; Miyoshi, Keiko; Munesue, Seiichi; Nakada, Hiroshi; Okayama, Minoru; Matsuo, Takashi; Noma, Takafumi

2007-08-01

Syndecans are transmembranous heparan sulfate proteoglycans (HSPGs) with covalently attached glycosaminoglycan side-chains located on the cell surface. The mammalian syndecan family is composed of four types of syndecans (syndecan-1 to -4). Syndecans interact with the intracellular cytoskeleton through the cytoplasmic domains of their core proteins and membrane proteins, extracellular enzymes, growth factors, and matrix components, through their heparan-sulfate chains, to regulate developmental processes.Here, as a first step to assess the possible roles of syndecan proteins in amelogenesis, we examined the expression patterns of all syndecan isoforms in continuously growing mouse incisors, in which we can overview major differentiation stages of amelogenesis at a glance. Understanding the expression domain of each syndecan isoform during specific developmental stages seems useful for investigating their physiological roles in amelogenesis. Immunohistochemical analysis of syndecan core proteins in the lower incisors from postnatal day 1 mice revealed spatially and temporally specific expression patterns, with syndecan-1 expressed in undifferentiated epithelial and mesenchymal cells, and syndecan-2, -3, and -4 in more differentiated cells. These findings suggest that each syndecan isoform functions distinctly during the amelogenesis of the incisors of mice.

Arrayed water-in-oil droplet bilayers for membrane transport analysis.

PubMed

Watanabe, R; Soga, N; Hara, M; Noji, H

2016-08-02

The water-in-oil droplet bilayer is a simple and useful lipid bilayer system for membrane transport analysis. The droplet interface bilayer is readily formed by the contact of two water-in-oil droplets enwrapped by a phospholipid monolayer. However, the size of individual droplets with femtoliter volumes in a high-throughput manner is difficult to control, resulting in low sensitivity and throughput of membrane transport analysis. To overcome this drawback, in this study, we developed a novel micro-device in which a large number of droplet interface bilayers (>500) are formed at a time by using femtoliter-sized droplet arrays immobilized on a hydrophobic/hydrophilic substrate. The droplet volume was controllable from 3.5 to 350 fL by changing the hydrophobic/hydrophilic pattern on the device, allowing high-throughput analysis of membrane transport mechanisms including membrane permeability to solutes (e.g., ions or small molecules) with or without the aid of transport proteins. Thus, this novel platform broadens the versatility of water-in-oil droplet bilayers and will pave the way for novel analytical and pharmacological applications such as drug screening.

Rooster sperm plasma membrane protein and phospholipid organization and reorganization attributed to cooling and cryopreservation

USDA-ARS?s Scientific Manuscript database

Cholesterol to phospholipid ratio is used as a representation for membrane fluidity, and predictor of cryopreservation success but results are not consistent across species and ignore the impact of membrane proteins. Therefore, this research explored the modulation of membrane fluidity and protein ...

TOF-SIMS imaging of protein adsorption on dialysis membrane

NASA Astrophysics Data System (ADS)

Aoyagi, Satoka; Hayama, Msayo; Hasegawa, Urara; Sakai, Kiyotaka; Hoshi, Takahiro; Kudo, Masahiro

2004-06-01

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is capable of chemical imaging of proteins on insulated samples such as hollow-fiber dialysis membranes. Albumin loss and a lowering of diffusive permeability caused by protein adsorption on dialysis membranes should be reduced in order to enhance dialysis adequacy of the patients. Bovine serum albumin (BSA)-adsorbed hollow-fiber dialysis membranes were tested in the present study. TOF-SIMS images and spectra of both native membranes and BSA-adsorbed membranes were compared in order to identify secondary ions related to BSA and membranes. Peaks of secondary ions related to BSA and each membrane were selected by means of information theory, and they are characterized by principal component analysis (PCA). Chemical images of BSA adsorption on both native and treated membranes were obtained to find that BSA permeability and interaction between the membranes and BSA definitely depend on the properties of a membrane. TOF-SIMS imaging obtained with information theory is a powerful tool to estimate protein adsorption on the dialysis membranes.

Comparative study of two protocols for quantitative image-analysis of serotonin transporter clustering in lymphocytes, a putative biomarker of therapeutic efficacy in major depression.

PubMed

Romay-Tallon, Raquel; Rivera-Baltanas, Tania; Allen, Josh; Olivares, Jose M; Kalynchuk, Lisa E; Caruncho, Hector J

2017-01-01

The pattern of serotonin transporter clustering on the plasma membrane of lymphocytes extracted from human whole blood samples has been identified as a putative biomarker of therapeutic efficacy in major depression. Here we evaluated the possibility of performing a similar analysis using blood smears obtained from rats, and from control human subjects and depression patients. We hypothesized that we could optimize a protocol to make the analysis of serotonin protein clustering in blood smears comparable to the analysis of serotonin protein clustering using isolated lymphocytes. Our data indicate that blood smears require a longer fixation time and longer times of incubation with primary and secondary antibodies. In addition, one needs to optimize the image analysis settings for the analysis of smears. When these steps are followed, the quantitative analysis of both the number and size of serotonin transporter clusters on the plasma membrane of lymphocytes is similar using both blood smears and isolated lymphocytes. The development of this novel protocol will greatly facilitate the collection of appropriate samples by eliminating the necessity and cost of specialized personnel for drawing blood samples, and by being a less invasive procedure. Therefore, this protocol will help us advance the validation of membrane protein clustering in lymphocytes as a biomarker of therapeutic efficacy in major depression, and bring it closer to its clinical application.

Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane.

PubMed

Elazar, Assaf; Weinstein, Jonathan; Biran, Ido; Fridman, Yearit; Bibi, Eitan; Fleishman, Sarel Jacob

2016-01-29

Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers.

Thylakoid membrane landscape in the sixties: a tribute to Andrew Benson.

PubMed

Anderson, Jan M

2007-05-01

Prior to the 1960s, the model for the molecular structure of cell membranes consisted of a lipid bilayer held in place by a thin film of electrostatically-associated protein stretched over the bilayer surface: (the Danielli-Davson-Robertson "unit membrane" model). Andrew Benson, an expert in the lipids of chloroplast thylakoid membranes, questioned the relevance of the unit membrane model for biological membranes, especially for thylakoid membranes, instead of emphasizing evidence in favour of hydrophobic interactions of membrane lipids within complementary hydrophobic regions of membrane-spanning proteins. With Elliot Weier, Benson postulated a remarkable subunit lipoprotein monolayer model for thylakoids. Following the advent of freeze fracture microscopy and the fluid lipid-protein mosaic model by Singer and Nicolson, the subunits, membrane-spanning integral proteins, span a dynamic lipid bilayer. Now that high resolution X-ray structures of photosystems I and II are being revealed, the seminal contribution of Andrew Benson can be appreciated.

Active Nuclear Import of Membrane Proteins Revisited

PubMed Central

Laba, Justyna K.; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M.

2015-01-01

It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker’s yeast. PMID:26473931

F-BAR family proteins, emerging regulators for cell membrane dynamic changes-from structure to human diseases.

PubMed

Liu, Suxuan; Xiong, Xinyu; Zhao, Xianxian; Yang, Xiaofeng; Wang, Hong

2015-05-09

Eukaryotic cell membrane dynamics change in curvature during physiological and pathological processes. In the past ten years, a novel protein family, Fes/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain proteins, has been identified to be the most important coordinators in membrane curvature regulation. The F-BAR domain family is a member of the Bin/Amphiphysin/Rvs (BAR) domain superfamily that is associated with dynamic changes in cell membrane. However, the molecular basis in membrane structure regulation and the biological functions of F-BAR protein are unclear. The pathophysiological role of F-BAR protein is unknown. This review summarizes the current understanding of structure and function in the BAR domain superfamily, classifies F-BAR family proteins into nine subfamilies based on domain structure, and characterizes F-BAR protein structure, domain interaction, and functional relevance. In general, F-BAR protein binds to cell membrane via F-BAR domain association with membrane phospholipids and initiates membrane curvature and scission via Src homology-3 (SH3) domain interaction with its partner proteins. This process causes membrane dynamic changes and leads to seven important cellular biological functions, which include endocytosis, phagocytosis, filopodium, lamellipodium, cytokinesis, adhesion, and podosome formation, via distinct signaling pathways determined by specific domain-binding partners. These cellular functions play important roles in many physiological and pathophysiological processes. We further summarize F-BAR protein expression and mutation changes observed in various diseases and developmental disorders. Considering the structure feature and functional implication of F-BAR proteins, we anticipate that F-BAR proteins modulate physiological and pathophysiological processes via transferring extracellular materials, regulating cell trafficking and mobility, presenting antigens, mediating extracellular matrix degradation, and transmitting signaling for cell proliferation.

Modular assembly of synthetic proteins that span the plasma membrane in mammalian cells.

PubMed

Qudrat, Anam; Truong, Kevin

2016-12-09

To achieve synthetic control over how a cell responds to other cells or the extracellular environment, it is important to reliably engineer proteins that can traffic and span the plasma membrane. Using a modular approach to assemble proteins, we identified the minimum necessary components required to engineer such membrane-spanning proteins with predictable orientation in mammalian cells. While a transmembrane domain (TM) fused to the N-terminus of a protein is sufficient to traffic it to the endoplasmic reticulum (ER), an additional signal peptidase cleavage site downstream of this TM enhanced sorting out of the ER. Next, a second TM in the synthetic protein helped anchor and accumulate the membrane-spanning protein on the plasma membrane. The orientation of the components of the synthetic protein were determined through measuring intracellular Ca 2+ signaling using the R-GECO biosensor and through measuring extracellular quenching of yellow fluorescent protein variants by saturating acidic and salt conditions. This work forms the basis of engineering novel proteins that span the plasma membrane to potentially control intracellular responses to extracellular conditions.

An ESIPT fluorescent probe sensitive to protein α-helix structures.

PubMed

Jiang, Nan; Yang, Chanli; Dong, Xiongwei; Sun, Xianglang; Zhang, Dan; Liu, Changlin

2014-07-28

A large majority of membrane proteins have one or more transmembrane regions consisting of α-helices. Membrane protein levels differ from one type of cell to another, and the expression of membrane proteins also changes from normal to diseased cells. For example, prostate cancer cells have been reported to have downregulated expression of membrane proteins, including zinc transporters, compared with normal prostate cells. These reports inspired us to design a fluorescence probe sensitive to protein α-helical structures to discriminate individual prostate cancer cells from normal ones. A benzazole derivative ( in this study) was observed to emit strong fluorescence resulting from an excited-state intramolecular proton transfer (ESIPT) in protein α-helical environments. The intensity of ESIPT fluorescence of was observed to be positively correlated with the α-helix content of proteins. The molecular docking simulation suggested that it had low energy for the binding of to proteins when the binding sites were localized within the α-helical regions of protein via H-bonds. Furthermore, was found to be localized in cell membranes through binding to transmembrane α-helical regions of membrane proteins, and was capable of probing differences in the α-helix contents of membrane proteins between normal and cancerous prostate cells through changes in the ESIPT emission intensity. These results indicated that could distinguish individual prostate cancer cells from normal ones, as the changes in the ESIPT fluorescence intensity of could reflect the regulation in expression of the membrane proteins including zinc transporters. This recognition strategy of individual prostate cancer cells might contribute to early diagnosis techniques for prostate cancer.

d-myo-Inositol-3-Phosphate Affects Phosphatidylinositol-Mediated Endomembrane Function in Arabidopsis and Is Essential for Auxin-Regulated Embryogenesis[W][OA

PubMed Central

Luo, Yu; Qin, Genji; Zhang, Jun; Liang, Yuan; Song, Yingqi; Zhao, Meiping; Tsuge, Tomohiko; Aoyama, Takashi; Liu, Jingjing; Gu, Hongya; Qu, Li-Jia

2011-01-01

In animal cells, myo-inositol is an important regulatory molecule in several physiological and biochemical processes, including signal transduction and membrane biogenesis. However, the fundamental biological functions of myo-inositol are still far from clear in plants. Here, we report the genetic characterization of three Arabidopsis thaliana genes encoding d-myo-inositol-3-phosphate synthase (MIPS), which catalyzes the rate-limiting step in de novo synthesis of myo-inositol. Each of the three MIPS genes rescued the yeast ino1 mutant, which is defective in yeast MIPS gene INO1, and they had different dynamic expression patterns during Arabidopsis embryo development. Although single mips mutants showed no obvious phenotypes, the mips1 mips2 double mutant and the mips1 mips2 mips3 triple mutant were embryo lethal, whereas the mips1 mips3 and mips1 mips2+/− double mutants had abnormal embryos. The mips phenotypes resembled those of auxin mutants. Indeed, the double and triple mips mutants displayed abnormal expression patterns of DR5:green fluorescent protein, an auxin-responsive fusion protein, and they had altered PIN1 subcellular localization. Also, membrane trafficking was affected in mips1 mips3. Interestingly, overexpression of PHOSPHATIDYLINOSITOL SYNTHASE2, which converts myo-inositol to membrane phosphatidylinositol (PtdIns), largely rescued the cotyledon and endomembrane defects in mips1 mips3. We conclude that myo-inositol serves as the main substrate for synthesizing PtdIns and phosphatidylinositides, which are essential for endomembrane structure and trafficking and thus for auxin-regulated embryogenesis. PMID:21505066

Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork

PubMed Central

Lenne, Pierre-François; Wawrezinieck, Laure; Conchonaud, Fabien; Wurtz, Olivier; Boned, Annie; Guo, Xiao-Jun; Rigneault, Hervé; He, Hai-Tao; Marguet, Didier

2006-01-01

It is by now widely recognized that cell membranes show complex patterns of lateral organization. Two mechanisms involving either a lipid-dependent (microdomain model) or cytoskeleton-based (meshwork model) process are thought to be responsible for these plasma membrane organizations. In the present study, fluorescence correlation spectroscopy measurements on various spatial scales were performed in order to directly identify and characterize these two processes in live cells with a high temporal resolution, without any loss of spatial information. Putative raft markers were found to be dynamically compartmented within tens of milliseconds into small microdomains (∅<120 nm) that are sensitive to the cholesterol and sphingomyelin levels, whereas actin-based cytoskeleton barriers are responsible for the confinement of the transferrin receptor protein. A free-like diffusion was observed when both the lipid-dependent and cytoskeleton-based organizations were disrupted, which suggests that these are two main compartmentalizing forces at work in the plasma membrane. PMID:16858413

Membrane and luminal proteins reach the apicoplast by different trafficking pathways in the malaria parasite Plasmodium falciparum

PubMed Central

Chaudhari, Rahul; Dey, Vishakha; Narayan, Aishwarya; Sharma, Shobhona

2017-01-01

The secretory pathway in Plasmodium falciparum has evolved to transport proteins to the host cell membrane and to an endosymbiotic organelle, the apicoplast. The latter can occur via the ER or the ER-Golgi route. Here, we study these three routes using proteins Erythrocyte Membrane Protein-1 (PfEMP1), Acyl Carrier Protein (ACP) and glutathione peroxidase-like thioredoxin peroxidase (PfTPxGl) and inhibitors of vesicular transport. As expected, the G protein-dependent vesicular fusion inhibitor AlF4− and microtubule destabilizing drug vinblastine block the trafficking of PfEMP-1, a protein secreted to the host cell membrane. However, while both PfTPxGl and ACP are targeted to the apicoplast, only ACP trafficking remains unaffected by these treatments. This implies that G protein-dependent vesicles do not play a role in classical apicoplast protein targeting. Unlike the soluble protein ACP, we show that PfTPxGl is localized to the outermost membrane of the apicoplast. Thus, the parasite apicoplast acquires proteins via two different pathways: first, the vesicular trafficking pathway appears to handle not only secretory proteins, but an apicoplast membrane protein, PfTPxGl; second, trafficking of apicoplast luminal proteins appear to be independent of G protein-coupled vesicles. PMID:28462015

Membrane and luminal proteins reach the apicoplast by different trafficking pathways in the malaria parasite Plasmodium falciparum.

PubMed

Chaudhari, Rahul; Dey, Vishakha; Narayan, Aishwarya; Sharma, Shobhona; Patankar, Swati

2017-01-01

The secretory pathway in Plasmodium falciparum has evolved to transport proteins to the host cell membrane and to an endosymbiotic organelle, the apicoplast. The latter can occur via the ER or the ER-Golgi route. Here, we study these three routes using proteins Erythrocyte Membrane Protein-1 (PfEMP1), Acyl Carrier Protein (ACP) and glutathione peroxidase-like thioredoxin peroxidase (PfTPx Gl ) and inhibitors of vesicular transport. As expected, the G protein-dependent vesicular fusion inhibitor AlF 4 - and microtubule destabilizing drug vinblastine block the trafficking of PfEMP-1, a protein secreted to the host cell membrane. However, while both PfTPx Gl and ACP are targeted to the apicoplast, only ACP trafficking remains unaffected by these treatments. This implies that G protein-dependent vesicles do not play a role in classical apicoplast protein targeting. Unlike the soluble protein ACP, we show that PfTPx Gl is localized to the outermost membrane of the apicoplast. Thus, the parasite apicoplast acquires proteins via two different pathways: first, the vesicular trafficking pathway appears to handle not only secretory proteins, but an apicoplast membrane protein, PfTPx Gl ; second, trafficking of apicoplast luminal proteins appear to be independent of G protein-coupled vesicles.