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Sample records for arabidopsis transmembrane bzip

  1. Identification of an Arabidopsis transmembrane bZIP transcription factor involved in the endoplasmic reticulum stress response

    SciTech Connect

    Tajima, Hiromi; Iwata, Yuji; Iwano, Megumi; Takayama, Seiji; Koizumi, Nozomu

    2008-09-19

    Among 75 bZIP transcription factors identified in Arabidopsis, 3 (AtbZIP17, AtbZIP28, and AtbZIP49) possess a putative transmembrane domain (TMD) in addition to AtbZIP60, which was characterized previously. In the present study, cDNAs of AtbZIP17 and AtbZIP28 were isolated. Truncated forms of AtbZIP17 and AtbZIP28 lacking the C-terminal domain including TMD were examined as putative active forms. One of them, AtbZIP28{delta}C, activated BiP1 and BiP3 promoters through the cis-elements P-UPRE and ERSE responsible for the ER stress response. Subsequently, a fusion protein of green fluorescent protein (GFP) and AtbZIP28 was expressed in Arabidopsis cultured cells. Under non-stress conditions, GFP fluorescence localization almost overlapped with an ER marker; however, tunicamycin and dithiothreitol treatment clearly increased GFP fluorescence in the nucleus suggesting that the N-terminal fragment of AtbZIP28 translocates to the nucleus in response to ER stress.

  2. Crosstalk between Two bZIP Signaling Pathways Orchestrates Salt-Induced Metabolic Reprogramming in Arabidopsis Roots

    PubMed Central

    Hartmann, Laura; Pedrotti, Lorenzo; Weiste, Christoph; Fekete, Agnes; Schierstaedt, Jasper; Göttler, Jasmin; Kempa, Stefan; Krischke, Markus; Dietrich, Katrin; Mueller, Martin J.; Vicente-Carbajosa, Jesus; Hanson, Johannes; Dröge-Laser, Wolfgang

    2015-01-01

    Soil salinity increasingly causes crop losses worldwide. Although roots are the primary targets of salt stress, the signaling networks that facilitate metabolic reprogramming to induce stress tolerance are less understood than those in leaves. Here, a combination of transcriptomic and metabolic approaches was performed in salt-treated Arabidopsis thaliana roots, which revealed that the group S1 basic leucine zipper transcription factors bZIP1 and bZIP53 reprogram primary C- and N-metabolism. In particular, gluconeogenesis and amino acid catabolism are affected by these transcription factors. Importantly, bZIP1 expression reflects cellular stress and energy status in roots. In addition to the well-described abiotic stress response pathway initiated by the hormone abscisic acid (ABA) and executed by SnRK2 (Snf1-RELATED-PROTEIN-KINASE2) and AREB-like bZIP factors, we identify a structurally related ABA-independent signaling module consisting of SnRK1s and S1 bZIPs. Crosstalk between these signaling pathways recruits particular bZIP factor combinations to establish at least four distinct gene expression patterns. Understanding this signaling network provides a framework for securing future crop productivity. PMID:26276836

  3. Crosstalk between Two bZIP Signaling Pathways Orchestrates Salt-Induced Metabolic Reprogramming in Arabidopsis Roots.

    PubMed

    Hartmann, Laura; Pedrotti, Lorenzo; Weiste, Christoph; Fekete, Agnes; Schierstaedt, Jasper; Göttler, Jasmin; Kempa, Stefan; Krischke, Markus; Dietrich, Katrin; Mueller, Martin J; Vicente-Carbajosa, Jesus; Hanson, Johannes; Dröge-Laser, Wolfgang

    2015-08-01

    Soil salinity increasingly causes crop losses worldwide. Although roots are the primary targets of salt stress, the signaling networks that facilitate metabolic reprogramming to induce stress tolerance are less understood than those in leaves. Here, a combination of transcriptomic and metabolic approaches was performed in salt-treated Arabidopsis thaliana roots, which revealed that the group S1 basic leucine zipper transcription factors bZIP1 and bZIP53 reprogram primary C- and N-metabolism. In particular, gluconeogenesis and amino acid catabolism are affected by these transcription factors. Importantly, bZIP1 expression reflects cellular stress and energy status in roots. In addition to the well-described abiotic stress response pathway initiated by the hormone abscisic acid (ABA) and executed by SnRK2 (Snf1-RELATED-PROTEIN-KINASE2) and AREB-like bZIP factors, we identify a structurally related ABA-independent signaling module consisting of SnRK1s and S1 bZIPs. Crosstalk between these signaling pathways recruits particular bZIP factor combinations to establish at least four distinct gene expression patterns. Understanding this signaling network provides a framework for securing future crop productivity. PMID:26276836

  4. The lumen-facing domain is important for the biological function and organelle-to-organelle movement of bZIP28 during ER stress in Arabidopsis.

    PubMed

    Sun, Le; Lu, Sun-Jie; Zhang, Shuang-Shuang; Zhou, Shun-Fan; Sun, Ling; Liu, Jian-Xiang

    2013-09-01

    The membrane-associated transcription factor, bZIP28, is relocated from the endoplasmic reticulum (ER) to the Golgi and proteolytically released from the membrane mediated by two proteases, S1P and S2P, in response to ER stress in Arabidopsis. The activated N-terminal domain recruits nuclear factor Y (NF-Y) subunits in the nucleus to regulate ER stress downstream genes. Little is known about the functions of the bZIP28 C-terminal lumen-facing domain. Here, we provide novel insights into how the ER lumen-facing domain affects the biological function and organelle-to-organelle movement of bZIP28 in the ER stress response. First, we demonstrated the functional redundancy of bZIP28 and bZIP60 by generation and analysis of the bZIP28 and bZIP60 double mutant zip28zip60. Subsequent genetic complementation experiments in zip28zip60 background with deletions on bZIP28 lumen-facing domain highlighted the importance of lumen-facing domain for its in vivo function of bZIP28 in the ER stress response. The protein subcellular localization and Western blotting results further revealed that the bZIP28 lumen-facing domain contains ER retention signal which is important for the proteolytic activation of bZIP28. Thus, the bZIP28 lumen-facing C-terminus plays important roles in the ER-to-Golgi movement of bZIP28, which may contribute to the sensing of the ER stress.

  5. A bZIP Protein, VIP1, Is a Regulator of Osmosensory Signaling in Arabidopsis1[W

    PubMed Central

    Tsugama, Daisuke; Liu, Shenkui; Takano, Tetsuo

    2012-01-01

    Abscisic acid is a stress-related phytohormone that has roles in dehydration and rehydration. In Arabidopsis (Arabidopsis thaliana), two genes that inactivate abscisic acid, CYP707A1 and CYP707A3, are rapidly up-regulated upon rehydration. The factors that regulate CYP707A1/3 are not well characterized. We expressed a bZIP protein, VIP1, as a green fluorescent protein fusion protein in Arabidopsis and found that the nuclear localization of VIP1 was enhanced within 10 min after rehydration. A yeast one-hybrid assay revealed that the amino-terminal region of VIP1 has transcriptional activation potential. In a transient reporter assay using Arabidopsis protoplasts, VIP1 enhanced the promoter activities of CYP707A1/3. In gel shift and chromatin immunoprecipitation analyses, VIP1 directly bound to DNA fragments of the CYP707A1/3 promoters. Transgenic plants expressing VIP1-green fluorescent protein were found to overexpress CYP707A1/3 mRNAs. The time course of nuclear-cytoplasmic shuttling of VIP1 was consistent with the time courses of the expression of CYP707A1/3. These results suggest that VIP1 functions as a regulator of osmosensory signaling in Arabidopsis. PMID:22452852

  6. Functional characterization of the Arabidopsis transcription factor bZIP29 reveals its role in leaf and root development

    PubMed Central

    Van Leene, Jelle; Blomme, Jonas; Kulkarni, Shubhada R; Cannoot, Bernard; De Winne, Nancy; Eeckhout, Dominique; Persiau, Geert; Van De Slijke, Eveline; Vercruysse, Leen; Vanden Bossche, Robin; Heyndrickx, Ken S; Vanneste, Steffen; Goossens, Alain; Gevaert, Kris; Vandepoele, Klaas; Gonzalez, Nathalie; Inzé, Dirk; De Jaeger, Geert

    2016-01-01

    Plant bZIP group I transcription factors have been reported mainly for their role during vascular development and osmosensory responses. Interestingly, bZIP29 has been identified in a cell cycle interactome, indicating additional functions of bZIP29 in plant development. Here, bZIP29 was functionally characterized to study its role during plant development. It is not present in vascular tissue but is specifically expressed in proliferative tissues. Genome-wide mapping of bZIP29 target genes confirmed its role in stress and osmosensory responses, but also identified specific binding to several core cell cycle genes and to genes involved in cell wall organization. bZIP29 protein complex analyses validated interaction with other bZIP group I members and provided insight into regulatory mechanisms acting on bZIP dimers. In agreement with bZIP29 expression in proliferative tissues and with its binding to promoters of cell cycle regulators, dominant-negative repression of bZIP29 altered the cell number in leaves and in the root meristem. A transcriptome analysis on the root meristem, however, indicated that bZIP29 might regulate cell number through control of cell wall organization. Finally, ectopic dominant-negative repression of bZIP29 and redundant factors led to a seedling-lethal phenotype, pointing to essential roles for bZIP group I factors early in plant development. PMID:27660483

  7. Redox-mediated Mechanisms Regulate DNA Binding Activity of the G-group of Basic Region Leucine Zipper (bZIP) Transcription Factors in Arabidopsis*

    PubMed Central

    Shaikhali, Jehad; Norén, Louise; de Dios Barajas-López, Juan; Srivastava, Vaibhav; König, Janine; Sauer, Uwe H.; Wingsle, Gunnar; Dietz, Karl-Josef; Strand, Åsa

    2012-01-01

    Plant genes that contain the G-box in their promoters are responsive to a variety of environmental stimuli. Bioinformatics analysis of transcriptome data revealed that the G-box element is significantly enriched in promoters of high light-responsive genes. From nuclear extracts of high light-treated Arabidopsis plants, we identified the AtbZIP16 transcription factor as a component binding to the G-box-containing promoter fragment of light-harvesting chlorophyll a/b-binding protein2.4 (LHCB2.4). AtbZIP16 belongs to the G-group of Arabidopsis basic region leucine zipper (bZIP) type transcription factors. Although AtbZIP16 and its close homologues AtbZIP68 and AtGBF1 bind the G-box, they do not bind the mutated half-sites of the G-box palindrome. In addition, AtbZIP16 interacts with AtbZIP68 and AtGBF1 in the yeast two-hybrid system. A conserved Cys residue was shown to be necessary for redox regulation and enhancement of DNA binding activity in all three proteins. Furthermore, transgenic Arabidopsis lines overexpressing the wild type version of bZIP16 and T-DNA insertion mutants for bZIP68 and GBF1 demonstrated impaired regulation of LHCB2.4 expression. Finally, overexpression lines for the mutated Cys variant of bZIP16 provided support for the biological significance of Cys330 in redox regulation of gene expression. Thus, our results suggest that environmentally induced changes in the redox state regulate the activity of members of the G-group of bZIP transcription factors. PMID:22718771

  8. The bZIP Protein VIP1 Is Involved in Touch Responses in Arabidopsis Roots1[OPEN

    PubMed Central

    Liu, Shenkui; Takano, Tetsuo

    2016-01-01

    VIP1 is a bZIP transcription factor in Arabidopsis (Arabidopsis thaliana). VIP1 transiently accumulates in the nucleus when cells are exposed to hypoosmotic conditions, but its physiological relevance is unclear. This is possibly because Arabidopsis has approximately 10 close homologs of VIP1 and they function redundantly. To examine their physiological roles, transgenic plants overexpressing a repression domain-fused form of VIP1 (VIP1-SRDXox plants), in which the gene activation mediated by VIP1 is expected to be repressed, were generated. Because hypoosmotic stress can mimic mechanical stimuli (e.g. touch), the touch-induced root-waving phenotypes and gene expression patterns in those transgenic plants were examined. VIP1-SRDXox plants exhibited more severe root waving and lower expression of putative VIP1 target genes. The expression of the VIP1-green fluorescent protein (GFP) fusion protein partially suppressed the VIP1-SRDX-induced increase in root waving when expressed in the VIP1-SRDXox plants. These results suggest that VIP1 can suppress the touch-induced root waving. The VIP1-SRDX-induced increase in root waving was also suppressed when the synthetic auxin 2,4-dichlorophenoxy acetic acid or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, which is known to activate auxin biosynthesis, was present in the growth medium. Root cap cells with the auxin marker DR5rev::GFP were more abundant in the VIP1-SRDXox background than in the wild-type background. Auxin is transported via the root cap, and the conditions of outermost root cap layers were abnormal in VIP1-SRDXox plants. These results raise the possibility that VIP1 influences structures of the root cap and thereby regulates the local auxin responses in roots. PMID:27208231

  9. Genome-wide analysis of gene expression reveals function of the bZIP transcription factor HY5 in the UV-B response of Arabidopsis.

    PubMed

    Ulm, Roman; Baumann, Alexander; Oravecz, Attila; Máté, Zoltán; Adám, Eva; Oakeley, Edward J; Schäfer, Eberhard; Nagy, Ferenc

    2004-02-01

    The light environment is a key factor that governs a multitude of developmental processes during the entire life cycle of plants. An important and increasing part of the incident sunlight encompasses a segment of the UV-B region (280-320 nm) that is not entirely absorbed by the ozone layer in the stratosphere of the earth. This portion of the solar radiation, which inevitably reaches the sessile plants, can act both as an environmental stress factor and an informational signal. To identify Arabidopsis genes involved in the UV response, we monitored the gene expression profile of UV-B-irradiated seedlings by using high-density oligonucleotide microarrays comprising almost the full Arabidopsis genome (>24,000 genes). A robust set of early low-level UV-B-responsive genes, 100 activated and 7 repressed, was identified. In all cases analyzed, UV-B induction was found to be independent of known photoreceptors. This group of genes is suggested to represent the molecular readout of the signaling cascade triggered by the elusive UV-B photoreceptor(s). Moreover, our analysis identified interactions between cellular responses to different UV-B ranges that led us to postulate the presence of partially distinct but interacting UV-B perception and signaling mechanisms. Finally, we demonstrate that the bZIP transcription factor HY5 is required for UV-B-mediated regulation of a subset of genes.

  10. Hexokinase 1 is required for glucose-induced repression of bZIP63, At5g22920, and BT2 in Arabidopsis

    PubMed Central

    Kunz, Sabine; Gardeström, Per; Pesquet, Edouard; Kleczkowski, Leszek A.

    2015-01-01

    Simple sugars, like glucose (Glc) and sucrose (Suc), act as signals to modulate the expression of hundreds of genes in plants. Frequently, however, it remains unclear whether this regulation is induced by the sugars themselves or by their derivatives generated in the course of carbohydrate (CH) metabolism. In the present study, we tested the relevance of different CH metabolism and allocation pathways affecting expression patterns of five selected sugar-responsive genes (bZIP63, At5g22920, BT2, MGD2, and TPS9) in Arabidopsis thaliana. In general, the expression followed diurnal changes in the overall sugar availability. However, under steady growth conditions, this response was hardly impaired in the mutants for CH metabolizing/ transporting proteins (adg1, sex1, sus1-4, sus5/6, and tpt2), including also hexokinase1 (HXK1) loss- and gain-of-function plants—gin2.1 and oe3.2, respectively. In addition, transgenic plants carrying pbZIP63::GUS showed no changes in reporter-gene-expression when grown on sugar under steady-state conditions. In contrast, short-term treatments of agar-grown seedlings with 1% Glc or Suc induced pbZIP63::GUS repression, which became even more apparent in seedlings grown in liquid media. Subsequent analyses of liquid-grown gin2.1 and oe3.2 seedlings revealed that Glc -dependent regulation of the five selected genes was not affected in gin2.1, whereas it was enhanced in oe3.2 plants for bZIP63, At5g22920, and BT2. The sugar treatments had no effect on ATP/ADP ratio, suggesting that changes in gene expression were not linked to cellular energy status. Overall, the data suggest that HXK1 does not act as Glc sensor controlling bZIP63, At5g22920, and BT2 expression, but it is nevertheless required for the production of a downstream metabolic signal regulating their expression. PMID:26236323

  11. The Arabidopsis bZIP Gene AtbZIP63 Is a Sensitive Integrator of Transient Abscisic Acid and Glucose Signals1[W][OA

    PubMed Central

    Matiolli, Cleverson Carlos; Tomaz, Juarez Pires; Duarte, Gustavo Turqueto; Prado, Fernanda Manso; Del Bem, Luiz Eduardo Vieira; Silveira, Amanda Bortolini; Gauer, Luciane; Corrêa, Luiz Gustavo Guedes; Drumond, Rodrigo Duarte; Viana, Américo José Carvalho; Di Mascio, Paolo; Meyer, Christian; Vincentz, Michel

    2011-01-01

    Glucose modulates plant metabolism, growth, and development. In Arabidopsis (Arabidopsis thaliana), Hexokinase1 (HXK1) is a glucose sensor that may trigger abscisic acid (ABA) synthesis and sensitivity to mediate glucose-induced inhibition of seedling development. Here, we show that the intensity of short-term responses to glucose can vary with ABA activity. We report that the transient (2 h/4 h) repression by 2% glucose of AtbZIP63, a gene encoding a basic-leucine zipper (bZIP) transcription factor partially involved in the Snf1-related kinase KIN10-induced responses to energy limitation, is independent of HXK1 and is not mediated by changes in ABA levels. However, high-concentration (6%) glucose-mediated repression appears to be modulated by ABA, since full repression of AtbZIP63 requires a functional ABA biosynthetic pathway. Furthermore, the combination of glucose and ABA was able to trigger a synergistic repression of AtbZIP63 and its homologue AtbZIP3, revealing a shared regulatory feature consisting of the modulation of glucose sensitivity by ABA. The synergistic regulation of AtbZIP63 was not reproduced by an AtbZIP63 promoter-5′-untranslated region::β-glucuronidase fusion, thus suggesting possible posttranscriptional control. A transcriptional inhibition assay with cordycepin provided further evidence for the regulation of mRNA decay in response to glucose plus ABA. Overall, these results indicate that AtbZIP63 is an important node of the glucose-ABA interaction network. The mechanisms by which AtbZIP63 may participate in the fine-tuning of ABA-mediated abiotic stress responses according to sugar availability (i.e., energy status) are discussed. PMID:21844310

  12. UV-B-responsive association of the Arabidopsis bZIP transcription factor ELONGATED HYPOCOTYL5 with target genes, including its own promoter.

    PubMed

    Binkert, Melanie; Kozma-Bognár, László; Terecskei, Kata; De Veylder, Lieven; Nagy, Ferenc; Ulm, Roman

    2014-10-01

    In plants subjected to UV-B radiation, responses are activated that minimize damage caused by UV-B. The bZIP transcription factor ELONGATED HYPOCOTYL5 (HY5) acts downstream of the UV-B photoreceptor UV RESISTANCE LOCUS8 (UVR8) and promotes UV-B-induced photomorphogenesis and acclimation. Expression of HY5 is induced by UV-B; however, the transcription factor(s) that regulate HY5 transcription in response to UV-B and the impact of UV-B on the association of HY5 with its target promoters are currently unclear. Here, we show that HY5 binding to the promoters of UV-B-responsive genes is enhanced by UV-B in a UVR8-dependent manner in Arabidopsis thaliana. In agreement, overexpression of REPRESSOR OF UV-B PHOTOMORPHOGENESIS2, a negative regulator of UVR8 function, blocks UV-B-responsive HY5 enrichment at target promoters. Moreover, we have identified a T/G-box in the HY5 promoter that is required for its UV-B responsiveness. We show that HY5 and its homolog HYH bind to the T/G(HY5)-box cis-acting element and that they act redundantly in the induction of HY5 expression upon UV-B exposure. Therefore, HY5 is enriched at target promoters in response to UV-B in a UVR8 photoreceptor-dependent manner, and HY5 and HYH interact directly with a T/G-box cis-acting element of the HY5 promoter, mediating the transcriptional activation of HY5 in response to UV-B.

  13. UV-B-Responsive Association of the Arabidopsis bZIP Transcription Factor ELONGATED HYPOCOTYL5 with Target Genes, Including Its Own Promoter[W][OPEN

    PubMed Central

    Binkert, Melanie; Kozma-Bognár, László; Terecskei, Kata; De Veylder, Lieven; Nagy, Ferenc; Ulm, Roman

    2014-01-01

    In plants subjected to UV-B radiation, responses are activated that minimize damage caused by UV-B. The bZIP transcription factor ELONGATED HYPOCOTYL5 (HY5) acts downstream of the UV-B photoreceptor UV RESISTANCE LOCUS8 (UVR8) and promotes UV-B-induced photomorphogenesis and acclimation. Expression of HY5 is induced by UV-B; however, the transcription factor(s) that regulate HY5 transcription in response to UV-B and the impact of UV-B on the association of HY5 with its target promoters are currently unclear. Here, we show that HY5 binding to the promoters of UV-B-responsive genes is enhanced by UV-B in a UVR8-dependent manner in Arabidopsis thaliana. In agreement, overexpression of REPRESSOR OF UV-B PHOTOMORPHOGENESIS2, a negative regulator of UVR8 function, blocks UV-B-responsive HY5 enrichment at target promoters. Moreover, we have identified a T/G-box in the HY5 promoter that is required for its UV-B responsiveness. We show that HY5 and its homolog HYH bind to the T/GHY5-box cis-acting element and that they act redundantly in the induction of HY5 expression upon UV-B exposure. Therefore, HY5 is enriched at target promoters in response to UV-B in a UVR8 photoreceptor-dependent manner, and HY5 and HYH interact directly with a T/G-box cis-acting element of the HY5 promoter, mediating the transcriptional activation of HY5 in response to UV-B. PMID:25351492

  14. The IRE1/bZIP60 pathway and Bax inhibitor 1 suppress systemic accumulation of potyviruses and potexviruses in Arabidopsis and N. benthamiana plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The inositol requiring enzyme (IRE1) is an endoplasmic reticulum (ER) stress sensor and when activated it splices the bZIP60 mRNA producing a truncated transcription factor that upregulates expression of genes involved in the unfolded protein response (UPR). Bax inhibitor 1 (BI-1) is another ER stre...

  15. Transmembrane Topologies of Ca2+-permeable Mechanosensitive Channels MCA1 and MCA2 in Arabidopsis thaliana.

    PubMed

    Kamano, Shumpei; Kume, Shinichiro; Iida, Kazuko; Lei, Kai-Jian; Nakano, Masataka; Nakayama, Yoshitaka; Iida, Hidetoshi

    2015-12-25

    Sensing mechanical stresses, including touch, stretch, compression, and gravity, is crucial for growth and development in plants. A good mechanosensor candidate is the Ca(2+)-permeable mechanosensitive (MS) channel, the pore of which opens to permeate Ca(2+) in response to mechanical stresses. However, the structure-function relationships of plant MS channels are poorly understood. Arabidopsis MCA1 and MCA2 form a homotetramer and exhibit Ca(2+)-permeable MS channel activity; however, their structures have only been partially elucidated. The transmembrane topologies of these ion channels need to be determined in more detail to elucidate the underlying regulatory mechanisms. We herein determined the topologies of MCA1 and MCA2 using two independent methods, the Suc2C reporter and split-ubiquitin yeast two-hybrid methods, and found that both proteins are single-pass type I integral membrane proteins with extracellular N termini and intracellular C termini. These results imply that an EF hand-like motif, coiled-coil motif, and plac8 motif are all present in the cytoplasm. Thus, the activities of both channels can be regulated by intracellular Ca(2+) and protein interactions.

  16. Endocytosis of Seven-Transmembrane RGS Protein Activates G- protein Coupled Signaling in Arabidopsis

    PubMed Central

    Urano, Daisuke; Phan, Nguyen; Jones, Janice C.; Yang, Jing; Huang, Jirong; Grigston, Jeffrey; Taylor, J. Philip; Jones, Alan M.

    2012-01-01

    Signal transduction typically begins by ligand-dependent activation of a concomitant partner which is otherwise in its resting state. However, in cases where signal activation is constitutive by default, the mechanism of regulation is unknown. The Arabidopsis thaliana heterotrimeric Gα protein self-activates without accessory proteins, and is kept in its resting state by the negative regulator, AtRGS1 (Regulator of G protein Signaling 1), which is the prototype of a seven transmembrane receptor fused with an RGS domain. Endocytosis of AtRGS1 by ligand-dependent endocytosis physically uncouples the GTPase accelerating activity of AtRGS1 from the Gα protein, permitting sustained activation. Phosphorylation of AtRGS1 by AtWNK8 kinase causes AtRGS1 endocytosis, required both for G protein-mediated sugar signaling and cell proliferation. In animals, receptor endocytosis results in signal desensitization, whereas in plants, endocytosis results in signal activation. These findings reveal how different organisms rearrange a regulatory system to result in opposite outcomes using similar phosphorylation-dependent endocytosis. PMID:22940907

  17. Biochemical and molecular analysis of a transmembrane protein kinase from Arabidopsis thaliana. Progress report, January 1993

    SciTech Connect

    Bleecker, A.B.

    1993-06-01

    We have isolated genomic and cDNA clones encoding a novel receptor-like protein kinase from the higher plant Arabidopsis thaliana. This kinase is being studied by combining biochemical, molecular, and genetic approaches. Domain-specific antibodies immunodecorate a polypeptide with a molecular mass of 120,000 daltons in extracts of Arabidopsis, where it has been found in all portions of the plant examined including root, stem, leaf, flower, and silique. Cytochemical analysis and initial studies using the kinase promoter with the GUS reporter gene system also indicate that the kinase is present throughout the plant. The kinase is glycosylated, like animal receptor kinases, and has been partially purified from Arabidopsis by using lectin columns. The kinase has been expressed in E coli, purified, and found to autophosphorylate on serine and threonine residues, but not on tyrosine residues. As such, it belongs to the small family of receptor-like kinases with serine/threonine specificity. Transgenic plants are now being produced that either overexpress or carry altered forms of the protein kinase gene. These experiments will help determine the natural role the kinase plays in a pathway of signal transduction.

  18. Biochemical and molecular analysis of a transmembrane protein kinase from Arabidopsis thaliana

    SciTech Connect

    Bleecker, A.B.

    1993-01-01

    We have isolated genomic and cDNA clones encoding a novel receptor-like protein kinase from the higher plant Arabidopsis thaliana. This kinase is being studied by combining biochemical, molecular, and genetic approaches. Domain-specific antibodies immunodecorate a polypeptide with a molecular mass of 120,000 daltons in extracts of Arabidopsis, where it has been found in all portions of the plant examined including root, stem, leaf, flower, and silique. Cytochemical analysis and initial studies using the kinase promoter with the GUS reporter gene system also indicate that the kinase is present throughout the plant. The kinase is glycosylated, like animal receptor kinases, and has been partially purified from Arabidopsis by using lectin columns. The kinase has been expressed in E coli, purified, and found to autophosphorylate on serine and threonine residues, but not on tyrosine residues. As such, it belongs to the small family of receptor-like kinases with serine/threonine specificity. Transgenic plants are now being produced that either overexpress or carry altered forms of the protein kinase gene. These experiments will help determine the natural role the kinase plays in a pathway of signal transduction.

  19. Plant bZIP Transcription Factors Responsive to Pathogens: A Review

    PubMed Central

    Alves, Murilo S.; Dadalto, Silvana P.; Gonçalves, Amanda B.; De Souza, Gilza B.; Barros, Vanessa A.; Fietto, Luciano G.

    2013-01-01

    Transcription factors of the basic leucine zipper (bZIP) family control important processes in all eukaryotes. In plants, bZIPs are master regulators of many central developmental and physiological processes, including morphogenesis, seed formation, abiotic and biotic stress responses. Modulation of the expression patterns of bZIP genes and changes in their activity often contribute to the activation of various signaling pathways and regulatory networks of different physiological processes. However, most advances in the study of plant bZIP transcription factors are related to their involvement in abiotic stress and development. In contrast, there are few examples of functional research with regard to biotic stress, particularly in the defense against pathogens. In this review, we summarize the recent progress revealing the role of bZIP transcription factors in the biotic stress responses of several plant species, from Arabidopsis to cotton. Moreover, we summarize the interacting partners of bZIP proteins in molecular responses during pathogen attack and the key components of the signal transduction pathways with which they physically interact during plant defense responses. Lastly, we focus on the recent advances regarding research on the functional role of bZIPs in major agricultural cultivars and examine the studies performed in this field. PMID:23574941

  20. Two seven-transmembrane domain MILDEW RESISTANCE LOCUS O proteins cofunction in Arabidopsis root thigmomorphogenesis.

    PubMed

    Chen, Zhongying; Noir, Sandra; Kwaaitaal, Mark; Hartmann, H Andreas; Wu, Ming-Jing; Mudgil, Yashwanti; Sukumar, Poornima; Muday, Gloria; Panstruga, Ralph; Jones, Alan M

    2009-07-01

    Directional root expansion is governed by nutrient gradients, positive gravitropism and hydrotropism, negative phototropism and thigmotropism, as well as endogenous oscillations in the growth trajectory (circumnutation). Null mutations in phylogenetically related Arabidopsis thaliana genes MILDEW RESISTANCE LOCUS O 4 (MLO4) and MLO11, encoding heptahelical, plasma membrane-localized proteins predominantly expressed in the root tip, result in aberrant root thigmomorphogenesis. mlo4 and mlo11 mutant plants show anisotropic, chiral root expansion manifesting as tightly curled root patterns upon contact with solid surfaces. The defect in mlo4 and mlo11 mutants is nonadditive and dependent on light and nutrients. Genetic epistasis experiments demonstrate that the mutant phenotype is independently modulated by the Gbeta subunit of the heterotrimeric G-protein complex. Analysis of expressed chimeric MLO4/MLO2 proteins revealed that the C-terminal domain of MLO4 is necessary but not sufficient for MLO4 action in root thigmomorphogenesis. The expression of the auxin efflux carrier fusion, PIN1-green fluorescent protein, the pattern of auxin-induced gene expression, and acropetal as well as basipetal auxin transport are altered at the root tip of mlo4 mutant seedlings. Moreover, addition of auxin transport inhibitors or the loss of EIR1/AGR1/PIN2 function abolishes root curling of mlo4, mlo11, and wild-type seedlings. These results demonstrate that the exaggerated root curling phenotypes of the mlo4 and mlo11 mutants depend on auxin gradients and suggest that MLO4 and MLO11 cofunction as modulators of touch-induced root tropism.

  1. Characterization of transmembrane auxin transport in Arabidopsis suspension-cultured cells.

    PubMed

    Seifertová, Daniela; Skůpa, Petr; Rychtář, Jan; Laňková, Martina; Pařezová, Markéta; Dobrev, Petre I; Hoyerová, Klára; Petrášek, Jan; Zažímalová, Eva

    2014-03-15

    Polar auxin transport is a crucial process for control and coordination of plant development. Studies of auxin transport through plant tissues and organs showed that auxin is transported by a combination of phloem flow and the active, carrier-mediated cell-to-cell transport. Since plant organs and even tissues are too complex for determination of the kinetics of carrier-mediated auxin uptake and efflux on the cellular level, simplified models of cell suspension cultures are often used, and several tobacco cell lines have been established for auxin transport assays. However, there are very few data available on the specificity and kinetics of auxin transport across the plasma membrane for Arabidopsis thaliana suspension-cultured cells. In this report, the characteristics of carrier-mediated uptake (influx) and efflux for the native auxin indole-3-acetic acid and synthetic auxins, naphthalene-1-acetic and 2,4-dichlorophenoxyacetic acids (NAA and 2,4-D, respectively) in A. thaliana ecotype Landsberg erecta suspension-cultured cells (LE line) are provided. By auxin competition assays and inhibitor treatments, we show that, similarly to tobacco cells, uptake carriers have high affinity towards 2,4-D and that NAA is a good tool for studies of auxin efflux in LE cells. In contrast to tobacco cells, metabolic profiling showed that only a small proportion of NAA is metabolized in LE cells. These results show that the LE cell line is a useful experimental system for measurements of kinetics of auxin carriers on the cellular level that is complementary to tobacco cells.

  2. Identification of new ABA- and MEJA-activated sugarcane bZIP genes by data mining in the SUCEST database.

    PubMed

    Schlögl, Paulo Sérgio; Nogueira, Fábio Tebaldi S; Drummond, Rodrigo; Felix, Juliana M; De Rosa, Vicente E; Vicentini, Renato; Leite, Adilson; Ulian, Eugênio C; Menossi, Marcelo

    2008-02-01

    Sugarcane is generally propagated by cuttings of the stalk containing one or more lateral buds, which will develop into a new plant. The transition from the dormant into the active stage constitutes a complex phenomenon characterized by changes in accumulation of phytohormones and several other physiological aspects. Abscisic acid (ABA) and methyl-jasmonate (MeJA) are major signaling molecules, which influence plant development and stress responses. These plant regulators modulate gene expression with the participation of many transcriptional factors. Basic leucine zipper proteins (bZIPs) form a large family of transcriptional factors involved in a variety of plant physiological processes, such as development and responses to stress. Query sequences consisting of full-length protein sequence of each of the Arabidopsis bZIP families were utilized to screen the sugarcane EST database (SUCEST) and 86 sugarcane assembled sequences (SAS) coding for bZIPs were identified. cDNA arrays and RNA-gel blots were used to study the expression of these sugarcane bZIP genes during early plantlet development and in response to ABA and MeJA. Six bZIP genes were found to be differentially expressed during development. ABA and MeJA modulated the expression of eight sugarcane bZIP genes. Our findings provide novel insights into the expression of this large protein family of transcriptional factors in sugarcane.

  3. Membrane-associated transcription factor peptidase, site-2 protease, antagonizes ABA signaling in Arabidopsis.

    PubMed

    Zhou, Shun-Fan; Sun, Le; Valdés, Ana Elisa; Engström, Peter; Song, Ze-Ting; Lu, Sun-Jie; Liu, Jian-Xiang

    2015-10-01

    Abscisic acid plays important roles in maintaining seed dormancy while gibberellins (GA) and other phytohormones antagonize ABA to promote germination. However, how ABA signaling is desensitized during the transition from dormancy to germination is still poorly understood. We functionally characterized the role of membrane-associated transcription factor peptidase, site-2 protease (S2P), in ABA signaling during seed germination in Arabidopsis. Genetic analysis showed that loss-of-function of S2P conferred high ABA sensitivity during seed germination, and expression of the activated form of membrane-associated transcription factor bZIP17, in which the transmembrane domain and endoplasmic reticulum (ER) lumen-facing C-terminus were deleted, in the S2P mutant rescued its ABA-sensitive phenotype. MYC and green fluorescent protein (GFP)-tagged bZIP17 were processed and translocated from the ER to the nucleus in response to ABA treatment. Furthermore, genes encoding negative regulators of ABA signaling, such as the transcription factor ATHB7 and its target genes HAB1, HAB2, HAI1 and AHG3, were up-regulated in seeds of the wild-type upon ABA treatment; this up-regulation was impaired in seeds of S2P mutants. Our results suggest that S2P desensitizes ABA signaling during seed germination through regulating the activation of the membrane-associated transcription factor bZIP17 and therefore controlling the expression level of genes encoding negative regulators of ABA signaling. PMID:25919792

  4. IRE1/bZIP60-Mediated Unfolded Protein Response Plays Distinct Roles in Plant Immunity and Abiotic Stress Responses

    PubMed Central

    Blanco, Francisca; Boatwright, Jon Lucas; Moreno, Ignacio; Jordan, Melissa R.; Chen, Yani; Brandizzi, Federica; Dong, Xinnian

    2012-01-01

    Endoplasmic reticulum (ER)-mediated protein secretion and quality control have been shown to play an important role in immune responses in both animals and plants. In mammals, the ER membrane-located IRE1 kinase/endoribonuclease, a key regulator of unfolded protein response (UPR), is required for plasma cell development to accommodate massive secretion of immunoglobulins. Plant cells can secrete the so-called pathogenesis-related (PR) proteins with antimicrobial activities upon pathogen challenge. However, whether IRE1 plays any role in plant immunity is not known. Arabidopsis thaliana has two copies of IRE1, IRE1a and IRE1b. Here, we show that both IRE1a and IRE1b are transcriptionally induced during chemically-induced ER stress, bacterial pathogen infection and treatment with the immune signal salicylic acid (SA). However, we found that IRE1a plays a predominant role in the secretion of PR proteins upon SA treatment. Consequently, the ire1a mutant plants show enhanced susceptibility to a bacterial pathogen and are deficient in establishing systemic acquired resistance (SAR), whereas ire1b is unaffected in these responses. We further demonstrate that the immune deficiency in ire1a is due to a defect in SA- and pathogen-triggered, IRE1-mediated cytoplasmic splicing of the bZIP60 mRNA, which encodes a transcription factor involved in the expression of UPR-responsive genes. Consistently, IRE1a is preferentially required for bZIP60 splicing upon pathogen infection, while IRE1b plays a major role in bZIP60 processing upon Tunicamycin (Tm)-induced stress. We also show that SA-dependent induction of UPR-responsive genes is altered in the bzip60 mutant resulting in a moderate susceptibility to a bacterial pathogen. These results indicate that the IRE1/bZIP60 branch of UPR is a part of the plant response to pathogens for which the two Arabidopsis IRE1 isoforms play only partially overlapping roles and that IRE1 has both bZIP60-dependent and bZIP60-independent functions in

  5. SnRK1-triggered switch of bZIP63 dimerization mediates the low-energy response in plants

    PubMed Central

    Mair, Andrea; Pedrotti, Lorenzo; Wurzinger, Bernhard; Anrather, Dorothea; Simeunovic, Andrea; Weiste, Christoph; Valerio, Concetta; Dietrich, Katrin; Kirchler, Tobias; Nägele, Thomas; Vicente Carbajosa, Jesús; Hanson, Johannes; Baena-González, Elena; Chaban, Christina; Weckwerth, Wolfram; Dröge-Laser, Wolfgang; Teige, Markus

    2015-01-01

    Metabolic adjustment to changing environmental conditions, particularly balancing of growth and defense responses, is crucial for all organisms to survive. The evolutionary conserved AMPK/Snf1/SnRK1 kinases are well-known metabolic master regulators in the low-energy response in animals, yeast and plants. They act at two different levels: by modulating the activity of key metabolic enzymes, and by massive transcriptional reprogramming. While the first part is well established, the latter function is only partially understood in animals and not at all in plants. Here we identified the Arabidopsis transcription factor bZIP63 as key regulator of the starvation response and direct target of the SnRK1 kinase. Phosphorylation of bZIP63 by SnRK1 changed its dimerization preference, thereby affecting target gene expression and ultimately primary metabolism. A bzip63 knock-out mutant exhibited starvation-related phenotypes, which could be functionally complemented by wild type bZIP63, but not by a version harboring point mutations in the identified SnRK1 target sites. DOI: http://dx.doi.org/10.7554/eLife.05828.001 PMID:26263501

  6. Transcriptome analysis of newly classified bZIP transcription factors of Brassica rapa in cold stress response.

    PubMed

    Hwang, Indeok; Jung, Hee-Jeong; Park, Jong-In; Yang, Tae-Jin; Nou, Ill-Sup

    2014-09-01

    Plant bZIP transcription factors play crucial roles in biological processes. In this study, 136 putative bZIP transcription members were identified in Brassica rapa. The bZIP family can be divided into nine groups according to the specific amino acid rich domain in B. rapa and Arabidopsis thaliana. To screen the cold stress responsive BrbZIP genes, we evaluated whether the transcription patterns of the BrbZIP genes were enhanced by cold treatment in the inbred lines, Chiifu and Kenshin, by microarray data analysis and qRT-PCR. The expression level of six genes increased significantly in Kenshin, but these genes were unchanged in Chiifu. These findings suggest that the six genes that encoded proteins containing N-rich regions might be involved in cold stress response. The results presented herein provide valuable information regarding the molecular basis of the bZIP transcription factors and their potential function in regulation growth and development, particularly in cold stress response.

  7. Transcriptome analysis of newly classified bZIP transcription factors of Brassica rapa in cold stress response.

    PubMed

    Hwang, Indeok; Jung, Hee-Jeong; Park, Jong-In; Yang, Tae-Jin; Nou, Ill-Sup

    2014-09-01

    Plant bZIP transcription factors play crucial roles in biological processes. In this study, 136 putative bZIP transcription members were identified in Brassica rapa. The bZIP family can be divided into nine groups according to the specific amino acid rich domain in B. rapa and Arabidopsis thaliana. To screen the cold stress responsive BrbZIP genes, we evaluated whether the transcription patterns of the BrbZIP genes were enhanced by cold treatment in the inbred lines, Chiifu and Kenshin, by microarray data analysis and qRT-PCR. The expression level of six genes increased significantly in Kenshin, but these genes were unchanged in Chiifu. These findings suggest that the six genes that encoded proteins containing N-rich regions might be involved in cold stress response. The results presented herein provide valuable information regarding the molecular basis of the bZIP transcription factors and their potential function in regulation growth and development, particularly in cold stress response. PMID:25075938

  8. The Transmembrane Region of Guard Cell SLAC1 Channels Perceives CO2 Signals via an ABA-Independent Pathway in Arabidopsis.

    PubMed

    Yamamoto, Yoshiko; Negi, Juntaro; Wang, Cun; Isogai, Yasuhiro; Schroeder, Julian I; Iba, Koh

    2016-02-01

    The guard cell S-type anion channel, SLOW ANION CHANNEL1 (SLAC1), a key component in the control of stomatal movements, is activated in response to CO2 and abscisic acid (ABA). Several amino acids existing in the N-terminal region of SLAC1 are involved in regulating its activity via phosphorylation in the ABA response. However, little is known about sites involved in CO2 signal perception. To dissect sites that are necessary for the stomatal CO2 response, we performed slac1 complementation experiments using transgenic plants expressing truncated SLAC1 proteins. Measurements of gas exchange and stomatal apertures in the truncated transgenic lines in response to CO2 and ABA revealed that sites involved in the stomatal CO2 response exist in the transmembrane region and do not require the SLAC1 N and C termini. CO2 and ABA regulation of S-type anion channel activity in guard cells of the transgenic lines confirmed these results. In vivo site-directed mutagenesis experiments targeted to amino acids within the transmembrane region of SLAC1 raise the possibility that two tyrosine residues exposed on the membrane are involved in the stomatal CO2 response.

  9. Phosphorylation Affects DNA-Binding of the Senescence-Regulating bZIP Transcription Factor GBF1

    PubMed Central

    Smykowski, Anja; Fischer, Stefan M.; Zentgraf, Ulrike

    2015-01-01

    Massive changes in the transcriptome of Arabidopsis thaliana during onset and progression of leaf senescence imply a central role for transcription factors. While many transcription factors are themselves up- or down-regulated during senescence, the bZIP transcription factor G-box-binding factor 1 (GBF1/bZIP41) is constitutively expressed in Arabidopsis leaf tissue but at the same time triggers the onset of leaf senescence, suggesting posttranscriptional mechanisms for senescence-specific GBF1 activation. Here we show that GBF1 is phosphorylated by the threonine/serine CASEIN KINASE II (CKII) in vitro and that CKII phosphorylation had a negative effect on GBF1 DNA-binding to G-boxes of two direct target genes, CATALASE2 and RBSCS1a. Phosphorylation mimicry at three serine positions in the basic region of GBF1 also had a negative effect on DNA-binding. Kinase assays revealed that CKII phosphorylates at least one serine in the basic domain but has additional phosphorylation sites outside this domain. Two different ckII α subunit1 and one α subunit2 T-DNA insertion lines showed no visible senescence phenotype, but in all lines the expression of the senescence marker gene SAG12 was remarkably diminished. A model is presented suggesting that senescence-specific GBF1 activation might be achieved by lowering the phosphorylation of GBF1 by CKII. PMID:27135347

  10. Dominant gain-of-function mutations in transmembrane domain III of ERS1 and ETR1 suggest a novel role for this domain in regulating the magnitude of ethylene response in Arabidopsis.

    PubMed

    Deslauriers, Stephen D; Alvarez, Ashley A; Lacey, Randy F; Binder, Brad M; Larsen, Paul B

    2015-10-01

    Prior work resulted in identification of an Arabidopsis mutant, eer5-1, with extreme ethylene response in conjunction with failure to induce a subset of ethylene-responsive genes, including AtEBP. EER5, which is a TREX-2 homolog that is part of a nucleoporin complex, functions as part of a cryptic aspect of the ethylene signaling pathway that is required for regulating the magnitude of ethylene response. A suppressor mutagenesis screen was carried out to identify second site mutations that could restore the growth of ethylene-treated eer5-1 to wild-type levels. A dominant gain-of-function mutation in the ethylene receptor ETHYLENE RESPONSE SENSOR 1 (ERS1) was identified, with the ers1-4 mutation being located in transmembrane domain III at a point nearly equivalent to the previously described etr1-2 mutation in the other Arabidopsis subfamily I ethylene receptor, ETHYLENE RESPONSE 1 (ETR1). Although both ers1-4 and etr1-2 partially suppress the ethylene hypersensitivity of eer5-1 and are at least in part REVERSION TO ETHYLENE SENSITIVITY 1 (RTE1)-dependent, ers1-4 was additionally found to restore the expression of AtEBP in ers1-4;eer5-1 etiolated seedlings after ethylene treatment in an EIN3-dependent manner. Our work indicates that ERS1-regulated expression of a subset of ethylene-responsive genes is related to controlling the magnitude of ethylene response, with hyperinduction of these genes correlated with reduced ethylene-dependent growth inhibition.

  11. Identification of Important Regions for Ethylene Binding and Signaling in the Transmembrane Domain of the ETR1 Ethylene Receptor of Arabidopsis[W][OA

    PubMed Central

    Wang, Wuyi; Esch, Jeff J.; Shiu, Shin-Han; Agula, Hasi; Binder, Brad M.; Chang, Caren; Patterson, Sara E.; Bleecker, Anthony B.

    2006-01-01

    The ethylene binding domain (EBD) of the Arabidopsis thaliana ETR1 receptor is modeled as three membrane-spanning helices. We surveyed ethylene binding activity in different kingdoms and performed a bioinformatic analysis of the EBD. Ethylene binding is confined to land plants, Chara, and a group of cyanobacteria but is largely absent in other organisms, consistent with our finding that EBD-like sequences are overrepresented among plant and cyanobacterial species. We made amino acid substitutions in 37 partially or completely conserved residues of the EBD and assayed their effects on ethylene binding and signaling. Mutations primarily in residues in Helices I and II midregions eliminated ethylene binding and conferred constitutive signaling, consistent with the inverse-agonist model of ethylene receptor signaling and indicating that these residues define the ethylene binding pocket. The largest class of mutations, clustered near the cytoplasmic ends of Helices I and III, gave normal ethylene binding activity yet still conferred constitutive signaling. Therefore, these residues may play a role in turning off the signal transmitter domain of the receptor. By contrast, only two mutations were loss of function with respect to signaling. These findings yield insight into the structure and function of the EBD and suggest a conserved role of the EBD as a negative regulator of the signal transmitter domain. PMID:17189345

  12. Genome-Wide Analysis of the bZIP Gene Family Identifies Two ABI5-Like bZIP Transcription Factors, BrABI5a and BrABI5b, as Positive Modulators of ABA Signalling in Chinese Cabbage.

    PubMed

    Bai, Yili; Zhu, Wenbo; Hu, Xiaochen; Sun, Congcong; Li, Yanlin; Wang, Dandan; Wang, Qinhu; Pei, Guoliang; Zhang, Yanfeng; Guo, Aiguang; Zhao, Huixian; Lu, Haibin; Mu, Xiaoqian; Hu, Jingjiang; Zhou, Xiaona; Xie, Chang Gen

    2016-01-01

    bZIP (basic leucine zipper) transcription factors coordinate plant growth and development and control responses to environmental stimuli. The genome of Chinese cabbage (Brassica rapa) encodes 136 putative bZIP transcription factors. The bZIP transcription factors in Brassica rapa (BrbZIP) are classified into 10 subfamilies. Phylogenetic relationship analysis reveals that subfamily A consists of 23 BrbZIPs. Two BrbZIPs within subfamily A, Bra005287 and Bra017251, display high similarity to ABI5 (ABA Insensitive 5). Expression of subfamily A BrbZIPs, like BrABI5a (Bra005287/BrbZIP14) and BrABI5b (Bra017251/BrbZIP13), are significantly induced by the plant hormone ABA. Subcellular localization assay reveal that both BrABI5a and BrABI5b have a nuclear localization. BrABI5a and BrABI5b could directly stimulate ABA Responsive Element-driven HIS (a HIS3 reporter gene, which confers His prototrophy) or LUC (LUCIFERASE) expression in yeast and Arabidopsis protoplast. Deletion of the bZIP motif abolished BrABI5a and BrABI5b transcriptional activity. The ABA insensitive phenotype of Arabidopsis abi5-1 is completely suppressed in transgenic lines expressing BrABI5a or BrABI5b. Overall, these results suggest that ABI5 orthologs, BrABI5a and BrABI5b, have key roles in ABA signalling in Chinese cabbage.

  13. Genome-Wide Analysis of the bZIP Gene Family Identifies Two ABI5-Like bZIP Transcription Factors, BrABI5a and BrABI5b, as Positive Modulators of ABA Signalling in Chinese Cabbage

    PubMed Central

    Hu, Xiaochen; Sun, Congcong; Li, Yanlin; Wang, Dandan; Wang, Qinhu; Pei, Guoliang; Zhang, Yanfeng; Guo, Aiguang; Zhao, Huixian; Lu, Haibin; Mu, Xiaoqian; Hu, Jingjiang; Zhou, Xiaona; Xie, Chang Gen

    2016-01-01

    bZIP (basic leucine zipper) transcription factors coordinate plant growth and development and control responses to environmental stimuli. The genome of Chinese cabbage (Brassica rapa) encodes 136 putative bZIP transcription factors. The bZIP transcription factors in Brassica rapa (BrbZIP) are classified into 10 subfamilies. Phylogenetic relationship analysis reveals that subfamily A consists of 23 BrbZIPs. Two BrbZIPs within subfamily A, Bra005287 and Bra017251, display high similarity to ABI5 (ABA Insensitive 5). Expression of subfamily A BrbZIPs, like BrABI5a (Bra005287/BrbZIP14) and BrABI5b (Bra017251/BrbZIP13), are significantly induced by the plant hormone ABA. Subcellular localization assay reveal that both BrABI5a and BrABI5b have a nuclear localization. BrABI5a and BrABI5b could directly stimulate ABA Responsive Element-driven HIS (a HIS3 reporter gene, which confers His prototrophy) or LUC (LUCIFERASE) expression in yeast and Arabidopsis protoplast. Deletion of the bZIP motif abolished BrABI5a and BrABI5b transcriptional activity. The ABA insensitive phenotype of Arabidopsis abi5-1 is completely suppressed in transgenic lines expressing BrABI5a or BrABI5b. Overall, these results suggest that ABI5 orthologs, BrABI5a and BrABI5b, have key roles in ABA signalling in Chinese cabbage. PMID:27414644

  14. Genome-Wide Analysis of the bZIP Gene Family Identifies Two ABI5-Like bZIP Transcription Factors, BrABI5a and BrABI5b, as Positive Modulators of ABA Signalling in Chinese Cabbage.

    PubMed

    Bai, Yili; Zhu, Wenbo; Hu, Xiaochen; Sun, Congcong; Li, Yanlin; Wang, Dandan; Wang, Qinhu; Pei, Guoliang; Zhang, Yanfeng; Guo, Aiguang; Zhao, Huixian; Lu, Haibin; Mu, Xiaoqian; Hu, Jingjiang; Zhou, Xiaona; Xie, Chang Gen

    2016-01-01

    bZIP (basic leucine zipper) transcription factors coordinate plant growth and development and control responses to environmental stimuli. The genome of Chinese cabbage (Brassica rapa) encodes 136 putative bZIP transcription factors. The bZIP transcription factors in Brassica rapa (BrbZIP) are classified into 10 subfamilies. Phylogenetic relationship analysis reveals that subfamily A consists of 23 BrbZIPs. Two BrbZIPs within subfamily A, Bra005287 and Bra017251, display high similarity to ABI5 (ABA Insensitive 5). Expression of subfamily A BrbZIPs, like BrABI5a (Bra005287/BrbZIP14) and BrABI5b (Bra017251/BrbZIP13), are significantly induced by the plant hormone ABA. Subcellular localization assay reveal that both BrABI5a and BrABI5b have a nuclear localization. BrABI5a and BrABI5b could directly stimulate ABA Responsive Element-driven HIS (a HIS3 reporter gene, which confers His prototrophy) or LUC (LUCIFERASE) expression in yeast and Arabidopsis protoplast. Deletion of the bZIP motif abolished BrABI5a and BrABI5b transcriptional activity. The ABA insensitive phenotype of Arabidopsis abi5-1 is completely suppressed in transgenic lines expressing BrABI5a or BrABI5b. Overall, these results suggest that ABI5 orthologs, BrABI5a and BrABI5b, have key roles in ABA signalling in Chinese cabbage. PMID:27414644

  15. REPRESSION OF SHOOT GROWTH, a bZIP Transcriptional Activator, Regulates Cell Elongation by Controlling the Level of Gibberellins

    PubMed Central

    Fukazawa, Jutarou; Sakai, Tatsuya; Ishida, Sarahmi; Yamaguchi, Isomaro; Kamiya, Yuji; Takahashi, Yohsuke

    2000-01-01

    Cell expansion, a developmental process regulated by both endogenous programs and environmental stimuli, is critically important for plant growth. Here, we report the isolation and characterization of RSG (for repression of shoot growth), a transcriptional activator with a basic leucine zipper (bZIP) domain. To examine the role of RSG in plant development, we generated transgenic tobacco plants expressing a dominant-negative form of RSG, which repressed the activity of full-length RSG. In transgenic plants, this expression severely inhibited stem internode growth, specifically cell elongation. These plants also had less endogenous amounts of the major active gibberellin (GA) in tobacco, GA1. Applying GAs restored the dwarf phenotypes of transgenic tobacco plants that expressed the dominant-negative form of RSG. To investigate the function of RSG in the regulation of the endogenous amounts of GAs, we identified a target for RSG. RSG bound and activated the promoter of Arabidopsis GA3, one of the genes encoding enzymes involved in GA biosynthesis. Moreover, the dominant-negative form of RSG decreased expression of the GA3 homolog in transgenic tobacco plants. Our results show that RSG, a bZIP transcriptional activator, regulates the morphology of plants by controlling the endogenous amounts of GAs. PMID:10852936

  16. GhABF2, a bZIP transcription factor, confers drought and salinity tolerance in cotton (Gossypium hirsutum L.)

    PubMed Central

    Liang, Chengzhen; Meng, Zhaohong; Meng, Zhigang; Malik, Waqas; Yan, Rong; Lwin, Khin Myat; Lin, Fazhuang; Wang, Yuan; Sun, Guoqing; Zhou, Tao; Zhu, Tao; Li, Jianying; Jin, Shuangxia; Guo, Sandui; Zhang, Rui

    2016-01-01

    The bZIP transcription factor (TF) act as an important regulator for the abscisic acid (ABA) mediated abiotic stresses signaling pathways in plants. Here, we reported the cloning and characterization of GhABF2, encoding for typical cotton bZIP TF. Overexpression of GhABF2 significantly improved drought and salt stress tolerance both in Arabidopsis and cotton. However, silencing of GhABF2 made transgenic cotton sensitive to PEG osmotic and salt stress. Expression of GhABF2 was induced by drought and ABA treatments but repressed by high salinity. Transcriptome analysis indicated that GhABF2 increases drought and salt tolerance by regulating genes related to ABA, drought and salt response. The proline contents, activity of superoxide dismutase (SOD) and catalase (CAT) were also significantly increased in GhABF2-overexpression cottons in comparison to wild type after drought and salt treatment. Further, an increase in fiber yield under drought and saline-alkali wetland exhibited the important role of GhABF2 in enhancing the drought and salt tolerance in transgenic lines. In conclusion, manipulation of GhABF2 by biotechnological tools could be a sustainable strategy to deploy drought and salt tolerance in cotton. PMID:27713524

  17. Transcriptomic profiling of Arabidopsis gene expression in response to varying micronutrient zinc supply

    PubMed Central

    Azevedo, Herlânder; Azinheiro, Sarah Gaspar; Muñoz-Mérida, Antonio; Castro, Pedro Humberto; Huettel, Bruno; Aarts, Mark G.M.; Assunção, Ana G.L.

    2016-01-01

    Deficiency of the micronutrient zinc is a widespread condition in agricultural soils, causing a negative impact on crop quality and yield. Nevertheless, there is an insufficient knowledge on the regulatory and molecular mechanisms underlying the plant response to inadequate zinc nutrition [1]. This information should contribute to the development of plant-based solutions with improved nutrient-use-efficiency traits in crops. Previously, the transcription factors bZIP19 and bZIP23 were identified as essential regulators of the response to zinc deficiency in Arabidopsis thaliana [2]. A microarray experiment comparing gene expression between roots of wild-type and the mutant bzip19 bzip23, exposed to zinc deficiency, led to the identification of differentially expressed genes related with zinc homeostasis, namely its transport and plant internal translocation [2]. Here, we provide the detailed methodology, bioinformatics analysis and quality controls related to the microarray gene expression profiling published by Assunção and co-workers [2]. Most significantly, the present dataset comprises new experimental variables, including analysis of shoot tissue, and zinc sufficiency and excess supply. Thus, it expands from 8 to 42 microarrays hybridizations, which have been deposited at the Gene Expression Omnibus (GEO) under the accession number GSE77286. Overall, it provides a resource for research on the molecular basis and regulatory events of the plant response to zinc supply, emphasizing the importance of Arabidopsis bZIP19 and bZIP23 transcription factors. PMID:26981422

  18. HTLV-1 Tax Protein Stimulation of DNA Binding of bZIP Proteins by Enhancing Dimerization

    NASA Astrophysics Data System (ADS)

    Wagner, Susanne; Green, Michael R.

    1993-10-01

    The Tax protein of human T cell leukemia virus type-1 (HTLV-I) transcriptionally activates the HTLV-I promoter. This activation requires binding sites for activating transcription factor (ATF) proteins, a family of cellular proteins that contain basic region-leucine zipper (bZIP) DNA binding domains. Data are presented showing that Tax increases the in vitro DNA binding activity of multiple ATF proteins. Tax also stimulated DNA binding by other bZIP proteins, but did not affect DNA binding proteins that lack a bZIP domain. The increase in DNA binding occurred because Tax promotes dimerization of the bZIP domain in the absence of DNA, and the elevated concentration of the bZIP homodimer then facilitates the DNA binding reaction. These results help explain how Tax activates viral transcription and transforms cells.

  19. FASCIATED EAR4 encodes a bZIP transcription factor that regulates shoot meristem size in maize.

    PubMed

    Pautler, Michael; Eveland, Andrea L; LaRue, Therese; Yang, Fang; Weeks, Rebecca; Lunde, China; Je, Byoung Il; Meeley, Robert; Komatsu, Mai; Vollbrecht, Erik; Sakai, Hajime; Jackson, David

    2015-01-01

    Plant architecture is dictated by precise control of meristematic activity. In the shoot, an imbalance in positive or negative maintenance signals can result in a fasciated or enlarged meristem phenotype. fasciated ear4 (fea4) is a semidwarfed mutant with fasciated ears and tassels as well as greatly enlarged vegetative and inflorescence meristems. We identified FEA4 as a bZIP transcription factor, orthologous to Arabidopsis thaliana PERIANTHIA. FEA4 was expressed in the peripheral zone of the vegetative shoot apical meristem and in the vasculature of immature leaves and conspicuously excluded from the stem cell niche at the tip of the shoot apical meristem and from incipient leaf primordia. Following the transition to reproductive fate, FEA4 was expressed throughout the entire inflorescence and floral meristems. Native expression of a functional YFP:FEA4 fusion recapitulated this pattern of expression. We used chromatin immunoprecipitation-sequencing to identify 4060 genes proximal to FEA4 binding sites, including ones that were potentially bound and modulated by FEA4 based on transcriptional changes in fea4 mutant ears. Our results suggest that FEA4 promotes differentiation in the meristem periphery by regulating auxin-based responses and genes associated with leaf differentiation and polarity, potentially in opposition to factors such as KNOTTED1 and WUSCHEL. PMID:25616871

  20. Identification of Two bZIP Transcription Factors Interacting with the Promoter of Soybean Rubisco Activase Gene (GmRCAα)

    PubMed Central

    Zhang, Jinyu; Du, Hongyang; Chao, Maoni; Yin, Zhitong; Yang, Hui; Li, Yakai; Huang, Fang; Yu, Deyue

    2016-01-01

    Rubisco activase (RCA), a key photosynthetic protein, catalyses the activation of Rubisco and thus plays an important role in photosynthesis. Although the RCA gene has been characterized in a variety of species, the molecular mechanism regulating its transcription remains unclear. Our previous studies on RCA gene expression in soybean suggested that expression of this gene is regulated by trans-acting factors. In the present study, we verified activity of the GmRCAα promoter in both soybean and Arabidopsis and used a yeast one-hybrid (Y1H) system for screening a leaf cDNA expression library to identify transcription factors (TFs) interacting with the GmRCAα promoter. Four basic leucine zipper (bZIP) TFs, GmbZIP04g, GmbZIP07g, GmbZIP1, and GmbZIP71, were isolated, and GmbZIP04g and GmbZIP07g were confirmed as able to bind to a 21-nt G-box-containing sequence. Additionally, the expression patterns of GmbZIP04g, GmbZIp07g, and GmRCAα were analyzed in response to abiotic stresses and during a 24-h period. Our study will help to advance elucidation of the network regulating GmRCAα transcription. PMID:27242832

  1. Differential expression of four soybean bZIP genes during Phakopsora pachyrhizi infection.

    PubMed

    Alves, Murilo S; Soares, Zamira G; Vidigal, Pedro M P; Barros, Everaldo G; Poddanosqui, Adriana M P; Aoyagi, Luciano N; Abdelnoor, Ricardo V; Marcelino-Guimarães, Francismar C; Fietto, Luciano G

    2015-11-01

    Asian soybean rust (ASR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, is one of most important diseases in the soybean (Glycine max (L.) Merr.) agribusiness. The identification and characterization of genes related to plant defense responses to fungal infection are essential to develop ASR-resistant plants. In this work, we describe four soybean genes, GmbZIP62, GmbZIP105, GmbZIPE1, and GmbZIPE2, which encode transcription factors containing a basic leucine zipper (bZIP) domain from two divergent classes, and that are responsive to P. pachyrhizi infection. Molecular phylogenetic analyses demonstrated that these genes encode proteins similar to bZIP factors responsive to pathogens. Yeast transactivation assays showed that only GmbZIP62 has strong transactivation activity in yeast. In addition, three of the bZIP transcription factors analyzed were also differentially expressed by plant defense hormones, and all were differentially expressed by fungal attack, indicating that these proteins might participate in response to ASR infection. The results suggested that these bZIP proteins are part of the plant defense response to P. pachyrhizi infection, by regulating the gene expression related to ASR infection responses. These bZIP genes are potential targets to obtain new soybean genotypes resistant to ASR.

  2. Transmembrane signaling proteoglycans.

    PubMed

    Couchman, John R

    2010-01-01

    Virtually all metazoan cells contain at least one and usually several types of transmembrane proteoglycans. These are varied in protein structure and type of polysaccharide, but the total number of vertebrate genes encoding transmembrane proteoglycan core proteins is less than 10. Some core proteins, including those of the syndecans, always possess covalently coupled glycosaminoglycans; others do not. Syndecan has a long evolutionary history, as it is present in invertebrates, but many other transmembrane proteoglycans are vertebrate inventions. The variety of proteins and their glycosaminoglycan chains is matched by diverse functions. However, all assume roles as coreceptors, often working alongside high-affinity growth factor receptors or adhesion receptors such as integrins. Other common themes are an ability to signal through their cytoplasmic domains, often to the actin cytoskeleton, and linkage to PDZ protein networks. Many transmembrane proteoglycans associate on the cell surface with metzincin proteases and can be shed by them. Work with model systems in vivo and in vitro reveals roles in growth, adhesion, migration, and metabolism. Furthermore, a wide range of phenotypes for the core proteins has been obtained in mouse knockout experiments. Here some of the latest developments in the field are examined in hopes of stimulating further interest in this fascinating group of molecules. PMID:20565253

  3. Expression and Functional Roles of the Pepper Pathogen-Induced bZIP Transcription Factor CabZIP2 in Enhanced Disease Resistance to Bacterial Pathogen Infection.

    PubMed

    Lim, Chae Woo; Baek, Woonhee; Lim, Sohee; Han, Sang-Wook; Lee, Sung Chul

    2015-07-01

    A pepper bZIP transcription factor gene, CabZIP2, was isolated from pepper leaves infected with a virulent strain of Xanthomonas campestris pv. vesicatoria. Transient expression analysis of the CabZIP2-GFP fusion protein in Nicotiana benthamiana revealed that the CabZIP2 protein is localized in the cytoplasm as well as the nucleus. The acidic domain in the N-terminal region of CabZIP2 that is fused to the GAL4 DNA-binding domain is required to activate the transcription of reporter genes in yeast. Transcription of CabZIP2 is induced in pepper plants inoculated with virulent or avirulent strains of X. campestris pv. vesicatoria. The CabZIP2 gene is also induced by defense-related hormones such as salicylic acid, methyl jasmonate, and ethylene. To elucidate the in vivo function of the CabZIP2 gene in plant defense, virus-induced gene silencing in pepper and overexpression in Arabidopsis were used. CabZIP2-silenced pepper plants were susceptible to infection by the virulent strain of X. campestris pv. vesicatoria, which was accompanied by reduced expression of defense-related genes such as CaBPR1 and CaAMP1. CabZIP2 overexpression in transgenic Arabidopsis plants conferred enhanced resistance to Pseudomonas syringae pv. tomato DC3000. Together, these results suggest that CabZIP2 is involved in bacterial disease resistance.

  4. Data-Driven Prediction and Design of bZIP Coiled-Coil Interactions

    PubMed Central

    Potapov, Vladimir; Kaplan, Jenifer B.; Keating, Amy E.

    2015-01-01

    Selective dimerization of the basic-region leucine-zipper (bZIP) transcription factors presents a vivid example of how a high degree of interaction specificity can be achieved within a family of structurally similar proteins. The coiled-coil motif that mediates homo- or hetero-dimerization of the bZIP proteins has been intensively studied, and a variety of methods have been proposed to predict these interactions from sequence data. In this work, we used a large quantitative set of 4,549 bZIP coiled-coil interactions to develop a predictive model that exploits knowledge of structurally conserved residue-residue interactions in the coiled-coil motif. Our model, which expresses interaction energies as a sum of interpretable residue-pair and triplet terms, achieves a correlation with experimental binding free energies of R = 0.68 and significantly out-performs other scoring functions. To use our model in protein design applications, we devised a strategy in which synthetic peptides are built by assembling 7-residue native-protein heptad modules into new combinations. An integer linear program was used to find the optimal combination of heptads to bind selectively to a target human bZIP coiled coil, but not to target paralogs. Using this approach, we designed peptides to interact with the bZIP domains from human JUN, XBP1, ATF4 and ATF5. Testing more than 132 candidate protein complexes using a fluorescence resonance energy transfer assay confirmed the formation of tight and selective heterodimers between the designed peptides and their targets. This approach can be used to make inhibitors of native proteins, or to develop novel peptides for applications in synthetic biology or nanotechnology. PMID:25695764

  5. VIP1 is very important/interesting protein 1 regulating touch responses of Arabidopsis

    PubMed Central

    Tsugama, Daisuke; Liu, Shenkui; Takano, Tetsuo

    2016-01-01

    ABSTRACT VIP1 (VIRE2-INTERACTING PROTEIN 1) is a bZIP transcription factor in Arabidopsis thaliana. VIP1 and its close homologs (i.e., Arabidopsis group I bZIP proteins) are present in the cytoplasm under steady conditions, but are transiently localized to the nucleus when cells are exposed to hypo-osmotic conditions, which mimic mechanical stimuli such as touch. Recently we have reported that overexpression of a repression domain-fused form of VIP1 represses the expression of some touch-responsive genes, changes structures and/or local auxin responses of the root cap cells, and enhances the touch-induced root waving. This raises the possibility that VIP1 suppresses touch-induced responses. VIP1 should be useful to further characterize touch responses of plants. Here we discuss 2 seemingly interesting perspectives about VIP1: (1) What factors are involved in regulating the nuclear localization of VIP1?; (2) What can be done to further characterize the physiological functions of VIP1 and other Arabidopsis group I bZIP proteins? PMID:27171129

  6. The Role of bZIP Transcription Factors in Green Plant Evolution: Adaptive Features Emerging from Four Founder Genes

    PubMed Central

    Schrago, Carlos Guerra; dos Santos, Renato Vicentini; Mueller-Roeber, Bernd; Vincentz, Michel

    2008-01-01

    Background Transcription factors of the basic leucine zipper (bZIP) family control important processes in all eukaryotes. In plants, bZIPs are regulators of many central developmental and physiological processes including photomorphogenesis, leaf and seed formation, energy homeostasis, and abiotic and biotic stress responses. Here we performed a comprehensive phylogenetic analysis of bZIP genes from algae, mosses, ferns, gymnosperms and angiosperms. Methodology/Principal Findings We identified 13 groups of bZIP homologues in angiosperms, three more than known before, that represent 34 Possible Groups of Orthologues (PoGOs). The 34 PoGOs may correspond to the complete set of ancestral angiosperm bZIP genes that participated in the diversification of flowering plants. Homologous genes dedicated to seed-related processes and ABA-mediated stress responses originated in the common ancestor of seed plants, and three groups of homologues emerged in the angiosperm lineage, of which one group plays a role in optimizing the use of energy. Conclusions/Significance Our data suggest that the ancestor of green plants possessed four bZIP genes functionally involved in oxidative stress and unfolded protein responses that are bZIP-mediated processes in all eukaryotes, but also in light-dependent regulations. The four founder genes amplified and diverged significantly, generating traits that benefited the colonization of new environments. PMID:18698409

  7. Novel DNA binding specificities of a putative herpesvirus bZIP oncoprotein.

    PubMed Central

    Qian, Z; Brunovskis, P; Lee, L; Vogt, P K; Kung, H J

    1996-01-01

    Marek's disease virus is a highly oncogenic herpesvirus that can cause T lymphomas and peripheral nerve demyelination in chickens. meq, a candidate oncogene of Marek's disease virus, encodes a basic leucine zipper (bZIP) transcription factor which contains a large proline-rich domain in its C terminus. On the basis of its bZIP structural homology, meq is perhaps the only member of the jun-fos gene family completely viral in origin. We previously showed that Meq's C-terminal domain has potent transactivation activity and that its bZIP domain can dimerize with itself and with c-Jun also. In an effort to identify viral and cellular targets of Meq, we have determined the optimal binding sites for Meq-Jun heterodimers and Meq-Meq homodimers. By a PCR-based approach using cyclic amplification of selected targets, Meq-Jun heterodimers were found to optimally bind tetradecanoylphorbol acetate response element (TRE) and cyclic AMP response element (CRE) consensus sequences. This result was consistent with the results of our previous functional analysis implicating Meq-Jun heterodimers in the transactivation of the Meq promoter through a TRE- or CRE-like sequence. Interestingly, Meq-Meq homodimers were found to bind two distinct motif elements. The first [GAGTGATG AC(G)TCATC] has a consensus which includes a TRE or CRE core flanked by additional nucleotides critical for tight binding. Methylation interference and mutational analyses confirmed the importance of the flanking residues. The sequences of a subset of TRE and CRE sites selected by Meq-Meq are closely related to the binding motif of Maf, another bZIP oncoprotein. The second putative Meq binding site (RACACACAY) bears a completely different consensus not shared by other bZIP proteins. Binding to this consensus sequence also requires secondary structure characteristics associated with DNA bending. CACA motifs are known to promote DNA curvature and function in a number of special biological processes. Our results lend

  8. Disordered regions in transmembrane proteins.

    PubMed

    Tusnády, Gábor E; Dobson, László; Tompa, Peter

    2015-11-01

    The functions of transmembrane proteins in living cells are widespread; they range from various transport processes to energy production, from cell-cell adhesion to communication. Structurally, they are highly ordered in their membrane-spanning regions, but may contain disordered regions in the cytosolic and extra-cytosolic parts. In this study, we have investigated the disordered regions in transmembrane proteins by a stringent definition of disordered residues on the currently available largest experimental dataset, and show a significant correlation between the spatial distributions of positively charged residues and disordered regions. This finding suggests a new role of disordered regions in transmembrane proteins by providing structural flexibility for stabilizing interactions with negatively charged head groups of the lipid molecules. We also find a preference of structural disorder in the terminal--as opposed to loop--regions in transmembrane proteins, and survey the respective functions involved in recruiting other proteins or mediating allosteric signaling effects. Finally, we critically compare disorder prediction methods on our transmembrane protein set. While there are no major differences between these methods using the usual statistics, such as per residue accuracies, Matthew's correlation coefficients, etc.; substantial differences can be found regarding the spatial distribution of the predicted disordered regions. We conclude that a predictor optimized for transmembrane proteins would be of high value to the field of structural disorder. PMID:26275590

  9. The stability of the three transmembrane and the four transmembrane human vitamin K epoxide reductase models

    NASA Astrophysics Data System (ADS)

    Wu, Sangwook

    2016-04-01

    The three transmembrane and the four transmembrane helix models are suggested for human vitamin K epoxide reductase (VKOR). In this study, we investigate the stability of the human three transmembrane/four transmembrane VKOR models by employing a coarse-grained normal mode analysis and molecular dynamics simulation. Based on the analysis of the mobility of each transmembrane domain, we suggest that the three transmembrane human VKOR model is more stable than the four transmembrane human VKOR model.

  10. The bZIP transcription factor HY5 interacts with the promoter of the monoterpene synthase gene QH6 in modulating its rhythmic expression

    PubMed Central

    Zhou, Fei; Sun, Tian-Hu; Zhao, Lei; Pan, Xi-Wu; Lu, Shan

    2015-01-01

    The Artemisia annua L. β-pinene synthase QH6 was previously determined to be circadian-regulated at the transcriptional level, showing a rhythmic fluctuation of steady-state transcript abundances. Here we isolated both the genomic sequence and upstream promoter region of QH6. Different regulatory elements, such as G-box (TGACACGTGGCA, −421 bp from the translation initiation site) which might have effects on rhythmic gene expression, were found. Using the yeast one-hybrid and electrophoretic mobility shift assay (EMSA), we confirmed that the bZIP transcription factor HY5 binds to this motif of QH6. Studies with promoter truncations before and after this motif suggested that this G-box was important for the diurnal fluctuation of the transgenic β-glucuronidase gene (GUS) transcript abundance in Arabidopsis thaliana. GUS gene driven by the promoter region immediately after G-box showed an arrhythmic expression in both light/dark (LD) and constant dark (DD) conditions, whereas the control with G-box retained its fluctuation in both LD and DD. We further transformed A. thaliana with the luciferase gene (LUC) driven by an 1400 bp fragment upstream QH6 with its G-box intact or mutated, respectively. The luciferase activity assay showed that a peak in the early morning disappeared in the mutant. Gene expression analysis also demonstrated that the rhythmic expression of LUC was abolished in the hy5-1 mutant. PMID:25983739

  11. The bZIP transcription factor HY5 interacts with the promoter of the monoterpene synthase gene QH6 in modulating its rhythmic expression.

    PubMed

    Zhou, Fei; Sun, Tian-Hu; Zhao, Lei; Pan, Xi-Wu; Lu, Shan

    2015-01-01

    The Artemisia annua L. β-pinene synthase QH6 was previously determined to be circadian-regulated at the transcriptional level, showing a rhythmic fluctuation of steady-state transcript abundances. Here we isolated both the genomic sequence and upstream promoter region of QH6. Different regulatory elements, such as G-box (TGACACGTGGCA, -421 bp from the translation initiation site) which might have effects on rhythmic gene expression, were found. Using the yeast one-hybrid and electrophoretic mobility shift assay (EMSA), we confirmed that the bZIP transcription factor HY5 binds to this motif of QH6. Studies with promoter truncations before and after this motif suggested that this G-box was important for the diurnal fluctuation of the transgenic β-glucuronidase gene (GUS) transcript abundance in Arabidopsis thaliana. GUS gene driven by the promoter region immediately after G-box showed an arrhythmic expression in both light/dark (LD) and constant dark (DD) conditions, whereas the control with G-box retained its fluctuation in both LD and DD. We further transformed A. thaliana with the luciferase gene (LUC) driven by an 1400 bp fragment upstream QH6 with its G-box intact or mutated, respectively. The luciferase activity assay showed that a peak in the early morning disappeared in the mutant. Gene expression analysis also demonstrated that the rhythmic expression of LUC was abolished in the hy5-1 mutant. PMID:25983739

  12. Simulations of temperature and salt concentration effects on bZIP, a basic region leucine zipper.

    PubMed

    Cukier, Robert I

    2012-05-31

    Basic region leucine zipper (bZIP) transcription factors are dimeric proteins that recognize DNA. The monomers consist of a leucine zipper subdomain responsible for dimerization and a highly basic DNA recognition subdomain. Twelve explicit solvent molecular dynamics (MD) trajectories were run on the GCN4 bZIP transcriptional factor in the absence of DNA at three temperatures and two ion concentrations (0 mM with Cl(-) ions to neutralize the bZIP and 200 mM with additional Na(+) and Cl(-) ions) to probe the conformational ensemble that the basic region samples. In most trajectories, the basic region exhibits an alligator-jaw-like opening and closing (only one monomer moves), versus scissor-like motion, by a mainly rigid body, hinge motion centered on three "fork" residues that span the basic region to the coiled coil. In this motion, the α-helical character of the basic region monomers is mostly maintained. A broad range of distances is accessed, consistent with the absence of particular interactions for the basic region monomers. In two of the trajectories, the basic region monomers "collapse" to form a stable state. The coiled coil, leucine zipper subdomain is very stable for all of the trajectories. Ion solvation of the charged residue side chains is transient, on the scale of a few picoseconds. There is no evidence for persistent specific ion salt bridges to charged residues. For 0 mM, only certain basic region positively charged residues are substantially Cl(-) ion salt bridged. For 200 mM, in addition, some basic region negatively (positively) charged residues are salt bridged to Na(+) (Cl(-)) ions. The different ion solvation patterns at the two ion concentrations are not greatly temperature sensitive, and the conformational sampling found in the MD is remarkably unperturbed by ion concentration and/or temperature. PMID:22559083

  13. Wheat Transcription Factor TaAREB3 Participates in Drought and Freezing Tolerances in Arabidopsis

    PubMed Central

    Wang, Jingyi; Li, Qian; Mao, Xinguo; Li, Ang; Jing, Ruilian

    2016-01-01

    AREB (ABA response element binding) proteins in plants play direct regulatory roles in response to multiple stresses, but their functions in wheat (Triticum aestivum L.) are not clear. In the present study, TaAREB3, a new member of the AREB transcription factor family, was isolated from wheat. Sequence analysis showed that the TaAREB3 protein is composed of three parts, a conserved N-terminal, a variable M region, and a conserved C-terminal with a bZIP domain. It belongs to the group A subfamily of bZIP transcription factors. TaAREB3 was constitutively expressed in stems, leaves, florets, anthers, pistils, seeds, and most highly, in roots. TaAREB3 gene expression was induced with abscisic acid (ABA) and low temperature stress, and its protein was localized in the nucleus when transiently expressed in tobacco epidermal cells and stably expressed in transgenic Arabidopsis. TaAREB3 protein has transcriptional activation activity, and can bind to the ABRE cis-element in vitro. Overexpression of TaAREB3 in Arabidopsis not only enhanced ABA sensitivity, but also strengthened drought and freezing tolerances. TaAREB3 also activated RD29A, RD29B, COR15A, and COR47 by binding to their promoter regions in transgenic Arabidopsis. These results demonstrated that TaAREB3 plays an important role in drought and freezing tolerances in Arabidopsis. PMID:26884722

  14. Expression analysis of bZIP transcription factor encoding genes in response to water deficit stress in rice.

    PubMed

    Ali, Kishwar; Rai, R D; Tyagi, Aruna

    2016-05-01

    In plants, basic region/leucine zipper motif (bZIP) transcription factors regulate several developmental processes and activate genes in response to biotic and abiotic stresses. Role of stress responsive bZIP transcription factors was studied in paddy in relation to different stages of development and water deficit stress (WDS) in a drought tolerant cultivar N22 and susceptible IR 64. Further, relative water content (RWC), membrane stability index (MSI) and abscisic acid (ABA) content were measured as indices of WDS at different stages of development and levels of stress. Expression of stress responsive bZIP transcription factors was directly correlated to developmental stage and WDS and indirectly to RWC, MSI and ABA content. PMID:27319052

  15. Functional dissection of a small anaerobically induced bZIP transcription factor from tomato.

    PubMed

    Sell, Simone; Hehl, Reinhard

    2004-11-01

    A small anaerobically induced tomato transcription factor was isolated from a subtractive library. This factor, designated ABZ1 (anaerobic basic leucine zipper), is anaerobically induced in fruits, leaves and roots and encodes a nuclear localized protein. ABZ1 shares close structural and sequence homology with the S-family of small basic leucine zipper (bZIP) transcription factors that are implicated in stress response. Nuclear localization of ABZ1 is mediated by the basic region and occurs under normoxic conditions. ABZ1 binds to G-box-like target sites as a dimer. Binding can be abolished by heterodimerization with a truncated protein retaining the leucine zipper but lacking the DNA binding domain. The protein binds in a sequence specific manner to the CaMV 35S promoter which is down regulated when ABZ1 is coexpressed. This correlates with the anaerobic down regulation of the 35S promoter in tomato and tobacco. These results may suggest that small bZIP proteins are involved in the negative regulation of gene expression under anaerobic conditions. PMID:15560794

  16. Transcriptional regulation of secretory capacity by bZip transcription factors

    PubMed Central

    FOX, Rebecca M.

    2015-01-01

    Cells of specialized secretory organs expand their secretory pathways to accommodate the increased protein load necessary for their function. The endoplasmic reticulum (ER), the Golgi apparatus and the secretory vesicles, expand not only the membrane components but also the protein machinery required for increased protein production and transport. Increased protein load causes an ER stress response akin to the Unfolded Protein Response (UPR). Recent work has implicated several bZip transcription factors in the regulation of protein components of the early secretory pathway necessary to alleviate this stress. Here, we highlight eight bZip transcription factors in regulating secretory pathway component genes. These include components of the three canonical branches of the UPR–ATF4, XBP1, and ATF6, as well as the five members of the Creb3 family of transcription factors. We review findings from both invertebrate and vertebrate model systems suggesting that all of these proteins increase secretory capacity in response to increased protein load. Finally, we propose that the Creb3 family of factors may have a dual role in secretory cell differentiation by also regulating the pathways necessary for cell cycle exit during terminal differentiation. PMID:25821458

  17. Characterization of a bZIP gene highly expressed during ripening of the peach fruit.

    PubMed

    Lovisetto, Alessandro; Guzzo, Flavia; Tadiello, Alice; Confortin, Enrico; Pavanello, Anna; Botton, Alessandro; Casadoro, Giorgio

    2013-09-01

    A ripening specific bZIP gene of peach was studied by ectopically expressing it in tomato. Two lines, with either a mild or a strong phenotype, respectively, were analyzed in detail. Transgenic fruit morphology was normal, yet the time spent to proceed through the various ripening stages was longer compared to wild type. In agreement with this finding the transgenic berries produced less ethylene, and also had a modified expression of some ripening-related genes that was particularly evident in berries with a strong phenotype. In particular, in the latter fruits polygalacturonase and lipoxygenase genes, but also genes coding for transcription factors (TFs) important for tomato ripening (i.e. TAGL1, CNR, APETALA2a, NOR) did not show the expected decreased expression in the red berries. As regards the RIN gene, its expression continued to increase in both mild and strong lines, and this is in agreement with the dilated ripening times. Interestingly, a metabolomic analysis of berries at various stages of ripening showed that the longer time spent by the transgenic berries to proceed from a stage to another was not due to a slackened metabolism. In fact, the differences in amount of stage-specific marker metabolites indicated that the transgenic berries had a very active metabolism. Therefore, the dilated ripening and the enhanced metabolism of the berries over-expressing the bZIP gene suggest that such gene might regulate ripening by acting as a pacemaker for some of the ripening metabolic pathways.

  18. Divergence of the bZIP Gene Family in Strawberry, Peach, and Apple Suggests Multiple Modes of Gene Evolution after Duplication

    PubMed Central

    Wang, Xiao-Long; Zhong, Yan; Cheng, Zong-Ming; Xiong, Jin-Song

    2015-01-01

    The basic leucine zipper (bZIP) transcription factors are the most diverse members of dimerizing transcription factors. In the present study, 50, 116, and 47 bZIP genes were identified in Malus domestica (apple), Prunus persica (peach), and Fragaria vesca (strawberry), respectively. Species-specific duplication was the main contributor to the large number of bZIPs observed in apple. After WGD in apple genome, orthologous bZIP genes corresponding to strawberry on duplicated regions in apple genome were retained. However, in peach ancestor, these syntenic regions were quickly lost or deleted. Maybe the positive selection contributed to the expansion of clade S to adapt to the development and environment stresses. In addition, purifying selection was mainly responsible for bZIP sequence-specific DNA binding. The analysis of orthologous pairs between chromosomes indicates that these orthologs derived from one gene duplication located on one of the nine ancient chromosomes in the Rosaceae. The comparative analysis of bZIP genes in three species provides information on the evolutionary fate of bZIP genes in apple and peach after they diverged from strawberry. PMID:26770968

  19. The phylogeny of C/S1 bZIP transcription factors reveals a shared algal ancestry and the pre-angiosperm translational regulation of S1 transcripts

    PubMed Central

    Peviani, Alessia; Lastdrager, Jeroen; Hanson, Johannes; Snel, Berend

    2016-01-01

    Basic leucine zippers (bZIPs) form a large plant transcription factor family. C and S1 bZIP groups can heterodimerize, fulfilling crucial roles in seed development and stress response. S1 sequences also harbor a unique regulatory mechanism, termed Sucrose-Induced Repression of Translation (SIRT). The conservation of both C/S1 bZIP interactions and SIRT remains poorly characterized in non-model species, leaving their evolutionary origin uncertain and limiting crop research. In this work, we explored recently published plant sequencing data to establish a detailed phylogeny of C and S1 bZIPs, investigating their intertwined role in plant evolution, and the origin of SIRT. Our analyses clarified C and S1 bZIP orthology relationships in angiosperms, and identified S1 sequences in gymnosperms. We experimentally showed that the gymnosperm orthologs are regulated by SIRT, tracing back the origin of this unique regulatory mechanism to the ancestor of seed plants. Additionally, we discovered an earlier S ortholog in the charophyte algae Klebsormidium flaccidum, together with a C ortholog. This suggests that C and S groups originated by duplication from a single algal proto-C/S ancestor. Based on our observations, we propose a model wherein the C/S1 bZIP dimer network evolved in seed plants from pre-existing C/S bZIP interactions. PMID:27457880

  20. The THERMOSENSITIVE MALE STERILE 1 Interacts with the BiPs via DnaJ Domain and Stimulates Their ATPase Enzyme Activities in Arabidopsis.

    PubMed

    Ma, Zhao-Xia; Leng, Ya-Jun; Chen, Guang-Xia; Zhou, Peng-Min; Ye, De; Chen, Li-Qun

    2015-01-01

    The Arabidopsis TMS1 encodes a heat shock protein identical to the Hsp40 protein AtERdj3A and plays important roles in the thermotolerance of pollen tubes and other plant tissues. Despite its importance to plant growth and reproduction, little has been known about its mechanisms underlying thermotolerance of plants. In this study, the relationship between TMS1 and the Hsp70 proteins, Binding Immunoglobulin Proteins (BiPs) was explored to understand the molecular mechanisms of TMS1 in thermotolerance of plants. The expression of TMS1 was induced not only by heat shock, but also by dithiothreitol (DTT) and L-azetidine-2-carboxylic acid (AZC), similarly to the three BiP genes, indicating that TMS1 may be involved in unfolded protein response (UPR). The firefly luciferase complementary imaging (LCI), GST pull-down and ATPase enzyme activity assays demonstrated that the DnaJ domain of TMS1 could interact with BiP1 and BiP3, and could stimulate their ATPase enzyme activities. In addition, the expression level of TMS1 was reduced in the bzip28 bzip60 double mutant. These results suggest that TMS1 may function at the downstream of bZIP28 and bZIP60 and be involved in termotolerance of plants, possibly by participating in refolding or degradation of unfolded and misfolded proteins through interaction with the BiPs. PMID:26186593

  1. A novel strategy to produce sweeter tomato fruits with high sugar contents by fruit-specific expression of a single bZIP transcription factor gene.

    PubMed

    Sagor, G H M; Berberich, Thomas; Tanaka, Shun; Nishiyama, Manabu; Kanayama, Yoshinori; Kojima, Seiji; Muramoto, Koji; Kusano, Tomonobu

    2016-04-01

    Enhancement of sugar content and sweetness is desirable in some vegetables and in almost all fruits; however, biotechnological methods to increase sugar content are limited. Here, a completely novel methodological approach is presented that produces sweeter tomato fruits but does not have any negative effects on plant growth. Sucrose-induced repression of translation (SIRT), which is mediated by upstream open reading frames (uORFs), was initially reported in Arabidopsis AtbZIP11, a class S basic region leucine zipper (bZIP) transcription factor gene. Here, two AtbZIP11 orthologous genes, SlbZIP1 and SlbZIP2, were identified in tomato (Solanum lycopersicum). SlbZIP1 and SlbZIP2 contained four and three uORFs, respectively, in the cDNA 5'-leader regions. The second uORFs from the 5' cDNA end were conserved and involved in SIRT. Tomato plants were transformed with binary vectors in which only the main open reading frames (ORFs) of SlbZIP1 and SlbZIP2, without the SIRT-responsive uORFs, were placed under the control of the fruit-specific E8 promoter. Growth and morphology of the resulting transgenic tomato plants were comparable to those of wild-type plants. Transgenic fruits were approximately 1.5-fold higher in sugar content (sucrose/glucose/fructose) than nontransgenic tomato fruits. In addition, the levels of several amino acids, such as asparagine and glutamine, were higher in transgenic fruits than in wild-type fruits. This was expected because SlbZIP transactivates the asparagine synthase and proline dehydrogenase genes. This 'sweetening' technology is broadly applicable to other plants that utilize sucrose as a major translocation sugar. PMID:26402509

  2. Transmembrane helix: simple or complex.

    PubMed

    Wong, Wing-Cheong; Maurer-Stroh, Sebastian; Schneider, Georg; Eisenhaber, Frank

    2012-07-01

    Transmembrane helical segments (TMs) can be classified into two groups of so-called 'simple' and 'complex' TMs. Whereas the first group represents mere hydrophobic anchors with an overrepresentation of aliphatic hydrophobic residues that are likely attributed to convergent evolution in many cases, the complex ones embody ancestral information and tend to have structural and functional roles beyond just membrane immersion. Hence, the sequence homology concept is not applicable on simple TMs. In practice, these simple TMs can attract statistically significant but evolutionarily unrelated hits during similarity searches (whether through BLAST- or HMM-based approaches). This is especially problematic for membrane proteins that contain both globular segments and TMs. As such, we have developed the transmembrane helix: simple or complex (TMSOC) webserver for the identification of simple and complex TMs. By masking simple TM segments in seed sequences prior to sequence similarity searches, the false-discovery rate decreases without sacrificing sensitivity. Therefore, TMSOC is a novel and necessary sequence analytic tool for both the experimentalists and the computational biology community working on membrane proteins. It is freely accessible at http://tmsoc.bii.a-star.edu.sg or available for download.

  3. Molecular cloning of a putative novel human bZIP transcription factor on chromosome 17q22

    SciTech Connect

    Luna, L.; Johnsen, O.; Skartlien, A.H.

    1994-08-01

    We have cloned and characterized cDNA clones representing several mRNA isoforms generated by alternative splicing of a single gene localized to chromosome 17q22. Sequence analysis showed that the predicted translational product of the longest open reading frame (2316 nucleotides, 772 amino acids) is related to transcription factors of the basic elucine zipper (bZIP) class. The sequence contained several regions characteristic of transcriptional regulatory domains. A cluster of amino acids flanking the bZIP region on both sides was highly conserved between TCF11 and p45 NF-E2, a subunit of the human globin locus control region-binding protein, NF-E2. These same regions showed remarkable homology to two invertebrate proteins, CNC and skn-1, postulated to regulate embryonic development in Drosophila melanogaster and Caenorhabditis elegans, respectively. 46 refs., 7 figs., 1 tab.

  4. DNA binding of Jun and Fos bZip domains: homodimers and heterodimers induce a DNA conformational change in solution.

    PubMed Central

    John, M; Leppik, R; Busch, S J; Granger-Schnarr, M; Schnarr, M

    1996-01-01

    We constructed plasmids encoding the sequences for the bZip modules of c-Jun and c-Fos which could then be expressed as soluble proteins in Escherichia coli. The purified bZip modules were tested for their binding capacities of synthetic oligonucleotides containing either TRE or CRE recognition sites in electrophoretic mobility shift assays and circular dichroism (CD). Electrophoretic mobility shift assays showed that bZip Jun homodimers and bZip Jun/Fos heterodimers bind a collagenase-like TRE (CTGACTCAT) with dissociation constants of respectively 1.4 x 10(-7) M and 5 x 10(-8) M. As reported earlier [Patel et al. (1990) Nature 347, 572-575], DNA binding induces a marked change of the protein structure. However, we found that the DNA also undergoes a conformational change. This is most clearly seen with small oligonucleotides of 13 or 14 bp harboring respectively a TRE (TGACTCA) or a CRE (TGACGTCA) sequence. In this case, the positive DNA CD signal at 280 nm increases almost two-fold with a concomitant blue-shift of 3-4 nm. Within experimental error the same spectral changes are observed for TRE and CRE containing DNA fragments. The spectral changes observed with a non-specific DNA fragment are weaker and the signal of free DNA is recovered upon addition of much smaller salt concentrations than required for a specific DNA fragment. Surprisingly the spectral changes induced by Jun/Jun homodimers are not identical to those induced by Jun/Fos heterodimers. However, in both cases the increase of the positive CD band and the concomitant blue shift would be compatible with a B to A-transition of part of the binding site or a DNA conformation intermediate between the canonical A and B structures. PMID:8948639

  5. Divergence and Conservation of the Major UPR Branch IRE1-bZIP Signaling Pathway across Eukaryotes.

    PubMed

    Zhang, Lingrui; Zhang, Changwei; Wang, Aiming

    2016-01-01

    The unfolded protein response (UPR) is crucial to life by regulating the cellular response to the stress in the endoplasmic reticulum (ER) imposed by abiotic and biotic cues such as heat shock and viral infection. The inositol requiring enzyme 1 (IRE1) signaling pathway activated by the IRE1-mediated unconventional splicing of HAC1 in yeast, bZIP60 in plants and XBP1 in metazoans, is the most ancient branch of the UPR. In this study, we systematically examined yeast IRE1p-HAC1, plant IRE1A/IRE1B-bZIP60 and human hIRE1-XBP1 pairs. We found that, unlike bZIP60, XBP1 is unable to functionally swap HAC1p in yeast, and that the inter-species heterotypic interactions among HAC1p, bZIP60 and XBP1 are not permitted. These data demonstrate evolutionary divergence of the downstream signaling of IRE1-bZIP. We also discovered that the dual cytosolic domains of plant IRE1s act in vivo in a mechanism consistent with IRE1p and hIRE1, and that plant IRE1B not only interacts with IRE1p but also forms typical IRE1 dynamic foci in yeast. Thus, the upstream components of the IRE1 signaling branch including IRE1 activation and action mechanisms are highly conserved. Taken together these data advance the molecular understanding of evolutionary divergence and conservation of the IRE1 signaling pathway across kingdoms. PMID:27256815

  6. Genome-wide characterization and analysis of bZIP transcription factor gene family related to abiotic stress in cassava.

    PubMed

    Hu, Wei; Yang, Hubiao; Yan, Yan; Wei, Yunxie; Tie, Weiwei; Ding, Zehong; Zuo, Jiao; Peng, Ming; Li, Kaimian

    2016-01-01

    The basic leucine zipper (bZIP) transcription factor family plays crucial roles in various aspects of biological processes. Currently, no information is available regarding the bZIP family in the important tropical crop cassava. Herein, 77 bZIP genes were identified from cassava. Evolutionary analysis indicated that MebZIPs could be divided into 10 subfamilies, which was further supported by conserved motif and gene structure analyses. Global expression analysis suggested that MebZIPs showed similar or distinct expression patterns in different tissues between cultivated variety and wild subspecies. Transcriptome analysis of three cassava genotypes revealed that many MebZIP genes were activated by drought in the root of W14 subspecies, indicating the involvement of these genes in the strong resistance of cassava to drought. Expression analysis of selected MebZIP genes in response to osmotic, salt, cold, ABA, and H2O2 suggested that they might participate in distinct signaling pathways. Our systematic analysis of MebZIPs reveals constitutive, tissue-specific and abiotic stress-responsive candidate MebZIP genes for further functional characterization in planta, yields new insights into transcriptional regulation of MebZIP genes, and lays a foundation for understanding of bZIP-mediated abiotic stress response. PMID:26947924

  7. Genome-wide characterization and analysis of bZIP transcription factor gene family related to abiotic stress in cassava.

    PubMed

    Hu, Wei; Yang, Hubiao; Yan, Yan; Wei, Yunxie; Tie, Weiwei; Ding, Zehong; Zuo, Jiao; Peng, Ming; Li, Kaimian

    2016-03-07

    The basic leucine zipper (bZIP) transcription factor family plays crucial roles in various aspects of biological processes. Currently, no information is available regarding the bZIP family in the important tropical crop cassava. Herein, 77 bZIP genes were identified from cassava. Evolutionary analysis indicated that MebZIPs could be divided into 10 subfamilies, which was further supported by conserved motif and gene structure analyses. Global expression analysis suggested that MebZIPs showed similar or distinct expression patterns in different tissues between cultivated variety and wild subspecies. Transcriptome analysis of three cassava genotypes revealed that many MebZIP genes were activated by drought in the root of W14 subspecies, indicating the involvement of these genes in the strong resistance of cassava to drought. Expression analysis of selected MebZIP genes in response to osmotic, salt, cold, ABA, and H2O2 suggested that they might participate in distinct signaling pathways. Our systematic analysis of MebZIPs reveals constitutive, tissue-specific and abiotic stress-responsive candidate MebZIP genes for further functional characterization in planta, yields new insights into transcriptional regulation of MebZIP genes, and lays a foundation for understanding of bZIP-mediated abiotic stress response.

  8. Genome-wide characterization and analysis of bZIP transcription factor gene family related to abiotic stress in cassava

    PubMed Central

    Hu, Wei; Yang, Hubiao; Yan, Yan; Wei, Yunxie; Tie, Weiwei; Ding, Zehong; Zuo, Jiao; Peng, Ming; Li, Kaimian

    2016-01-01

    The basic leucine zipper (bZIP) transcription factor family plays crucial roles in various aspects of biological processes. Currently, no information is available regarding the bZIP family in the important tropical crop cassava. Herein, 77 bZIP genes were identified from cassava. Evolutionary analysis indicated that MebZIPs could be divided into 10 subfamilies, which was further supported by conserved motif and gene structure analyses. Global expression analysis suggested that MebZIPs showed similar or distinct expression patterns in different tissues between cultivated variety and wild subspecies. Transcriptome analysis of three cassava genotypes revealed that many MebZIP genes were activated by drought in the root of W14 subspecies, indicating the involvement of these genes in the strong resistance of cassava to drought. Expression analysis of selected MebZIP genes in response to osmotic, salt, cold, ABA, and H2O2 suggested that they might participate in distinct signaling pathways. Our systematic analysis of MebZIPs reveals constitutive, tissue-specific and abiotic stress-responsive candidate MebZIP genes for further functional characterization in planta, yields new insights into transcriptional regulation of MebZIP genes, and lays a foundation for understanding of bZIP-mediated abiotic stress response. PMID:26947924

  9. Genomic identification of group A bZIP transcription factors and their responses to abiotic stress in carrot.

    PubMed

    Que, F; Wang, G L; Huang, Y; Xu, Z S; Wang, F; Xiong, A S

    2015-01-01

    The basic-region/leucine-zipper (bZIP) family is one of the major transcription factor (TF) families associated with responses to abiotic stresses. Many members of group A in this family have been extensively examined and are reported to perform significant functions in ABA signaling as well as in responses to abiotic stresses. In this study, 10 bZIP factors in carrot were classified into group A based on their DNA-binding domains. The cis-acting regulatory elements and folding states of these 10 factors were analyzed. Evolutionary analysis of the group A members suggested their importance during the course of evolution in plants. In addition, cis-acting elements and the folding state of proteins were important for DNA binding and could affect gene expression. Quantitative RT-PCR was conducted to investigate the stress response of 10 genes encoding the group A factors. Six genes showed responses to abiotic stresses, while four genes showed other special phenomenon. The current analysis on group A bZIP family TFs in carrot is the first to investigate the TFs of Apiaceae via genome analysis. These results provide new information for future studies on carrot. PMID:26535641

  10. CD studies of interaction of a bZIP oligopeptide model with DNA

    NASA Astrophysics Data System (ADS)

    Votavová, Hana; Točík, Zdeněk; Šponar, Jaroslav

    1999-05-01

    A leucine zipper (bZIP) binding peptide BP1 was constructed based on the DNA binding sequence of the GCN4 protein, slightly modified to make it more similar to the sequence of other bZIP proteins (Jun) with related DNA binding specificity. Selfcomplementary DNA hexadecanucleotides containing modified ATF/CRE target sites were used to study peptide-DNA complex formation. Four oligonucleotides contained substitutions of two GC or AT pairs by IC pairs in the ATF/CRE target sequence. In two other oligonucleotides there was a substitution of A by I in two AT pairs (mismatch IT pairs were presumably formed in the duplex) and one oligonucleotide contained I instead of C in two base pairs (IG mismatch in the duplex). Conformation changes of BP1 that occur on complex formation were studied by circular dichroism spectroscopy. The binding of peptide BP1 to oligonucleotides is accompanied by an increase of the α-helix content, which depends strongly on the oligonucleotide sequence. The substitution of two GC pairs within the specific binding site has either none or only a small effect. However, the substitution of two AT pairs within the binding site by IC strongly decreases the specificity of binding to a level observed with an oligonucleotide containing the C/EBP binding site, differing from the ATF/CRE site at four positions (Votavová et al., J. Biomol. Struct. Dyn. 3 (1997) 587). Similar results were obtained also with an oligonucleotide containing I instead of C in two base pairs (IG mismatch in the duplex). Two oligonucleotides with two substitutions of A by I but with unchanged T in the AT pairs (IT mismatch) showed smaller decrease in the α-helix formation on peptide binding than oligonucleotides, in which the whole AT pair was replaced by IC. The effect of such a substitution depends on the position of the original AT pairs in the target sequence, but the presence of T appears to be essential for specific peptide binding.

  11. An Autonomously Reciprocating Transmembrane Nanoactuator.

    PubMed

    Watson, Matthew A; Cockroft, Scott L

    2016-01-22

    Biological molecular machines operate far from equilibrium by coupling chemical potential to repeated cycles of dissipative nanomechanical motion. This principle has been exploited in supramolecular systems that exhibit true machine behavior in solution and on surfaces. However, designed membrane-spanning assemblies developed to date have been limited to simple switches or stochastic shuttles, and true machine behavior has remained elusive. Herein, we present a transmembrane nanoactuator that turns over chemical fuel to drive autonomous reciprocating (back-and-forth) nanomechanical motion. Ratcheted reciprocating motion of a DNA/PEG copolymer threaded through a single α-hemolysin pore was induced by a combination of DNA strand displacement processes and enzyme-catalyzed reactions. Ion-current recordings revealed saw-tooth patterns, indicating that the assemblies operated in autonomous, asymmetric cycles of conformational change at rates of up to one cycle per minute. PMID:26661295

  12. Type II Transmembrane Serine Proteases*

    PubMed Central

    Bugge, Thomas H.; Antalis, Toni M.; Wu, Qingyu

    2009-01-01

    Analysis of genome and expressed sequence tag data bases at the turn of the millennium unveiled a new protease family named the type II transmembrane serine proteases (TTSPs) in a Journal of Biological Chemistry minireview (Hooper, J. D., Clements, J. A., Quigley, J. P., and Antalis, T. M. (2001) J. Biol. Chem. 276, 857–860). Since then, the number of known TTSPs has more than doubled, and more importantly, our understanding of the physiological functions of individual TTSPs and their contribution to human disease has greatly increased. Progress has also been made in identifying molecular substrates and endogenous inhibitors. This minireview summarizes the current knowledge of the rapidly advancing TTSP field. PMID:19487698

  13. Cooperative Transmembrane Penetration of Nanoparticles

    PubMed Central

    Zhang, Haizhen; Ji, Qiuju; Huang, Changjin; Zhang, Sulin; Yuan, Bing; Yang, Kai; Ma, Yu-qiang

    2015-01-01

    Physical penetration of lipid bilayer membranes presents an alternative pathway for cellular delivery of nanoparticles (NPs) besides endocytosis. NPs delivered through this pathway could reach the cytoplasm, thereby opening the possibility of organelle-specific targeting. Herein we perform dissipative particle dynamics simulations to elucidate the transmembrane penetration mechanisms of multiple NPs. Our simulations demonstrate that NPs’ translocation proceeds in a cooperative manner, where the interplay of the quantity and surface chemistry of the NPs regulates the translocation efficiency. For NPs with hydrophilic surfaces, the increase of particle quantity facilitates penetration, while for NPs with partly or totally hydrophobic surfaces, the opposite highly possibly holds. Moreover, a set of interesting cooperative ways, such as aggregation, aggregation-dispersion, and aggregation-dispersion-reaggregation of the NPs, are observed during the penetration process. We find that the penetration behaviors of multiple NPs are mostly dominated by the changes of the NP-membrane force components in the membrane plane direction, in addition to that in the penetration direction, suggesting a different interaction mechanism between the multiple NPs and the membrane compared with the one-NP case. These results provide a fundamental understanding in the underlying mechanisms of cooperative penetration of NPs, and shed light on the NP-based drug and gene delivery. PMID:26013284

  14. Stomatal Development in Arabidopsis

    PubMed Central

    Pillitteri, Lynn Jo; Dong, Juan

    2013-01-01

    Stomata consist of two guard cells that function as turgor-operated valves that regulate gas exchange in plants. In Arabidopsis, a dedicated cell lineage is initiated and undergoes a series of cell divisions and cell-state transitions to produce a stoma. A set of basic helix-loop-helix (bHLH) transcription factors regulates the transition and differentiation events through the lineage, while the placement of stomata relative to each other is controlled by intercellular signaling via peptide ligands, transmembrane receptors, and mitogen-activated protein kinase (MAPK) modules. Some genes involved in regulating stomatal differentiation or density are also involved in hormonal and environmental stress responses, which may provide a link between modulation of stomatal development or function in response to changes in the environment. Premitotic polarlylocalized proteins provide an added layer of regulation, which can be addressed more thoroughly with the identification of additional proteins in this pathway. Linking the networks that control stomatal development promises to bring advances to our understanding of signal transduction, cell polarity, and cell-fate specification in plants. PMID:23864836

  15. The bZIP Protein MeaB Mediates Virulence Attributes in Aspergillus flavus

    PubMed Central

    Yin, Wen-Bing; Franke, Stephen; Choithani, Anjali; Keller, Nancy P.

    2013-01-01

    LaeA is a fungal specific virulence factor of both plant and human pathogenic fungi. Transcriptional profiles of laeA mutants have been successfully exploited to identify regulatory mechanisms of secondary metabolism in fungi; here we use laeA mutants as tools to elucidate virulence attributes in Aspergillus flavus. Microarray expression profiles of ΔlaeA and over-expression laeA (OE::laeA) were compared to wild type A. flavus. Strikingly, several nitrogen metabolism genes are oppositely mis-regulated in the ΔlaeA and OE::laeA mutants. One of the nitrogen regulatory genes, the bZIP encoding meaB, is up-regulated in ΔlaeA. Significantly, over-expression of meaB (OE::meaB) phenocopies the decreased virulence attributes of a ΔlaeA phenotype including decreased colonization of host seed, reduced lipase activity and loss of aflatoxin B1 production in seed. However, a double knock-down of laeA and meaB (KD::laeA,meaB) demonstrated that KD::laeA,meaB closely resembled ΔlaeA rather than wild type or ΔmeaB in growth, aflatoxin biosynthesis and sclerotia production thus suggesting that meaB does not contribute to the ΔlaeA phenotype. MeaB and LaeA appear to be part of regulatory networks that allow them to have both shared and distinct roles in fungal biology. PMID:24040154

  16. Transmembrane signaling in kidney health and disease.

    PubMed

    Hack, N; Schultz, A; Clayman, P; Goldberg, H; Skorecki, K L

    1995-08-01

    Transmembrane signal transduction is the process whereby a ligand binds to the external surface of the cell membrane and elicits a physiological response specific for that ligand and cell type. It is now appreciated that numerous disease states represent disturbances in normal transmembrane signaling mechanisms. In the current paper, we focus our attention on the mesangial cell of the glomerular microcirculation as a prototypical model system for understanding normal and abnormal transmembrane signaling processes. Among the major receptor and effector mechanisms for transmembrane signal transduction in the mesangial cell, this paper emphasizes the phospholipase effector response to growth factors and vasoactive hormones. The post-translational and transcriptional pathways for regulation of phospholipase C and phospholipase A2 are described, including consideration of perturbations in these systems that characterize two disease models, namely: acute cyclosporine nephrotoxicity and early diabetic glomerulopathy.

  17. Genomic surveys and expression analysis of bZIP gene family in castor bean (Ricinus communis L.).

    PubMed

    Jin, Zhengwei; Xu, Wei; Liu, Aizhong

    2014-02-01

    The basic leucine zipper (bZIP) transcription factors comprise a family of transcriptional regulators present extensively in plants, involved in regulating diverse biological processes such as flower and vascular development, seed maturation, stress signaling and pathogen defense. Castor bean (Ricinus communis L. Euphorbiaceae) is one of the most important non-edible oilseed crops and its seed oil is broadly used for industrial applications. We performed a comprehensive genome-wide identification and analysis of the bZIP transcription factors that exist in the castor bean genome in this study. In total, 49 RcbZIP transcription factors were identified, characterized and categorized into 11 groups (I-XI) based on their gene structure, DNA-binding sites, conserved motifs, and phylogenetic relationships. The dimerization properties of 49 RcbZIP proteins were predicted on the basis of the characteristic features in the leucine zipper. Global expression profiles of 49 RcbZIP genes among different tissues were examined using high-throughput sequencing of digital gene expression profiles, and resulted in diverse expression patterns that may provide basic information to further reveal the function of the 49 RcbZIP genes in castor bean. The results obtained from this study would provide valuable information in understanding the molecular basis of the RcbZIP transcription factor family and their potential function in regulating the growth and development, particularly in seed filling of castor bean.

  18. Genome-Wide Identification and Characterization of bZIP Transcription Factors in Brassica oleracea under Cold Stress.

    PubMed

    Hwang, Indeok; Manoharan, Ranjith Kumar; Kang, Jong-Goo; Chung, Mi-Young; Kim, Young-Wook; Nou, Ill-Sup

    2016-01-01

    Cabbages (Brassica oleracea L.) are an important vegetable crop around world, and cold temperature is among the most significant abiotic stresses causing agricultural losses, especially in cabbage crops. Plant bZIP transcription factors play diverse roles in biotic/abiotic stress responses. In this study, 119 putative BolbZIP transcription factors were identified using amino acid sequences from several bZIP domain consensus sequences. The BolbZIP members were classified into 63 categories based on amino acid sequence similarity and were also compared with BrbZIP and AtbZIP transcription factors. Based on this BolbZIP identification and classification, cold stress-responsive BolbZIP genes were screened in inbred lines, BN106 and BN107, using RNA sequencing data and qRT-PCR. The expression level of the 3 genes, Bol008071, Bol033132, and Bol042729, was significantly increased in BN107 under cold conditions and was unchanged in BN106. The upregulation of these genes in BN107, a cold-susceptible inbred line, suggests that they might be significant components in the cold response. Among three identified genes, Bol033132 has 97% sequence similarity to Bra020735, which was identified in a screen for cold-related genes in B. rapa and a protein containing N-rich regions in LCRs. The results obtained in this study provide valuable information for understanding the potential function of BolbZIP transcription factors in cold stress responses.

  19. Genome-Wide Identification and Characterization of bZIP Transcription Factors in Brassica oleracea under Cold Stress

    PubMed Central

    Hwang, Indeok; Manoharan, Ranjith Kumar; Kang, Jong-Goo; Chung, Mi-Young; Kim, Young-Wook; Nou, Ill-Sup

    2016-01-01

    Cabbages (Brassica oleracea L.) are an important vegetable crop around world, and cold temperature is among the most significant abiotic stresses causing agricultural losses, especially in cabbage crops. Plant bZIP transcription factors play diverse roles in biotic/abiotic stress responses. In this study, 119 putative BolbZIP transcription factors were identified using amino acid sequences from several bZIP domain consensus sequences. The BolbZIP members were classified into 63 categories based on amino acid sequence similarity and were also compared with BrbZIP and AtbZIP transcription factors. Based on this BolbZIP identification and classification, cold stress-responsive BolbZIP genes were screened in inbred lines, BN106 and BN107, using RNA sequencing data and qRT-PCR. The expression level of the 3 genes, Bol008071, Bol033132, and Bol042729, was significantly increased in BN107 under cold conditions and was unchanged in BN106. The upregulation of these genes in BN107, a cold-susceptible inbred line, suggests that they might be significant components in the cold response. Among three identified genes, Bol033132 has 97% sequence similarity to Bra020735, which was identified in a screen for cold-related genes in B. rapa and a protein containing N-rich regions in LCRs. The results obtained in this study provide valuable information for understanding the potential function of BolbZIP transcription factors in cold stress responses. PMID:27314020

  20. Genome-Wide Identification and Characterization of bZIP Transcription Factors in Brassica oleracea under Cold Stress.

    PubMed

    Hwang, Indeok; Manoharan, Ranjith Kumar; Kang, Jong-Goo; Chung, Mi-Young; Kim, Young-Wook; Nou, Ill-Sup

    2016-01-01

    Cabbages (Brassica oleracea L.) are an important vegetable crop around world, and cold temperature is among the most significant abiotic stresses causing agricultural losses, especially in cabbage crops. Plant bZIP transcription factors play diverse roles in biotic/abiotic stress responses. In this study, 119 putative BolbZIP transcription factors were identified using amino acid sequences from several bZIP domain consensus sequences. The BolbZIP members were classified into 63 categories based on amino acid sequence similarity and were also compared with BrbZIP and AtbZIP transcription factors. Based on this BolbZIP identification and classification, cold stress-responsive BolbZIP genes were screened in inbred lines, BN106 and BN107, using RNA sequencing data and qRT-PCR. The expression level of the 3 genes, Bol008071, Bol033132, and Bol042729, was significantly increased in BN107 under cold conditions and was unchanged in BN106. The upregulation of these genes in BN107, a cold-susceptible inbred line, suggests that they might be significant components in the cold response. Among three identified genes, Bol033132 has 97% sequence similarity to Bra020735, which was identified in a screen for cold-related genes in B. rapa and a protein containing N-rich regions in LCRs. The results obtained in this study provide valuable information for understanding the potential function of BolbZIP transcription factors in cold stress responses. PMID:27314020

  1. Arabidopsis thaliana

    PubMed Central

    Strzalka, Wojciech; Aggarwal, Chhavi

    2013-01-01

    The proliferating cell nuclear antigen (PCNA) is a key component of the eukaryotic DNA replication machinery. It also plays an important role in DNA repair mechanisms. Despite the intense scientific research on yeast and human PCNA, information describing the function of this protein in plants is still very limited. In the previous study Arabidopsis PCNA2 but not PCNA1 was proposed to be functionally important in DNA polymerase η-dependent postreplication repair. In addition to the above study, PCNA2 but not PCNA1 was also shown to be necessary for Arabidopsis DNA polymerase λ-dependent oxidative DNA damage bypass. Taking into account the reported differences between PCNA1 and PCNA2, we tested the idea of a possible cooperation between PCNA1 and PCNA2 in the plant cell. In a bimolecular fluorescence complementation assay an interaction between PCNA1 and PCNA2 was observed in the nucleus, as well as in the cytoplasm. This finding, together with our previous results, indicates that PCNA1 and PCNA2 may cooperate in planta by forming homo- and heterotrimeric rings. The observed interaction might be relevant when distinct functions for PCNA1 and PCNA2 are considered. PMID:23656863

  2. Assembly of transmembrane proteins on oil-water interfaces

    NASA Astrophysics Data System (ADS)

    Yunker, Peter; Landry, Corey; Chong, Shaorong; Weitz, David

    2015-03-01

    Transmembrane proteins are difficult to handle by aqueous solution-based biochemical and biophysical approaches, due to the hydrophobicity of transmembrane helices. Detergents can solubilize transmembrane proteins; however, surfactant coated transmembrane proteins are not always functional, and purifying detergent coated proteins in a micellar solution can be difficult. Motivated by this problem, we study the self-assembly of transmembrane proteins on oil-water interfaces. We found that the large water-oil interface of oil drops prevents nascent transmembrane proteins from forming non-functional aggregates. The oil provides a hydrophobic environment for the transmembrane helix, allowing the ectodomain to fold into its natural structure and orientation. Further, modifying the strength or valency of hydrophobic interactions between transmembrane proteins results in the self-assembly of spatially clustered, active proteins on the oil-water interface. Thus, hydrophobic interactions can facilitate, rather than inhibit, the assembly of transmembrane proteins.

  3. Both the hydrophobicity and a positively charged region flanking the C-terminal region of the transmembrane domain of signal-anchored proteins play critical roles in determining their targeting specificity to the endoplasmic reticulum or endosymbiotic organelles in Arabidopsis cells.

    PubMed

    Lee, Junho; Lee, Hyunkyung; Kim, Jinho; Lee, Sumin; Kim, Dae Heon; Kim, Sanguk; Hwang, Inhwan

    2011-04-01

    Proteins localized to various cellular and subcellular membranes play pivotal roles in numerous cellular activities. Accordingly, in eukaryotic cells, the biogenesis of organellar proteins is an essential process requiring their correct localization among various cellular and subcellular membranes. Localization of these proteins is determined by either cotranslational or posttranslational mechanisms, depending on the final destination. However, it is not fully understood how the targeting specificity of membrane proteins is determined in plant cells. Here, we investigate the mechanism by which signal-anchored (SA) proteins are differentially targeted to the endoplasmic reticulum (ER) or endosymbiotic organelles using in vivo targeting, subcellular fractionation, and bioinformatics approaches. For targeting SA proteins to endosymbiotic organelles, the C-terminal positively charged region (CPR) flanking the transmembrane domain (TMD) is necessary but not sufficient. The hydrophobicity of the TMD in CPR-containing proteins also plays a critical role in determining targeting specificity; TMDs with a hydrophobicity value >0.4 on the Wimley and White scale are targeted primarily to the ER, whereas TMDs with lower values are targeted to endosymbiotic organelles. Based on these data, we propose that the CPR and the hydrophobicity of the TMD play a critical role in determining the targeting specificity between the ER and endosymbiotic organelles.

  4. Transmembrane START domain proteins: in silico identification, characterization and expression analysis under stress conditions in chickpea (Cicer arietinum L.).

    PubMed

    Satheesh, Viswanathan; Chidambaranathan, Parameswaran; Jagannadham, Prasanth Tejkumar; Kumar, Vajinder; Jain, Pradeep K; Chinnusamy, Viswanathan; Bhat, Shripad R; Srinivasan, R

    2016-01-01

    Steroidogenic acute regulatory related transfer (StART) proteins that are involved in transport of lipid molecules, play a myriad of functions in insects, mammals and plants. These proteins consist of a modular START domain of approximately 200 amino acids which binds and transfers the lipids. In the present study we have performed a genome-wide search for all START domain proteins in chickpea. The search identified 36 chickpea genes belonging to the START domain family. Through a phylogenetic tree reconstructed with Arabidopsis, rice, chickpea, and soybean START proteins, we were able to identify four transmembrane START (TM-START) proteins in chickpea. These four proteins are homologous to the highly conserved mammalian phosphatidylcholine transfer proteins. Multiple sequence alignment of all the transmembrane containing START proteins from Arabidopsis, rice, chickpea, and soybean revealed that the amino acid residues to which phosphatidylcholine binds in mammals, is also conserved in all these plant species, implying an important functional role and a very similar mode of action of all these proteins across dicots and monocots. This study characterizes a few of the not so well studied transmembrane START superfamily genes that may be involved in stress signaling. Expression analysis in various tissues showed that these genes are predominantly expressed in flowers and roots of chickpea. Three of the chickpea TM-START genes showed induced expression in response to drought, salt, wound and heat stress, suggesting their role in stress response.

  5. TRAMPLE: the transmembrane protein labelling environment.

    PubMed

    Fariselli, Piero; Finelli, Michele; Rossi, Ivan; Amico, Mauro; Zauli, Andrea; Martelli, Pier Luigi; Casadio, Rita

    2005-07-01

    TRAMPLE (http://gpcr.biocomp.unibo.it/biodec/) is a web application server dedicated to the detection and the annotation of transmembrane protein sequences. TRAMPLE includes different state-of-the-art algorithms for the prediction of signal peptides, transmembrane segments (both beta-strands and alpha-helices), secondary structure and fast fold recognition. TRAMPLE also includes a complete content management system to manage the results of the predictions. Each user of the server has his/her own workplace, where the data can be stored, organized, accessed and annotated with documents through a simple web-based interface. In this manner, TRAMPLE significantly improves usability with respect to other more traditional web servers. PMID:15980454

  6. Crystallizing Transmembrane Peptides in Lipidic Mesophases

    SciTech Connect

    Höfer, Nicole; Aragão, David; Caffrey, Martin

    2011-09-28

    Structure determination of membrane proteins by crystallographic means has been facilitated by crystallization in lipidic mesophases. It has been suggested, however, that this so-called in meso method, as originally implemented, would not apply to small protein targets having {le}4 transmembrane crossings. In our study, the hypothesis that the inherent flexibility of the mesophase would enable crystallogenesis of small proteins was tested using a transmembrane pentadecapeptide, linear gramicidin, which produced structure-grade crystals. This result suggests that the in meso method should be considered as a viable means for high-resolution structure determination of integral membrane peptides, many of which are predicted to be coded for in the human genome.

  7. Ion fluxes through nanopores and transmembrane channels

    NASA Astrophysics Data System (ADS)

    Bordin, J. R.; Diehl, A.; Barbosa, M. C.; Levin, Y.

    2012-03-01

    We introduce an implicit solvent Molecular Dynamics approach for calculating ionic fluxes through narrow nanopores and transmembrane channels. The method relies on a dual-control-volume grand-canonical molecular dynamics (DCV-GCMD) simulation and the analytical solution for the electrostatic potential inside a cylindrical nanopore recently obtained by Levin [Europhys. Lett.EULEEJ0295-507510.1209/epl/i2006-10240-4 76, 163 (2006)]. The theory is used to calculate the ionic fluxes through an artificial transmembrane channel which mimics the antibacterial gramicidin A channel. Both current-voltage and current-concentration relations are calculated under various experimental conditions. We show that our results are comparable to the characteristics associated to the gramicidin A pore, especially the existence of two binding sites inside the pore and the observed saturation in the current-concentration profiles.

  8. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

    PubMed Central

    Corradi, Valentina; Vergani, Paola; Tieleman, D. Peter

    2015-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily. CFTR controls the flow of anions through the apical membrane of epithelia. Dysfunctional CFTR causes the common lethal genetic disease cystic fibrosis. Transitions between open and closed states of CFTR are regulated by ATP binding and hydrolysis on the cytosolic nucleotide binding domains, which are coupled with the transmembrane (TM) domains forming the pathway for anion permeation. Lack of structural data hampers a global understanding of CFTR and thus the development of “rational” approaches directly targeting defective CFTR. In this work, we explored possible conformational states of the CFTR gating cycle by means of homology modeling. As templates, we used structures of homologous ABC transporters, namely TM(287–288), ABC-B10, McjD, and Sav1866. In the light of published experimental results, structural analysis of the transmembrane cavity suggests that the TM(287–288)-based CFTR model could correspond to a commonly occupied closed state, whereas the McjD-based model could represent an open state. The models capture the important role played by Phe-337 as a filter/gating residue and provide structural information on the conformational transition from closed to open channel. PMID:26229102

  9. The bZIP transcription factor PfZipA regulates secondary metabolism and oxidative stress response in the plant endophytic fungus Pestalotiopsis fici.

    PubMed

    Wang, Xiuna; Wu, Fan; Liu, Ling; Liu, Xingzhong; Che, Yongsheng; Keller, Nancy P; Guo, Liyun; Yin, Wen-Bing

    2015-08-01

    The bZIP transcription factors are conserved in all eukaryotes and play critical roles in organismal responses to environmental challenges. In filamentous fungi, several lines of evidence indicate that secondary metabolism (SM) is associated with oxidative stress mediated by bZIP proteins. Here we uncover a connection with a bZIP protein and oxidative stress induction of SM in the plant endophytic fungus Pestalotiopsis fici. A homology search of the P. fici genome with the bZIP protein RsmA, involved in SM and the oxidative stress response in Aspergillus nidulans, identified PfZipA. Deletion of PfzipA resulted in a strain that displayed resistant to the oxidative reagents tert-butylhydroperoxide (tBOOH), diamide, and menadione sodium bisulfite (MSB), but increased sensitivity to H2O2 as compared to wild type (WT). Secondary metabolite production presented a complex pattern dependent on PfzipA and oxidative reagents. Without oxidative treatment, the ΔPfzipA strain produced less isosulochrin and ficipyroneA than WT; addition of tBOOH further decreased production of iso-A82775C and pestaloficiol M in ΔPfzipA; diamide treatment resulted in equivalent production of isosulochrin and ficipyroneA in the two strains; MSB treatment further decreased production of RES1214-1 and iso-A82775C but increased pestaloficiol M production in the mutant; and H2O2 treatment resulted in enhanced production of isosulochrin, RES1214-1 and pestheic acid but decreased ficipyroneA and pestaloficiol M in ΔPfzipA compared to WT. Our results suggest that PfZipA regulation of SM is modified by oxidative stress pathways and provide insights into a possible role of PfZipA in mediating SM synthesis in the endophytic lifestyle of P. fici. PMID:25847004

  10. Endoplasmic reticulum stress triggers ROS signalling, changes the redox state, and regulates the antioxidant defence of Arabidopsis thaliana

    PubMed Central

    Turkan, Ismail

    2014-01-01

    Inefficient chaperone activity in endoplasmic reticulum (ER) causes accumulation of unfolded proteins and is called ER stress, which triggers the unfolded protein response. For proper oxidative protein folding, reactive oxygen species (ROS) such as H2O2 are produced in the ER. Although the role of ROS during abiotic stresses such as salinity is well documented, the role of ER-related ROS production and its signalling is not yet known. Moreover, how H2O2 production, redox regulation, and antioxidant defence are affected in salt-treated plants when ER protein-folding machinery is impaired needs to be elucidated. For this aim, changes in NADPH-oxidase-dependent ROS signalling and H2O2 content at sequential time intervals and after 48h of ER stress, induced by tunicamycin (Tm), salinity, and their combination were determined in Arabidopsis thaliana. The main root growth was inhibited by ER stress, while low levels of Tm caused an increase in lateral root density. Salt stress and Tm induced the expression of ER-stress-related genes (bZIP17, bZIP28, bZIP60, TIN1, BiP1, BiP3) and ERO1. Tm induced expression of RBOHD and RBOHF, which led to an early increase in H2O2 and triggered ROS signalling. This study is the first report that ER stress induces the antioxidant system and the Asada–Halliwell pathway of A. thaliana in a similar way to salinity. ER stress caused oxidative damage, as evident by increased H2O2 accumulation, lipid peroxidation, and protein oxidation. As a result, this study shows that ER stress triggers ROS signalling, changes the redox state, and regulates the antioxidant defence of A. thaliana. PMID:24558072

  11. Genome-wide analyses of the bZIP family reveal their involvement in the development, ripening and abiotic stress response in banana

    PubMed Central

    Hu, Wei; Wang, Lianzhe; Tie, Weiwei; Yan, Yan; Ding, Zehong; Liu, Juhua; Li, Meiying; Peng, Ming; Xu, Biyu; Jin, Zhiqiang

    2016-01-01

    The leucine zipper (bZIP) transcription factors play important roles in multiple biological processes. However, less information is available regarding the bZIP family in the important fruit crop banana. In this study, 121 bZIP transcription factor genes were identified in the banana genome. Phylogenetic analysis showed that MabZIPs were classified into 11 subfamilies. The majority of MabZIP genes in the same subfamily shared similar gene structures and conserved motifs. The comprehensive transcriptome analysis of two banana genotypes revealed the differential expression patterns of MabZIP genes in different organs, in various stages of fruit development and ripening, and in responses to abiotic stresses, including drought, cold, and salt. Interaction networks and co-expression assays showed that group A MabZIP-mediated networks participated in various stress signaling, which was strongly activated in Musa ABB Pisang Awak. This study provided new insights into the complicated transcriptional control of MabZIP genes and provided robust tissue-specific, development-dependent, and abiotic stress-responsive candidate MabZIP genes for potential applications in the genetic improvement of banana cultivars. PMID:27445085

  12. A light-regulated bZIP module, photozipper, induces the binding of fused proteins to the target DNA sequence in a blue light-dependent manner.

    PubMed

    Hisatomi, Osamu; Furuya, Keigo

    2015-11-01

    Aureochrome-1 (AUREO1) has been identified as a blue light (BL) receptor responsible for the BL-induced blanching of a stramenopile alga, Vaucheria frigida. BL induces the dimerization of monomeric AUREO1, which subsequently increases its affinity for the target sequence. We made a synthetic gene encoding N-terminally truncated monomeric AUREO1 (Photozipper protein) containing a basic region/leucine zipper (bZIP) domain and a light-oxygen-voltage-sensing domain. In the present study, yellow fluorescent protein or mCherry protein was fused with the Photozipper (PZ) protein, and their oligomeric structures and DNA-binding were compared in the dark and light states. Dynamic light scattering and size exclusion chromatography demonstrated that the hydrodynamic radii and molecular masses of the fusion proteins increased upon BL illumination, suggesting that fusion PZs underwent BL-induced dimerization. Moreover, BL-induced dimerization enhanced their affinities for the target sequence. Taken together, PZ likely functions as a BL-regulated bZIP module in fusion proteins, and can possibly provide a new approach for controlling bZIP transcription factors. PMID:26441326

  13. The Vrille Gene of Drosophila Is a Maternal Enhancer of Decapentaplegic and Encodes a New Member of the Bzip Family of Transcription Factors

    PubMed Central

    George, H.; Terracol, R.

    1997-01-01

    We report here the genetical and molecular characterization of a new Drosophila zygotic lethal locus, vrille (vri). vri alleles act not only as dominant maternal enhancers of embryonic dorsoventral patterning defects caused by easter and decapentaplegic (dpp) mutations, but also as dominant zygotic enhancers of dpp alleles for phenotypes in wing. The vri gene encodes a new member of the bZIP family of transcription factors closely related to gene 9 of Xenopus laevis; induced by thyroid hormone during the tadpole tail resorption program, and NF-IL3A, a human T cell transcription factor that transactivates the interleukin3 promoter. NF-IL3A shares 93% similarity and 60% identity with Vri for a stretch of 68 amino acids that includes the bZIP domain. Although all the alleles tested behave like antimorphs, the dominant enhancement is also seen with a nonsense mutation allele that prevents translation of the bZIP domain. Because of the strong dominant enhancement of dpp phenotypes by vri alleles in both embryo and wing, and also the similarity between the wing vein phenotypes caused by the vri and shortvein dpp alleles, we postualte that vri interacts either directly or indirectly with certain components of the dpp (a TGFβ homologue) signal transduction pathway. PMID:9258679

  14. Genome-wide analyses of the bZIP family reveal their involvement in the development, ripening and abiotic stress response in banana.

    PubMed

    Hu, Wei; Wang, Lianzhe; Tie, Weiwei; Yan, Yan; Ding, Zehong; Liu, Juhua; Li, Meiying; Peng, Ming; Xu, Biyu; Jin, Zhiqiang

    2016-01-01

    The leucine zipper (bZIP) transcription factors play important roles in multiple biological processes. However, less information is available regarding the bZIP family in the important fruit crop banana. In this study, 121 bZIP transcription factor genes were identified in the banana genome. Phylogenetic analysis showed that MabZIPs were classified into 11 subfamilies. The majority of MabZIP genes in the same subfamily shared similar gene structures and conserved motifs. The comprehensive transcriptome analysis of two banana genotypes revealed the differential expression patterns of MabZIP genes in different organs, in various stages of fruit development and ripening, and in responses to abiotic stresses, including drought, cold, and salt. Interaction networks and co-expression assays showed that group A MabZIP-mediated networks participated in various stress signaling, which was strongly activated in Musa ABB Pisang Awak. This study provided new insights into the complicated transcriptional control of MabZIP genes and provided robust tissue-specific, development-dependent, and abiotic stress-responsive candidate MabZIP genes for potential applications in the genetic improvement of banana cultivars. PMID:27445085

  15. The yapA Encodes bZIP Transcription Factor Involved in Stress Tolerance in Pathogenic Fungus Talaromyces marneffei

    PubMed Central

    Dankai, Wiyada; Pongpom, Monsicha; Youngchim, Sirida; Cooper, Chester R.; Vanittanakom, Nongnuch

    2016-01-01

    Talaromyces marneffei, formerly Penicillium marneffei, is a thermally dimorphic fungus. It causes a fatal disseminated disease in patients infected with the human immunodeficiency virus (HIV). Studies on the stress defense mechanism of T. marneffei can lead to a better understanding of the pathogenicity and the progression of the disease due to this fungus. The basic leucine-zipper (bZip) transcription factor gene in Saccharomyces cerevisiae, named yap1 (yeast activating protein-1), is known as a crucial central regulator of stress responses including those caused by oxidative agents, cadmium, and drugs. An ortholog of yap1, designated yapA, was identified in T. marneffei. We found that the yapA gene was involved in growth and fungal cell development. The yapA deletion mutant exhibited delays in the rate of growth, germination, and conidiation. Surprisingly, the yapA gene was also involved in the pigmentation of T. marneffei. Moreover, the mutant was sensitive to oxidative stressors such as H2O2 and menadione, similar to S. cerevisiae yap1 mutant, as well as the nitrosative stressor NaNO2. In addition, the yapA mutant demonstrated significantly decreased survival in human macrophage THP-1 compared to wild-type and complemented strains. This study reveals the role of yapA in fungal growth, cell development, stress response, and potential virulence in T. marneffei. PMID:27706212

  16. Rite of passage: a bZIP transcription factor must transit the cell apex to become competent.

    PubMed

    Momany, Michelle

    2015-11-01

    In the filamentous fungus Aspergillus nidulans BrlA triggers the central developmental pathway that controls the transition from vegetative growth to asexual reproduction. Upstream regulators including the bZIP transcription factor FlbB activate the expression of brlA. Previous work has established that FlbB localizes to both the apex of the hypha, where it interacts with and is anchored by FlbE, and to nuclei, with highest levels in the nucleus closest to the apex and successively lower levels in nuclei further away from the apex. In this issue, Herrero-Garcia et al. dissect the roles of these two FlbB pools and the mechanisms underlying their localization and activity. Using a photoactivatable tag, they demonstrate that FlbB moves from the tip into the apical nucleus. Through a series of deletion constructs, they show that import of FlbB into the nucleus requires a bipartite NLS, that FlbB localization at the tip requires actin and that the FlbB tip-high gradient appears to be mass action dependent as the gradient is lost with FlbB constitutive upregulation. They show that while the pool of FlbB at the apex is required for triggering asexual development, the tip high nuclear gradient is not required. PMID:26387769

  17. Development of T cell lymphoma in HTLV-1 bZIP factor and Tax double transgenic mice.

    PubMed

    Zhao, Tiejun; Satou, Yorifumi; Matsuoka, Masao

    2014-07-01

    Adult T-cell leukemia (ATL) is an aggressive T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). ATL cells possess a CD4+ CD25+ phenotype, similar to that of regulatory T cells (Tregs). Tax has been reported to play a crucial role in the leukemogenesis of HTLV-1. The HTLV-1 bZIP factor (HBZ), which is encoded by the minus strand of the viral genomic RNA, is expressed in all ATL cases and induces neoplastic and inflammatory disease in vivo. To test whether HBZ and Tax are both required for T cell malignancy, we generated HBZ/Tax double transgenic mice in which HBZ and Tax are expressed exclusively in CD4+ T cells. Survival was much reduced in HBZ/Tax double-transgenic mice compared with wild type littermates. Transgenic expression of HBZ and Tax induced skin lesions and T-cell lymphoma in mice, resembling diseases observed in HTLV-1 infected individuals. However, Tax single transgenic mice did not develop major health problems. In addition, memory CD4+ T cells and Foxp3+ Treg cells counts were increased in HBZ/Tax double transgenic mice, and their proliferation was enhanced. There was very little difference between HBZ single and HBZ/Tax double transgenic mice. Taken together, these results show that HBZ, in addition to Tax, plays a critical role in T-cell lymphoma arising from HTLV-1 infection.

  18. Nuclear Import of the Parsley bZIP Transcription Factor CPRF2 Is Regulated by Phytochrome Photoreceptors

    PubMed Central

    Kircher, Stefan; Wellmer, Frank; Nick, Peter; Rügner, Alexander; Schäfer, Eberhard; Harter, Klaus

    1999-01-01

    In plants, light perception by photoreceptors leads to differential expression of an enormous number of genes. An important step for differential gene expression is the regulation of transcription factor activities. To understand these processes in light signal transduction we analyzed the three well-known members of the common plant regulatory factor (CPRF) family from parsley (Petroselinum crispum). Here, we demonstrate that these CPRFs, which belong to the basic- region leucine-zipper (bZIP) domain-containing transcription factors, are differentially distributed within parsley cells, indicating different regulatory functions within the regulatory networks of the plant cell. In particular, we show by cell fractionation and immunolocalization approaches that CPRF2 is transported from the cytosol into the nucleus upon irradiation due to action of phytochrome photoreceptors. Two NH2-terminal domains responsible for cytoplasmic localization of CPRF2 in the dark were characterized by deletion analysis using a set of CPRF2-green fluorescent protein (GFP) gene fusion constructs transiently expressed in parsley protoplasts. We suggest that light-induced nuclear import of CPRF2 is an essential step in phytochrome signal transduction. PMID:9922448

  19. Predicting transmembrane beta-barrels in proteomes

    PubMed Central

    Bigelow, Henry R.; Petrey, Donald S.; Liu, Jinfeng; Przybylski, Dariusz; Rost, Burkhard

    2004-01-01

    Very few methods address the problem of predicting beta-barrel membrane proteins directly from sequence. One reason is that only very few high-resolution structures for transmembrane beta-barrel (TMB) proteins have been determined thus far. Here we introduced the design, statistics and results of a novel profile-based hidden Markov model for the prediction and discrimination of TMBs. The method carefully attempts to avoid over-fitting the sparse experimental data. While our model training and scoring procedures were very similar to a recently published work, the architecture and structure-based labelling were significantly different. In particular, we introduced a new definition of beta- hairpin motifs, explicit state modelling of transmembrane strands, and a log-odds whole-protein discrimination score. The resulting method reached an overall four-state (up-, down-strand, periplasmic-, outer-loop) accuracy as high as 86%. Furthermore, accurately discriminated TMB from non-TMB proteins (45% coverage at 100% accuracy). This high precision enabled the application to 72 entirely sequenced Gram-negative bacteria. We found over 164 previously uncharacterized TMB proteins at high confidence. Database searches did not implicate any of these proteins with membranes. We challenge that the vast majority of our 164 predictions will eventually be verified experimentally. All proteome predictions and the PROFtmb prediction method are available at http://www.rostlab.org/services/PROFtmb/. PMID:15141026

  20. The Arabidopsis Circadian System

    PubMed Central

    McClung, C. Robertson; Salomé, Patrice A.; Michael, Todd P.

    2002-01-01

    Rhythms with periods of approximately 24 hr are widespread in nature. Those that persist in constant conditions are termed circadian rhythms and reflect the activity of an endogenous biological clock. Plants, including Arabidopsis, are richly rhythmic. Expression analysis, most recently on a genomic scale, indicates that the Arabidopsis circadian clock regulates a number of key metabolic pathways and stress responses. A number of sensitive and high-throughput assays have been developed to monitor the Arabidopsis clock. These assays have facilitated the identification of components of plant circadian systems through genetic and molecular biological studies. Although much remains to be learned, the framework of the Arabidopsis circadian system is coming into focus. Dedication This review is dedicated to the memory of DeLill Nasser, a wonderful mentor and an unwavering advocate of both Arabidopsis and circadian rhythms research. PMID:22303209

  1. Transmembrane protein sorting driven by membrane curvature

    PubMed Central

    Strahl, H.; Ronneau, S.; González, B. Solana; Klutsch, D.; Schaffner-Barbero, C.; Hamoen, L. W.

    2015-01-01

    The intricate structure of prokaryotic and eukaryotic cells depends on the ability to target proteins to specific cellular locations. In most cases, we have a poor understanding of the underlying mechanisms. A typical example is the assembly of bacterial chemoreceptors at cell poles. Here we show that the classical chemoreceptor TlpA of Bacillus subtilis does not localize according to the consensus stochastic nucleation mechanism but accumulates at strongly curved membrane areas generated during cell division. This preference was confirmed by accumulation at non-septal curved membranes. Localization appears to be an intrinsic property of the protein complex and does not rely on chemoreceptor clustering, as was previously shown for Escherichia coli. By constructing specific amino-acid substitutions, we demonstrate that the preference for strongly curved membranes arises from the curved shape of chemoreceptor trimer of dimers. These findings demonstrate that the intrinsic shape of transmembrane proteins can determine their cellular localization. PMID:26522943

  2. Transmembrane protein sorting driven by membrane curvature

    NASA Astrophysics Data System (ADS)

    Strahl, H.; Ronneau, S.; González, B. Solana; Klutsch, D.; Schaffner-Barbero, C.; Hamoen, L. W.

    2015-11-01

    The intricate structure of prokaryotic and eukaryotic cells depends on the ability to target proteins to specific cellular locations. In most cases, we have a poor understanding of the underlying mechanisms. A typical example is the assembly of bacterial chemoreceptors at cell poles. Here we show that the classical chemoreceptor TlpA of Bacillus subtilis does not localize according to the consensus stochastic nucleation mechanism but accumulates at strongly curved membrane areas generated during cell division. This preference was confirmed by accumulation at non-septal curved membranes. Localization appears to be an intrinsic property of the protein complex and does not rely on chemoreceptor clustering, as was previously shown for Escherichia coli. By constructing specific amino-acid substitutions, we demonstrate that the preference for strongly curved membranes arises from the curved shape of chemoreceptor trimer of dimers. These findings demonstrate that the intrinsic shape of transmembrane proteins can determine their cellular localization.

  3. Isolation and characterization of a gene from Medicago sativa L., encoding a bZIP transcription factor.

    PubMed

    Li, Yan; Sun, Yan; Yang, Qingchuan; Fang, Feng; Kang, Junmei; Zhang, Tiejun

    2013-02-01

    A full-length cDNA of 1,537 nucleotides was cloned from Medicago sativa L. cv. "Zhongmu No. 1" by rapid amplification of cDNA ends. It was designated as MsZIP, encoding a protein of 340 amino acids. The protein molecular weight was 36.43 kDa, and the theoretical isoelectric point was 5.72. The MsZIP preferentially localized in nucleus and have signal peptide. Blast analysis revealed that MsZIP shared the highest homology with some bZIP proteins of M. truncatula. The transcript of MsZIP was strongly enriched in leaf compared with root and stem of mature alfalfa plants. MsZIP was strongly induced by 15 % PEG6000 (polyethylene glycol), 50 μM abscisic acid, 200 mM NaCl, 70 μM gibberellic acid, 5 mM salicylic acid and 200 μM methyl jasmonate. Physiological resistance parameters were measured in the transgenic tobacco. Malondialdehyde content, relative water content, soluble sugar content, soluble protein content and proline content in transgenic tobacco increased compared with non-transgenic tobacco under salt stress or drought stress. The results showed that accumulation of the MsZIP protein in the vegetative tissues of transgenic plants enhanced their tolerance to osmotic pressure stress. These results demonstrate a role for the MsZIP protein in stress protection and suggest the potential of the MsZIP gene for genetic engineering of salt tolerance and drought tolerance.

  4. Molecular mechanisms for generating transmembrane proton gradients.

    PubMed

    Gunner, M R; Amin, Muhamed; Zhu, Xuyu; Lu, Jianxun

    2013-01-01

    Membrane proteins use the energy of light or high energy substrates to build a transmembrane proton gradient through a series of reactions leading to proton release into the lower pH compartment (P-side) and proton uptake from the higher pH compartment (N-side). This review considers how the proton affinity of the substrates, cofactors and amino acids are modified in four proteins to drive proton transfers. Bacterial reaction centers (RCs) and photosystem II (PSII) carry out redox chemistry with the species to be oxidized on the P-side while reduction occurs on the N-side of the membrane. Terminal redox cofactors are used which have pKas that are strongly dependent on their redox state, so that protons are lost on oxidation and gained on reduction. Bacteriorhodopsin is a true proton pump. Light activation triggers trans to cis isomerization of a bound retinal. Strong electrostatic interactions within clusters of amino acids are modified by the conformational changes initiated by retinal motion leading to changes in proton affinity, driving transmembrane proton transfer. Cytochrome c oxidase (CcO) catalyzes the reduction of O2 to water. The protons needed for chemistry are bound from the N-side. The reduction chemistry also drives proton pumping from N- to P-side. Overall, in CcO the uptake of 4 electrons to reduce O2 transports 8 charges across the membrane, with each reduction fully coupled to removal of two protons from the N-side, the delivery of one for chemistry and transport of the other to the P-side.

  5. Molecular mechanisms for generating transmembrane proton gradients.

    PubMed

    Gunner, M R; Amin, Muhamed; Zhu, Xuyu; Lu, Jianxun

    2013-01-01

    Membrane proteins use the energy of light or high energy substrates to build a transmembrane proton gradient through a series of reactions leading to proton release into the lower pH compartment (P-side) and proton uptake from the higher pH compartment (N-side). This review considers how the proton affinity of the substrates, cofactors and amino acids are modified in four proteins to drive proton transfers. Bacterial reaction centers (RCs) and photosystem II (PSII) carry out redox chemistry with the species to be oxidized on the P-side while reduction occurs on the N-side of the membrane. Terminal redox cofactors are used which have pKas that are strongly dependent on their redox state, so that protons are lost on oxidation and gained on reduction. Bacteriorhodopsin is a true proton pump. Light activation triggers trans to cis isomerization of a bound retinal. Strong electrostatic interactions within clusters of amino acids are modified by the conformational changes initiated by retinal motion leading to changes in proton affinity, driving transmembrane proton transfer. Cytochrome c oxidase (CcO) catalyzes the reduction of O2 to water. The protons needed for chemistry are bound from the N-side. The reduction chemistry also drives proton pumping from N- to P-side. Overall, in CcO the uptake of 4 electrons to reduce O2 transports 8 charges across the membrane, with each reduction fully coupled to removal of two protons from the N-side, the delivery of one for chemistry and transport of the other to the P-side. PMID:23507617

  6. Identification of Novel Components of the Unfolded Protein Response in Arabidopsis.

    PubMed

    Hossain, Md Amir; Henríquez-Valencia, Carlos; Gómez-Páez, Marcela; Medina, Joaquín; Orellana, Ariel; Vicente-Carbajosa, Jesús; Zouhar, Jan

    2016-01-01

    Unfavorable environmental and developmental conditions may cause disturbances in protein folding in the endoplasmic reticulum (ER) that are recognized and counteracted by components of the Unfolded Protein Response (UPR) signaling pathways. The early cellular responses include transcriptional changes to increase the folding and processing capacity of the ER. In this study, we systematically screened a collection of inducible transgenic Arabidopsis plants expressing a library of transcription factors for resistance toward UPR-inducing chemicals. We identified 23 candidate genes that may function as novel regulators of the UPR and of which only three genes (bZIP10, TBF1, and NF-YB3) were previously associated with the UPR. The putative role of identified candidate genes in the UPR signaling is supported by favorable expression patterns in both developmental and stress transcriptional analyses. We demonstrated that WRKY75 is a genuine regulator of the ER-stress cellular responses as its expression was found to be directly responding to ER stress-inducing chemicals. In addition, transgenic Arabidopsis plants expressing WRKY75 showed resistance toward salt stress, connecting abiotic and ER-stress responses. PMID:27242851

  7. A systematic survey in Arabidopsis thaliana of transcription factors that modulate circadian parameters

    PubMed Central

    Hanano, Shigeru; Stracke, Ralf; Jakoby, Marc; Merkle, Thomas; Domagalska, Malgorzata A; Weisshaar, Bernd; Davis, Seth J

    2008-01-01

    Background Plant circadian systems regulate various biological processes in harmony with daily environmental changes. In Arabidopsis thaliana, the underlying clock mechanism is comprised of multiple integrated transcriptional feedbacks, which collectively lead to global patterns of rhythmic gene expression. The transcriptional networks are essential within the clock itself and in its output pathway. Results Here, to expand understanding of transcriptional networks within and associated to the clock, we performed both an in silico analysis of transcript rhythmicity of transcription factor genes, and a pilot assessment of functional phenomics on the MYB, bHLH, and bZIP families. In our in silico analysis, we defined which members of these families express a circadian waveform of transcript abundance. Up to 20% of these families were over-represented as clock-controlled genes. To detect members that contribute to proper oscillator function, we systematically measured rhythmic growth via an imaging system in hundreds of misexpression lines targeting members of the transcription-factor families. Three transcription factors were found that conferred aberrant circadian rhythms when misexpressed: MYB3R2, bHLH69, and bHLH92. Conclusion Transcript abundance of many transcription factors in Arabidopsis oscillates in a circadian manner. Further, a developed pipeline assessed phenotypic contribution of a panel of transcriptional regulators in the circadian system. PMID:18426557

  8. Identification of Novel Components of the Unfolded Protein Response in Arabidopsis

    PubMed Central

    Hossain, Md. Amir; Henríquez-Valencia, Carlos; Gómez-Páez, Marcela; Medina, Joaquín; Orellana, Ariel; Vicente-Carbajosa, Jesús; Zouhar, Jan

    2016-01-01

    Unfavorable environmental and developmental conditions may cause disturbances in protein folding in the endoplasmic reticulum (ER) that are recognized and counteracted by components of the Unfolded Protein Response (UPR) signaling pathways. The early cellular responses include transcriptional changes to increase the folding and processing capacity of the ER. In this study, we systematically screened a collection of inducible transgenic Arabidopsis plants expressing a library of transcription factors for resistance toward UPR-inducing chemicals. We identified 23 candidate genes that may function as novel regulators of the UPR and of which only three genes (bZIP10, TBF1, and NF-YB3) were previously associated with the UPR. The putative role of identified candidate genes in the UPR signaling is supported by favorable expression patterns in both developmental and stress transcriptional analyses. We demonstrated that WRKY75 is a genuine regulator of the ER-stress cellular responses as its expression was found to be directly responding to ER stress-inducing chemicals. In addition, transgenic Arabidopsis plants expressing WRKY75 showed resistance toward salt stress, connecting abiotic and ER-stress responses. PMID:27242851

  9. Screen Identifying Arabidopsis Transcription Factors Involved in the Response to 9-Lipoxygenase-Derived Oxylipins.

    PubMed

    Walper, Elisabeth; Weiste, Christoph; Mueller, Martin J; Hamberg, Mats; Dröge-Laser, Wolfgang

    2016-01-01

    13-Lipoxygenase-derived oxylipins, such as jasmonates act as potent signaling molecules in plants. Although experimental evidence supports the impact of oxylipins generated by the 9-Lipoxygenase (9-LOX) pathway in root development and pathogen defense, their signaling function in plants remains largely elusive. Based on the root growth inhibiting properties of the 9-LOX-oxylipin 9-HOT (9-hydroxy-10,12,15-octadecatrienoic acid), we established a screening approach aiming at identifying transcription factors (TFs) involved in signaling and/or metabolism of this oxylipin. Making use of the AtTORF-Ex (Arabidopsis thaliana Transcription Factor Open Reading Frame Expression) collection of plant lines overexpressing TF genes, we screened for those TFs which restore root growth on 9-HOT. Out of 6,000 lines, eight TFs were recovered at least three times and were therefore selected for detailed analysis. Overexpression of the basic leucine Zipper (bZIP) TF TGA5 and its target, the monoxygenase CYP81D11 reduced the effect of added 9-HOT, presumably due to activation of a detoxification pathway. The highly related ETHYLENE RESPONSE FACTORs ERF106 and ERF107 induce a broad detoxification response towards 9-LOX-oxylipins and xenobiotic compounds. From a set of 18 related group S-bZIP factors isolated in the screen, bZIP11 is known to participate in auxin-mediated root growth and may connect oxylipins to root meristem function. The TF candidates isolated in this screen provide starting points for further attempts to dissect putative signaling pathways involving 9-LOX-derived oxylipins. PMID:27073862

  10. Screen Identifying Arabidopsis Transcription Factors Involved in the Response to 9-Lipoxygenase-Derived Oxylipins

    PubMed Central

    Walper, Elisabeth; Weiste, Christoph; Mueller, Martin J.; Hamberg, Mats; Dröge-Laser, Wolfgang

    2016-01-01

    13-Lipoxygenase-derived oxylipins, such as jasmonates act as potent signaling molecules in plants. Although experimental evidence supports the impact of oxylipins generated by the 9-Lipoxygenase (9-LOX) pathway in root development and pathogen defense, their signaling function in plants remains largely elusive. Based on the root growth inhibiting properties of the 9-LOX-oxylipin 9-HOT (9-hydroxy-10,12,15-octadecatrienoic acid), we established a screening approach aiming at identifying transcription factors (TFs) involved in signaling and/or metabolism of this oxylipin. Making use of the AtTORF-Ex (Arabidopsis thaliana Transcription Factor Open Reading Frame Expression) collection of plant lines overexpressing TF genes, we screened for those TFs which restore root growth on 9-HOT. Out of 6,000 lines, eight TFs were recovered at least three times and were therefore selected for detailed analysis. Overexpression of the basic leucine Zipper (bZIP) TF TGA5 and its target, the monoxygenase CYP81D11 reduced the effect of added 9-HOT, presumably due to activation of a detoxification pathway. The highly related ETHYLENE RESPONSE FACTORs ERF106 and ERF107 induce a broad detoxification response towards 9-LOX-oxylipins and xenobiotic compounds. From a set of 18 related group S-bZIP factors isolated in the screen, bZIP11 is known to participate in auxin-mediated root growth and may connect oxylipins to root meristem function. The TF candidates isolated in this screen provide starting points for further attempts to dissect putative signaling pathways involving 9-LOX-derived oxylipins. PMID:27073862

  11. Stress-related function of bHLH109 in somatic embryo induction in Arabidopsis.

    PubMed

    Nowak, Katarzyna; Gaj, Małgorzata D

    2016-04-01

    The bHLH109 gene of the bHLH family was identified among the transcription factor encoding genes that were differentially expressed in an embryogenic culture of Arabidopsis. A strong activation of bHLH109 expression was found to be associated with somatic embryogenesis (SE) induction. Several pieces of evidence suggested the involvement of bHLH109 in SE, including the high stimulation of the gene expression in SE-induced explants, which contrasts to the drastically lower level of the gene transcripts in the non-embryogenic callus and in tissue that is induced towards shoot regeneration via organogenesis. Moreover, in contrast to the overexpression of bHLH109, which has been indicated to enhance SE induction in a culture, the bhlh109 knock-out mutation was found to impair the embryogenic potential of explants. In order to identify the genes interacting with the bHLH109, the candidate co-expressed genes were identified in a yeast one hybrid assay. The in vitro regulatory interactions that were identified were verified through mutant and expression analysis. The results suggest that in SE bHLH109 acts as an activator of ECP63, a member of the LEA (LATE EMBRYOGENESIS ABUNDANT) family. Among the potential regulators of bHLH109, three candidates (At5g61620, bZIP4 and bZIP43) were indicated to possibly control bHLH109. The functions of all of the genes that are assumed to interact with bHLH109 are annotated to stress responses. Collectively, the results of the study provide new evidence that cell responses to stress that is imposed under in vitro conditions underlies the promotion of SE. bHLH109 may play a central role in the stress-related mechanism of SE induction via an increased accumulation of the LEA protein (ECP63), which results in the enhanced tolerance of the cells to stress. PMID:26973252

  12. Transmembrane beta-barrel protein structure prediction

    NASA Astrophysics Data System (ADS)

    Randall, Arlo; Baldi, Pierre

    Transmembrane β-barrel (TMB) proteins are embedded in the outer membranes of mitochondria, Gram-negative bacteria, and chloroplasts. These proteins perform critical functions, including active ion-transport and passive nutrient intake. Therefore, there is a need for accurate prediction of secondary and tertiary structures of TMB proteins. A variety of methods have been developed for predicting the secondary structure and these predictions are very useful for constructing a coarse topology of TMB structure; however, they do not provide enough information to construct a low-resolution tertiary structure for a TMB protein. In addition, while the overall structural architecture is well conserved among TMB proteins, the amino acid sequences are highly divergent. Thus, traditional homology modeling methods cannot be applied to many putative TMB proteins. Here, we describe the TMBpro: a pipeline of methods for predicting TMB secondary structure, β-residue contacts, and finally tertiary structure. The tertiary prediction method relies on the specific construction rules that TMB proteins adhere to and on the predicted β-residue contacts to dramatically reduce the search space for the model building procedure.

  13. The Origins of Transmembrane Ion Channels

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; Wilson, Michael A.

    2012-01-01

    Even though membrane proteins that mediate transport of ions and small molecules across cell walls are among the largest and least understood biopolymers in contemporary cells, it is still possible to shed light on their origins and early evolution. The central observation is that transmembrane portions of most ion channels are simply bundles of -helices. By combining results of experimental and computer simulation studies on synthetic models and natural channels, mostly of non-genomic origin, we show that the emergence of -helical channels was protobiologically plausible, and did not require highly specific amino acid sequences. Despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. Specifically, we explain how the antiamoebin channels, which are made of identical helices, 16 amino acids in length, achieve efficiency comparable to that of highly evolved channels. We further show that antiamoebin channels are extremely flexible, compared to modern, genetically coded channels. On the basis of our results, we propose that channels evolved further towards high structural complexity because they needed to acquire stable rigid structures and mechanisms for precise regulation rather than improve efficiency. In general, even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during evolution.

  14. Video imaging of cardiac transmembrane activity

    NASA Astrophysics Data System (ADS)

    Baxter, William T.; Davidenko, Jorge; Cabo, Candido; Jalife, Jose

    1994-05-01

    High resolution movies of transmembrane electrical activity in thin (0.5 mm) slices of sheep epicardial muscle were recorded by optical imaging with voltage-sensitive dyes and a CCD video camera. Activity was monitored at approximately 65,000 picture elements per 2 cm2 tissue for several seconds at a 16 msec sampling rate. Simple image processing operations permitted visualization and analysis of the optical signal, while isochrome maps depicted complex patterns of propagation. Maps of action potential duration and regional intermittent conduction block showed that even these small preparations may exhibit considerable spatial heterogeneity. Self-sustaining reentrant activity in the form of spiral waves was consistently initiated and observed either drifting across the tissue or anchored to small heterogeneities. The current limitations of video optical mappings are a low signal-to- noise ratio and low temporal resolution. The advantages include high spatial resolution and direct correlation of electrical activity with anatomy. Video optical mapping permits the analysis of the electrophysiological properties of any region of the preparation during both regular stimulation and reentrant activation, providing a useful tool for studying cardiac arrhythmias.

  15. Interface connections of a transmembrane voltage sensor.

    PubMed

    Freites, J Alfredo; Tobias, Douglas J; von Heijne, Gunnar; White, Stephen H

    2005-10-18

    Voltage-sensitive ion channels open and close in response to changes in transmembrane (TM) potential caused by the motion of the S4 voltage sensors. These sensors are alpha-helices that include four or more positively charged amino acids, most commonly arginine. The so-called paddle model, based on the high-resolution structure of the KvAP K+ channel [Jiang, et al. (2003) Nature 423, 33-41], posits that the S4 sensors move within the membrane bilayer in response to TM voltage changes. Direct exposure of S4 sensors to lipid is contrary to the classical expectation that the dielectric contrast between the membrane hydrocarbon core and water presents an insurmountable energetic penalty to burial of electric charges. Nevertheless, recent experiments have shown that a helix with the sequence of KvAP S4 can be inserted across the endoplasmic reticulum membrane. To reconcile this result with the classical energetics argument, we have carried out a molecular dynamics simulation of an isolated TM S4 helix in a lipid bilayer. The simulation reveals a stabilizing hydrogen-bonded network of water and lipid phosphates around the arginines that reduces the effective thickness of the bilayer hydrocarbon core to approximately 10 A in the vicinity of the helix. It suggests that bilayer phospholipids can adapt locally to strongly perturbing protein elements, causing the phospholipids to become a structural extension of the protein.

  16. A conserved proline residue in the leucine zipper region of AtbZIP34 and AtbZIP61 in Arabidopsis thaliana interferes with the formation of homodimer

    SciTech Connect

    Shen Huaishun; Cao Kaiming; Wang Xiping

    2007-10-19

    Two putative Arabidopsis E group bZIP transcript factors, AtbZIP34 and AtbZIP61, are nuclear-localized and their transcriptional activation domain is in their N-terminal region. By searching GenBank, we found other eight plant homologues of AtbZIP34 and AtbZIP61. All of them have a proline residue in the third heptad of zipper region. Yeast two-hybrid assay and EMSA showed that AtbZIP34 and AtbZIP61 could not form homodimer while their mutant forms, AtbZIP34m and AtbZIP61m, which the proline residue was replaced by an alanine residue in the zipper region, could form homodimer and bind G-box element. These results suggest that the conserved proline residue interferes with the homodimer formation. However, both AtbZIP34 and AtbZIP61 could form heterodimers with members of I group and S group transcription factors in which some members involved in vascular development. So we speculate that AtbZIP34 and AtbZIP61 may participate in plant development via interacting with other group bZIP transcription factors.

  17. A conserved proline residue in the leucine zipper region of AtbZIP34 and AtbZIP61 in Arabidopsis thaliana interferes with the formation of homodimer.

    PubMed

    Shen, Huaishun; Cao, Kaiming; Wang, Xiping

    2007-10-19

    Two putative Arabidopsis E group bZIP transcript factors, AtbZIP34 and AtbZIP61, are nuclear-localized and their transcriptional activation domain is in their N-terminal region. By searching GenBank, we found other eight plant homologues of AtbZIP34 and AtbZIP61. All of them have a proline residue in the third heptad of zipper region. Yeast two-hybrid assay and EMSA showed that AtbZIP34 and AtbZIP61 could not form homodimer while their mutant forms, AtbZIP34m and AtbZIP61m, which the proline residue was replaced by an alanine residue in the zipper region, could form homodimer and bind G-box element. These results suggest that the conserved proline residue interferes with the homodimer formation. However, both AtbZIP34 and AtbZIP61 could form heterodimers with members of I group and S group transcription factors in which some members involved in vascular development. So we speculate that AtbZIP34 and AtbZIP61 may participate in plant development via interacting with other group bZIP transcription factors. PMID:17719007

  18. Are Aquaporins the Missing Transmembrane Osmosensors?

    PubMed

    Hill, A E; Shachar-Hill, Y

    2015-08-01

    Regulation of cell volume is central to homeostasis. It is assumed to begin with the detection of a change in water potential across the bounding membrane, but it is not clear how this is accomplished. While examples of general osmoreceptors (which sense osmotic pressure in one phase) and stretch-activated ion channels (which require swelling of a cell or organelle) are known, effective volume regulation requires true transmembrane osmosensors (TMOs) which directly detect a water potential difference spanning a membrane. At present, no TMO molecule has been unambiguously identified, and clear evidence for mammalian TMOs is notably lacking. In this paper, we set out a theory of TMOs which requires a water channel spanning the membrane that excludes the major osmotic solutes, responds directly without the need for any other process such as swelling, and signals to other molecules associated with the magnitude of changing osmotic differences. The most likely molecules that are fit for this purpose and which are also ubiquitous in eukaryotic cells are aquaporins (AQPs). We review experimental evidence from several systems which indicates that AQPs are essential elements in regulation and may be functioning as TMOs; i.e. the first step in an osmosensing sequence that signals osmotic imbalance in a cell or organelle. We extend this concept to several systems of current interest in which the cellular involvement of AQPs as simple water channels is puzzling or counter-intuitive. We suggest that, apart from regulatory volume changes in cells, AQPs may also be acting as TMOs in red cells, secretory granules and microorganisms. PMID:25791748

  19. Lipid bilayer microarray for parallel recording of transmembrane ion currents.

    PubMed

    Le Pioufle, Bruno; Suzuki, Hiroaki; Tabata, Kazuhito V; Noji, Hiroyuki; Takeuchi, Shoji

    2008-01-01

    This paper describes a multiwell biochip for simultaneous parallel recording of ion current through transmembrane pores reconstituted in planar lipid bilayer arrays. Use of a thin poly(p-xylylene) (parylene) film having micrometer-sized apertures (phi=15-50 microm, t=20 microm) led to formation of highly stable bilayer lipid membranes (BLMs) for incorporation of transmembrane pores; thus, a large number of BLMs could be arrayed without any skillful technique. We optically confirmed the simultaneous formation of BLMs in a 5x5 matrix, and in our durability test, the BLM lasted more than 15 h. Simultaneous parallel recording of alamethicin and gramicidin transmembrane pores in multiple contiguous recording sites demonstrated the feasibility of high-throughput screening of transmembrane ion currents in artificial lipid bilayers.

  20. A deterministic algorithm for constrained enumeration of transmembrane protein folds.

    SciTech Connect

    Brown, William Michael; Young, Malin M.; Sale, Kenneth L.; Faulon, Jean-Loup Michel; Schoeniger, Joseph S.

    2004-07-01

    A deterministic algorithm for enumeration of transmembrane protein folds is presented. Using a set of sparse pairwise atomic distance constraints (such as those obtained from chemical cross-linking, FRET, or dipolar EPR experiments), the algorithm performs an exhaustive search of secondary structure element packing conformations distributed throughout the entire conformational space. The end result is a set of distinct protein conformations, which can be scored and refined as part of a process designed for computational elucidation of transmembrane protein structures.

  1. Characterization of Disease-Associated Mutations in Human Transmembrane Proteins

    PubMed Central

    Molnár, János; Szakács, Gergely; Tusnády, Gábor E.

    2016-01-01

    Transmembrane protein coding genes are commonly associated with human diseases. We characterized disease causing mutations and natural polymorphisms in transmembrane proteins by mapping missense genetic variations from the UniProt database on the transmembrane protein topology listed in the Human Transmembrane Proteome database. We found characteristic differences in the spectrum of amino acid changes within transmembrane regions: in the case of disease associated mutations the non-polar to non-polar and non-polar to charged amino acid changes are equally frequent. In contrast, in the case of natural polymorphisms non-polar to charged amino acid changes are rare while non-polar to non-polar changes are common. The majority of disease associated mutations result in glycine to arginine and leucine to proline substitutions. Mutations to positively charged amino acids are more common in the center of the lipid bilayer, where they cause more severe structural and functional anomalies. Our analysis contributes to the better understanding of the effect of disease associated mutations in transmembrane proteins, which can help prioritize genetic variations in personal genomic investigations. PMID:26986070

  2. Interfacial water screens the protein-induced transmembrane voltage.

    PubMed

    Zarzycki, Piotr

    2015-01-29

    Transmembrane proteins are crucial in cellular traffic, signal transduction, and energy storage in a form of transmembrane voltage. These proteins are stabilized by hydrophobic and hydrophilic interactions, in which cytoplasmic and exoplasmic water plays a special role. Water structural ordering generates the dipole potential that typically overcompensates for an intrinsic membrane-protein potential gradient, and thus it modifies and sustains an overall cellular electrostatics. Although the transmembrane voltage has been extensively studied, the dipole potential has attracted very little attention. Here, by using molecular dynamics, we examined water electrostatic response to the transmembrane charge, field, and potential asymmetry introduced by the presence of four integral membrane proteins: typical of inner (α-helix) and outer membrane (β-barrel). In all cases, the protein presence introduces electrostatic directionality in the transmembrane dipole field and voltage. In particular, water generates a deep potential sink if strongly polar residues are densely packed on one side of bilayer, as frequently occurs in a selectivity filter of the K(+) channel. We also found that protein secondary structure is less important than the polar residue distribution along the protein channel. Our findings are relevant for understanding the driving force behind biomembrane conductivity: the ability of biological water to electrostatically screen the transmembrane voltage.

  3. Tight interconnection and multi-level control of Arabidopsis MYB44 in MAPK cascade signalling.

    PubMed

    Persak, Helene; Pitzschke, Andrea

    2013-01-01

    Abiotic stress poses a huge, ever-increasing problem to plants and agriculture. The dissection of signalling pathways mediating stress tolerance is a prerequisite to develop more resistant plant species. Mitogen-activated protein kinase (MAPK) cascades are universal signalling modules. In Arabidopsis, the MAPK MPK3 and its upstream regulator MAPK kinase MKK4 initiate the adaptation response to numerous abiotic and biotic stresses. Yet, molecular steps directly linked with MKK4-MPK3 activation are largely unknown. Starting with a yeast-two-hybrid screen for interacting partners of MKK4, we identified a transcription factor, MYB44. MYB44 is controlled at multiple levels by and strongly inter-connected with MAPK signalling. As we had shown earlier, stress-induced expression of the MYB44 gene is regulated by a MPK3-targeted bZIP transcription factor VIP1. At the protein level, MYB44 interacts with MPK3 in vivo. MYB44 is phosphorylated by MPK3 in vitro at a single residue, Ser145. Although replacement of Ser145 by a non-phosphorylatable (S145A) or phosphomimetic (S145D) residue did not alter MYB44 subcellular localisation, dimerization behaviour nor DNA-binding characteristics, abiotic stress tolerance tests in stable transgenic Arabidopsis plants clearly related S145 phosphorylation to MYB44 function: Compared to Arabidopsis wild type plants, MYB44 overexpressing lines exhibit an enhanced tolerance to osmotic stress and are slightly more sensitive to abscisic acid. Interestingly, overexpression of the S145A variant revealed that impaired phosphorylation does not render the MYB44 protein non-functional. Instead, S145A lines are highly sensitive to abiotic stress, and thereby remarkably similar to mpk3-deficient plants. Its in vivo interaction with the nuclear sub-pools of both MPK3 and MKK4 renders MYB44 the first plant transcription factor to have a second function as putative MAPK cascade scaffolding protein.

  4. [Orthologs of arabidopsis CLAVATA 1 gene in cultivated Brassicaceae plants].

    PubMed

    Martynov, V V; Tsvetkov, I L; Khavkin, E E

    2004-01-01

    In arabidopsis (Arabidopsis thaliana), the CLAVATA1 (CLV1) gene is involved in maintaining the balance between the stem cells in the central zone of the stem apical meristem and the determined cells at its periphery. However, CLV1 has not been previously characterized in other Brassicaceae. Using the direct amplification of genomic DNA, we obtained a full-length CLV1 ortholog from canola plants (Brassica napus), and also three CLV1 fragments from rape (B. rapa), canola (B. napus), and false flax (Camelina sativa), which corresponded to the transmembrane domain and a part of the kinase domain of the CLAVATA1 protein. The nucleotide and deduced amino acid sequences of the full-size CLV1 ortholog from B. napus were similar by 81 and 87% to the prototype gene from arabidopsis; in the case of shorter gene fragments, the similarity was as high as 91-93 and 98%, respectively. By their primary structure, the CLV1 genes in the Brassicaceae considerably differ from its putative structural homologs beyond this family.

  5. Starch Metabolism in Arabidopsis

    PubMed Central

    Streb, Sebastian; Zeeman, Samuel C.

    2012-01-01

    Starch is the major non-structural carbohydrate in plants. It serves as an important store of carbon that fuels plant metabolism and growth when they are unable to photosynthesise. This storage can be in leaves and other green tissues, where it is degraded during the night, or in heterotrophic tissues such as roots, seeds and tubers, where it is stored over longer time periods. Arabidopsis accumulates starch in many of its tissues, but mostly in its leaves during the day. It has proven to be a powerful genetic system for discovering how starch is synthesised and degraded, and new proteins and processes have been discovered. Such work has major significance for our starch crops, whose yield and quality could be improved by the application of this knowledge. Research into Arabidopsis starch metabolism has begun to reveal how its daily turnover is integrated into the rest of metabolism and adapted to the environmental conditions. Furthermore, Arabidopsis mutant lines deficient in starch metabolism have been employed as tools to study other biological processes ranging from sugar sensing to gravitropism and flowering time control. This review gives a detailed account of the use of Arabidopsis to study starch metabolism. It describes the major discoveries made and presents an overview of our understanding today, together with some as-yet unresolved questions. PMID:23393426

  6. Suppressor Screens in Arabidopsis.

    PubMed

    Li, Xin; Zhang, Yuelin

    2016-01-01

    Genetic screens have proven to be a useful tool in the dissection of biological processes in plants. Specifically, suppressor screens have been widely used to study signal transduction pathways. Here we provide a detailed protocol for ethyl methanesulfonate (EMS) mutagenesis used in our suppressor screens in Arabidopsis and discuss the basic principles behind suppressor screen design and downstream analyses. PMID:26577776

  7. Transmembrane domain interactions control biological functions of neuropilin-1.

    PubMed

    Roth, Lise; Nasarre, Cécile; Dirrig-Grosch, Sylvie; Aunis, Dominique; Crémel, Gérard; Hubert, Pierre; Bagnard, Dominique

    2008-02-01

    Neuropilin-1 (NRP1) is a transmembrane receptor playing a pivotal role in the control of semaphorins and VEGF signaling pathways. The exact mechanism controlling semaphorin receptor complex formation is unknown. A structural analysis and modeling of NRP1 revealed a putative dimerization GxxxG motif potentially important for NRP1 dimerization and oligomerization. Our data show that this motif mediates the dimerization of the transmembrane domain of NRP1 as demonstrated by a dimerization assay (ToxLuc assay) performed in natural membrane and FRET analysis. A synthetic peptide derived from the transmembrane segment of NRP1 abolished the inhibitory effect of Sema3A. This effect depends on the capacity of the peptide to interfere with NRP1 dimerization and the formation of oligomeric complexes. Mutation of the GxxxG dimerization motif in the transmembrane domain of NRP1 confirmed its biological importance for Sema3A signaling. Overall, our results shed light on an essential step required for semaphorin signaling and provide novel evidence for the crucial role of transmembrane domain of bitopic protein containing GxxxG motif in the formation of receptor complexes that are a prerequisite for cell signaling.

  8. PDBTM: Protein Data Bank of transmembrane proteins after 8 years.

    PubMed

    Kozma, Dániel; Simon, István; Tusnády, Gábor E

    2013-01-01

    The PDBTM database (available at http://pdbtm.enzim.hu), the first comprehensive and up-to-date transmembrane protein selection of the Protein Data Bank, was launched in 2004. The database was created and has been continuously updated by the TMDET algorithm that is able to distinguish between transmembrane and non-transmembrane proteins using their 3D atomic coordinates only. The TMDET algorithm can locate the spatial positions of transmembrane proteins in lipid bilayer as well. During the last 8 years not only the size of the PDBTM database has been steadily growing from ∼400 to 1700 entries but also new structural elements have been identified, in addition to the well-known α-helical bundle and β-barrel structures. Numerous 'exotic' transmembrane protein structures have been solved since the first release, which has made it necessary to define these new structural elements, such as membrane loops or interfacial helices in the database. This article reports the new features of the PDBTM database that have been added since its first release, and our current efforts to keep the database up-to-date and easy to use so that it may continue to serve as a fundamental resource for the scientific community.

  9. Molecular Signatures in Arabidopsis thaliana in Response to Insect Attack and Bacterial Infection

    PubMed Central

    Barah, Pankaj; Winge, Per; Kusnierczyk, Anna; Tran, Diem Hong; Bones, Atle M.

    2013-01-01

    Background Under the threat of global climatic change and food shortages, it is essential to take the initiative to obtain a comprehensive understanding of common and specific defence mechanisms existing in plant systems for protection against different types of biotic invaders. We have implemented an integrated approach to analyse the overall transcriptomic reprogramming and systems-level defence responses in the model plant species Arabidopsis thaliana (A. thaliana henceforth) during insect Brevicoryne brassicae (B. brassicae henceforth) and bacterial Pseudomonas syringae pv. tomato strain DC3000 (P. syringae henceforth) attacks. The main aim of this study was to identify the attacker-specific and general defence response signatures in A. thaliana when attacked by phloem-feeding aphids or pathogenic bacteria. Results The obtained annotated networks of differentially expressed transcripts indicated that members of transcription factor families, such as WRKY, MYB, ERF, BHLH and bZIP, could be crucial for stress-specific defence regulation in Arabidopsis during aphid and P. syringae attack. The defence response pathways, signalling pathways and metabolic processes associated with aphid attack and P. syringae infection partially overlapped. Components of several important biosynthesis and signalling pathways, such as salicylic acid (SA), jasmonic acid (JA), ethylene (ET) and glucosinolates, were differentially affected during the two the treatments. Several stress-regulated transcription factors were known to be associated with stress-inducible microRNAs. The differentially regulated gene sets included many signature transcription factors, and our co-expression analysis showed that they were also strongly co-expressed during 69 other biotic stress experiments. Conclusions Defence responses and functional networks that were unique and specific to aphid or P. syringae stresses were identified. Furthermore, our analysis revealed a probable link between biotic stress and

  10. Prediction of three-dimensional transmembrane helical protein structures

    NASA Astrophysics Data System (ADS)

    Barth, Patrick

    Membrane proteins are critical to living cells and their dysfunction can lead to serious diseases. High-resolution structures of these proteins would provide very valuable information for designing eficient therapies but membrane protein crystallization is a major bottleneck. As an important alternative approach, methods for predicting membrane protein structures have been developed in recent years. This chapter focuses on the problem of modeling the structure of transmembrane helical proteins, and describes recent advancements, current limitations, and future challenges facing de novo modeling, modeling with experimental constraints, and high-resolution comparative modeling of these proteins. Abbreviations: MP, membrane protein; SP, water-soluble protein; RMSD, root-mean square deviation; Cα RMSD, root-mean square deviation over Cα atoms; TM, transmembrane; TMH, transmembrane helix; GPCR, G protein-coupled receptor; 3D, three dimensional; NMR, nuclear magnetic resonance spectroscopy; EPR, electron paramagnetic resonance spectroscopy; FTIR, Fourier transform infrared spectroscopy.

  11. Optimizing an emperical scoring function for transmembrane protein structure determination.

    SciTech Connect

    Young, Malin M.; Sale, Kenneth L.; Gray, Genetha Anne; Kolda, Tamara Gibson

    2003-10-01

    We examine the problem of transmembrane protein structure determination. Like many other questions that arise in biological research, this problem cannot be addressed by traditional laboratory experimentation alone. An approach that integrates experiment and computation is required. We investigate a procedure which states the transmembrane protein structure determination problem as a bound constrained optimization problem using a special empirical scoring function, called Bundler, as the objective function. In this paper, we describe the optimization problem and some of its mathematical properties. We compare and contrast results obtained using two different derivative free optimization algorithms.

  12. Overexpression of a mitochondrial ATP synthase small subunit gene (AtMtATP6) confers tolerance to several abiotic stresses in Saccharomyces cerevisiae and Arabidopsis thaliana.

    PubMed

    Zhang, Xinxin; Liu, Shenkui; Takano, Tetsuo

    2008-07-01

    Mitochondrial F(1)F(0)-ATPase is a key enzyme in plant metabolism, providing cells with ATP that uses the transmembrane electrochemical proton gradient to drive synthesis of ATP. A 6 kDa protein (At3g46430) has been previously purified from Arabidopsis thaliana mitochondrial F(1)F(0)-ATPase. In this study, the gene (AtMtATP6; GenBank accession no. AK117680) encoding this protein was isolated from Arabidopsis and characterized. Northern blot analyses showed that the expression of AtMtATP6 gene in Arabidopsis suspension-cultured cells was induced by several abiotic stresses from salts, drought, and cold. Over-expression of AtMtATP6 gene in transgenic yeast and Arabidopsis plants increased the resistance to salts, drought, oxidative and cold stresses. Taken together, our data raise the possibility that induction of the F(1)F(0)-ATPase plays a role in stress tolerance.

  13. Trichome morphogenesis in Arabidopsis.

    PubMed Central

    Schwab, B; Folkers, U; Ilgenfritz, H; Hülskamp, M

    2000-01-01

    Trichomes (plant hairs) in Arabidopsis thaliana are large non-secreting epidermal cells with a characteristic three-dimensional architecture. Because trichomes are easily accessible to a combination of genetic, cell biological and molecular methods they have become an ideal model system to study various aspects of plant cell morphogenesis. In this review we will summarize recent progress in the understanding of trichome morphogenesis. PMID:11128981

  14. Marginally hydrophobic transmembrane α-helices shaping membrane protein folding

    PubMed Central

    De Marothy, Minttu T; Elofsson, Arne

    2015-01-01

    Cells have developed an incredible machinery to facilitate the insertion of membrane proteins into the membrane. While we have a fairly good understanding of the mechanism and determinants of membrane integration, more data is needed to understand the insertion of membrane proteins with more complex insertion and folding pathways. This review will focus on marginally hydrophobic transmembrane helices and their influence on membrane protein folding. These weakly hydrophobic transmembrane segments are by themselves not recognized by the translocon and therefore rely on local sequence context for membrane integration. How can such segments reside within the membrane? We will discuss this in the light of features found in the protein itself as well as the environment it resides in. Several characteristics in proteins have been described to influence the insertion of marginally hydrophobic helices. Additionally, the influence of biological membranes is significant. To begin with, the actual cost for having polar groups within the membrane may not be as high as expected; the presence of proteins in the membrane as well as characteristics of some amino acids may enable a transmembrane helix to harbor a charged residue. The lipid environment has also been shown to directly influence the topology as well as membrane boundaries of transmembrane helices—implying a dynamic relationship between membrane proteins and their environment. PMID:25970811

  15. Computational approaches to detect allosteric pathways in transmembrane molecular machines.

    PubMed

    Stolzenberg, Sebastian; Michino, Mayako; LeVine, Michael V; Weinstein, Harel; Shi, Lei

    2016-07-01

    Many of the functions of transmembrane proteins involved in signal processing and transduction across the cell membrane are determined by allosteric couplings that propagate the functional effects well beyond the original site of activation. Data gathered from breakthroughs in biochemistry, crystallography, and single molecule fluorescence have established a rich basis of information for the study of molecular mechanisms in the allosteric couplings of such transmembrane proteins. The mechanistic details of these couplings, many of which have therapeutic implications, however, have only become accessible in synergy with molecular modeling and simulations. Here, we review some recent computational approaches that analyze allosteric coupling networks (ACNs) in transmembrane proteins, and in particular the recently developed Protein Interaction Analyzer (PIA) designed to study ACNs in the structural ensembles sampled by molecular dynamics simulations. The power of these computational approaches in interrogating the functional mechanisms of transmembrane proteins is illustrated with selected examples of recent experimental and computational studies pursued synergistically in the investigation of secondary active transporters and GPCRs. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

  16. Metabolomic analysis reveals the relationship between AZI1 and sugar signaling in systemic acquired resistance of Arabidopsis.

    PubMed

    Wang, Xiao-Yan; Li, Dian-Zhen; Li, Qi; Ma, Yan-Qin; Yao, Jing-Wen; Huang, Xuan; Xu, Zi-Qin

    2016-10-01

    The function of AZI1 in systemic acquired resistance of Arabidopsis was confirmed by investigation of the phenotypic features of wild-type Col-0, AZI1 T-DNA knockout and AZI1 overexpressing plants after infection with virulent and avirulent Pseudomonas syringae. Real-time quantitative PCR and Northern blotting analyses showed that the transcript abundances of PR genes increased significantly in local and systemic leaves of wild-type Col-0 and AZI1 overexpressing plants challenged with avirulent P. syringae, whereas the mRNA accumulation of PR genes was obviously attenuated in local and systemic leaves of AZI1 T-DNA knockout plants after localized infiltration with avirulent Psm avrRpm1. The changes of metabolomic profiles in distal leaves of three types of materials infected with avirulent P. syringae were determined by (1)H NMR spectrometry and data mining showed that the soluble carbonhydrates might function as signal substances in the systemic immunity of Arabidopsis. At the same time, the expression of the sugar signaling genes in local and distal leaves after infection of avirulent P. syringae was compared. As a result, it was found that the transcript abundances of sugar signaling genes, including SUS1, SUS2, SUS3, SUS6, SUT1, HXK1, HXK2, SNRK1.2, ERD6, TPS1, TOR, SNRK1.1, SNRK1.3 and bZIP11, were obviously changed in distal leaves of different materials with the modulated AZI1 activities, indicating sugar-related genes are involved in regulation of the systemic immunity mediated by AZI1. These results also illustrated that the immune system associated with sugar molecules probably was an important part of the systemic acquired resistance in Arabidopsis.

  17. Maize and Arabidopsis ARGOS Proteins Interact with Ethylene Receptor Signaling Complex, Supporting a Regulatory Role for ARGOS in Ethylene Signal Transduction.

    PubMed

    Shi, Jinrui; Drummond, Bruce J; Wang, Hongyu; Archibald, Rayeann L; Habben, Jeffrey E

    2016-08-01

    The phytohormone ethylene regulates plant growth and development as well as plant response to environmental cues. ARGOS genes reduce plant sensitivity to ethylene when overexpressed in transgenic Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). A previous genetic study suggested that the endoplasmic reticulum and Golgi-localized maize ARGOS1 targets the ethylene signal transduction components at or upstream of CONSTITUTIVE TRIPLE RESPONSE1, but the mechanism of ARGOS modulating ethylene signaling is unknown. Here, we demonstrate in Arabidopsis that ZmARGOS1, as well as the Arabidopsis ARGOS homolog ORGAN SIZE RELATED1, physically interacts with Arabidopsis REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1), an ethylene receptor interacting protein that regulates the activity of ETHYLENE RESPONSE1. The protein-protein interaction was also detected with the yeast split-ubiquitin two-hybrid system. Using the same yeast assay, we found that maize RTE1 homolog REVERSION-TO-ETHYLENE SENSITIVITY1 LIKE4 (ZmRTL4) and ZmRTL2 also interact with maize and Arabidopsis ARGOS proteins. Like AtRTE1 in Arabidopsis, ZmRTL4 and ZmRTL2 reduce ethylene responses when overexpressed in maize, indicating a similar mechanism for ARGOS regulating ethylene signaling in maize. A polypeptide fragment derived from ZmARGOS8, consisting of a Pro-rich motif flanked by two transmembrane helices that are conserved among members of the ARGOS family, can interact with AtRTE1 and maize RTL proteins in Arabidopsis. The conserved domain is necessary and sufficient to reduce ethylene sensitivity in Arabidopsis and maize. Overall, these results suggest a physical association between ARGOS and the ethylene receptor signaling complex via AtRTE1 and maize RTL proteins, supporting a role for ARGOS in regulating ethylene perception and the early steps of signal transduction in Arabidopsis and maize.

  18. Maize and Arabidopsis ARGOS Proteins Interact with Ethylene Receptor Signaling Complex, Supporting a Regulatory Role for ARGOS in Ethylene Signal Transduction[OPEN

    PubMed Central

    Shi, Jinrui; Wang, Hongyu; Habben, Jeffrey E.

    2016-01-01

    The phytohormone ethylene regulates plant growth and development as well as plant response to environmental cues. ARGOS genes reduce plant sensitivity to ethylene when overexpressed in transgenic Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). A previous genetic study suggested that the endoplasmic reticulum and Golgi-localized maize ARGOS1 targets the ethylene signal transduction components at or upstream of CONSTITUTIVE TRIPLE RESPONSE1, but the mechanism of ARGOS modulating ethylene signaling is unknown. Here, we demonstrate in Arabidopsis that ZmARGOS1, as well as the Arabidopsis ARGOS homolog ORGAN SIZE RELATED1, physically interacts with Arabidopsis REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1), an ethylene receptor interacting protein that regulates the activity of ETHYLENE RESPONSE1. The protein-protein interaction was also detected with the yeast split-ubiquitin two-hybrid system. Using the same yeast assay, we found that maize RTE1 homolog REVERSION-TO-ETHYLENE SENSITIVITY1 LIKE4 (ZmRTL4) and ZmRTL2 also interact with maize and Arabidopsis ARGOS proteins. Like AtRTE1 in Arabidopsis, ZmRTL4 and ZmRTL2 reduce ethylene responses when overexpressed in maize, indicating a similar mechanism for ARGOS regulating ethylene signaling in maize. A polypeptide fragment derived from ZmARGOS8, consisting of a Pro-rich motif flanked by two transmembrane helices that are conserved among members of the ARGOS family, can interact with AtRTE1 and maize RTL proteins in Arabidopsis. The conserved domain is necessary and sufficient to reduce ethylene sensitivity in Arabidopsis and maize. Overall, these results suggest a physical association between ARGOS and the ethylene receptor signaling complex via AtRTE1 and maize RTL proteins, supporting a role for ARGOS in regulating ethylene perception and the early steps of signal transduction in Arabidopsis and maize. PMID:27268962

  19. Influence of the valine zipper region on the structure and aggregation of the basic leucine zipper (bZIP) domain of activating transcription factor 5 (ATF5).

    PubMed

    Ciaccio, Natalie A; Reynolds, T Steele; Middaugh, C Russell; Laurence, Jennifer S

    2012-11-01

    Protein aggregation is a major problem for biopharmaceuticals. While the control of aggregation is critically important for the future of protein pharmaceuticals, mechanisms of aggregate assembly, particularly the role that structure plays, are still poorly understood. Increasing evidence indicates that partially folded intermediates critically influence the aggregation pathway. We have previously reported the use of the basic leucine zipper (bZIP) domain of activating transcription factor 5 (ATF5) as a partially folded model system to investigate protein aggregation. This domain contains three regions with differing structural propensity: a N-terminal polybasic region, a central helical leucine zipper region, and a C-terminal extended valine zipper region. Additionally, a centrally positioned cysteine residue readily forms an intermolecular disulfide bond that reduces aggregation. Computational analysis of ATF5 predicts that the valine zipper region facilitates self-association. Here we test this hypothesis using a truncated mutant lacking the C-terminal valine zipper region. We compare the structure and aggregation of this mutant to the wild-type (WT) form under both reducing and nonreducing conditions. Our data indicate that removal of this region results in a loss of α-helical structure in the leucine zipper and a change in the mechanism of self-association. The mutant form displays increased association at low temperature but improved resistance to thermally induced aggregation.

  20. Influence of the valine zipper region on the structure and aggregation of the basic leucine zipper (bZIP) domain of activating transcription factor 5 (ATF5).

    PubMed

    Ciaccio, Natalie A; Reynolds, T Steele; Middaugh, C Russell; Laurence, Jennifer S

    2012-11-01

    Protein aggregation is a major problem for biopharmaceuticals. While the control of aggregation is critically important for the future of protein pharmaceuticals, mechanisms of aggregate assembly, particularly the role that structure plays, are still poorly understood. Increasing evidence indicates that partially folded intermediates critically influence the aggregation pathway. We have previously reported the use of the basic leucine zipper (bZIP) domain of activating transcription factor 5 (ATF5) as a partially folded model system to investigate protein aggregation. This domain contains three regions with differing structural propensity: a N-terminal polybasic region, a central helical leucine zipper region, and a C-terminal extended valine zipper region. Additionally, a centrally positioned cysteine residue readily forms an intermolecular disulfide bond that reduces aggregation. Computational analysis of ATF5 predicts that the valine zipper region facilitates self-association. Here we test this hypothesis using a truncated mutant lacking the C-terminal valine zipper region. We compare the structure and aggregation of this mutant to the wild-type (WT) form under both reducing and nonreducing conditions. Our data indicate that removal of this region results in a loss of α-helical structure in the leucine zipper and a change in the mechanism of self-association. The mutant form displays increased association at low temperature but improved resistance to thermally induced aggregation. PMID:23067245

  1. Molecular cloning and characterization of a tomato cDNA encoding a systemically wound-inducible bZIP DNA-binding protein

    NASA Technical Reports Server (NTRS)

    Stankovic, B.; Vian, A.; Henry-Vian, C.; Davies, E.

    2000-01-01

    Localized wounding of one leaf in intact tomato (Lycopersicon esculentum Mill.) plants triggers rapid systemic transcriptional responses that might be involved in defense. To better understand the mechanism(s) of intercellular signal transmission in wounded tomatoes, and to identify the array of genes systemically up-regulated by wounding, a subtractive cDNA library for wounded tomato leaves was constructed. A novel cDNA clone (designated LebZIP1) encoding a DNA-binding protein was isolated and identified. This clone appears to be encoded by a single gene, and belongs to the family of basic leucine zipper domain (bZIP) transcription factors shown to be up-regulated by cold and dark treatments. Analysis of the mRNA levels suggests that the transcript for LebZIP1 is both organ-specific and up-regulated by wounding. In wounded wild-type tomatoes, the LebZIP1 mRNA levels in distant tissue were maximally up-regulated within only 5 min following localized wounding. Exogenous abscisic acid (ABA) prevented the rapid wound-induced increase in LebZIP1 mRNA levels, while the basal levels of LebZIP1 transcripts were higher in the ABA mutants notabilis (not), sitiens (sit), and flacca (flc), and wound-induced increases were greater in the ABA-deficient mutants. Together, these results suggest that ABA acts to curtail the wound-induced synthesis of LebZIP1 mRNA.

  2. Basic leucine zipper (bZIP) domain transcription factor MBZ1 regulates cell wall integrity, spore adherence, and virulence in Metarhizium robertsii.

    PubMed

    Huang, Wei; Shang, Yanfang; Chen, Peilin; Cen, Kai; Wang, Chengshu

    2015-03-27

    Transcription factors (TFs) containing the basic leucine zipper (bZIP) domain are widely distributed in eukaryotes and display an array of distinct functions. In this study, a bZIP-type TF gene (MBZ1) was deleted and functionally characterized in the insect pathogenic fungus Metarhizium robertsii. The deletion mutant (ΔMBZ1) showed defects in cell wall integrity, adhesion to hydrophobic surfaces, and topical infection of insects. Relative to the WT, ΔMBZ1 was also impaired in growth and conidiogenesis. Examination of putative target gene expression indicated that the genes involved in chitin biosynthesis were differentially transcribed in ΔMBZ1 compared with the WT, which led to the accumulation of a higher level of chitin in mutant cell walls. MBZ1 exhibited negative regulation of subtilisin proteases, but positive control of an adhesin gene, which is consistent with the observation of effects on cell autolysis and a reduction in spore adherence to hydrophobic surfaces in ΔMBZ1. Promoter binding assays indicated that MBZ1 can bind to different target genes and suggested the possibility of heterodimer formation to increase the diversity of the MBZ1 regulatory network. The results of this study advance our understanding of the divergence of bZIP-type TFs at both intra- and interspecific levels.

  3. Influence of the Valine Zipper Region on the Structure and Aggregation of the Basic Leucine Zipper (bZIP) Domain of Activating Transcription Factor 5 (ATF5)

    PubMed Central

    Ciaccio, Natalie A.; Reynolds, T. Steele; Middaugh, C. Russell; Laurence, Jennifer S.

    2012-01-01

    Protein aggregation is a major problem for biopharmaceuticals. While the control of aggregation is critically important for the future of protein pharmaceuticals, mechanisms of aggregate assembly, particularly the role that structure plays, are still poorly understood. Increasing evidence indicates that partially folded intermediates critically influence the aggregation pathway. We have previously reported the use of the basic leucine zipper (bZIP) domain of Activating Transcription Factor 5 (ATF5) as a partially folded model system to investigate protein aggregation. This domain contains three regions with differing structural propensity: a N-terminal polybasic region, a central helical leucine zipper region, and a C-terminal extended valine zipper region. Additionally, a centrally positioned cysteine residue readily forms an intermolecular disulfide bond that reduces aggregation. Computational analysis of ATF5 predicts that the valine zipper region facilitates self-association. Here we test this hypothesis using a truncated mutant lacking the C-terminal valine zipper region. We compare the structure and aggregation of this mutant to the wild-type (WT) form under both reducing and non-reducing conditions. Our data indicate that removal of this region results in a loss of alpha-helical structure in the leucine zipper and a change in the mechanism of self-association. The mutant form displays increased association at low temperature but improved resistance to thermally induced aggregation. PMID:23067245

  4. Basic leucine zipper (bZIP) domain transcription factor MBZ1 regulates cell wall integrity, spore adherence, and virulence in Metarhizium robertsii.

    PubMed

    Huang, Wei; Shang, Yanfang; Chen, Peilin; Cen, Kai; Wang, Chengshu

    2015-03-27

    Transcription factors (TFs) containing the basic leucine zipper (bZIP) domain are widely distributed in eukaryotes and display an array of distinct functions. In this study, a bZIP-type TF gene (MBZ1) was deleted and functionally characterized in the insect pathogenic fungus Metarhizium robertsii. The deletion mutant (ΔMBZ1) showed defects in cell wall integrity, adhesion to hydrophobic surfaces, and topical infection of insects. Relative to the WT, ΔMBZ1 was also impaired in growth and conidiogenesis. Examination of putative target gene expression indicated that the genes involved in chitin biosynthesis were differentially transcribed in ΔMBZ1 compared with the WT, which led to the accumulation of a higher level of chitin in mutant cell walls. MBZ1 exhibited negative regulation of subtilisin proteases, but positive control of an adhesin gene, which is consistent with the observation of effects on cell autolysis and a reduction in spore adherence to hydrophobic surfaces in ΔMBZ1. Promoter binding assays indicated that MBZ1 can bind to different target genes and suggested the possibility of heterodimer formation to increase the diversity of the MBZ1 regulatory network. The results of this study advance our understanding of the divergence of bZIP-type TFs at both intra- and interspecific levels. PMID:25673695

  5. In vivo binding of hot pepper bZIP transcription factor CabZIP1 to the G-box region of pathogenesis-related protein 1 promoter

    SciTech Connect

    Lee, Boo-Ja; Park, Chang-Jin; Kim, Sung-Kyu; Kim, Ki-Jeong; Paek, Kyung-Hee . E-mail: khpaek95@korea.ac.kr

    2006-05-26

    We find that salicylic acid and ethephon treatment in hot pepper increases the expression of a putative basic/leucine zipper (bZIP) transcription factor gene, CabZIP1. CabZIP1 mRNA is expressed ubiquitously in various organs. The green fluorescent protein-fused transcription factor, CabZIP1::GFP, can be specifically localized to the nucleus, an action that is consistent with the presence of a nuclear localization signal in its protein sequence. Transient overexpression of the CabZIP1 transcription factor results in an increase in PR-1 transcripts level in Nicotiana benthamiana leaves. Using chromatin immunoprecipitation, we demonstrate that CabZIP1 binds to the G-box elements in native promoter of the hot pepper pathogenesis-related protein 1 (CaPR-1) gene in vivo. Taken together, our results suggest that CabZIP1 plays a role as a transcriptional regulator of the CaPR-1 gene.

  6. Biological insertion of computationally designed short transmembrane segments.

    PubMed

    Baeza-Delgado, Carlos; von Heijne, Gunnar; Marti-Renom, Marc A; Mingarro, Ismael

    2016-01-01

    The great majority of helical membrane proteins are inserted co-translationally into the ER membrane through a continuous ribosome-translocon channel. The efficiency of membrane insertion depends on transmembrane (TM) helix amino acid composition, the helix length and the position of the amino acids within the helix. In this work, we conducted a computational analysis of the composition and location of amino acids in transmembrane helices found in membrane proteins of known structure to obtain an extensive set of designed polypeptide segments with naturally occurring amino acid distributions. Then, using an in vitro translation system in the presence of biological membranes, we experimentally validated our predictions by analyzing its membrane integration capacity. Coupled with known strategies to control membrane protein topology, these findings may pave the way to de novo membrane protein design. PMID:26987712

  7. Biological insertion of computationally designed short transmembrane segments

    PubMed Central

    Baeza-Delgado, Carlos; von Heijne, Gunnar; Marti-Renom, Marc A.; Mingarro, Ismael

    2016-01-01

    The great majority of helical membrane proteins are inserted co-translationally into the ER membrane through a continuous ribosome-translocon channel. The efficiency of membrane insertion depends on transmembrane (TM) helix amino acid composition, the helix length and the position of the amino acids within the helix. In this work, we conducted a computational analysis of the composition and location of amino acids in transmembrane helices found in membrane proteins of known structure to obtain an extensive set of designed polypeptide segments with naturally occurring amino acid distributions. Then, using an in vitro translation system in the presence of biological membranes, we experimentally validated our predictions by analyzing its membrane integration capacity. Coupled with known strategies to control membrane protein topology, these findings may pave the way to de novo membrane protein design. PMID:26987712

  8. Quantification of structural distortions in the transmembrane helices of GPCRs.

    PubMed

    Deupi, Xavier

    2012-01-01

    A substantial part of the structural and much of the functional information about G protein-coupled receptors (GPCRs) comes from studies on rhodopsin. Thus, analysis tools for detailed structure comparison are key to see to what extent this information can be extended to other GPCRs. Among the methods to evaluate protein structures and, in particular, helix distortions, HELANAL has the advantage that it provides data (local bend and twist angles) that can be easily translated to structural effects, as a local opening/tightening of the helix.In this work I show how HELANAL can be used to extract detailed structural information of the transmembrane bundle of GPCRs, and I provide some examples on how these data can be interpreted to study basic principles of protein structure, to compare homologous proteins and to study mechanisms of receptor activation. Also, I show how in combination with the sequence analysis tools provided by the program GMoS, distortions in individual receptors can be put in the context of the whole Class A GPCR family. Specifically, quantification of the strong proline-induced distortions in the transmembrane bundle of rhodopsin shows that they are not standard proline kinks. Moreover, the helix distortions in transmembrane helix (TMH) 5 and TMH 6 of rhodopsin are also present in the rest of GPCR crystal structures obtained so far, and thus, rhodopsin-based homology models have modeled correctly these strongly distorted helices. While in some cases the inherent "rhodopsin bias" of many of the GPCR models to date has not been a disadvantage, the availability of more templates will clearly result in better homology models. This type of analysis can be, of course, applied to any protein, and it may be particularly useful for the structural analysis of other membrane proteins. A detailed knowledge of the local structural changes related to ligand binding and how they are translated into larger-scale movements of transmembrane domains is key to

  9. Transmembrane current imaging in the heart during pacing and fibrillation.

    PubMed

    Gray, Richard A; Mashburn, David N; Sidorov, Veniamin Y; Roth, Bradley J; Pathmanathan, Pras; Wikswo, John P

    2013-10-01

    Recently, we described a method to quantify the time course of total transmembrane current (Im) and the relative role of its two components, a capacitive current (Ic) and a resistive current (Iion), corresponding to the cardiac action potential during stable propagation. That approach involved recording high-fidelity (200 kHz) transmembrane potential (Vm) signals with glass microelectrodes at one site using a spatiotemporal coordinate transformation via measured conduction velocity. Here we extend our method to compute these transmembrane currents during stable and unstable propagation from fluorescence signals of Vm at thousands of sites (3 kHz), thereby introducing transmembrane current imaging. In contrast to commonly used linear Laplacians of extracellular potential (Ve) to compute Im, we utilized nonlinear image processing to compute the required second spatial derivatives of Vm. We quantified the dynamic spatial patterns of current density of Im and Iion for both depolarization and repolarization during pacing (including nonplanar patterns) by calibrating data with the microelectrode signals. Compared to planar propagation, we found that the magnitude of Iion was significantly reduced at sites of wave collision during depolarization but not repolarization. Finally, we present uncalibrated dynamic patterns of Im during ventricular fibrillation and show that Im at singularity sites was monophasic and positive with a significant nonzero charge (Im integrated over 10 ms) in contrast with nonsingularity sites. Our approach should greatly enhance the understanding of the relative roles of functional (e.g., rate-dependent membrane dynamics and propagation patterns) and static spatial heterogeneities (e.g., spatial differences in tissue resistance) via recordings during normal and compromised propagation, including arrhythmias. PMID:24094412

  10. Transmembrane Current Imaging in the Heart during Pacing and Fibrillation

    PubMed Central

    Gray, Richard A.; Mashburn, David N.; Sidorov, Veniamin Y.; Roth, Bradley J.; Pathmanathan, Pras; Wikswo, John P.

    2013-01-01

    Recently, we described a method to quantify the time course of total transmembrane current (Im) and the relative role of its two components, a capacitive current (Ic) and a resistive current (Iion), corresponding to the cardiac action potential during stable propagation. That approach involved recording high-fidelity (200 kHz) transmembrane potential (Vm) signals with glass microelectrodes at one site using a spatiotemporal coordinate transformation via measured conduction velocity. Here we extend our method to compute these transmembrane currents during stable and unstable propagation from fluorescence signals of Vm at thousands of sites (3 kHz), thereby introducing transmembrane current imaging. In contrast to commonly used linear Laplacians of extracellular potential (Ve) to compute Im, we utilized nonlinear image processing to compute the required second spatial derivatives of Vm. We quantified the dynamic spatial patterns of current density of Im and Iion for both depolarization and repolarization during pacing (including nonplanar patterns) by calibrating data with the microelectrode signals. Compared to planar propagation, we found that the magnitude of Iion was significantly reduced at sites of wave collision during depolarization but not repolarization. Finally, we present uncalibrated dynamic patterns of Im during ventricular fibrillation and show that Im at singularity sites was monophasic and positive with a significant nonzero charge (Im integrated over 10 ms) in contrast with nonsingularity sites. Our approach should greatly enhance the understanding of the relative roles of functional (e.g., rate-dependent membrane dynamics and propagation patterns) and static spatial heterogeneities (e.g., spatial differences in tissue resistance) via recordings during normal and compromised propagation, including arrhythmias. PMID:24094412

  11. CREST - a large and diverse superfamily of putative transmembrane hydrolases

    PubMed Central

    2011-01-01

    Background A number of membrane-spanning proteins possess enzymatic activity and catalyze important reactions involving proteins, lipids or other substrates located within or near lipid bilayers. Alkaline ceramidases are seven-transmembrane proteins that hydrolyze the amide bond in ceramide to form sphingosine. Recently, a group of putative transmembrane receptors called progestin and adipoQ receptors (PAQRs) were found to be distantly related to alkaline ceramidases, raising the possibility that they may also function as membrane enzymes. Results Using sensitive similarity search methods, we identified statistically significant sequence similarities among several transmembrane protein families including alkaline ceramidases and PAQRs. They were unified into a large and diverse superfamily of putative membrane-bound hydrolases called CREST (alkaline ceramidase, PAQR receptor, Per1, SID-1 and TMEM8). The CREST superfamily embraces a plethora of cellular functions and biochemical activities, including putative lipid-modifying enzymes such as ceramidases and the Per1 family of putative phospholipases involved in lipid remodeling of GPI-anchored proteins, putative hormone receptors, bacterial hemolysins, the TMEM8 family of putative tumor suppressors, and the SID-1 family of putative double-stranded RNA transporters involved in RNA interference. Extensive similarity searches and clustering analysis also revealed several groups of proteins with unknown function in the CREST superfamily. Members of the CREST superfamily share seven predicted core transmembrane segments with several conserved sequence motifs. Conclusions Universal conservation of a set of histidine and aspartate residues across all groups in the CREST superfamily, coupled with independent discoveries of hydrolase activities in alkaline ceramidases and the Per1 family as well as results from previous mutational studies of Per1, suggests that the majority of CREST members are metal-dependent hydrolases

  12. Grafting PNIPAAm from β-barrel shaped transmembrane nanopores.

    PubMed

    Charan, Himanshu; Kinzel, Julia; Glebe, Ulrich; Anand, Deepak; Garakani, Tayebeh Mirzaei; Zhu, Leilei; Bocola, Marco; Schwaneberg, Ulrich; Böker, Alexander

    2016-11-01

    The research on protein-polymer conjugates by grafting from the surface of proteins has gained significant interest in the last decade. While there are many studies with globular proteins, membrane proteins have remained untouched to the best of our knowledge. In this study, we established the conjugate formation with a class of transmembrane proteins and grow polymer chains from the ferric hydroxamate uptake protein component A (FhuA; a β-barrel transmembrane protein of Escherichia coli). As the lysine residues of naturally occurring FhuA are distributed over the whole protein, FhuA was reengineered to have up to 11 lysines, distributed symmetrically in a rim on the membrane exposed side (outside) of the protein channel and exclusively above the hydrophobic region. Reengineering of FhuA ensures a polymer growth only on the outside of the β-barrel and prevents blockage of the channel as a result of the polymerization. A water-soluble initiator for controlled radical polymerization (CRP) was consecutively linked to the lysine residues of FhuA and N-isopropylacrylamide (NIPAAm) polymerized under copper-mediated CRP conditions. The conjugate formation was analyzed by using MALDI-ToF mass spectrometry, SDS-PAGE, circular dichroism spectroscopy, analytical ultracentrifugation, dynamic light scattering, transmission electron microscopy and size exclusion chromatography. Such conjugates combine the specific functions of the transmembrane proteins, like maintaining membrane potential gradients or translocation of substrates with the unique properties of synthetic polymers such as temperature and pH stimuli handles. FhuA-PNIPAAm conjugates will serve as functional nanosized building blocks for applications in targeted drug delivery, self-assembly systems, functional membranes and transmembrane protein gated nanoreactors. PMID:27614163

  13. Transmembrane chloride flux in tissue-cultured chick heart cells

    SciTech Connect

    Piwnica-Worms, D.; Jacob, R.; Horres, C.R.; Lieberman, M.

    1983-05-01

    To evaluate the transmembrane movement of chloride in a preparation of cardiac muscle lacking the extracellular diffusion limitations of natural specimens, intracellular chloride concentration ( (Cl) i) and transmembrane /sup 36/Cl efflux have been determined in growth-oriented embryonic chick heart cells in tissue culture. Using the method of isotopic equilibrium, (Cl)i was 25.1 +/- 7.3 mmol x (liter cell water)-1, comparable to the value of 24.9 +/- 5.4 mmol x (liter cell water)-1 determined by coulometric titration. Two cellular /sup 36/Cl compartments were found; one exchanged with a rate constant of 0.67 +/- 0.12 min-1 and was associated with the cardiac muscle cells; the other, attributed to the fibroblasts, exchanged with a rate constant of 0.18 +/- 0.05 min-1. At 37 degrees C, transmembrane Cl flux of cardiac muscle under steady-state conditions was 30 pmol x cm-2 x s-1. In K-free, normal, or high-Ko solutions, the responses of the membrane potential to changes in external Cl concentration suggested that chloride conductance was low. These results indicate that Cl transport across the myocardial cell membrane is more rapid than K transport and is largely electrically silent.

  14. Transmembrane Helix Assembly by Max-Min Ant System Algorithm.

    PubMed

    Sujaree, Kanon; Kitjaruwankul, Sunan; Boonamnaj, Panisak; Supunyabut, Chirayut; Sompornpisut, Pornthep

    2015-12-01

    Because of the rapid progress in biochemical and structural studies of membrane proteins, considerable attention has been given on developing efficient computational methods for solving low-to-medium resolution structures using sparse structural data. In this study, we demonstrate a novel algorithm, max-min ant system (MMAS), designed to find an assembly of α-helical transmembrane proteins using a rigid helix arrangement guided by distance constraints. The new algorithm generates a large variety with finite number of orientations of transmembrane helix bundle and finds the solution that is matched with the provided distance constraints based on the behavior of ants to search for the shortest possible path between their nest and the food source. To demonstrate the efficiency of the novel search algorithm, MMAS is applied to determine the transmembrane packing of KcsA and MscL ion channels from a limited distance information extracted from the crystal structures, and the packing of KvAP voltage sensor domain using a set of 10 experimentally determined constraints, and the results are compared with those of two popular used stochastic methods, simulated annealing Monte Carlo method and genetic algorithm. PMID:26058409

  15. Probing transmembrane mechanical coupling and cytomechanics using magnetic twisting cytometry

    NASA Technical Reports Server (NTRS)

    Wang, N.; Ingber, D. E.

    1995-01-01

    We recently developed a magnetic twisting cytometry technique that allows us to apply controlled mechanical stresses to specific cell surface receptors using ligand-coated ferromagnetic microbeads and to simultaneously measure the mechanical response in living cells. Using this technique, we have previously shown the following: (i) beta 1 integrin receptors mediate mechanical force transfer across the cell surface and to the cytoskeleton, whereas other transmembrane receptors (e.g., scavenger receptors) do not; (ii) cytoskeletal stiffness increases in direct proportion to the level of stress applied to integrins; and (iii) the slope of this linear stiffening response differs depending on the shape of the cell. We now show that different integrins (beta 1, alpha V beta 3, alpha V, alpha 5, alpha 2) and other transmembrane receptors (scavenger receptor, platelet endothelial cell adhesion molecule) differ in their ability to mediate force transfer across the cell surface. In addition, the linear stiffening behavior previously observed in endothelial cells was found to be shared by other cell types. Finally, we demonstrate that dynamic changes in cell shape that occur during both cell spreading and retraction are accompanied by coordinate changes in cytoskeletal stiffness. Taken together, these results suggest that the magnetic twisting cytometry technique may be a powerful and versatile tool for studies analyzing the molecular basis of transmembrane mechanical coupling to the cytoskeleton as well as dynamic relations between changes in cytoskeletal structure and alterations in cell form and function.

  16. Transcriptome analysis reveals transmembrane targets on transplantable midbrain dopamine progenitors.

    PubMed

    Bye, Chris R; Jönsson, Marie E; Björklund, Anders; Parish, Clare L; Thompson, Lachlan H

    2015-04-14

    An important challenge for the continued development of cell therapy for Parkinson's disease (PD) is the establishment of procedures that better standardize cell preparations for use in transplantation. Although cell sorting has been an anticipated strategy, its application has been limited by lack of knowledge regarding transmembrane proteins that can be used to target and isolate progenitors for midbrain dopamine (mDA) neurons. We used a "FACS-array" approach to identify 18 genes for transmembrane proteins with high expression in mDA progenitors and describe the utility of four of these targets (Alcam, Chl1, Gfra1, and Igsf8) for isolating mDA progenitors from rat primary ventral mesencephalon through flow cytometry. Alcam and Chl1 facilitated a significant enrichment of mDA neurons following transplantation, while targeting of Gfra1 allowed for robust separation of dopamine and serotonin neurons. Importantly, we also show that mDA progenitors isolated on the basis of transmembrane proteins are capable of extensive, functional innervation of the host striatum and correction of motor impairment in a unilateral model of PD. These results are highly relevant for current efforts to establish safe and effective stem cell-based procedures for PD, where clinical translation will almost certainly require safety and standardization measures in order to deliver well-characterized cell preparations.

  17. Basic amino-acid side chains regulate transmembrane integrin signalling.

    PubMed

    Kim, Chungho; Schmidt, Thomas; Cho, Eun-Gyung; Ye, Feng; Ulmer, Tobias S; Ginsberg, Mark H

    2011-12-18

    Side chains of Lys/Arg near transmembrane domain (TMD) membrane-water interfaces can 'snorkel', placing their positive charge near negatively charged phospholipid head groups; however, snorkelling's functional effects are obscure. Integrin β TMDs have such conserved basic amino acids. Here we use NMR spectroscopy to show that integrin β(3)(Lys 716) helps determine β(3) TMD topography. The α(ΙΙb)β(3) TMD structure indicates that precise β(3) TMD crossing angles enable the assembly of outer and inner membrane 'clasps' that hold the αβ TMD together to limit transmembrane signalling. Mutation of β(3)(Lys 716) caused dissociation of α(ΙΙb)β(3) TMDs and integrin activation. To confirm that altered topography of β(3)(Lys 716) mutants activated α(ΙΙb)β(3), we used directed evolution of β(3)(K716A) to identify substitutions restoring default state. Introduction of Pro(711) at the midpoint of β(3) TMD (A711P) increased α(ΙΙb)β(3) TMD association and inactivated integrin α(ΙΙb)β(3)(A711P,K716A). β(3)(Pro 711) introduced a TMD kink of 30 ± 1° precisely at the border of the outer and inner membrane clasps, thereby decoupling the tilt between these segments. Thus, widely occurring snorkelling residues in TMDs can help maintain TMD topography and membrane-embedding, thereby regulating transmembrane signalling.

  18. Molecular genetics of root gravitropism and waving in Arabidopsis thaliana

    NASA Technical Reports Server (NTRS)

    Sedbrook, J.; Boonsirichai, K.; Chen, R.; Hilson, P.; Pearlman, R.; Rosen, E.; Rutherford, R.; Batiza, A.; Carroll, K.; Schulz, T.; Masson, P. H.

    1998-01-01

    When Arabidopsis thaliana seedlings grow embedded in an agar-based medium, their roots grow vertically downward. This reflects their ability to sense the gravity vector and to position their tip parallel to it (gravitropism). We have isolated a number of mutations affecting root gravitropism in Arabidopsis thaliana. One of these mutations, named arg1, affects root and hypocotyl gravitropism without promoting defects in starch content or in the ability of seedlings' organs to respond to plant hormones. The ARG1 gene was cloned and shown to code for a protein with a J domain at its amino terminus and a second sequence motif found in several cytoskeleton binding proteins. Mutations in the AGR1 locus promote a strong defect in root gravitropism. Some alleles also confer an increased root resistance to exogenous ethylene and an increased sensitivity to auxin. AGR1 was cloned and found to encode a putative transmembrane protein which might be involved in polar auxin transport, or in regulating the differential growth response to gravistimulation. When Arabidopsis seedlings grow on the surface of agar-based media tilted backward, their roots wave. That wavy pattern of root growth derives from a combined response to gravity, touch and other surface-derived stimuli. It is accompanied by a reversible rotation of the root tip about its axis. A number of mutations affect the presence or the shape of root waves on tilted agar-based surfaces. One of them, wvc1, promotes the formation of compressed root waves under these conditions. The physiological and molecular analyses of this mutant suggest that a tryptophan-derived molecule other than IAA might be an important regulator of the curvature responsible for root waving.

  19. Molecular genetics of root gravitropism and waving in Arabidopsis thaliana.

    PubMed

    Sedbrook, J; Boonsirichai, K; Chen, R; Hilson, P; Pearlman, R; Rosen, E; Rutherford, R; Batiza, A; Carroll, K; Schulz, T; Masson, P H

    1998-05-01

    When Arabidopsis thaliana seedlings grow embedded in an agar-based medium, their roots grow vertically downward. This reflects their ability to sense the gravity vector and to position their tip parallel to it (gravitropism). We have isolated a number of mutations affecting root gravitropism in Arabidopsis thaliana. One of these mutations, named arg1, affects root and hypocotyl gravitropism without promoting defects in starch content or in the ability of seedlings' organs to respond to plant hormones. The ARG1 gene was cloned and shown to code for a protein with a J domain at its amino terminus and a second sequence motif found in several cytoskeleton binding proteins. Mutations in the AGR1 locus promote a strong defect in root gravitropism. Some alleles also confer an increased root resistance to exogenous ethylene and an increased sensitivity to auxin. AGR1 was cloned and found to encode a putative transmembrane protein which might be involved in polar auxin transport, or in regulating the differential growth response to gravistimulation. When Arabidopsis seedlings grow on the surface of agar-based media tilted backward, their roots wave. That wavy pattern of root growth derives from a combined response to gravity, touch and other surface-derived stimuli. It is accompanied by a reversible rotation of the root tip about its axis. A number of mutations affect the presence or the shape of root waves on tilted agar-based surfaces. One of them, wvc1, promotes the formation of compressed root waves under these conditions. The physiological and molecular analyses of this mutant suggest that a tryptophan-derived molecule other than IAA might be an important regulator of the curvature responsible for root waving.

  20. Transgenic Arabidopsis Gene Expression System

    NASA Technical Reports Server (NTRS)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  1. Interferon-γ Promotes Inflammation and Development of T-Cell Lymphoma in HTLV-1 bZIP Factor Transgenic Mice

    PubMed Central

    Mitagami, Yu; Yasunaga, Jun-ichirou; Kinosada, Haruka; Ohshima, Koichi; Matsuoka, Masao

    2015-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) is an etiological agent of several inflammatory diseases and a T-cell malignancy, adult T-cell leukemia (ATL). HTLV-1 bZIP factor (HBZ) is the only viral gene that is constitutively expressed in HTLV-1-infected cells, and it has multiple functions on T-cell signaling pathways. HBZ has important roles in HTLV-1-mediated pathogenesis, since HBZ transgenic (HBZ-Tg) mice develop systemic inflammation and T-cell lymphomas, which are similar phenotypes to HTLV-1-associated diseases. We showed previously that in HBZ-Tg mice, HBZ causes unstable Foxp3 expression, leading to an increase in regulatory T cells (Tregs) and the consequent induction of IFN-γ-producing cells, which in turn leads to the development of inflammation in the mice. In this study, we show that the severity of inflammation is correlated with the development of lymphomas in HBZ-Tg mice, suggesting that HBZ-mediated inflammation is closely linked to oncogenesis in CD4+ T cells. In addition, we found that IFN-γ-producing cells enhance HBZ-mediated inflammation, since knocking out IFN-γ significantly reduced the incidence of dermatitis as well as lymphoma. Recent studies show the critical roles of the intestinal microbiota in the development of Tregs in vivo. We found that even germ-free HBZ-Tg mice still had an increased number of Tregs and IFN-γ-producing cells, and developed dermatitis, indicating that an intrinsic activity of HBZ evokes aberrant T-cell differentiation and consequently causes inflammation. These results show that immunomodulation by HBZ is implicated in both inflammation and oncogenesis, and suggest a causal connection between HTLV-1-associated inflammation and ATL. PMID:26296091

  2. Overexpression of the bZIP transcription factor OsbZIP79 suppresses the production of diterpenoid phytoalexin in rice cells.

    PubMed

    Miyamoto, Koji; Nishizawa, Yoko; Minami, Eiichi; Nojiri, Hideaki; Yamane, Hisakazu; Okada, Kazunori

    2015-01-15

    Phytoalexins are antimicrobial specialised metabolites that are produced by plants in response to pathogen attack. Momilactones and phytocassanes are major diterpenoid phytoalexins in rice that are synthesised from geranylgeranyl diphosphate that is derived from the methylerythritol phosphate (MEP) pathway. We have previously reported that rice cells overexpressing the basic leucine zipper (bZIP) transcription factor OsTGAP1 exhibit a hyperaccumulation of momilactones and phytocassanes, with hyperinductive expression of momilactone and phytocassane biosynthetic genes and MEP pathway genes, upon response to a chitin oligosaccharide elicitor. For a better understanding of OsTGAP1-mediated regulation of diterpenoid phytoalexin production, we identified OsTGAP1-interacting proteins using yeast two-hybrid screening. Among the OsTGAP1-interacting protein candidates, a TGA factor OsbZIP79 was investigated to verify its physical interaction with OsTGAP1 and involvement in the regulation of phytoalexin production. An in vitro pull-down assay demonstrated that OsTGAP1 and OsbZIP79 exhibited a heterodimeric as well as a homodimeric interaction. A bimolecular fluorescence complementation analysis also showed the interaction between OsTGAP1 and OsbZIP79 in vivo. Intriguingly, whereas OsbZIP79 transactivation activity was observed in a transient reporter assay, the overexpression of OsbZIP79 resulted in suppression of the elicitor-inducible expression of diterpenoid phytoalexin biosynthetic genes, and thus caused a decrease in the accumulation of phytoalexin in rice cells. These results suggest that OsbZIP79 functions as a negative regulator of phytoalexin production triggered by a chitin oligosaccharide elicitor in rice cells, although it remains open under which conditions OsbZIP79 can work with OsTGAP1.

  3. Molecular Insights into the Transmembrane Domain of the Thyrotropin Receptor

    PubMed Central

    Chantreau, Vanessa; Taddese, Bruck; Munier, Mathilde; Gourdin, Louis; Henrion, Daniel; Rodien, Patrice; Chabbert, Marie

    2015-01-01

    The thyrotropin receptor (TSHR) is a G protein-coupled receptor (GPCR) that is member of the leucine-rich repeat subfamily (LGR). In the absence of crystal structure, the success of rational design of ligands targeting the receptor internal cavity depends on the quality of the TSHR models built. In this subfamily, transmembrane helices (TM) 2 and 5 are characterized by the absence of proline compared to most receptors, raising the question of the structural conformation of these helices. To gain insight into the structural properties of these helices, we carried out bioinformatics and experimental studies. Evolutionary analysis of the LGR family revealed a deletion in TM5 but provided no information on TM2. Wild type residues at positions 2.58, 2.59 or 2.60 in TM2 and/or at position 5.50 in TM5 were substituted to proline. Depending on the position of the proline substitution, different effects were observed on membrane expression, glycosylation, constitutive cAMP activity and responses to thyrotropin. Only proline substitution at position 2.59 maintained complex glycosylation and high membrane expression, supporting occurrence of a bulged TM2. The TSHR transmembrane domain was modeled by homology with the orexin 2 receptor, using a protocol that forced the deletion of one residue in the TM5 bulge of the template. The stability of the model was assessed by molecular dynamics simulations. TM5 straightened during the equilibration phase and was stable for the remainder of the simulations. Our data support a structural model of the TSHR transmembrane domain with a bulged TM2 and a straight TM5 that is specific of glycoprotein hormone receptors. PMID:26545118

  4. Molecular Insights into the Transmembrane Domain of the Thyrotropin Receptor.

    PubMed

    Chantreau, Vanessa; Taddese, Bruck; Munier, Mathilde; Gourdin, Louis; Henrion, Daniel; Rodien, Patrice; Chabbert, Marie

    2015-01-01

    The thyrotropin receptor (TSHR) is a G protein-coupled receptor (GPCR) that is member of the leucine-rich repeat subfamily (LGR). In the absence of crystal structure, the success of rational design of ligands targeting the receptor internal cavity depends on the quality of the TSHR models built. In this subfamily, transmembrane helices (TM) 2 and 5 are characterized by the absence of proline compared to most receptors, raising the question of the structural conformation of these helices. To gain insight into the structural properties of these helices, we carried out bioinformatics and experimental studies. Evolutionary analysis of the LGR family revealed a deletion in TM5 but provided no information on TM2. Wild type residues at positions 2.58, 2.59 or 2.60 in TM2 and/or at position 5.50 in TM5 were substituted to proline. Depending on the position of the proline substitution, different effects were observed on membrane expression, glycosylation, constitutive cAMP activity and responses to thyrotropin. Only proline substitution at position 2.59 maintained complex glycosylation and high membrane expression, supporting occurrence of a bulged TM2. The TSHR transmembrane domain was modeled by homology with the orexin 2 receptor, using a protocol that forced the deletion of one residue in the TM5 bulge of the template. The stability of the model was assessed by molecular dynamics simulations. TM5 straightened during the equilibration phase and was stable for the remainder of the simulations. Our data support a structural model of the TSHR transmembrane domain with a bulged TM2 and a straight TM5 that is specific of glycoprotein hormone receptors. PMID:26545118

  5. Transmembrane peptides influence the affinity of sterols for phospholipid bilayers.

    PubMed

    Nyström, Joel H; Lönnfors, Max; Nyholm, Thomas K M

    2010-07-21

    Cholesterol is distributed unevenly between different cellular membrane compartments, and the cholesterol content increases from the inner bilayers toward the plasma membrane. It has been suggested that this cholesterol gradient is important in the sorting of transmembrane proteins. Cholesterol has also been to shown play an important role in lateral organization of eukaryotic cell membranes. In this study the aim was to determine how transmembrane proteins influence the lateral distribution of cholesterol in phospholipid bilayers. Insight into this can be obtained by studying how cholesterol interacts with bilayer membranes of different composition in the presence of designed peptides that mimic the transmembrane helices of proteins. For this purpose we developed an assay in which the partitioning of the fluorescent cholesterol analog CTL between LUVs and mbetaCD can be measured. Comparison of how cholesterol and CTL partitioning between mbetaCD and phospholipid bilayers with different composition suggests that CTL sensed changes in bilayer composition similarly as cholesterol. Therefore, the results obtained with CTL can be used to understand cholesterol distribution in lipid bilayers. The effect of WALP23 on CTL partitioning between DMPC bilayers and mbetaCD was measured. From the results it was clear that WALP23 increased both the order in the bilayers (as seen from CTL and DPH anisotropy) and the affinity of the sterol for the bilayer in a concentration dependent way. Although WALP23 also increased the order in DLPC and POPC bilayers the effects on CTL partitioning was much smaller with these lipids. This indicates that proteins have the largest effect on sterol interactions with phospholipids that have longer and saturated acyl chains. KALP23 did not significantly affect the acyl chain order in the phospholipid bilayers, and inclusion of KALP23 into DMPC bilayers slightly decreased CTL partitioning into the bilayer. This shows that transmembrane proteins can

  6. Cystic fibrosis transmembrane conductance regulator (ABCC7) structure.

    PubMed

    Hunt, John F; Wang, Chi; Ford, Robert C

    2013-02-01

    Structural studies of the cystic fibrosis transmembrane conductance regulator (CFTR) are reviewed. Like many membrane proteins, full-length CFTR has proven to be difficult to express and purify, hence much of the structural data available is for the more tractable, independently expressed soluble domains. Therefore, this chapter covers structural data for individual CFTR domains in addition to the sparser data available for the full-length protein. To set the context for these studies, we will start by reviewing structural information on model proteins from the ATP-binding cassette (ABC) transporter superfamily, to which CFTR belongs.

  7. Cystic Fibrosis Transmembrane Conductance Regulator (ABCC7) Structure

    PubMed Central

    Hunt, John F.; Wang, Chi; Ford, Robert C.

    2013-01-01

    Structural studies of the cystic fibrosis transmembrane conductance regulator (CFTR) are reviewed. Like many membrane proteins, full-length CFTR has proven to be difficult to express and purify, hence much of the structural data available is for the more tractable, independently expressed soluble domains. Therefore, this chapter covers structural data for individual CFTR domains in addition to the sparser data available for the full-length protein. To set the context for these studies, we will start by reviewing structural information on model proteins from the ATP-binding cassette (ABC) transporter superfamily, to which CFTR belongs. PMID:23378596

  8. NUCLEAR FACTOR Y, Subunit C (NF-YC) Transcription Factors Are Positive Regulators of Photomorphogenesis in Arabidopsis thaliana

    PubMed Central

    Siriwardana, Chamindika L.; Holt III, Ben F.

    2016-01-01

    Recent reports suggest that NF-Y transcription factors are positive regulators of skotomorphogenesis in Arabidopsis thaliana. Three NF-YC genes (NF-YC3, NF-YC4, and NF-YC9) are known to have overlapping functions in photoperiod dependent flowering and previous studies demonstrated that they interact with basic leucine zipper (bZIP) transcription factors. This included ELONGATED HYPOCOTYL 5 (HY5), which has well-demonstrated roles in photomorphogenesis. Similar to hy5 mutants, we report that nf-yc3 nf-yc4 nf-yc9 triple mutants failed to inhibit hypocotyl elongation in all tested light wavelengths. Surprisingly, nf-yc3 nf-yc4 nf-yc9 hy5 mutants had synergistic defects in light perception, suggesting that NF-Ys represent a parallel light signaling pathway. As with other photomorphogenic transcription factors, nf-yc3 nf-yc4 nf-yc9 triple mutants also partially suppressed the short hypocotyl and dwarf rosette phenotypes of CONSTITUTIVE PHOTOMORPHOGENIC 1 (cop1) mutants. Thus, our data strongly suggest that NF-Y transcription factors have important roles as positive regulators of photomorphogenesis, and in conjunction with other recent reports, implies that the NF-Y are multifaceted regulators of early seedling development. PMID:27685091

  9. Genome-Wide Transcriptome Profiling Revealed Cotton Fuzz Fiber Development Having a Similar Molecular Model as Arabidopsis Trichome

    PubMed Central

    Wan, Qun; Zhang, Hua; Ye, Wenxue; Wu, Huaitong; Zhang, Tianzhen

    2014-01-01

    The cotton fiber, as a single-celled trichome, is a biological model system for studying cell differentiation and elongation. However, the complexity of gene expression and regulation in the fiber complicates genetic research. In this study, we investigated the genome-wide transcriptome profiling in Texas Marker-1 (TM-1) and five naked seed or fuzzless mutants (three dominant and two recessive) during the fuzz initial development stage. More than three million clean tags were generated from each sample representing the expression data for 27,325 genes, which account for 72.8% of the annotated Gossypium raimondii primary transcript genes. Thousands of differentially expressed genes (DEGs) were identified between TM-1 and the mutants. Based on functional enrichment analysis, the DEGs downregulated in the mutants were enriched in protein synthesis-related genes and transcription factors, while DEGs upregulated in the mutants were enriched in DNA/chromatin structure-related genes and transcription factors. Pathway analysis showed that ATP synthesis, and sugar and lipid metabolism-related pathways play important roles in fuzz initial development. Also, we identified a large number of transcription factors such as MYB, bHLH, HB, WRKY, AP2/EREBP, bZIP and C2H2 zinc finger families that were differently expressed between TM-1 and the mutants, and were also related to trichome development in Arabidopsis. PMID:24823367

  10. Incorporating significant amino acid pairs to identify O-linked glycosylation sites on transmembrane proteins and non-transmembrane proteins

    PubMed Central

    2010-01-01

    Background While occurring enzymatically in biological systems, O-linked glycosylation affects protein folding, localization and trafficking, protein solubility, antigenicity, biological activity, as well as cell-cell interactions on membrane proteins. Catalytic enzymes involve glycotransferases, sugar-transferring enzymes and glycosidases which trim specific monosaccharides from precursors to form intermediate structures. Due to the difficulty of experimental identification, several works have used computational methods to identify glycosylation sites. Results By investigating glycosylated sites that contain various motifs between Transmembrane (TM) and non-Transmembrane (non-TM) proteins, this work presents a novel method, GlycoRBF, that implements radial basis function (RBF) networks with significant amino acid pairs (SAAPs) for identifying O-linked glycosylated serine and threonine on TM proteins and non-TM proteins. Additionally, a membrane topology is considered for reducing the false positives on glycosylated TM proteins. Based on an evaluation using five-fold cross-validation, the consideration of a membrane topology can reduce 31.4% of the false positives when identifying O-linked glycosylation sites on TM proteins. Via an independent test, GlycoRBF outperforms previous O-linked glycosylation site prediction schemes. Conclusion A case study of Cyclic AMP-dependent transcription factor ATF-6 alpha was presented to demonstrate the effectiveness of GlycoRBF. Web-based GlycoRBF, which can be accessed at http://GlycoRBF.bioinfo.tw, can identify O-linked glycosylated serine and threonine effectively and efficiently. Moreover, the structural topology of Transmembrane (TM) proteins with glycosylation sites is provided to users. The stand-alone version of GlycoRBF is also available for high throughput data analysis. PMID:21034461

  11. Structure of Staphylococcal α-Hemolysin, a Heptameric Transmembrane Pore

    NASA Astrophysics Data System (ADS)

    Song, Langzhou; Hobaugh, Michael R.; Shustak, Christopher; Cheley, Stephen; Bayley, Hagan; Gouaux, J. Eric

    1996-12-01

    The structure of the Staphylococcus aureus α-hemolysin pore has been determined to 1.9 overset{circ}{mathrm A} resolution. Contained within the mushroom-shaped homo-oligomeric heptamer is a solvent-filled channel, 100 overset{circ}{mathrm A} in length, that runs along the sevenfold axis and ranges from 14 overset{circ}{mathrm A} to 46 overset{circ}{mathrm A} in diameter. The lytic, transmembrane domain comprises the lower half of a 14-strand antiparallel β barrel, to which each protomer contributes two β strands, each 65 overset{circ}{mathrm A} long. The interior of the β barrel is primarily hydrophilic, and the exterior has a hydrophobic belt 28 overset{circ}{mathrm A} wide. The structure proves the heptameric subunit stoichiometry of the α-hemolysin oligomer, shows that a glycine-rich and solvent-exposed region of a water-soluble protein can self-assemble to form a transmembrane pore of defined structure, and provides insight into the principles of membrane interaction and transport activity of β barrel pore-forming toxins.

  12. Stability analysis of the inverse transmembrane potential problem in electrocardiography

    NASA Astrophysics Data System (ADS)

    Burger, Martin; Mardal, Kent-André; Nielsen, Bjørn Fredrik

    2010-10-01

    In this paper we study some mathematical properties of an inverse problem arising in connection with electrocardiograms (ECGs). More specifically, we analyze the possibility for recovering the transmembrane potential in the heart from ECG recordings, a challenge currently investigated by a growing number of groups. Our approach is based on the bidomain model for the electrical activity in the myocardium, and leads to a parameter identification problem for elliptic partial differential equations (PDEs). It turns out that this challenge can be split into two subproblems: the task of recovering the potential at the heart surface from body surface recordings; the problem of computing the transmembrane potential inside the heart from the potential determined at the heart surface. Problem (1), which can be formulated as the Cauchy problem for an elliptic PDE, has been extensively studied and is well known to be severely ill-posed. The main purpose of this paper is to prove that problem (2) is stable and well posed if a suitable prior is available. Moreover, our theoretical findings are illuminated by a series of numerical experiments. Finally, we discuss some aspects of uniqueness related to the anisotropy in the heart.

  13. Synaptobrevin Transmembrane Domain Dimerization Studied by Multiscale Molecular Dynamics Simulations

    PubMed Central

    Han, Jing; Pluhackova, Kristyna; Wassenaar, Tsjerk A.; Böckmann, Rainer A.

    2015-01-01

    Synaptic vesicle fusion requires assembly of the SNARE complex composed of SNAP-25, syntaxin-1, and synaptobrevin-2 (sybII) proteins. The SNARE proteins found in vesicle membranes have previously been shown to dimerize via transmembrane (TM) domain interactions. While syntaxin homodimerization is supposed to promote the transition from hemifusion to complete fusion, the role of synaptobrevin’s TM domain association in the fusion process remains poorly understood. Here, we combined coarse-grained and atomistic simulations to model the homodimerization of the sybII transmembrane domain and of selected TM mutants. The wild-type helix is shown to form a stable, right-handed dimer with the most populated helix-helix interface, including key residues predicted in a previous mutagenesis study. In addition, two alternative binding interfaces were discovered, which are essential to explain the experimentally observed higher-order oligomerization of sybII. In contrast, only one dimerization interface was found for a fusion-inactive poly-Leu mutant. Moreover, the association kinetics found for this mutant is lower as compared to the wild-type. These differences in dimerization between the wild-type and the poly-Leu mutant are suggested to be responsible for the reported differences in fusogenic activity between these peptides. This study provides molecular insight into the role of TM sequence specificity for peptide aggregation in membranes. PMID:26287628

  14. Forming transmembrane channels using end-functionalized nanotubes

    NASA Astrophysics Data System (ADS)

    Dutt, Meenakshi; Kuksenok, Olga; Little, Steven R.; Balazs, Anna C.

    2011-01-01

    Using dissipative particle dynamics (DPD) simulations, we examine the interaction between amphiphilic nanotubes and lipid bilayer membranes. The nanotubes are represented by a hydrophobic shaft that is end-functionalized with hydrophilic groups. Nanotubes that are capped by a monolayer of hydrophilic beads or also encompass hydrophilic ``hairs'' on just one end of the shaft are found to spontaneously penetrate and assume a transmembrane position; the process, however, depends critically on the membrane tension. On the other hand, nanotubes that include hydrophilic hairs at both ends of the hydrophobic shaft are not observed to spontaneously self-organize into the bilayer. When the membrane is stretched to form a pore, the nanotubes with two hairy ends adsorb on the edge of the pore and become localized in the membrane, thus forming a transmembrane channel. The findings from these studies provide guidelines for creating biomimetic nanotube channels that are capable of selectively transporting molecules through the membrane in response to changes in the local environment.

  15. Bioenergetics and mitochondrial transmembrane potential during differentiation of cultured osteoblasts

    NASA Technical Reports Server (NTRS)

    Komarova, S. V.; Ataullakhanov, F. I.; Globus, R. K.

    2000-01-01

    To evaluate the relationship between osteoblast differentiation and bioenergetics, cultured primary osteoblasts from fetal rat calvaria were grown in medium supplemented with ascorbate to induce differentiation. Before ascorbate treatment, the rate of glucose consumption was 320 nmol. h(-1). 10(6) cells(-1), respiration was 40 nmol. h(-1). 10(6) cells(-1), and the ratio of lactate production to glucose consumption was approximately 2, indicating that glycolysis was the main energy source for immature osteoblasts. Ascorbate treatment for 14 days led to a fourfold increase in respiration, a threefold increase in ATP production, and a fivefold increase in ATP content compared with that shown in immature cells. Confocal imaging of mitochondria stained with a transmembrane potential-sensitive vital dye showed that mature cells possessed abundant amounts of high-transmembrane-potential mitochondria, which were concentrated near the culture medium-facing surface. Acute treatment of mature osteoblasts with metabolic inhibitors showed that the rate of glycolysis rose to maintain the cellular energy supply constant. Thus progressive differentiation coincided with changes in cellular metabolism and mitochondrial activity, which are likely to play key roles in osteoblast function.

  16. Repression of the Arabidopsis thaliana jasmonic acid/ethylene-induced defense pathway by TGA-interacting glutaredoxins depends on their C-terminal ALWL motif.

    PubMed

    Zander, Mark; Chen, Shuxia; Imkampe, Julia; Thurow, Corinna; Gatz, Christiane

    2012-07-01

    Glutaredoxins are small heat-stable oxidoreductases that transfer electrons from glutathione (GSH) to oxidized cysteine residues, thereby contributing to protein integrity and regulation. In Arabidopsis thaliana, floral glutaredoxins ROXY1 and ROXY2 and pathogen-induced ROXY19/GRX480 interact with bZIP transcription factors of the TGACG (TGA) motif-binding family. ROXY1, ROXY2, and TGA factors PERIANTHIA, TGA9, and TGA10 play essential roles in floral development. In contrast, ectopically expressed ROXY19/GRX480 negatively regulates expression of jasmonic acid (JA)/ethylene (ET)-induced defense genes through an unknown mechanism that requires clade II transcription factors TGA2, TGA5, and/or TGA6. Here, we report that at least 17 of the 21 land plant-specific glutaredoxins encoded in the Arabidopsis genome interact with TGA2 in a yeast-two-hybrid system. To investigate their capacity to interfere with the expression of JA/ET-induced genes, we developed a transient expression system. Activation of the ORA59 (OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF-domain protein 59) promoter by transcription factor EIN3 (ETHYLENE INSENSITVE 3) was suppressed by co-expressed ROXY19/GRX480. Suppression depended on the L**LL motif in the C-terminus of ROXY19/GRX480. This putative protein interaction domain was recently described as being essential for the TGA/ROXY interaction. Ten of the 17 tested ROXY proteins suppressed ORA59 promoter activity, which correlated with the presence of the C-terminal ALWL motif, which is essential for ROXY1 function in flower development. ROXY19/GRX480-mediated repression depended on the GSH binding site, suggesting that redox modification of either TGA factors or as yet unknown target proteins is important for the suppression of ORA59 promoter activity.

  17. AtTMEM18 plays important roles in pollen tube and vegetative growth in Arabidopsis

    PubMed Central

    Dou, Xiao‐Ying; Yang, Ke‐Zhen; Ma, Zhao‐Xia; Chen, Li‐Qun; Zhang, Xue‐Qin; Bai, Jin‐Rong

    2016-01-01

    Abstract In flowering plants, pollen tube growth is essential for delivery of male gametes into the female gametophyte or embryo sac for double fertilization. Although many genes have been identified as being involved in the process, the molecular mechanisms of pollen tube growth remains poorly understood. In this study, we identified that the Arabidopsis Transmembrane Protein 18 (AtTMEM18) gene played important roles in pollen tube growth. The AtTMEM18 shares a high similarity with the Transmembrane 18 proteins (TMEM18s) that are conserved in most eukaryotes and may play important roles in obesity in humans. Mutation in the AtTMEM18 by a Ds insertion caused abnormal callose deposition in the pollen grains and had a significant impact on pollen germination and pollen tube growth. AtTMEM18 is expressed in pollen grains, pollen tubes, root tips and other vegetative tissues. The pollen‐rescued assays showed that the mutation in AtTMEM18 also caused defects in roots, stems, leaves and transmitting tracts. AtTMEM18‐GFP was located around the nuclei. Genetic assays demonstrated that the localization of AtTMEM18 around the nuclei in the generative cells of pollen grains was essential for the male fertility. Furthermore, expression of the rice TMEM18‐homologous protein (OsTMEM18) driven by LAT52 promoter could recover the fertility of the Arabidopsis attmem18 mutant. These results suggested that the TMEM18 is important for plant growth in Arabidopsis. PMID:26699939

  18. Arabidopsis thaliana—Aphid Interaction

    PubMed Central

    Louis, Joe; Singh, Vijay; Shah, Jyoti

    2012-01-01

    Aphids are important pests of plants that use their stylets to tap into the sieve elements to consume phloem sap. Besides the removal of photosynthates, aphid infestation also alters source-sink patterns. Most aphids also vector viral diseases. In this chapter, we will summarize on recent significant findings in plant-aphid interaction, and how studies involving Arabidopsis thaliana and Myzus persicae (Sülzer), more commonly known as the green peach aphid (GPA), are beginning to provide important insights into the molecular basis of plant defense and susceptibility to aphids. The recent demonstration that expression of dsRNA in Arabidopsis can be used to silence expression of genes in GPA has further expanded the utility of Arabidopsis for evaluating the contribution of the aphid genome-encoded proteins to this interaction. PMID:22666177

  19. Arabidopsis thaliana CML25 mediates the Ca(2+) regulation of K(+) transmembrane trafficking during pollen germination and tube elongation.

    PubMed

    Wang, Shuang-Shuang; Diao, Wen-Zhu; Yang, Xue; Qiao, Zhu; Wang, Mei; Acharya, Biswa R; Zhang, Wei

    2015-11-01

    The concentration alteration of cytosolic-free calcium ([Ca(2+) ]cyt ) is a well-known secondary messenger in plants and plays important roles during pollen grain germination and tube elongation. Here we demonstrate that CML25, a member of calmodulin-like proteins, has Ca(2+) -binding activity and plays a role in pollen grain germination, tube elongation and seed setting. CML25 transcript was abundant in mature pollen grains and pollen tubes, and its product CML25 protein was primarily directed to the cytoplasm. Two independent CML25 loss-of-function T-DNA insertion mutants suffered a major reduction in both the rate of pollen germination and the elongation of the pollen tube. Also, pollen grains of cml25 mutants were less sensitive to the external K(+) and Ca(2+) concentration than wild-type pollen. The disruption of CML25 increased the [Ca(2+) ]cyt in both the pollen grain and the pollen tube, which in turn impaired the Ca(2+) -dependent inhibition of whole-cell inward K(+) currents in protoplasts prepared from these materials (pollen grain and pollen tube). Complementation of cml25-1 mutant resulted in the recovery of wild-type phenotype. Our findings indicate that CML25 is an important transducer in the Ca(2+) -mediated regulation of K(+) influx during pollen germination and tube elongation. PMID:25923414

  20. Arabidopsis thaliana CML25 mediates the Ca(2+) regulation of K(+) transmembrane trafficking during pollen germination and tube elongation.

    PubMed

    Wang, Shuang-Shuang; Diao, Wen-Zhu; Yang, Xue; Qiao, Zhu; Wang, Mei; Acharya, Biswa R; Zhang, Wei

    2015-11-01

    The concentration alteration of cytosolic-free calcium ([Ca(2+) ]cyt ) is a well-known secondary messenger in plants and plays important roles during pollen grain germination and tube elongation. Here we demonstrate that CML25, a member of calmodulin-like proteins, has Ca(2+) -binding activity and plays a role in pollen grain germination, tube elongation and seed setting. CML25 transcript was abundant in mature pollen grains and pollen tubes, and its product CML25 protein was primarily directed to the cytoplasm. Two independent CML25 loss-of-function T-DNA insertion mutants suffered a major reduction in both the rate of pollen germination and the elongation of the pollen tube. Also, pollen grains of cml25 mutants were less sensitive to the external K(+) and Ca(2+) concentration than wild-type pollen. The disruption of CML25 increased the [Ca(2+) ]cyt in both the pollen grain and the pollen tube, which in turn impaired the Ca(2+) -dependent inhibition of whole-cell inward K(+) currents in protoplasts prepared from these materials (pollen grain and pollen tube). Complementation of cml25-1 mutant resulted in the recovery of wild-type phenotype. Our findings indicate that CML25 is an important transducer in the Ca(2+) -mediated regulation of K(+) influx during pollen germination and tube elongation.

  1. HTLV-1 bZIP Factor Impairs Anti-viral Immunity by Inducing Co-inhibitory Molecule, T Cell Immunoglobulin and ITIM Domain (TIGIT).

    PubMed

    Yasuma, Keiko; Yasunaga, Jun-ichirou; Takemoto, Keiko; Sugata, Kenji; Mitobe, Yuichi; Takenouchi, Norihiro; Nakagawa, Masanori; Suzuki, Yutaka; Matsuoka, Masao

    2016-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) infects CD4+ T cells and induces proliferation of infected cells in vivo, which leads to the onset of adult T-cell leukemia (ATL) in some infected individuals. The HTLV-1 bZIP factor (HBZ) gene, which is encoded in the minus strand of HTLV-1, plays critical roles in pathogenesis. In this study, RNA-seq and ChIP-seq analyses using HBZ transduced T cells revealed that HBZ upregulates the expression and promoter acetylation levels of a co-inhibitory molecule, T cell immunoglobulin and ITIM domain (TIGIT), in addition to those of regulatory T cells related genes, Foxp3 and Ccr4. TIGIT was expressed on CD4+ T cells from HBZ-transgenic (HBZ-Tg) mice, and on ATL cells and HTLV-1 infected CD4+ T cells of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in vivo. Expression of Blimp1 and IL-10 was upregulated in TIGIT+CD4+ cells of HBZ-Tg mice compared with TIGIT-CD4+ T cells, suggesting the correlation between TIGIT expression and IL-10 production. When CD4+ T cells from HBZ-Tg mice were stimulated with TIGIT's ligand, CD155, their production of the inhibitory cytokine IL-10 was enhanced. Furthermore, dendritic cells from HBZ-Tg mice produced high levels of IL-10 after stimulation. These data suggest that HBZ alters immune system to suppressive state via TIGIT and IL-10. Importantly, TIGIT suppressed T-cell responses to another HTLV-1 virus protein, Tax, in vitro. Blocking of TIGIT and PD-1 slightly increased anti-Tax T-cell activity in some HAM/TSP patients. These results suggest that HBZ-induced TIGIT on HTLV-1 infected cells impairs T-cell responses to viral antigens. This study shows that HBZ-induced TIGIT plays a pivotal role in attenuating host immune responses and shaping a microenvironment favorable to HTLV-1. PMID:26735971

  2. HTLV-1 bZIP Factor Impairs Anti-viral Immunity by Inducing Co-inhibitory Molecule, T Cell Immunoglobulin and ITIM Domain (TIGIT)

    PubMed Central

    Yasuma, Keiko; Yasunaga, Jun-ichirou; Takemoto, Keiko; Sugata, Kenji; Mitobe, Yuichi; Takenouchi, Norihiro; Nakagawa, Masanori; Suzuki, Yutaka; Matsuoka, Masao

    2016-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) infects CD4+ T cells and induces proliferation of infected cells in vivo, which leads to the onset of adult T-cell leukemia (ATL) in some infected individuals. The HTLV-1 bZIP factor (HBZ) gene, which is encoded in the minus strand of HTLV-1, plays critical roles in pathogenesis. In this study, RNA-seq and ChIP-seq analyses using HBZ transduced T cells revealed that HBZ upregulates the expression and promoter acetylation levels of a co-inhibitory molecule, T cell immunoglobulin and ITIM domain (TIGIT), in addition to those of regulatory T cells related genes, Foxp3 and Ccr4. TIGIT was expressed on CD4+ T cells from HBZ-transgenic (HBZ-Tg) mice, and on ATL cells and HTLV-1 infected CD4+ T cells of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in vivo. Expression of Blimp1 and IL-10 was upregulated in TIGIT+CD4+ cells of HBZ-Tg mice compared with TIGIT-CD4+ T cells, suggesting the correlation between TIGIT expression and IL-10 production. When CD4+ T cells from HBZ-Tg mice were stimulated with TIGIT’s ligand, CD155, their production of the inhibitory cytokine IL-10 was enhanced. Furthermore, dendritic cells from HBZ-Tg mice produced high levels of IL-10 after stimulation. These data suggest that HBZ alters immune system to suppressive state via TIGIT and IL-10. Importantly, TIGIT suppressed T-cell responses to another HTLV-1 virus protein, Tax, in vitro. Blocking of TIGIT and PD-1 slightly increased anti-Tax T-cell activity in some HAM/TSP patients. These results suggest that HBZ-induced TIGIT on HTLV-1 infected cells impairs T-cell responses to viral antigens. This study shows that HBZ-induced TIGIT plays a pivotal role in attenuating host immune responses and shaping a microenvironment favorable to HTLV-1. PMID:26735971

  3. The Ankyrin-Repeat Transmembrane Protein BDA1 Functions Downstream of the Receptor-Like Protein SNC2 to Regulate Plant Immunity1[C][OA

    PubMed Central

    Yang, Yuanai; Zhang, Yaxi; Ding, Pingtao; Johnson, Kaeli; Li, Xin; Zhang, Yuelin

    2012-01-01

    Plants utilize a large number of immune receptors to recognize pathogens and activate defense responses. A small number of these receptors belong to the receptor-like protein family. Previously, we showed that a gain-of-function mutation in the receptor-like protein SNC2 (for Suppressor of NPR1, Constitutive2) leads to constitutive activation of defense responses in snc2-1D mutant plants. To identify defense signaling components downstream of SNC2, we carried out a suppressor screen in the snc2-1D mutant background of Arabidopsis (Arabidopsis thaliana). Map-based cloning of one of the suppressor genes, BDA1 (for bian da; “becoming big” in Chinese), showed that it encodes a protein with amino-terminal ankyrin repeats and carboxyl-terminal transmembrane domains. Loss-of-function mutations in BDA1 suppress the dwarf morphology and constitutive defense responses in snc2-1D npr1-1 (for nonexpressor of pathogenesis-related genes1,1) and also result in enhanced susceptibility to bacterial pathogens. In contrast, a gain-of-function allele of bda1 isolated from a separate genetic screen to search for mutants with enhanced pathogen resistance was found to constitutively activate cell death and defense responses. These data suggest that BDA1 is a critical signaling component that functions downstream of SNC2 to regulate plant immunity. PMID:22740615

  4. Routes to the Tonoplast: The Sorting of Tonoplast Transporters in Arabidopsis Mesophyll Protoplasts[W

    PubMed Central

    Wolfenstetter, Susanne; Wirsching, Petra; Dotzauer, Dorina; Schneider, Sabine; Sauer, Norbert

    2012-01-01

    Vacuoles perform a multitude of functions in plant cells, including the storage of amino acids and sugars. Tonoplast-localized transporters catalyze the import and release of these molecules. The mechanisms determining the targeting of these transporters to the tonoplast are largely unknown. Using the paralogous Arabidopsis thaliana inositol transporters INT1 (tonoplast) and INT4 (plasma membrane), we performed domain swapping and mutational analyses and identified a C-terminal di-leucine motif responsible for the sorting of higher plant INT1-type transporters to the tonoplast in Arabidopsis mesophyll protoplasts. We demonstrate that this motif can reroute other proteins, such as INT4, SUCROSE TRANSPORTER2 (SUC2), or SWEET1, to the tonoplast and that the position of the motif relative to the transmembrane helix is critical. Rerouted INT4 is functionally active in the tonoplast and complements the growth phenotype of an int1 mutant. In Arabidopsis plants defective in the β-subunit of the AP-3 adaptor complex, INT1 is correctly localized to the tonoplast, while sorting of the vacuolar sucrose transporter SUC4 is blocked in cis-Golgi stacks. Moreover, we demonstrate that both INT1 and SUC4 trafficking to the tonoplast is sensitive to brefeldin A. Our data show that plants possess at least two different Golgi-dependent targeting mechanisms for newly synthesized transporters to the tonoplast. PMID:22253225

  5. Routes to the tonoplast: the sorting of tonoplast transporters in Arabidopsis mesophyll protoplasts.

    PubMed

    Wolfenstetter, Susanne; Wirsching, Petra; Dotzauer, Dorina; Schneider, Sabine; Sauer, Norbert

    2012-01-01

    Vacuoles perform a multitude of functions in plant cells, including the storage of amino acids and sugars. Tonoplast-localized transporters catalyze the import and release of these molecules. The mechanisms determining the targeting of these transporters to the tonoplast are largely unknown. Using the paralogous Arabidopsis thaliana inositol transporters INT1 (tonoplast) and INT4 (plasma membrane), we performed domain swapping and mutational analyses and identified a C-terminal di-leucine motif responsible for the sorting of higher plant INT1-type transporters to the tonoplast in Arabidopsis mesophyll protoplasts. We demonstrate that this motif can reroute other proteins, such as INT4, SUCROSE TRANSPORTER2 (SUC2), or SWEET1, to the tonoplast and that the position of the motif relative to the transmembrane helix is critical. Rerouted INT4 is functionally active in the tonoplast and complements the growth phenotype of an int1 mutant. In Arabidopsis plants defective in the β-subunit of the AP-3 adaptor complex, INT1 is correctly localized to the tonoplast, while sorting of the vacuolar sucrose transporter SUC4 is blocked in cis-Golgi stacks. Moreover, we demonstrate that both INT1 and SUC4 trafficking to the tonoplast is sensitive to brefeldin A. Our data show that plants possess at least two different Golgi-dependent targeting mechanisms for newly synthesized transporters to the tonoplast.

  6. Large-Conductance Transmembrane Porin Made from DNA Origami.

    PubMed

    Göpfrich, Kerstin; Li, Chen-Yu; Ricci, Maria; Bhamidimarri, Satya Prathyusha; Yoo, Jejoong; Gyenes, Bertalan; Ohmann, Alexander; Winterhalter, Mathias; Aksimentiev, Aleksei; Keyser, Ulrich F

    2016-09-27

    DNA nanotechnology allows for the creation of three-dimensional structures at nanometer scale. Here, we use DNA to build the largest synthetic pore in a lipid membrane to date, approaching the dimensions of the nuclear pore complex and increasing the pore-area and the conductance 10-fold compared to previous man-made channels. In our design, 19 cholesterol tags anchor a megadalton funnel-shaped DNA origami porin in a lipid bilayer membrane. Confocal imaging and ionic current recordings reveal spontaneous insertion of the DNA porin into the lipid membrane, creating a transmembrane pore of tens of nanosiemens conductance. All-atom molecular dynamics simulations characterize the conductance mechanism at the atomic level and independently confirm the DNA porins' large ionic conductance.

  7. Large-Conductance Transmembrane Porin Made from DNA Origami.

    PubMed

    Göpfrich, Kerstin; Li, Chen-Yu; Ricci, Maria; Bhamidimarri, Satya Prathyusha; Yoo, Jejoong; Gyenes, Bertalan; Ohmann, Alexander; Winterhalter, Mathias; Aksimentiev, Aleksei; Keyser, Ulrich F

    2016-09-27

    DNA nanotechnology allows for the creation of three-dimensional structures at nanometer scale. Here, we use DNA to build the largest synthetic pore in a lipid membrane to date, approaching the dimensions of the nuclear pore complex and increasing the pore-area and the conductance 10-fold compared to previous man-made channels. In our design, 19 cholesterol tags anchor a megadalton funnel-shaped DNA origami porin in a lipid bilayer membrane. Confocal imaging and ionic current recordings reveal spontaneous insertion of the DNA porin into the lipid membrane, creating a transmembrane pore of tens of nanosiemens conductance. All-atom molecular dynamics simulations characterize the conductance mechanism at the atomic level and independently confirm the DNA porins' large ionic conductance. PMID:27504755

  8. Large-Conductance Transmembrane Porin Made from DNA Origami

    PubMed Central

    2016-01-01

    DNA nanotechnology allows for the creation of three-dimensional structures at nanometer scale. Here, we use DNA to build the largest synthetic pore in a lipid membrane to date, approaching the dimensions of the nuclear pore complex and increasing the pore-area and the conductance 10-fold compared to previous man-made channels. In our design, 19 cholesterol tags anchor a megadalton funnel-shaped DNA origami porin in a lipid bilayer membrane. Confocal imaging and ionic current recordings reveal spontaneous insertion of the DNA porin into the lipid membrane, creating a transmembrane pore of tens of nanosiemens conductance. All-atom molecular dynamics simulations characterize the conductance mechanism at the atomic level and independently confirm the DNA porins’ large ionic conductance. PMID:27504755

  9. Transmembrane helix dimerization: beyond the search for sequence motifs.

    PubMed

    Li, Edwin; Wimley, William C; Hristova, Kalina

    2012-02-01

    Studies of the dimerization of transmembrane (TM) helices have been ongoing for many years now, and have provided clues to the fundamental principles behind membrane protein (MP) folding. Our understanding of TM helix dimerization has been dominated by the idea that sequence motifs, simple recognizable amino acid sequences that drive lateral interaction, can be used to explain and predict the lateral interactions between TM helices in membrane proteins. But as more and more unique interacting helices are characterized, it is becoming clear that the sequence motif paradigm is incomplete. Experimental evidence suggests that the search for sequence motifs, as mediators of TM helix dimerization, cannot solve the membrane protein folding problem alone. Here we review the current understanding in the field, as it has evolved from the paradigm of sequence motifs into a view in which the interactions between TM helices are much more complex. This article is part of a Special Issue entitled: Membrane protein structure and function.

  10. The first transmembrane domain (TM1) of β2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels

    PubMed Central

    Morera, Francisco J.; Alioua, Abderrahmane; Kundu, Pallob; Salazar, Marcelo; Gonzalez, Carlos; Martinez, Agustin D.; Stefani, Enrico; Toro, Ligia; Latorre, Ramon

    2012-01-01

    The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca2+-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein–protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit. PMID:22710124

  11. Magnetic resonance study of the transmembrane nitrite diffusion.

    PubMed

    Samouilov, A; Woldman, Ya Yu; Zweier, J L; Khramtsov, V V

    2007-05-01

    Nitrite (NO(2)-), being a product of metabolism of both nitric oxide (NO(*)) and nitrate (NO(3)-), can accumulate in tissues and regenerate NO() by several mechanisms. The effect of NO(2)- on ischemia/reperfusion injury was also reported. Nevertheless, the mechanisms of intracellular NO(2)- accumulation are poorly understood. We suggested significant role of nitrite penetration through biological membranes in the form of undissociated nitrous acid (HNO(2)). This hypothesis has been tested using large unilamellar phosphatidylcholine liposomes and several spectroscopic techniques. HNO(2) transport across the phospholipid bilayer of liposomes facilitates proton transfer resulting in intraliposomal acidification, which was measured using pH-sensitive probes. NO(2)(-)-mediated intraliposomal acidification was confirmed by EPR spectroscopy using membrane-impermeable pH-sensitive nitroxide, AMC (2,2,5,5-tetramethyl-1-yloxy-2,5-dihydro-1H-imidazol-3-ium-4-yl)-aminomethanesulfonic acid (pK 5.25), and by (31)P NMR spectroscopy using inorganic phosphate (pK 6.9). Nitrite accumulates inside liposomes in concentration exceeding its concentration in the bulk solution, when initial transmembrane pH gradient (alkaline inside) is applied. Intraliposomal accumulation of NO(2)- was observed by direct measurement using chemiluminescence technique. Perfusion of isolated rat hearts with buffer containing 4 microM NO(2)- was performed. The nitrite concentrations in the effluent and in the tissue, measured after 1 min perfusion, were close, supporting fast penetration of the nitrite through the tissue. Measurements of the nitrite/nitrate showed that total concentration of NO(x) in myocardium increased from initial 7.8 to 24.7 microM after nitrite perfusion. Physiological significance of passive transmembrane transport of NO(2)- and its coupling with intraliposomal acidification are discussed.

  12. Transmembrane Pores Formed by Human Antimicrobial Peptide LL-37

    SciTech Connect

    Qian, Shuo

    2011-01-01

    Human LL-37 is a multifunctional cathelicidin peptide that has shown a wide spectrum of antimicrobial activity by permeabilizing microbial membranes similar to other antimicrobial peptides; however, its molecular mechanism has not been clarified. Two independent experiments revealed LL-37 bound to membranes in the {alpha}-helical form with the axis lying in the plane of membrane. This led to the conclusion that membrane permeabilization by LL-37 is a nonpore carpet-like mechanism of action. Here we report the detection of transmembrane pores induced by LL-37. The pore formation coincided with LL-37 helices aligning approximately normal to the plane of the membrane. We observed an unusual phenomenon of LL-37 embedded in stacked membranes, which are commonly used in peptide orientation studies. The membrane-bound LL-37 was found in the normal orientation only when the membrane spacing in the multilayers exceeded its fully hydrated value. This was achieved by swelling the stacked membranes with excessive water to a swollen state. The transmembrane pores were detected and investigated in swollen states by means of oriented circular dichroism, neutron in-plane scattering, and x-ray lamellar diffraction. The results are consistent with the effect of LL-37 on giant unilamellar vesicles. The detected pores had a water channel of radius 2333 {angstrom}. The molecular mechanism of pore formation by LL-37 is consistent with the two-state model exhibited by magainin and other small pore-forming peptides. The discovery that peptide-membrane interactions in swollen states are different from those in less hydrated states may have implications for other large membrane-active peptides and proteins studied in stacked membranes.

  13. Photometric recording of transmembrane potential in outer hair cells

    NASA Astrophysics Data System (ADS)

    Nakagawa, Takashi; Oghalai, John S.; Saggau, Peter; Rabbitt, Richard D.; Brownell, William E.

    2006-06-01

    Cochlear outer hair cells (OHCs) are polarized epithelial cells that have mechanoelectrical transduction channels within their apical stereocilia and produce electromotile force along their lateral wall. Phase shifts, or time delays, in the transmembrane voltage occurring at different axial locations along the cell may contribute to our understanding of how these cells operate at auditory frequencies. We developed a method to optically measure the phase of the OHC transmembrane potential using the voltage-sensitive dye (VSD) di-8-ANEPPS. The exit aperture of a fibre-optic light source was driven in two dimensions so that a 24 µm spot of excitation light could be positioned along the length of the OHC. We used the whole-cell patch-clamp technique in the current-clamp mode to stimulate the OHC at the base. The photometric response and the voltage response were monitored with a photodetector and patch-clamp amplifier, respectively. The photometric response was used to measure the regional changes in the membrane potential in response to maintained (dc) and sinusoidal (ac) current stimuli applied at the base of the cell. We used a neutral density filter to lower the excitation light intensity and reduce phototoxicity. A sensitive detector and lock-in amplifier were used to measure the small ac VSD signal. This permitted measurements of the ac photometric response below the noise floor of the static fluorescence. The amplitude and phase components of the photometric response were recorded for stimuli up to 800 Hz. VSD data at 400-800 Hz show the presence of a small phase delay between the stimulus voltage at the base of the cell and the local membrane potential measured along the lateral wall. Results are consistent with the hypothesis that OHCs exhibit inhomogeneous membrane potentials that vary with position in analogy with the voltage in nerve axons.

  14. Araport: the Arabidopsis Information Portal

    PubMed Central

    Krishnakumar, Vivek; Hanlon, Matthew R.; Contrino, Sergio; Ferlanti, Erik S.; Karamycheva, Svetlana; Kim, Maria; Rosen, Benjamin D.; Cheng, Chia-Yi; Moreira, Walter; Mock, Stephen A.; Stubbs, Joseph; Sullivan, Julie M.; Krampis, Konstantinos; Miller, Jason R.; Micklem, Gos; Vaughn, Matthew; Town, Christopher D.

    2015-01-01

    The Arabidopsis Information Portal (https://www.araport.org) is a new online resource for plant biology research. It houses the Arabidopsis thaliana genome sequence and associated annotation. It was conceived as a framework that allows the research community to develop and release ‘modules’ that integrate, analyze and visualize Arabidopsis data that may reside at remote sites. The current implementation provides an indexed database of core genomic information. These data are made available through feature-rich web applications that provide search, data mining, and genome browser functionality, and also by bulk download and web services. Araport uses software from the InterMine and JBrowse projects to expose curated data from TAIR, GO, BAR, EBI, UniProt, PubMed and EPIC CoGe. The site also hosts ‘science apps,’ developed as prototypes for community modules that use dynamic web pages to present data obtained on-demand from third-party servers via RESTful web services. Designed for sustainability, the Arabidopsis Information Portal strategy exploits existing scientific computing infrastructure, adopts a practical mixture of data integration technologies and encourages collaborative enhancement of the resource by its user community. PMID:25414324

  15. 21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cystic fibrosis transmembrane conductance... DEVICES Immunological Test Systems § 866.5900 Cystic fibrosis transmembrane conductance regulator (CFTR... intended as an aid in confirmatory diagnostic testing of individuals with suspected cystic fibrosis...

  16. Activation of transmembrane cell‐surface receptors via a common mechanism? The “rotation model”

    PubMed Central

    2015-01-01

    It has long been thought that transmembrane cell‐surface receptors, such as receptor tyrosine kinases and cytokine receptors, among others, are activated by ligand binding through ligand‐induced dimerization of the receptors. However, there is growing evidence that prior to ligand binding, various transmembrane receptors have a preformed, yet inactive, dimeric structure on the cell surface. Various studies also demonstrate that during transmembrane signaling, ligand binding to the extracellular domain of receptor dimers induces a rotation of transmembrane domains, followed by rearrangement and/or activation of intracellular domains. The paper here describes transmembrane cell‐surface receptors that are known or proposed to exist in dimeric form prior to ligand binding, and discusses how these preformed dimers are activated by ligand binding. PMID:26241732

  17. Assignment of Oriented Sample NMR Resonances from a Three Transmembrane Helix Protein

    PubMed Central

    Murray, D. T.; Hung, I.; Cross, T. A.

    2014-01-01

    Oriented sample solid state NMR techniques have been routinely employed to determine the structures of membrane proteins with one or two transmembrane helices. For larger proteins the technique has been limited by spectral resolution and lack of assignment strategies. Here, a strategy for resonance assignment is devised and applied to a three transmembrane helix protein. Sequence specific assignments for all labeled transmembrane amino acid sites are obtained, which provide a set of orientational restraints and helix orientation in the bilayer. Our experiments expand the utility of solid state NMR in membrane protein structure characterization to three transmembrane helix proteins and represent a straightforward strategy for routinely characterizing multiple transmembrane helix protein structures. PMID:24509383

  18. Transmembrane Signaling Characterized in Bacterial Chemoreceptors by Using Sulfhydryl Cross-Linking in vivo

    NASA Astrophysics Data System (ADS)

    Lee, Geoffrey F.; Lebert, Michael R.; Lilly, Angela A.; Hazelbauer, Gerald L.

    1995-04-01

    Transmembrane signaling by bacterial chemoreceptors is thought to involve conformational changes within a stable homodimer. We investigated the functional consequences of constraining movement between pairs of helices in the four-helix structure of the transmembrane domain of chemoreceptor Trg. Using a family of cysteine-containing receptors, we identified oxidation treatments for intact cells that catalyzed essentially complete sulfhydryl cross-linking at selected positions and yet left flagellar and sensory functions largely unperturbed. Constraining movement by cross-links between subunits had little effect on tactic response, but constraining movement between transmembrane segments of the monomer drastically reduced function. We deduce that transmembrane signaling requires substantial movement between transmembrane helices of a monomer but not between interacting helices across the interface between subunits.

  19. Characterization of transmembrane movement of glucose and glucose analogs in Streptococcus mutants Ingbritt.

    PubMed Central

    Dashper, S G; Reynolds, E C

    1990-01-01

    The transmembrane movement of radiolabeled, nonmetabolizable glucose analogs in Streptococcus mutants Ingbritt was studied under conditions of differing transmembrane electrochemical potentials (delta psi) and pH gradients (delta pH). The delta pH and delta psi were determined from the transmembrane equilibration of radiolabeled benzoate and tetraphenylphosphonium ions, respectively. Growth conditions of S. mutants Ingbritt were chosen so that the cells had a low apparent phosphoenolpyruvate (PEP)-dependent glucose:phosphotransferase activity. Cells energized under different conditions produced transmembrane proton potentials ranging from -49 to -103 mV but did not accumulate 6-deoxyglucose intracellularly. An artificial transmembrane proton potential was generated in deenergized cells by creating a delta psi with a valinomycin-induced K+ diffusion potential and a delta pH by rapid acidification of the medium. Artificial transmembrane proton potentials up to -83 mV, although producing proton influx, could not accumulate 6-deoxyglucose in deenergized cells or 2-deoxyglucose or thiomethylgalactoside in deenergized, PEP-depleted cells. The transmembrane diffusion of glucose in PEP-depleted, KF-treated cells did not exhibit saturation kinetics or competitive inhibition by 6-deoxyglucose or 2-deoxyglucose, indicating that diffusion was not facilitated by a membrane carrier. As proton-linked membrane carriers have been shown to facilitate diffusion in the absence of a transmembrane proton potential, the results therefore are not consistent with a proton-linked glucose carrier in S. mutans Ingbritt. This together with the lack of proton-linked transport of the glucose analogs suggests that glucose transmembrane movement in S. mutans Ingbritt is not linked to the transmembrane proton potential. PMID:2298698

  20. Spike initiation by transmembrane current: a white-noise analysis.

    PubMed Central

    Bryant, H L; Segundo, J P

    1976-01-01

    1. Those features of a transmembrane current correlated with spike initiation were examined in Aplysia neurones using a Gaussian white-noise stimulus. This stimulus has the advantages that it presents numerous wave forms in random order without prejudgement as to their efficacies, and that it allows straightforward statistical calculations. 2. Stimulation with a repeating segment of Gaussian white-noise current revealed remarkable invariance in the firing times of the tested neurones and indicated a high degree of reliability of their response. 3. Frequencies (less than 5 Hz) involved in spike triggering propagated faithfully for up to several millimetres, justifying intrasomatic current injection to examine spike initiation at the trigger locus. 4. Examination of current wave forms preceding spikes indicated that a wide variety could be effective. Hence, a statistical analysis was performed, including computation of probability densities, averages, standard deviations and correlation coefficients of pairs of current values. Each statistic was displayed as a function of time before the spike. 5. The average current trajectory preceding a spike was multiphasic and depended on the presence and polarity of a d.c. bias. An early relatively small inward- or outward-going phase was followed by a large outward phase before the spike. The early phase tended to oppose the polarity of the d.c. bias. 6. The late outward phase of the average current trajectory reached a maximum 40--75 msec before triggering the action potential (AP) and returned to near zero values at the moment of triggering. The fact that the current peak occurs in advance of the AP may be partially explained by a phase delay between the transmembrane current and potential. The failure of the average current trajectory to return to control values immediately following the peak argues for a positive role of the declining phase in spike triggering. 7. Probability densities preceding spikes were Gaussian

  1. Molecular analysis of ethylene-insensitive mutants in arabidopsis

    SciTech Connect

    Meyerowitz, E.

    1991-01-01

    The subject of this study is the biochemical basis of ethylene reception. The Arabidopsis thaliana ETR gene codes for the ethylene receptor or is involved in transduction of the ethylene-generated signal. We have cloned an etr mutation which results in a decrease in the ethylene response of the plant, with a decrease in ethylene binding of about five-fold. Two genes have been found in the cloned region which confer resistance. By sequence analysis, the first protein contains three distinct regions: a transmembrane region, a serine/threonine protein kinase region, and a control region similar to the RAS-binding region of yeast adenylate cyclase. The second protein contains a zinc-finger; since sequence of the first protein shows no mutant-dependent changes, and transition metals have been implicated in ethylene binding, this protein could be the ETR gene product. However, no mutant dependent differences have been found in this protein, either. The mutation could be upstream of the coding region of either gene and involve regulatory elements, so we are continuing to sequence. (MHB)

  2. Topological Analysis of Hedgehog Acyltransferase, a Multipalmitoylated Transmembrane Protein*

    PubMed Central

    Konitsiotis, Antonio D.; Jovanović, Biljana; Ciepla, Paulina; Spitaler, Martin; Lanyon-Hogg, Thomas; Tate, Edward W.; Magee, Anthony I.

    2015-01-01

    Hedgehog proteins are secreted morphogens that play critical roles in development and disease. During maturation of the proteins through the secretory pathway, they are modified by the addition of N-terminal palmitic acid and C-terminal cholesterol moieties, both of which are critical for their correct function and localization. Hedgehog acyltransferase (HHAT) is the enzyme in the endoplasmic reticulum that palmitoylates Hedgehog proteins, is a member of a small subfamily of membrane-bound O-acyltransferase proteins that acylate secreted proteins, and is an important drug target in cancer. However, little is known about HHAT structure and mode of function. We show that HHAT is comprised of ten transmembrane domains and two reentrant loops with the critical His and Asp residues on opposite sides of the endoplasmic reticulum membrane. We further show that HHAT is palmitoylated on multiple cytosolic cysteines that maintain protein structure within the membrane. Finally, we provide evidence that mutation of the conserved His residue in the hypothesized catalytic domain results in a complete loss of HHAT palmitoylation, providing novel insights into how the protein may function in vivo. PMID:25505265

  3. Decreasing transmembrane segment length greatly decreases perfringolysin O pore size

    SciTech Connect

    Lin, Qingqing; Li, Huilin; Wang, Tong; London, Erwin

    2015-04-08

    Perfringolysin O (PFO) is a transmembrane (TM) β-barrel protein that inserts into mammalian cell membranes. Once inserted into membranes, PFO assembles into pore-forming oligomers containing 30–50 PFO monomers. These form a pore of up to 300 Å, far exceeding the size of most other proteinaceous pores. In this study, we found that altering PFO TM segment length can alter the size of PFO pores. A PFO mutant with lengthened TM segments oligomerized to a similar extent as wild-type PFO, and exhibited pore-forming activity and a pore size very similar to wild-type PFO as measured by electron microscopy and a leakage assay. In contrast, PFO with shortened TM segments exhibited a large reduction in pore-forming activity and pore size. This suggests that the interaction between TM segments can greatly affect the size of pores formed by TM β-barrel proteins. PFO may be a promising candidate for engineering pore size for various applications.

  4. Quantifying ligand bias at seven-transmembrane receptors.

    PubMed

    Rajagopal, Sudarshan; Ahn, Seungkirl; Rominger, David H; Gowen-MacDonald, William; Lam, Christopher M; Dewire, Scott M; Violin, Jonathan D; Lefkowitz, Robert J

    2011-09-01

    Seven transmembrane receptors (7TMRs), commonly referred to as G protein-coupled receptors, form a large part of the "druggable" genome. 7TMRs can signal through parallel pathways simultaneously, such as through heterotrimeric G proteins from different families, or, as more recently appreciated, through the multifunctional adapters, β-arrestins. Biased agonists, which signal with different efficacies to a receptor's multiple downstream pathways, are useful tools for deconvoluting this signaling complexity. These compounds may also be of therapeutic use because they have distinct functional and therapeutic profiles from "balanced agonists." Although some methods have been proposed to identify biased ligands, no comparison of these methods applied to the same set of data has been performed. Therefore, at this time, there are no generally accepted methods to quantify the relative bias of different ligands, making studies of biased signaling difficult. Here, we use complementary computational approaches for the quantification of ligand bias and demonstrate their application to two well known drug targets, the β2 adrenergic and angiotensin II type 1A receptors. The strategy outlined here allows a quantification of ligand bias and the identification of weakly biased compounds. This general method should aid in deciphering complex signaling pathways and may be useful for the development of novel biased therapeutic ligands as drugs.

  5. Decreasing transmembrane segment length greatly decreases perfringolysin O pore size

    DOE PAGESBeta

    Lin, Qingqing; Li, Huilin; Wang, Tong; London, Erwin

    2015-04-08

    Perfringolysin O (PFO) is a transmembrane (TM) β-barrel protein that inserts into mammalian cell membranes. Once inserted into membranes, PFO assembles into pore-forming oligomers containing 30–50 PFO monomers. These form a pore of up to 300 Å, far exceeding the size of most other proteinaceous pores. In this study, we found that altering PFO TM segment length can alter the size of PFO pores. A PFO mutant with lengthened TM segments oligomerized to a similar extent as wild-type PFO, and exhibited pore-forming activity and a pore size very similar to wild-type PFO as measured by electron microscopy and a leakagemore » assay. In contrast, PFO with shortened TM segments exhibited a large reduction in pore-forming activity and pore size. This suggests that the interaction between TM segments can greatly affect the size of pores formed by TM β-barrel proteins. PFO may be a promising candidate for engineering pore size for various applications.« less

  6. Evolution of a transcriptional regulator from a transmembrane nucleoporin

    PubMed Central

    Franks, Tobias M.; Benner, Chris; Narvaiza, Iñigo; Marchetto, Maria C.N.; Young, Janet M.; Malik, Harmit S.; Gage, Fred H.; Hetzer, Martin W.

    2016-01-01

    Nuclear pore complexes (NPCs) emerged as nuclear transport channels in eukaryotic cells ∼1.5 billion years ago. While the primary role of NPCs is to regulate nucleo–cytoplasmic transport, recent research suggests that certain NPC proteins have additionally acquired the role of affecting gene expression at the nuclear periphery and in the nucleoplasm in metazoans. Here we identify a widely expressed variant of the transmembrane nucleoporin (Nup) Pom121 (named sPom121, for “soluble Pom121”) that arose by genomic rearrangement before the divergence of hominoids. sPom121 lacks the nuclear membrane-anchoring domain and thus does not localize to the NPC. Instead, sPom121 colocalizes and interacts with nucleoplasmic Nup98, a previously identified transcriptional regulator, at gene promoters to control transcription of its target genes in human cells. Interestingly, sPom121 transcripts appear independently in several mammalian species, suggesting convergent innovation of Nup-mediated transcription regulation during mammalian evolution. Our findings implicate alternate transcription initiation as a mechanism to increase the functional diversity of NPC components. PMID:27198230

  7. Topological analysis of Hedgehog acyltransferase, a multipalmitoylated transmembrane protein.

    PubMed

    Konitsiotis, Antonio D; Jovanović, Biljana; Ciepla, Paulina; Spitaler, Martin; Lanyon-Hogg, Thomas; Tate, Edward W; Magee, Anthony I

    2015-02-01

    Hedgehog proteins are secreted morphogens that play critical roles in development and disease. During maturation of the proteins through the secretory pathway, they are modified by the addition of N-terminal palmitic acid and C-terminal cholesterol moieties, both of which are critical for their correct function and localization. Hedgehog acyltransferase (HHAT) is the enzyme in the endoplasmic reticulum that palmitoylates Hedgehog proteins, is a member of a small subfamily of membrane-bound O-acyltransferase proteins that acylate secreted proteins, and is an important drug target in cancer. However, little is known about HHAT structure and mode of function. We show that HHAT is comprised of ten transmembrane domains and two reentrant loops with the critical His and Asp residues on opposite sides of the endoplasmic reticulum membrane. We further show that HHAT is palmitoylated on multiple cytosolic cysteines that maintain protein structure within the membrane. Finally, we provide evidence that mutation of the conserved His residue in the hypothesized catalytic domain results in a complete loss of HHAT palmitoylation, providing novel insights into how the protein may function in vivo. PMID:25505265

  8. Role of GxxxG Motifs in Transmembrane Domain Interactions.

    PubMed

    Teese, Mark G; Langosch, Dieter

    2015-08-25

    Transmembrane (TM) helices of integral membrane proteins can facilitate strong and specific noncovalent protein-protein interactions. Mutagenesis and structural analyses have revealed numerous examples in which the interaction between TM helices of single-pass membrane proteins is dependent on a GxxxG or (small)xxx(small) motif. It is therefore tempting to use the presence of these simple motifs as an indicator of TM helix interactions. In this Current Topic review, we point out that these motifs are quite common, with more than 50% of single-pass TM domains containing a (small)xxx(small) motif. However, the actual interaction strength of motif-containing helices depends strongly on sequence context and membrane properties. In addition, recent studies have revealed several GxxxG-containing TM domains that interact via alternative interfaces involving hydrophobic, polar, aromatic, or even ionizable residues that do not form recognizable motifs. In multipass membrane proteins, GxxxG motifs can be important for protein folding, and not just oligomerization. Our current knowledge thus suggests that the presence of a GxxxG motif alone is a weak predictor of protein dimerization in the membrane. PMID:26244771

  9. Cystic fibrosis transmembrane regulator gene mutations in Bahrain.

    PubMed

    Eskandarani, H A

    2002-12-01

    A genotypic study was undertaken to characterize the cystic fibrosis transmembrane regulator gene mutations (CFTR) in the Bahraini cystic fibrosis (CF) population using a polymerase chain reaction-based direct gene test to search for 15 common CF mutations amongst Arabs. During the period October 2000 to May 2001, 19 patients (12 males and seven females; aged at time of study between 4 months and 14 years with a mean age of 5.4 +/- 4.3 years) from 13 families were recruited in the study. Patients were diagnosed as having CF, based on a typical clinical picture and sweat chloride levels > 60 mmol/l and were screened for CFTR mutations. The rate of consanguinity among the families was 77 per cent. Eight mutations were detected in 21 of the 26 alleles examined. The overall detection rate was approximately 81 per cent. The allele frequency of the eight mutations was estimated to be approximately 73 per cent. There was no specific phenotypic pattern that correlated with a specific genotype. All families except two were of Bahraini origin. Of the eight mutations detected, four were common among Bahrainis (2043delG > 548A --> T > 4041C --> G = deltaF508, in order of decreasing frequency), accounting for 66 per cent of the Bahraini CF alleles. However, we also detected four different heterozygous mutations, namely: 1161delC, 1756G -->T, 3120 + 1G --> A, and 3661A --> T, accounting for 16 per cent of the Bahraini CF alleles.

  10. LINKIN, a new transmembrane protein necessary for cell adhesion

    PubMed Central

    Kato, Mihoko; Chou, Tsui-Fen; Yu, Collin Z; DeModena, John; Sternberg, Paul W

    2014-01-01

    In epithelial collective migration, leader and follower cells migrate while maintaining cell–cell adhesion and tissue polarity. We have identified a conserved protein and interactors required for maintaining cell adhesion during a simple collective migration in the developing C. elegans male gonad. LINKIN is a previously uncharacterized, transmembrane protein conserved throughout Metazoa. We identified seven atypical FG–GAP domains in the extracellular domain, which potentially folds into a β-propeller structure resembling the α-integrin ligand-binding domain. C. elegans LNKN-1 localizes to the plasma membrane of all gonadal cells, with apical and lateral bias. We identified the LINKIN interactors RUVBL1, RUVBL2, and α-tubulin by using SILAC mass spectrometry on human HEK 293T cells and testing candidates for lnkn-1-like function in C. elegans male gonad. We propose that LINKIN promotes adhesion between neighboring cells through its extracellular domain and regulates microtubule dynamics through RUVBL proteins at its intracellular domain. DOI: http://dx.doi.org/10.7554/eLife.04449.001 PMID:25437307

  11. Evolution of a transcriptional regulator from a transmembrane nucleoporin.

    PubMed

    Franks, Tobias M; Benner, Chris; Narvaiza, Iñigo; Marchetto, Maria C N; Young, Janet M; Malik, Harmit S; Gage, Fred H; Hetzer, Martin W

    2016-05-15

    Nuclear pore complexes (NPCs) emerged as nuclear transport channels in eukaryotic cells ∼1.5 billion years ago. While the primary role of NPCs is to regulate nucleo-cytoplasmic transport, recent research suggests that certain NPC proteins have additionally acquired the role of affecting gene expression at the nuclear periphery and in the nucleoplasm in metazoans. Here we identify a widely expressed variant of the transmembrane nucleoporin (Nup) Pom121 (named sPom121, for "soluble Pom121") that arose by genomic rearrangement before the divergence of hominoids. sPom121 lacks the nuclear membrane-anchoring domain and thus does not localize to the NPC. Instead, sPom121 colocalizes and interacts with nucleoplasmic Nup98, a previously identified transcriptional regulator, at gene promoters to control transcription of its target genes in human cells. Interestingly, sPom121 transcripts appear independently in several mammalian species, suggesting convergent innovation of Nup-mediated transcription regulation during mammalian evolution. Our findings implicate alternate transcription initiation as a mechanism to increase the functional diversity of NPC components. PMID:27198230

  12. Structural Plasticity in Self-Assembling Transmembrane β-Sheets

    PubMed Central

    Bishop, Christopher M.; Wimley, William C.

    2011-01-01

    Here we test the hypothesis that membrane-spanning β-sheets can exhibit structural plasticity in membranes due to their ability to shift hydrogen-bonding patterns. Transmembrane β-sheet forming peptides of the sequence AcWLn, where n = 5, 6, or 7, which range from 21 to 27 Å in maximum length, were incorporated into bilayers made of phosphatidylcholine lipids with saturated acyl chains containing 14, 16, or 18 carbons, which are 36–50 Å in thickness. The effect of the peptide β-sheets on fluid- and gel-phase bilayers were studied with differential scanning calorimetry and circular dichroism spectroscopy. We show that AcWL5 forms a stable, peptide-rich gel phase in all three lipids. The whole family of AcWLn peptides appears to form similarly stable, nonmembrane-disrupting β-sheets in all bilayer phases and thicknesses. Bilayers containing up to 20 mol % peptide, which is the maximum concentration tested, formed gel phases with melting temperatures that were equal to, or slightly higher than, the pure lipid transitions. Given the range of peptide lengths and bilayer thicknesses tested, these experiments show that the AcWLn family of membrane-inserted β-sheets exhibit remarkable structural plasticity in membranes. PMID:21843473

  13. Toxoplasma gondii transmembrane microneme proteins and their modular design

    PubMed Central

    Sheiner, Lilach; Santos, Joana M.; Klages, Natacha; Parussini, Fabiola; Jemmely, Noelle; Friedrich, Nikolas; Ward, Gary E.; Soldati-Favre, Dominique

    2010-01-01

    Summary Host cell invasion by the Apicomplexa critically relies on regulated secretion of transmembrane micronemal proteins (TM-MICs). Toxoplasma gondii possesses functionally non-redundant MICs complexes that participate in gliding motility, host cell attachment, moving junction formation, rhoptry secretion and invasion. The TM-MICs are released onto the parasite’s surface as complexes capable of interacting with host cell receptors. Additionally, TgMIC2 simultaneously connects to the actomyosin system via binding to aldolase. During invasion these adhesive complexes are shed from the surface notably via intramembrane cleavage of the TM-MICs by a rhomboid protease. Some TM-MICs act as escorters and assure trafficking of the complexes to the micronemes. We have investigated the properties of TgMIC6, TgMIC8, TgMIC8.2, TgAMA1 and the new micronemal protein TgMIC16 with respect to interaction with aldolase, susceptibility to rhomboid cleavage and presence of trafficking signals. We conclude that several TM-MICs lack targeting information within their C-terminal domains, indicating that trafficking depends on yet unidentified proteins interacting with their ectodomains. Most TM-MICs serve as substrates for a rhomboid protease and some of them are able to bind to aldolase. We also show that the residues responsible for binding to aldolase are essential for TgAMA1 but dispensable forTgMIC6 function during invasion. PMID:20545864

  14. A small-molecule photoactivatable optical sensor of transmembrane potential

    PubMed Central

    Grenier, Vincent; Walker, Alison S.; Miller, Evan W.

    2015-01-01

    This paper discloses the design, synthesis, and imaging applications of the first member of a new class of SPOTs, small-molecule photoactivatable optical sensors of transmembrane potential. SPOT2.1.Cl features an established voltage-sensitive dye, VoltageFluor2.1.Cl—or—VF capped with a dimethoxy-o-nitrobenzyl (DMNB) caging group to effectively eliminate fluorescence of the VF dye prior to uncaging. SPOT2.1.Cl localizes to cell membranes and displays weak fluorescence until photoactivated. Illumination generates the parent VF dye which then optically reports on changes in the membrane voltage. After photoactivation with spatially restricted light, SPOT2.1.Cl-loaded cells display bright, voltage-sensitive fluorescence associated with the plasma membrane, while neighboring cells remain dark. Activated SPOT reports on action potentials in single trials. SPOT can be activated in neuron cell bodies or uncaged in dendrites to enable structural tracing via “backfilling” of the dye to the soma, followed by functional imaging in the labeled cell. The combination of cellular specificity achieved through spatially-defined patterns of illumination, coupled with the fast, sensitive, and non-capacitive voltage sensing characteristics of VF dyes makes SPOT2.1.Cl a useful tool for interrogating both structure and function of neuronal systems. PMID:26247778

  15. The N-Terminus of the Floral Arabidopsis TGA Transcription Factor PERIANTHIA Mediates Redox-Sensitive DNA-Binding

    PubMed Central

    Gutsche, Nora; Zachgo, Sabine

    2016-01-01

    The Arabidopsis TGA transcription factor (TF) PERIANTHIA (PAN) regulates the formation of the floral organ primordia as revealed by the pan mutant forming an abnormal pentamerous arrangement of the outer three floral whorls. The Arabidopsis TGA bZIP TF family comprises 10 members, of which PAN and TGA9/10 control flower developmental processes and TGA1/2/5/6 participate in stress-responses. For the TGA1 protein it was shown that several cysteines can be redox-dependently modified. TGA proteins interact in the nucleus with land plant-specific glutaredoxins, which may alter their activities posttranslationally. Here, we investigated the DNA-binding of PAN to the AAGAAT motif under different redox-conditions. The AAGAAT motif is localized in the second intron of the floral homeotic regulator AGAMOUS (AG), which controls stamen and carpel development as well as floral determinacy. Whereas PAN protein binds to this regulatory cis-element under reducing conditions, the interaction is strongly reduced under oxidizing conditions in EMSA studies. The redox-sensitive DNA-binding is mediated via a special PAN N-terminus, which is not present in other Arabidopsis TGA TFs and comprises five cysteines. Two N-terminal PAN cysteines, Cys68 and Cys87, were shown to form a disulfide bridge and Cys340, localized in a C-terminal putative transactivation domain, can be S-glutathionylated. Comparative land plant analyses revealed that the AAGAAT motif exists in asterid and rosid plant species. TGA TFs with N-terminal extensions of variable length were identified in all analyzed seed plants. However, a PAN-like N-terminus exists only in the rosids and exclusively Brassicaceae homologs comprise four to five of the PAN N-terminal cysteines. Redox-dependent modifications of TGA cysteines are known to regulate the activity of stress-related TGA TFs. Here, we show that the N-terminal PAN cysteines participate in a redox-dependent control of the PAN interaction with a highly conserved

  16. The N-Terminus of the Floral Arabidopsis TGA Transcription Factor PERIANTHIA Mediates Redox-Sensitive DNA-Binding.

    PubMed

    Gutsche, Nora; Zachgo, Sabine

    2016-01-01

    The Arabidopsis TGA transcription factor (TF) PERIANTHIA (PAN) regulates the formation of the floral organ primordia as revealed by the pan mutant forming an abnormal pentamerous arrangement of the outer three floral whorls. The Arabidopsis TGA bZIP TF family comprises 10 members, of which PAN and TGA9/10 control flower developmental processes and TGA1/2/5/6 participate in stress-responses. For the TGA1 protein it was shown that several cysteines can be redox-dependently modified. TGA proteins interact in the nucleus with land plant-specific glutaredoxins, which may alter their activities posttranslationally. Here, we investigated the DNA-binding of PAN to the AAGAAT motif under different redox-conditions. The AAGAAT motif is localized in the second intron of the floral homeotic regulator AGAMOUS (AG), which controls stamen and carpel development as well as floral determinacy. Whereas PAN protein binds to this regulatory cis-element under reducing conditions, the interaction is strongly reduced under oxidizing conditions in EMSA studies. The redox-sensitive DNA-binding is mediated via a special PAN N-terminus, which is not present in other Arabidopsis TGA TFs and comprises five cysteines. Two N-terminal PAN cysteines, Cys68 and Cys87, were shown to form a disulfide bridge and Cys340, localized in a C-terminal putative transactivation domain, can be S-glutathionylated. Comparative land plant analyses revealed that the AAGAAT motif exists in asterid and rosid plant species. TGA TFs with N-terminal extensions of variable length were identified in all analyzed seed plants. However, a PAN-like N-terminus exists only in the rosids and exclusively Brassicaceae homologs comprise four to five of the PAN N-terminal cysteines. Redox-dependent modifications of TGA cysteines are known to regulate the activity of stress-related TGA TFs. Here, we show that the N-terminal PAN cysteines participate in a redox-dependent control of the PAN interaction with a highly conserved

  17. Quantitative approaches to utilizing mutational analysis and disulfide crosslinking for modeling a transmembrane domain.

    PubMed Central

    Lee, G. F.; Hazelbauer, G. L.

    1995-01-01

    The transmembrane domain of chemoreceptor Trg from Escherichia coli contains four transmembrane segments in its native homodimer, two from each subunit. We had previously used mutational analysis and sulfhydryl cross-linking between introduced cysteines to obtain data relevant to the three-dimensional organization of this domain. In the current study we used Fourier analysis to assess these data quantitatively for periodicity along the sequences of the segments. The analyses provided a strong indication of alpha-helical periodicity in the first transmembrane segment and a substantial indication of that periodicity for the second segment. On this basis, we considered both segments as idealized alpha-helices and proceeded to model the transmembrane domain as a unit of four helices. For this modeling, we calculated helical crosslinking moments, parameters analogous to helical hydrophobic moments, as a quantitative way of condensing and utilizing a large body of crosslinking data. Crosslinking moments were used to define the relative separation and orientation of helical pairs, thus creating a quantitatively derived model for the transmembrane domain of Trg. Utilization of Fourier transforms to provide a quantitative indication of periodicity in data from analyses of transmembrane segments, in combination with helical crosslinking moments to position helical pairs should be useful in modeling other transmembrane domains. PMID:7549874

  18. Assessing Gravitropic Responses in Arabidopsis.

    PubMed

    Barker, Richard; Cox, Benjamin; Silber, Logan; Sangari, Arash; Assadi, Amir; Masson, Patrick

    2016-01-01

    Arabidopsis thaliana was the first higher organism to have its genome sequenced and is now widely regarded as the model dicot. Like all plants, Arabidopsis develops distinct growth patterns in response to different environmental stimuli. This can be seen in the gravitropic response of roots. Methods to investigate this particular tropism are presented here. First, we describe a high-throughput time-lapse photographic analysis of root growth and curvature response to gravistimulation allowing the quantification of gravitropic kinetics and growth rate at high temporal resolution. Second, we present a protocol that allows a quantitative evaluation of gravitropic sensitivity using a homemade 2D clinostat. Together, these approaches allow an initial comparative analysis of the key phenomena associated with root gravitropism between different genotypes and/or accessions. PMID:26867611

  19. Asparagine Metabolic Pathways in Arabidopsis.

    PubMed

    Gaufichon, Laure; Rothstein, Steven J; Suzuki, Akira

    2016-04-01

    Inorganic nitrogen in the form of ammonium is assimilated into asparagine via multiple steps involving glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AspAT) and asparagine synthetase (AS) in Arabidopsis. The asparagine amide group is liberated by the reaction catalyzed by asparaginase (ASPG) and also the amino group of asparagine is released by asparagine aminotransferase (AsnAT) for use in the biosynthesis of amino acids. Asparagine plays a primary role in nitrogen recycling, storage and transport in developing and germinating seeds, as well as in vegetative and senescence organs. A small multigene family encodes isoenzymes of each step of asparagine metabolism in Arabidopsis, except for asparagine aminotransferase encoded by a single gene. The aim of this study is to highlight the structure of the genes and encoded enzyme proteins involved in asparagine metabolic pathways; the regulation and role of different isogenes; and kinetic and physiological properties of encoded enzymes in different tissues and developmental stages. PMID:26628609

  20. AT14A mediates the cell wall-plasma membrane-cytoskeleton continuum in Arabidopsis thaliana cells.

    PubMed

    Lü, Bing; Wang, Juan; Zhang, Yu; Wang, Hongcheng; Liang, Jiansheng; Zhang, Jianhua

    2012-06-01

    AT14A has a small domain that has sequence similarities to integrins from animals. Integrins serve as a transmembrane linker between the extracellular matrix and the cytoskeleton, which play critical roles in a variety of biological processes. Because the function of AT14A is unknown, Arabidopsis thaliana AT14A, which is a transmembrane receptor for cell adhesion molecules and a middle member of the cell wall-plasma membrane-cytoskeleton continuum in plants, has been described. AT14A, co-expressed with green fluorescent protein (GFP), was found to localize mainly to the plasma membrane. The mutant Arabidopsis at14a-1 cells exhibit various phenotypes with cell shape, cell cluster size, thickness, and cellulose content of cell wall, the adhesion between cells, and the adhesion of plasma membrane to cell wall varied by plasmolysis. Using direct staining of filamentous actin and indirect immunofluorescence staining of microtubules, cortical actin filaments and microtubules arrays were significantly altered in cells, either where AT14A was absent or over-expressed. It is concluded that AT14A may be a substantial middle member of the cell wall-plasma membrane-cytoskeleton continuum and play an important role in the continuum by regulating cell wall and cortical cytoskeleton organization. PMID:22456678

  1. Changes of transmembrane Ca2+ gradient in the formation of macrophage-derived foam cells.

    PubMed

    Yang, X; Zhang, Y; Huang, Y; Yang, F

    2000-02-01

    Macrophages from C57BL/6J mice, an animal susceptible to atherosclerosis, were chosen as target cells to study changes in the transmembrane Ca2+ gradient during the formation of macrophage-derived foam cells. The transmembrane Ca2+ gradients of single living cells were examined-using Fura-2/AM combined with Fluo-3/AM by laser scanning confocal microscopy. Exposure to Oxidized Low Density Lipoprotein, decreases the Ca2+ gradient across macrophage plasma membrane, but increases that across the nuclear membrane. The altered transmembrane Ca2+ gradients could induce the up-regulation of scavenger receptor in macrophages, resulting in the formation of foam cells.

  2. A unique phenylalanine in the transmembrane domain strengthens homodimerization of the syndecan-2 transmembrane domain and functionally regulates syndecan-2.

    PubMed

    Kwon, Mi-Jung; Choi, Youngsil; Yun, Ji-Hye; Lee, Weontae; Han, Inn-Oc; Oh, Eok-Soo

    2015-02-27

    The syndecans are a type of cell surface adhesion receptor that initiates intracellular signaling events through receptor clustering mediated by their highly conserved transmembrane domains (TMDs). However, the exact function of the syndecan TMD is not yet fully understood. Here, we investigated the specific regulatory role of the syndecan-2 TMD. We found that syndecan-2 mutants in which the TMD had been replaced with that of syndecan-4 were defective in syndecan-2-mediated functions, suggesting that the TMD of syndecan-2 plays one or more specific roles. Interestingly, syndecan-2 has a stronger tendency to form sodium dodecyl sulfate (SDS)-resistant homodimers than syndecan-4. Our structural studies showed that a unique phenylalanine residue (Phe(167)) enables an additional molecular interaction between the TMDs of the syndecan-2 homodimer. The presence of Phe(167) was correlated with a higher tendency toward oligomerization, and its replacement with isoleucine significantly reduced the SDS-resistant dimer formation and cellular functions of syndecan-2 (e.g. cell migration). Conversely, replacement of isoleucine with phenylalanine at this position in the syndecan-4 TMD rescued the defects observed in a mutant syndecan-2 harboring the syndecan-4 TMD. Taken together, these data suggest that Phe(167) in the TMD of syndecan-2 endows the protein with specific functions. Our work offers new insights into the signaling mediated by the TMD of syndecan family members.

  3. BRASSINOSTEROID INSENSITIVE2 interacts with ABSCISIC ACID INSENSITIVE5 to mediate the antagonism of brassinosteroids to abscisic acid during seed germination in Arabidopsis.

    PubMed

    Hu, Yanru; Yu, Diqiu

    2014-11-01

    Seed germination and postgerminative growth are regulated by a delicate hormonal balance. Abscisic acid (ABA) represses Arabidopsis thaliana seed germination and postgerminative growth, while brassinosteroids (BRs) antagonize ABA-mediated inhibition and promote these processes. However, the molecular mechanism underlying BR-repressed ABA signaling remains largely unknown. Here, we show that the Glycogen Synthase Kinase 3-like kinase BRASSINOSTEROID INSENSITIVE2 (BIN2), a critical repressor of BR signaling, positively regulates ABA responses during seed germination and postgerminative growth. Mechanistic investigation revealed that BIN2 physically interacts with ABSCISIC ACID INSENSITIVE5 (ABI5), a bZIP transcription factor. Further genetic analysis demonstrated that the ABA-hypersensitive phenotype of BIN2-overexpressing plants requires ABI5. BIN2 was found to phosphorylate and stabilize ABI5 in the presence of ABA, while application of epibrassinolide (the active form of BRs) inhibited the regulation of ABI5 by BIN2. Consistently, the ABA-induced accumulation of ABI5 was affected in BIN2-related mutants. Moreover, mutations of the BIN2 phosphorylation sites on ABI5 made the mutant protein respond to ABA improperly. Additionally, the expression of several ABI5 regulons was positively modulated by BIN2. These results provide evidence that BIN2 phosphorylates and stabilizes ABI5 to mediate ABA response during seed germination, while BRs repress the BIN2-ABI5 cascade to antagonize ABA-mediated inhibition.

  4. BRASSINOSTEROID INSENSITIVE2 Interacts with ABSCISIC ACID INSENSITIVE5 to Mediate the Antagonism of Brassinosteroids to Abscisic Acid during Seed Germination in Arabidopsis[W

    PubMed Central

    Hu, Yanru; Yu, Diqiu

    2014-01-01

    Seed germination and postgerminative growth are regulated by a delicate hormonal balance. Abscisic acid (ABA) represses Arabidopsis thaliana seed germination and postgerminative growth, while brassinosteroids (BRs) antagonize ABA-mediated inhibition and promote these processes. However, the molecular mechanism underlying BR-repressed ABA signaling remains largely unknown. Here, we show that the Glycogen Synthase Kinase 3-like kinase BRASSINOSTEROID INSENSITIVE2 (BIN2), a critical repressor of BR signaling, positively regulates ABA responses during seed germination and postgerminative growth. Mechanistic investigation revealed that BIN2 physically interacts with ABSCISIC ACID INSENSITIVE5 (ABI5), a bZIP transcription factor. Further genetic analysis demonstrated that the ABA-hypersensitive phenotype of BIN2-overexpressing plants requires ABI5. BIN2 was found to phosphorylate and stabilize ABI5 in the presence of ABA, while application of epibrassinolide (the active form of BRs) inhibited the regulation of ABI5 by BIN2. Consistently, the ABA-induced accumulation of ABI5 was affected in BIN2-related mutants. Moreover, mutations of the BIN2 phosphorylation sites on ABI5 made the mutant protein respond to ABA improperly. Additionally, the expression of several ABI5 regulons was positively modulated by BIN2. These results provide evidence that BIN2 phosphorylates and stabilizes ABI5 to mediate ABA response during seed germination, while BRs repress the BIN2-ABI5 cascade to antagonize ABA-mediated inhibition. PMID:25415975

  5. Protein S-Acyltransferase 14: A Specific Role for Palmitoylation in Leaf Senescence in Arabidopsis1[OPEN

    PubMed Central

    Li, Yaxiao; Scott, Rod; Doughty, James; Grant, Murray

    2016-01-01

    The Asp-His-His-Cys-Cys-rich domain-containing Protein S-Acyl Transferases (PATs) are multipass transmembrane proteins that catalyze S-acylation (commonly known as S-palmitoylation), the reversible posttranslational lipid modification of proteins. Palmitoylation enhances the hydrophobicity of proteins, contributes to their membrane association, and plays roles in protein trafficking and signaling. In Arabidopsis (Arabidopsis thaliana), there are at least 24 PATs; previous studies on two PATs established important roles in growth, development, and stress responses. In this study, we identified a, to our knowledge, novel PAT, AtPAT14, in Arabidopsis. Complementation studies in yeast (Saccharomyces cerevisiae) and Arabidopsis demonstrate that AtPAT14 possesses PAT enzyme activity. Disruption of AtPAT14 by T-DNA insertion resulted in an accelerated senescence phenotype. This coincided with increased transcript levels of some senescence-specific and pathogen-resistant marker genes. We show that early senescence of pat14 does not involve the signaling molecules jasmonic acid and abscisic acid, or autophagy, but associates with salicylic acid homeostasis and signaling. This strongly suggests that AtPAT14 plays a pivotal role in regulating senescence via salicylic acid pathways. Senescence is a complex process required for normal plant growth and development and requires the coordination of many genes and signaling pathways. However, precocious senescence results in loss of biomass and seed production. The negative regulation of leaf senescence by AtPAT14 in Arabidopsis highlights, to our knowledge for the first time, a specific role for palmitoylation in leaf senescence. PMID:26537563

  6. Orientation and conformation of lipids in crystals of transmembrane proteins.

    PubMed

    Marsh, Derek; Páli, Tibor

    2013-03-01

    Orientational order parameters and individual dihedral torsion angles are evaluated for phospholipid and glycolipid molecules that are resolved in X-ray structures of integral transmembrane proteins in crystals. The order parameters of the lipid chains and glycerol backbones in protein crystals are characterised by a much wider distribution of orientational order than is found in fluid lipid bilayers and reconstituted lipid-protein membranes. This indicates that the lipids that are resolved in crystals of membrane proteins are mostly not representative of the entire lipid-protein interface. Much of the chain configurational disorder of the membrane-bound lipids in crystals arises from C-C bonds in energetically disallowed skew conformations. This suggests configurational heterogeneity of the lipids at a single binding site: eclipsed conformations occur also in the glycerol backbone torsion angles and the C-C torsion angles of the lipid head groups. Conformations of the lipid glycerol backbone in protein crystals are not restricted to the gauche C1-C2 rotamers found invariably in phospholipid bilayer crystals. Lipid head-group conformations in the protein crystals also do not conform solely to the bent-down conformation, with gauche-gauche configuration of the phosphodiester, that is characteristic of phospholipid bilayer membranes. Stereochemical violations in the protein-bound lipids are evidenced by ester carboxyl groups in non-planar configurations, and even in the cis configuration. Some lipids have the incorrect enantiomeric configuration of the glycerol backbone, and many of the branched methyl groups in the phytanyl chains associated with bacteriorhodopsin have the incorrect S configuration. PMID:22644500

  7. A Functional-Phylogenetic Classification System for Transmembrane Solute Transporters

    PubMed Central

    Saier, Milton H.

    2000-01-01

    A comprehensive classification system for transmembrane molecular transporters has been developed and recently approved by the transport panel of the nomenclature committee of the International Union of Biochemistry and Molecular Biology. This system is based on (i) transporter class and subclass (mode of transport and energy coupling mechanism), (ii) protein phylogenetic family and subfamily, and (iii) substrate specificity. Almost all of the more than 250 identified families of transporters include members that function exclusively in transport. Channels (115 families), secondary active transporters (uniporters, symporters, and antiporters) (78 families), primary active transporters (23 families), group translocators (6 families), and transport proteins of ill-defined function or of unknown mechanism (51 families) constitute distinct categories. Transport mode and energy coupling prove to be relatively immutable characteristics and therefore provide primary bases for classification. Phylogenetic grouping reflects structure, function, mechanism, and often substrate specificity and therefore provides a reliable secondary basis for classification. Substrate specificity and polarity of transport prove to be more readily altered during evolutionary history and therefore provide a tertiary basis for classification. With very few exceptions, a phylogenetic family of transporters includes members that function by a single transport mode and energy coupling mechanism, although a variety of substrates may be transported, sometimes with either inwardly or outwardly directed polarity. In this review, I provide cross-referencing of well-characterized constituent transporters according to (i) transport mode, (ii) energy coupling mechanism, (iii) phylogenetic grouping, and (iv) substrates transported. The structural features and distribution of recognized family members throughout the living world are also evaluated. The tabulations should facilitate familial and functional

  8. Mechanisms of Hop Inhibition Include the Transmembrane Redox Reaction▿

    PubMed Central

    Behr, Jürgen; Vogel, Rudi F.

    2010-01-01

    In this work, a novel mechanistic model of hop inhibition beyond the proton ionophore action toward (beer spoiling) bacteria was developed. Investigations were performed with model systems using cyclic voltammetry for the determination of redox processes/conditions in connection with growth challenges with hop-sensitive and -resistant Lactobacillus brevis strains in the presence of oxidants. Cyclic voltammetry identified a transmembrane redox reaction of hop compounds at low pH (common in beer) and in the presence of manganese (present in millimolar levels in lactic acid bacteria). The antibacterial action of hop compounds could be extended from the described proton ionophore activity, lowering the intracellular pH, to pronounced redox reactivity, causing cellular oxidative damage. Accordingly, a correlation between the resistance of L. brevis strains to a sole oxidant to their resistance to hop could not be expected and was not detected. However, in connection with our recent study concerning hop ionophore properties and the resistance of hop-sensitive and -tolerant L. brevis strains toward proton ionophores (J. Behr and R. F. Vogel, J. Agric. Food Chem. 57:6074-6081, 2009), we suggest that both ionophore and oxidant resistance are required for survival under hop stress conditions and confirmed this correlation according to the novel mechanistic model. In consequence, the expression of several published hop resistance mechanisms involved in manganese binding/transport and intracellular redox balance, as well as that of proteins involved in oxidative stress under “highly reducing” conditions (cf. anaerobic cultivation and “antioxidative” hop compounds in the growth medium), is now comprehensible. Accordingly, hop resistance as a multifactorial dynamic property at least implies distinct resistance levels against two different mechanisms of hop inhibition, namely, proton ionophore-induced and oxidative stress-induced mechanisms. Beyond this specific model of

  9. Hormone Action on Transmembrane Electron and H+ Transport 1

    PubMed Central

    Böttger, Michael; Hilgendorf, Frank

    1988-01-01

    A possible involvement of two different systems in proton translocation was investigated by simultaneous measurement of transmembrane electron flow and proton secretion in a pH-stat combined with a redoxstat. The pH gradient between cytoplasm and apoplast is probably maintained by an H+ -pumping ATPase and by a second proton extrusion system, which seems to be linked to a redox chain with NAD(P)H as electron donor. Indole acetic acid inhibits both e− and H+ efflux, but only if the `electron draw' from the outside is not too high. The electron draw depends on the hexacyanoferrate level at the plasmalemma surface and on the Ca2+ concentration. The inhibiting effect of auxin on e− and H+ efflux in the presence of hexacyanoferrate can be only detected at low levels of bivalent cations and of the artificial electron acceptor. The inhibition of e− and H+ efflux by auxin requires high oxygen levels. The influence of auxin on both e− and H+ transfer disappears below 2 kilopascals O2, a level which does not influence respiration. Ethanol and fusicoccin do not increase the e− flux, probably because the electron transfer from the plasma membrane to HCF III is the limiting step. If electron transfer is reduced by IAA pretreatment, ethanol increases e− flux. Fusicoccin decreases e− and increases H+ efflux if the rates have been lowered previously by indole acetic acid pretreatment. This effect depends on high oxygen levels and is reversible by lowering oxygen pressure. Auxin and Ca2+ change e− flow and H+ ejection in a 1:1 ratio. PMID:16666028

  10. Synaptobrevin Transmembrane Domain Influences Exocytosis by Perturbing Vesicle Membrane Curvature

    PubMed Central

    Chang, Che-Wei; Jackson, Meyer B.

    2015-01-01

    Membrane fusion requires that nearly flat lipid bilayers deform into shapes with very high curvature. This makes membrane bending a critical force in determining fusion mechanisms. A lipid bilayer will bend spontaneously when material is distributed asymmetrically between its two monolayers, and its spontaneous curvature (C0) will influence the stability of curved fusion intermediates. Prior work on Ca2+-triggered exocytosis revealed that fusion pore lifetime (τ) varies with vesicle content (Q), and showed that this relation reflects membrane bending energetics. Lipids that alter C0 change the dependence of τ on Q. These results suggested that the greater stability of an initial exocytotic fusion pore associated with larger vesicles reflects the need to bend more membrane during fusion pore dilation. In this study, we explored the possibility of manipulating C0 by mutating the transmembrane domain (TMD) of the vesicle membrane protein synaptobrevin 2 (syb2). Amperometric measurements of exocytosis in mouse chromaffin cells revealed that syb2 TMD mutations altered the relation between τ and Q. The effects of these mutations showed a striking periodicity, changing sign as the structural perturbation moved through the inner and outer leaflets. Some glycine and charge mutations also influenced the dependence of τ on Q in a manner consistent with expected changes in C0. These results suggest that side chains in the syb2 TMD influence the kinetics of exocytosis by perturbing the packing of the surrounding lipids. The present results support the view that membrane bending occurs during fusion pore expansion rather than during fusion pore formation. This supports the view of an initial fusion pore through two relatively flat membranes formed by protein. PMID:26153704

  11. Transmembrane tumor necrosis factor-alpha sensitizes adipocytes to insulin.

    PubMed

    Zhou, Wenjing; Yang, Peng; Liu, Li; Zheng, Shan; Zeng, Qingling; Liang, Huifang; Zhu, Yazhen; Zhang, Zunyue; Wang, Jing; Yin, Bingjiao; Gong, Feili; Wu, Yiping; Li, Zhuoya

    2015-05-01

    Transmembrane TNF-α (tmTNF-α) acts both as a ligand, delivering 'forward signaling' via TNFR, and as a receptor, transducing 'reverse signaling'. The contradiction of available data regarding the effect of tmTNF-α on insulin resistance may be due to imbalance in both signals. Here, we demonstrated that high glucose-induced impairment of insulin-stimulated glucose uptake by 3T3-L1 adipocytes was concomitant with decreased tmTNF-α expression and increased soluble TNF-α (sTNF-α) secretion. However, when TACE was inhibited, preventing the conversion of tmTNF-α to sTNF-α, this insulin resistance was partially reversed, indicating a salutary role of tmTNF-α. Treatment of 3T3-L1 adipocytes with exogenous tmTNF-α promoted insulin-induced phosphorylation of IRS-1 and Akt, facilitated GLUT4 expression and membrane translocation, and increased glucose uptake while addition of sTNF-α resulted in the opposite effect. Furthermore, tmTNF-α downregulated the production of IL-6 and MCP-1 via NF-κB inactivation, as silencing of A20, an inhibitor for NF-κB, by siRNA, abolished this effect of tmTNF-α. However, tmTNF-α upregulated adiponectin expression through the PPAR-γ pathway, as inhibition of PPAR-γ by GW9662 abrogated both tmTNF-α-induced adiponectin transcription and glucose uptake. Our data suggest that tmTNF-α functions as an insulin sensitizer via forward signaling.

  12. A functional-phylogenetic classification system for transmembrane solute transporters.

    PubMed

    Saier, M H

    2000-06-01

    A comprehensive classification system for transmembrane molecular transporters has been developed and recently approved by the transport panel of the nomenclature committee of the International Union of Biochemistry and Molecular Biology. This system is based on (i) transporter class and subclass (mode of transport and energy coupling mechanism), (ii) protein phylogenetic family and subfamily, and (iii) substrate specificity. Almost all of the more than 250 identified families of transporters include members that function exclusively in transport. Channels (115 families), secondary active transporters (uniporters, symporters, and antiporters) (78 families), primary active transporters (23 families), group translocators (6 families), and transport proteins of ill-defined function or of unknown mechanism (51 families) constitute distinct categories. Transport mode and energy coupling prove to be relatively immutable characteristics and therefore provide primary bases for classification. Phylogenetic grouping reflects structure, function, mechanism, and often substrate specificity and therefore provides a reliable secondary basis for classification. Substrate specificity and polarity of transport prove to be more readily altered during evolutionary history and therefore provide a tertiary basis for classification. With very few exceptions, a phylogenetic family of transporters includes members that function by a single transport mode and energy coupling mechanism, although a variety of substrates may be transported, sometimes with either inwardly or outwardly directed polarity. In this review, I provide cross-referencing of well-characterized constituent transporters according to (i) transport mode, (ii) energy coupling mechanism, (iii) phylogenetic grouping, and (iv) substrates transported. The structural features and distribution of recognized family members throughout the living world are also evaluated. The tabulations should facilitate familial and functional

  13. Mechanisms of hop inhibition include the transmembrane redox reaction.

    PubMed

    Behr, Jürgen; Vogel, Rudi F

    2010-01-01

    In this work, a novel mechanistic model of hop inhibition beyond the proton ionophore action toward (beer spoiling) bacteria was developed. Investigations were performed with model systems using cyclic voltammetry for the determination of redox processes/conditions in connection with growth challenges with hop-sensitive and -resistant Lactobacillus brevis strains in the presence of oxidants. Cyclic voltammetry identified a transmembrane redox reaction of hop compounds at low pH (common in beer) and in the presence of manganese (present in millimolar levels in lactic acid bacteria). The antibacterial action of hop compounds could be extended from the described proton ionophore activity, lowering the intracellular pH, to pronounced redox reactivity, causing cellular oxidative damage. Accordingly, a correlation between the resistance of L. brevis strains to a sole oxidant to their resistance to hop could not be expected and was not detected. However, in connection with our recent study concerning hop ionophore properties and the resistance of hop-sensitive and -tolerant L. brevis strains toward proton ionophores (J. Behr and R. F. Vogel, J. Agric. Food Chem. 57:6074-6081, 2009), we suggest that both ionophore and oxidant resistance are required for survival under hop stress conditions and confirmed this correlation according to the novel mechanistic model. In consequence, the expression of several published hop resistance mechanisms involved in manganese binding/transport and intracellular redox balance, as well as that of proteins involved in oxidative stress under "highly reducing" conditions (cf. anaerobic cultivation and "antioxidative" hop compounds in the growth medium), is now comprehensible. Accordingly, hop resistance as a multifactorial dynamic property at least implies distinct resistance levels against two different mechanisms of hop inhibition, namely, proton ionophore-induced and oxidative stress-induced mechanisms. Beyond this specific model of hop

  14. Using "Arabidopsis" Genetic Sequences to Teach Bioinformatics

    ERIC Educational Resources Information Center

    Zhang, Xiaorong

    2009-01-01

    This article describes a new approach to teaching bioinformatics using "Arabidopsis" genetic sequences. Several open-ended and inquiry-based laboratory exercises have been designed to help students grasp key concepts and gain practical skills in bioinformatics, using "Arabidopsis" leucine-rich repeat receptor-like kinase (LRR RLK) genetic…

  15. An International Bioinformatics Infrastructure to Underpin the Arabidopsis Community

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The future bioinformatics needs of the Arabidopsis community as well as those of other scientific communities that depend on Arabidopsis resources were discussed at a pair of recent meetings held by the Multinational Arabidopsis Steering Committee (MASC) and the North American Arabidopsis Steering C...

  16. Bidirectional Transformation of a Metamorphic Protein between the Water-Soluble and Transmembrane Native States.

    PubMed

    Tanaka, Koji; Caaveiro, Jose M M; Tsumoto, Kouhei

    2015-11-24

    The bidirectional transformation of a protein between its native water-soluble and integral transmembrane conformations is demonstrated for FraC, a hemolytic protein of the family of pore-forming toxins. In the presence of biological membranes, the water-soluble conformation of FraC undergoes a remarkable structural reorganization generating cytolytic transmembrane nanopores conducive to cell death. So far, the reverse transformation from the native transmembrane conformation to the native water-soluble conformation has not been reported. We describe the use of detergents with different physicochemical properties to achieve the spontaneous conversion of transmembrane pores of FraC back into the initial water-soluble state. Thermodynamic and kinetic stability data suggest that specific detergents cause an asymmetric change in the energy landscape of the protein, allowing the bidirectional transformation of a membrane protein.

  17. Transmembrane chemokines act as receptors in a novel mechanism termed inverse signaling

    PubMed Central

    Hattermann, Kirsten; Gebhardt, Henrike; Krossa, Sebastian; Ludwig, Andreas; Lucius, Ralph

    2016-01-01

    The transmembrane chemokines CX3CL1/fractalkine and CXCL16 are widely expressed in different types of tumors, often without an appropriate expression of their classical receptors. We observed that receptor-negative cancer cells could be stimulated by the soluble chemokines. Searching for alternative receptors we detected that all cells expressing or transfected with transmembrane chemokine ligands bound the soluble chemokines with high affinity and responded by phosphorylation of intracellular kinases, enhanced proliferation and anti-apoptosis. This activity requires the intracellular domain and apparently the dimerization of the transmembrane chemokine ligand. Thus, shed soluble chemokines can generate auto- or paracrine signals by binding and activating their transmembrane forms. We term this novel mechanism “inverse signaling”. We suppose that inverse signaling is an autocrine feedback and fine-tuning system in the communication between cells that in tumors supports stabilization and proliferation. DOI: http://dx.doi.org/10.7554/eLife.10820.001 PMID:26796342

  18. Modulation of Innate Immune Signalling by Lipid-Mediated MAVS Transmembrane Domain Oligomerization

    PubMed Central

    Ron, David; Volmer, Romain

    2015-01-01

    RIG-I-like receptors detect viral RNA in infected cells and promote oligomerization of the outer mitochondrial membrane protein MAVS to induce innate immunity to viral infection through type I interferon production. Mitochondrial reactive oxygen species (mROS) have been shown to enhance anti-viral MAVS signalling, but the mechanisms have remained obscure. Using a biochemical oligomerization-reporter fused to the transmembrane domain of MAVS, we found that mROS inducers promoted lipid-dependent MAVS transmembrane domain oligomerization in the plane of the outer mitochondrial membrane. These events were mirrored by Sendai virus infection, which similarly induced lipid peroxidation and promoted lipid-dependent MAVS transmembrane domain oligomerization. Our observations point to a role for mROS-induced changes in lipid bilayer properties in modulating antiviral innate signalling by favouring the oligomerization of MAVS transmembrane domain in the outer-mitochondrial membrane. PMID:26317833

  19. Highly effective yet simple transmembrane anion transporters based upon ortho-phenylenediamine bis-ureas.

    PubMed

    Karagiannidis, Louise E; Haynes, Cally J E; Holder, Katie J; Kirby, Isabelle L; Moore, Stephen J; Wells, Neil J; Gale, Philip A

    2014-10-18

    Simple, highly fluorinated receptors are shown to function as highly effective transmembrane anion antiporters with the most active transporters rivalling the transport efficacy of natural anion transporter prodigiosin for bicarbonate.

  20. Transmembrane Signaling by the Aspartate Receptor: Engineered Disulfides Reveal Static Regions of the Subunit Interface†

    PubMed Central

    Chervitz, Stephen A.; Lin, Christina M.; Falke, Joseph J.

    2010-01-01

    Ligand binding to the periplasmic domain of the transmembrane aspartate receptor generates an intramolecular conformational change which spans the bilayer and ultimately signals the cytoplasmic CheA histidine kinase, thereby triggering chemotaxis. The receptor is a homodimer stabilized by the interface between its two identical subunits: the present study investigates the role of the periplasmic and transmembrane regions of this interface in the mechanism of transmembrane signaling. Free cysteines and disulfide bonds are engineered into selected interfacial positions, and the resulting effects on the transmembrane signal are assayed by monitoring in vitro regulation of kinase activity. Three of the 14 engineered cysteine pairs examined, as well as six of the 14 engineered disulfides, cause perturbations of the interface structure which essentially destroy transmembrane regulation of the kinase. The remaining 11 cysteine pairs, and eight engineered disulfides covalently linking the two subunits at locations spanning positions 18–75, are observed to retain significant transmembrane kinase regulation. The eight functional disulfides positively identify adjacent faces of the two N-terminal helices in the native receptor dimer and indicate that large regions of the periplasmic and transmembrane subunit interface remain effectively static during the transmembrane signal. The results are consistent with a model in which the subunit interface plays a structural role, while the second membrane-spanning helix transmits the ligand-induced signal across the bilayer to the kinase binding domain. The effects of engineered cysteines and disulfides on receptor methylation in vitro are also measured, enabling direct comparison of the in vitro methylation and phosphorylation assays. PMID:7626643

  1. TMRPres2D: high quality visual representation of transmembrane protein models.

    PubMed

    Spyropoulos, Ioannis C; Liakopoulos, Theodore D; Bagos, Pantelis G; Hamodrakas, Stavros J

    2004-11-22

    The 'TransMembrane protein Re-Presentation in 2-Dimensions' (TMRPres2D) tool, automates the creation of uniform, two-dimensional, high analysis graphical images/models of alpha-helical or beta-barrel transmembrane proteins. Protein sequence data and structural information may be acquired from public protein knowledge bases, emanate from prediction algorithms, or even be defined by the user. Several important biological and physical sequence attributes can be embedded in the graphical representation. PMID:15201184

  2. A graph-theoretic approach for classification and structure prediction of transmembrane β-barrel proteins

    PubMed Central

    2012-01-01

    Background Transmembrane β-barrel proteins are a special class of transmembrane proteins which play several key roles in human body and diseases. Due to experimental difficulties, the number of transmembrane β-barrel proteins with known structures is very small. Over the years, a number of learning-based methods have been introduced for recognition and structure prediction of transmembrane β-barrel proteins. Most of these methods emphasize on homology search rather than any biological or chemical basis. Results We present a novel graph-theoretic model for classification and structure prediction of transmembrane β-barrel proteins. This model folds proteins based on energy minimization rather than a homology search, avoiding any assumption on availability of training dataset. The ab initio model presented in this paper is the first method to allow for permutations in the structure of transmembrane proteins and provides more structural information than any known algorithm. The model is also able to recognize β-barrels by assessing the pseudo free energy. We assess the structure prediction on 41 proteins gathered from existing databases on experimentally validated transmembrane β-barrel proteins. We show that our approach is quite accurate with over 90% F-score on strands and over 74% F-score on residues. The results are comparable to other algorithms suggesting that our pseudo-energy model is close to the actual physical model. We test our classification approach and show that it is able to reject α-helical bundles with 100% accuracy and β-barrel lipocalins with 97% accuracy. Conclusions We show that it is possible to design models for classification and structure prediction for transmembrane β-barrel proteins which do not depend essentially on training sets but on combinatorial properties of the structures to be proved. These models are fairly accurate, robust and can be run very efficiently on PC-like computers. Such models are useful for the genome

  3. Ion fluxes, transmembrane potential, and osmotic stabilization: a new dynamic electrophysiological model for eukaryotic cells.

    PubMed

    Poignard, Clair; Silve, Aude; Campion, Frederic; Mir, Lluis M; Saut, Olivier; Schwartz, Laurent

    2011-03-01

    Survival of mammalian cells is achieved by tight control of cell volume, while transmembrane potential has been known to control many cellular functions since the seminal work of Hodgkin and Huxley. Regulation of cell volume and transmembrane potential have a wide range of implications in physiology, from neurological and cardiac disorders to cancer and muscle fatigue. Therefore, understanding the relationship between transmembrane potential, ion fluxes, and cell volume regulation has become of great interest. In this paper we derive a system of differential equations that links transmembrane potential, ionic concentrations, and cell volume. In particular, we describe the dynamics of the cell within a few seconds after an osmotic stress, which cannot be done by the previous models in which either cell volume was constant or osmotic regulation instantaneous. This new model demonstrates that both membrane potential and cell volume stabilization occur within tens of seconds of changes in extracellular osmotic pressure. When the extracellular osmotic pressure is constant, the cell volume varies as a function of transmembrane potential and ion fluxes, thus providing an implicit link between transmembrane potential and cell volume. Experimental data provide results that corroborate the numerical simulations of the model in terms of time-related changes in cell volume and dynamics of the phenomena. This paper can be seen as a generalization of previous electrophysiological results, since under restrictive conditions they can be derived from our model. PMID:21079946

  4. How are exons encoding transmembrane sequences distributed in the exon-intron structure of genes?

    PubMed

    Sawada, Ryusuke; Mitaku, Shigeki

    2011-01-01

    The exon-intron structure of eukaryotic genes raises a question about the distribution of transmembrane regions in membrane proteins. Were exons that encode transmembrane regions formed simply by inserting introns into preexisting genes or by some kind of exon shuffling? To answer this question, the exon-per-gene distribution was analyzed for all genes in 40 eukaryotic genomes with a particular focus on exons encoding transmembrane segments. In 21 higher multicellular eukaryotes, the percentage of multi-exon genes (those containing at least one intron) within all genes in a genome was high (>70%) and with a mean of 87%. When genes were grouped by the number of exons per gene in higher eukaryotes, good exponential distributions were obtained not only for all genes but also for the exons encoding transmembrane segments, leading to a constant ratio of membrane proteins independent of the exon-per-gene number. The positional distribution of transmembrane regions in single-pass membrane proteins showed that they are generally located in the amino or carboxyl terminal regions. This nonrandom distribution of transmembrane regions explains the constant ratio of membrane proteins to the exon-per-gene numbers because there are always two terminal (i.e., the amino and carboxyl) regions - independent of the length of sequences.

  5. ERAD of proteins containing aberrant transmembrane domains requires ubiquitylation of cytoplasmic lysine residues

    PubMed Central

    Briant, Kit; Koay, Yee-Hui; Otsuka, Yuka; Swanton, Eileithyia

    2015-01-01

    ABSTRACT Clearance of misfolded proteins from the endoplasmic reticulum (ER) is mediated by the ubiquitin-proteasome system in a process known as ER-associated degradation (ERAD). The mechanisms through which proteins containing aberrant transmembrane domains are degraded by ERAD are poorly understood. To address this question, we generated model ERAD substrates based on CD8 with either a non-native transmembrane domain but a folded ER luminal domain (CD8TMD*), or the native transmembrane domain but a misfolded luminal domain (CD8LUM*). Although both chimeras were degraded by ERAD, we found that the location of the folding defect determined the initial site of ubiquitylation. Ubiquitylation of cytoplasmic lysine residues was required for the extraction of CD8TMD* from the ER membrane during ERAD, whereas CD8LUM* continued to be degraded in the absence of cytoplasmic lysine residues. Cytoplasmic lysine residues were also required for degradation of an additional ERAD substrate containing an unassembled transmembrane domain and when a non-native transmembrane domain was introduced into CD8LUM*. Our results suggest that proteins with defective transmembrane domains are removed from the ER through a specific ERAD mechanism that depends upon ubiquitylation of cytoplasmic lysine residues. PMID:26446255

  6. The pore architecture of the cystic fibrosis transmembrane conductance regulator channel revealed by co-mutation in pore-forming transmembrane regions.

    PubMed

    Qian, F; Liu, L; Liu, Z; Lu, C

    2016-07-18

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel contains 12 transmembrane (TM) regions that are presumed to form the channel pore. However, there is no direct evidence clearly illustrating the involvement of these transmembrane regions in the actual CFTR pore structure. To obtain insight into the architecture of the CFTR channel pore, we used patch clamp recording techniques and a strategy of co-mutagenesis of two potential pore-forming transmembrane regions (TM1 and TM6) to investigate the collaboration of these two TM regions. We performed a range of specific functional assays comparing the single channel conductance, anion binding, and anion selectivity properties of the co-mutated CFTR variants, and the results indicated that TM1 and TM6 play vital roles in forming the channel pore and, thus, determine the functional properties of the channel. Furthermore, we provided functional evidence that the amino acid threonine (T338) in TM6 has synergic effects with lysine (K95) in TM1. Therefore, we propose that these two residues have functional collaboration in the CFTR channel pore and may collectively form a selective filter. PMID:27070741

  7. Expression of the ZNT1 Zinc Transporter from the Metal Hyperaccumulator Noccaea caerulescens Confers Enhanced Zinc and Cadmium Tolerance and Accumulation to Arabidopsis thaliana.

    PubMed

    Lin, Ya-Fen; Hassan, Zeshan; Talukdar, Sangita; Schat, Henk; Aarts, Mark G M

    2016-01-01

    Prompt regulation of transition metal transporters is crucial for plant zinc homeostasis. NcZNT1 is one of such transporters, found in the metal hyperaccumulator Brassicaceae species Noccaea caerulescens. It is orthologous to AtZIP4 from Arabidopsis thaliana, an important actor in Zn homeostasis. We examined if the NcZNT1 function contributes to the metal hyperaccumulation of N. caerulescens. NcZNT1 was found to be a plasma-membrane located metal transporter. Constitutive overexpression of NcZNT1 in A. thaliana conferred enhanced tolerance to exposure to excess Zn and Cd supply, as well as increased accumulation of Zn and Cd and induction of the Fe deficiency response, when compared to non-transformed wild-type plants. Promoters of both genes were induced by Zn deficiency in roots and shoots of A. thaliana. In A. thaliana, the AtZIP4 and NcZNT1 promoters were mainly active in cortex, endodermis and pericycle cells under Zn deficient conditions. In N. caerulescens, the promoters were active in the same tissues, though the activity of the NcZNT1 promoter was higher and not limited to Zn deficient conditions. Common cis elements were identified in both promoters by 5' deletion analysis. These correspond to the previously determined Zinc Deficiency Responsive Elements found in A. thaliana to interact with two redundantly acting transcription factors, bZIP19 and bZIP23, controlling the Zn deficiency response. In conclusion, these results suggest that NcZNT1 is an important factor in contributing to Zn and Cd hyperaccumulation in N. caerulescens. Differences in cis- and trans-regulators are likely to account for the differences in expression between A. thaliana and N. caerulescens. The high, constitutive NcZNT1 expression in the stele of N. caerulescens roots implicates its involvement in long distance root-to-shoot metal transport by maintaining a Zn/Cd influx into cells responsible for xylem loading. PMID:26930473

  8. Expression of the ZNT1 Zinc Transporter from the Metal Hyperaccumulator Noccaea caerulescens Confers Enhanced Zinc and Cadmium Tolerance and Accumulation to Arabidopsis thaliana

    PubMed Central

    Schat, Henk; Aarts, Mark G. M.

    2016-01-01

    Prompt regulation of transition metal transporters is crucial for plant zinc homeostasis. NcZNT1 is one of such transporters, found in the metal hyperaccumulator Brassicaceae species Noccaea caerulescens. It is orthologous to AtZIP4 from Arabidopsis thaliana, an important actor in Zn homeostasis. We examined if the NcZNT1 function contributes to the metal hyperaccumulation of N. caerulescens. NcZNT1 was found to be a plasma-membrane located metal transporter. Constitutive overexpression of NcZNT1 in A. thaliana conferred enhanced tolerance to exposure to excess Zn and Cd supply, as well as increased accumulation of Zn and Cd and induction of the Fe deficiency response, when compared to non-transformed wild-type plants. Promoters of both genes were induced by Zn deficiency in roots and shoots of A. thaliana. In A. thaliana, the AtZIP4 and NcZNT1 promoters were mainly active in cortex, endodermis and pericycle cells under Zn deficient conditions. In N. caerulescens, the promoters were active in the same tissues, though the activity of the NcZNT1 promoter was higher and not limited to Zn deficient conditions. Common cis elements were identified in both promoters by 5’ deletion analysis. These correspond to the previously determined Zinc Deficiency Responsive Elements found in A. thaliana to interact with two redundantly acting transcription factors, bZIP19 and bZIP23, controlling the Zn deficiency response. In conclusion, these results suggest that NcZNT1 is an important factor in contributing to Zn and Cd hyperaccumulation in N. caerulescens. Differences in cis- and trans-regulators are likely to account for the differences in expression between A. thaliana and N. caerulescens. The high, constitutive NcZNT1 expression in the stele of N. caerulescens roots implicates its involvement in long distance root-to-shoot metal transport by maintaining a Zn/Cd influx into cells responsible for xylem loading. PMID:26930473

  9. Distinct Amino Acids in the C-Linker Domain of the Arabidopsis K+ Channel KAT2 Determine Its Subcellular Localization and Activity at the Plasma Membrane1[W

    PubMed Central

    Nieves-Cordones, Manuel; Chavanieu, Alain; Jeanguenin, Linda; Alcon, Carine; Szponarski, Wojciech; Estaran, Sebastien; Chérel, Isabelle; Zimmermann, Sabine; Sentenac, Hervé; Gaillard, Isabelle

    2014-01-01

    Shaker K+ channels form the major K+ conductance of the plasma membrane in plants. They are composed of four subunits arranged around a central ion-conducting pore. The intracellular carboxy-terminal region of each subunit contains several regulatory elements, including a C-linker region and a cyclic nucleotide-binding domain (CNBD). The C-linker is the first domain present downstream of the sixth transmembrane segment and connects the CNBD to the transmembrane core. With the aim of identifying the role of the C-linker in the Shaker channel properties, we performed subdomain swapping between the C-linker of two Arabidopsis (Arabidopsis thaliana) Shaker subunits, K+ channel in Arabidopsis thaliana2 (KAT2) and Arabidopsis thaliana K+ rectifying channel1 (AtKC1). These two subunits contribute to K+ transport in planta by forming heteromeric channels with other Shaker subunits. However, they display contrasting behavior when expressed in tobacco mesophyll protoplasts: KAT2 forms homotetrameric channels active at the plasma membrane, whereas AtKC1 is retained in the endoplasmic reticulum when expressed alone. The resulting chimeric/mutated constructs were analyzed for subcellular localization and functionally characterized. We identified two contiguous amino acids, valine-381 and serine-382, located in the C-linker carboxy-terminal end, which prevent KAT2 surface expression when mutated into the equivalent residues from AtKC1. Moreover, we demonstrated that the nine-amino acid stretch 312TVRAASEFA320 that composes the first C-linker α-helix located just below the pore is a crucial determinant of KAT2 channel activity. A KAT2 C-linker/CNBD three-dimensional model, based on animal HCN (for Hyperpolarization-activated, cyclic nucleotide-gated K+) channels as structure templates, has been built and used to discuss the role of the C-linker in plant Shaker inward channel structure and function. PMID:24406792

  10. Taxonomy and Phylogeny of Arabidopsis (Brassicaceae)

    PubMed Central

    Al-Shehbaz, Ihsan A.; O'Kane, Steve L.

    2002-01-01

    Detailed taxonomic, cytological, and phylogenetic accounts of Arabidopsis are presented. As currently delimited, the genus consists of nine species all of which are indigenous to Europe, with the ranges of two species extending into northern and eastern Asia and North American into central United States. A survey of chromosome numbers in the genus is presented, and the country of origin for each count is given. Detailed descriptions of all species and subspecies and keys to all taxa are provided. Generic assignments are updated for the 50 species previously included in Arabidopsis. A cladogram of the species of Arabidopsis based on molecular phylogenetic studies by the authors is given. PMID:22303187

  11. Sulfenome mining in Arabidopsis thaliana

    PubMed Central

    Waszczak, Cezary; Akter, Salma; Eeckhout, Dominique; Persiau, Geert; Wahni, Khadija; Bodra, Nandita; Van Molle, Inge; De Smet, Barbara; Vertommen, Didier; Gevaert, Kris; De Jaeger, Geert; Van Montagu, Marc; Messens, Joris; Van Breusegem, Frank

    2014-01-01

    Reactive oxygen species (ROS) have been shown to be potent signaling molecules. Today, oxidation of cysteine residues is a well-recognized posttranslational protein modification, but the signaling processes steered by such oxidations are poorly understood. To gain insight into the cysteine thiol-dependent ROS signaling in Arabidopsis thaliana, we identified the hydrogen peroxide (H2O2)-dependent sulfenome: that is, proteins with at least one cysteine thiol oxidized to a sulfenic acid. By means of a genetic construct consisting of a fusion between the C-terminal domain of the yeast (Saccharomyces cerevisiae) AP-1–like (YAP1) transcription factor and a tandem affinity purification tag, we detected ∼100 sulfenylated proteins in Arabidopsis cell suspensions exposed to H2O2 stress. The in vivo YAP1-based trapping of sulfenylated proteins was validated by a targeted in vitro analysis of DEHYDROASCORBATE REDUCTASE2 (DHAR2). In DHAR2, the active site nucleophilic cysteine is regulated through a sulfenic acid-dependent switch, leading to S-glutathionylation, a protein modification that protects the protein against oxidative damage. PMID:25049418

  12. Polyploidy in the Arabidopsis genus.

    PubMed

    Bomblies, Kirsten; Madlung, Andreas

    2014-06-01

    Whole genome duplication (WGD), which gives rise to polyploids, is a unique type of mutation that duplicates all the genetic material in a genome. WGD provides an evolutionary opportunity by generating abundant genetic "raw material," and has been implicated in diversification, speciation, adaptive radiation, and invasiveness, and has also played an important role in crop breeding. However, WGD at least initially challenges basic biological functions by increasing cell size, altering relationships between cell volume and DNA content, and doubling the number of homologous chromosome copies that must be sorted during cell division. Newly polyploid lineages often have extensive changes in gene regulation, genome structure, and may suffer meiotic or mitotic chromosome mis-segregation. The abundance of species that persist in nature as polyploids shows that these problems are surmountable and/or that advantages of WGD might outweigh drawbacks. The molecularly especially tractable Arabidopsis genus has several ancient polyploidy events in its history and contains several independent more recent polyploids. This genus can thus provide important insights into molecular aspects of polyploid formation, establishment, and genome evolution. The ability to integrate ecological and evolutionary questions with molecular and genetic understanding makes comparative analyses in this genus particularly attractive and holds promise for advancing our general understanding of polyploid biology. Here, we highlight some of the findings from Arabidopsis that have given us insights into the origin and evolution of polyploids. PMID:24788061

  13. UV-induced DNA damage promotes resistance to the biotrophic pathogen Hyaloperonospora parasitica in Arabidopsis.

    PubMed

    Kunz, Bernard A; Dando, Paige K; Grice, Desma M; Mohr, Peter G; Schenk, Peer M; Cahill, David M

    2008-10-01

    Plant innate immunity to pathogenic microorganisms is activated in response to recognition of extracellular or intracellular pathogen molecules by transmembrane receptors or resistance proteins, respectively. The defense signaling pathways share components with those involved in plant responses to UV radiation, which can induce expression of plant genes important for pathogen resistance. Such intriguing links suggest that UV treatment might activate resistance to pathogens in normally susceptible host plants. Here, we demonstrate that pre-inoculative UV (254 nm) irradiation of Arabidopsis (Arabidopsis thaliana) susceptible to infection by the biotrophic oomycete Hyaloperonospora parasitica, the causative agent of downy mildew, induces dose- and time-dependent resistance to the pathogen detectable up to 7 d after UV exposure. Limiting repair of UV photoproducts by postirradiation incubation in the dark, or mutational inactivation of cyclobutane pyrimidine dimer photolyase, (6-4) photoproduct photolyase, or nucleotide excision repair increased the magnitude of UV-induced pathogen resistance. In the absence of treatment with 254-nm UV, plant nucleotide excision repair mutants also defective for cyclobutane pyrimidine dimer or (6-4) photoproduct photolyase displayed resistance to H. parasitica, partially attributable to short wavelength UV-B (280-320 nm) radiation emitted by incubator lights. These results indicate UV irradiation can initiate the development of resistance to H. parasitica in plants normally susceptible to the pathogen and point to a key role for UV-induced DNA damage. They also suggest UV treatment can circumvent the requirement for recognition of H. parasitica molecules by Arabidopsis proteins to activate an immune response.

  14. Subcellular Localization of Arabidopsis 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase1

    PubMed Central

    Leivar, Pablo; González, Víctor M.; Castel, Susanna; Trelease, Richard N.; López-Iglesias, Carmen; Arró, Montserrat; Boronat, Albert; Campos, Narciso; Ferrer, Albert; Fernàndez-Busquets, Xavier

    2005-01-01

    Plants produce diverse isoprenoids, which are synthesized in plastids, mitochondria, endoplasmic reticulum (ER), and the nonorganellar cytoplasm. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate, a rate-limiting step in the cytoplasmic pathway. Several branches of the pathway lead to the synthesis of structurally and functionally varied, yet essential, isoprenoids. Several HMGR isoforms have been identified in all plants examined. Studies based on gene expression and on fractionation of enzyme activity suggested that subcellular compartmentalization of HMGR is an important intracellular channeling mechanism for the production of the specific classes of isoprenoids. Plant HMGR has been shown previously to insert in vitro into the membrane of microsomal vesicles, but the final in vivo subcellular localization(s) remains controversial. To address the latter in Arabidopsis (Arabidopsis thaliana) cells, we conducted a multipronged microscopy and cell fractionation approach that included imaging of chimeric HMGR green fluorescent protein localizations in transiently transformed cell leaves, immunofluorescence confocal microscopy in wild-type and stably transformed seedlings, immunogold electron microscopy examinations of endogenous HMGR in seedling cotyledons, and sucrose density gradient analyses of HMGR-containing organelles. Taken together, the results reveal that endogenous Arabidopsis HMGR is localized at steady state within ER as expected, but surprisingly also predominantly within spherical, vesicular structures that range from 0.2- to 0.6-μm diameter, located in the cytoplasm and within the central vacuole in differentiated cotyledon cells. The N-terminal region, including the transmembrane domain of HMGR, was found to be necessary and sufficient for directing HMGR to ER and the spherical structures. It is believed, although not directly demonstrated, that these vesicle-like structures are derived from segments of HMGR

  15. Intermolecular Headgroup Interaction and Hydration as Driving Forces for Lipid Transmembrane Asymmetry.

    PubMed

    Smolentsev, Nikolay; Lütgebaucks, Cornelis; Okur, Halil I; de Beer, Alex G F; Roke, Sylvie

    2016-03-30

    Variations between the inner and outer leaflets of cell membranes are crucial for cell functioning and signaling, drug-membrane interactions, and the formation of lipid domains. Transmembrane asymmetry can in principle be comprised of an asymmetric charge distribution, differences in hydration, specific headgroup/H-bonding interactions, or a difference in the number of lipids per leaflet. Here, we characterize the transmembrane asymmetry of small unilamellar liposomes consisting of zwitterionic and charged lipids in aqueous solution using vibrational sum frequency scattering and second harmonic scattering, label-free methods, specifically sensitive to lipid and water asymmetries. For single component liposomes, transmembrane asymmetry is present for the charge distribution and lipid hydration, but the leaflets are not detectably asymmetric in terms of the number of lipids per leaflet, even though geometrical packing arguments would predict so. Such a lipid transmembrane asymmetry can, however, be induced in binary lipid mixtures under conditions that enable H-bonding interactions between phosphate and amine groups. In this case, the measured asymmetry consists of a different number of lipids in the outer and inner leaflet, a difference in transmembrane headgroup hydration, and a different headgroup orientation for the interacting phosphate groups.

  16. Intrinsic Disorder in Transmembrane Proteins: Roles in Signaling and Topology Prediction

    PubMed Central

    Bürgi, Jérôme; Xue, Bin; Uversky, Vladimir N.

    2016-01-01

    Intrinsically disordered regions (IDRs) are peculiar stretches of amino acids that lack stable conformations in solution. Intrinsic Disorder containing Proteins (IDP) are defined by the presence of at least one large IDR and have been linked to multiple cellular processes including cell signaling, DNA binding and cancer. Here we used computational analyses and publicly available databases to deepen insight into the prevalence and function of IDRs specifically in transmembrane proteins, which are somewhat neglected in most studies. We found that 50% of transmembrane proteins have at least one IDR of 30 amino acids or more. Interestingly, these domains preferentially localize to the cytoplasmic side especially of multi-pass transmembrane proteins, suggesting that disorder prediction could increase the confidence of topology prediction algorithms. This was supported by the successful prediction of the topology of the uncharacterized multi-pass transmembrane protein TMEM117, as confirmed experimentally. Pathway analysis indicated that IDPs are enriched in cell projection and axons and appear to play an important role in cell adhesion, signaling and ion binding. In addition, we found that IDP are enriched in phosphorylation sites, a crucial post translational modification in signal transduction, when compared to fully ordered proteins and to be implicated in more protein-protein interaction events. Accordingly, IDPs were highly enriched in short protein binding regions called Molecular Recognition Features (MoRFs). Altogether our analyses strongly support the notion that the transmembrane IDPs act as hubs in cellular signal events. PMID:27391701

  17. The fifth international conference on Arabidopsis research

    SciTech Connect

    Hangarter, R.; Scholl, R.; Davis, K.; Feldmann, K.

    1993-12-31

    This volume contains abstracts of oral and poster presentations made in conjunction with the Fifth International Conference on Arabidopsis Research held August 19--22, 1993 at the Ohio State University, Columbus, Ohio.

  18. Cytological analysis of Arabidopsis thaliana meiotic chromosomes.

    PubMed

    Armstrong, Susan J; Sanchez-Moran, Eugenio; Franklin, F Chris H

    2009-01-01

    Advances in molecular biology and in the genetics of Arabidopsis thaliana have led to this organism becoming an important model for the analysis of meiosis in plants. Cytogenetic investigations are pivotal to meiotic studies and a number of technological improvements for Arabidopsis cytology have provided a range of tools to investigate chromosome behaviour during meiosis. This chapter includes protocols on basic cytology, FISH analysis, immunocytology, a procedure for a meiotic time course and electron microscopy.

  19. Autoinhibition of a calmodulin-dependent calcium pump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain

    NASA Technical Reports Server (NTRS)

    Curran, A. C.; Hwang, I.; Corbin, J.; Martinez, S.; Rayle, D.; Sze, H.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    2000-01-01

    The regulation of Ca(2+)-pumps is important for controlling [Ca(2+)] in the cytosol and organelles of all eukaryotes. Here, we report a genetic strategy to identify residues that function in autoinhibition of a novel calmodulin-activated Ca(2+)-pump with an N-terminal regulatory domain (isoform ACA2 from Arabidopsis). Mutant pumps with constitutive activity were identified by complementation of a yeast (K616) deficient in two Ca(2+)-pumps. Fifteen mutations were found that disrupted a segment of the N-terminal autoinhibitor located between Lys(23) and Arg(54). Three mutations (E167K, D219N, and E341K) were found associated with the stalk that connects the ATPase catalytic domain (head) and with the transmembrane domain. Enzyme assays indicated that the stalk mutations resulted in calmodulin-independent activity, with V(max), K(mATP), and K(mCa(2+)) similar to that of a pump in which the N-terminal autoinhibitor had been deleted. A highly conservative substitution at Asp(219) (D219E) still produced a deregulated pump, indicating that the autoinhibitory structure in the stalk is highly sensitive to perturbation. In plasma membrane H(+)-ATPases from yeast and plants, similarly positioned mutations resulted in hyperactive pumps. Together, these results suggest that a structural feature of the stalk is of general importance in regulating diverse P-type ATPases.

  20. Interactions between Transmembrane Helices within Monomers of the Aquaporin AtPIP2;1 Play a Crucial Role in Tetramer Formation.

    PubMed

    Yoo, Yun-Joo; Lee, Hyun Kyung; Han, Wonhee; Kim, Dae Heon; Lee, Myoung Hui; Jeon, Jouhyun; Lee, Dong Wook; Lee, Junho; Lee, Yongjik; Lee, Juhun; Kim, Jin Seok; Cho, Yunje; Han, Jin-Kwan; Hwang, Inhwan

    2016-07-01

    Aquaporin (AQP) is a water channel protein found in various subcellular membranes of both prokaryotic and eukaryotic cells. The physiological functions of AQPs have been elucidated in many organisms. However, understanding their biogenesis remains elusive, particularly regarding how they assemble into tetramers. Here, we investigated the amino acid residues involved in the tetramer formation of the Arabidopsis plasma membrane AQP AtPIP2;1 using extensive amino acid substitution mutagenesis. The mutant proteins V41A/E44A, F51A/L52A, F87A/I91A, F92A/I93A, V95A/Y96A, and H216A/L217A, harboring alanine substitutions in the transmembrane (TM) helices of AtPIP2;1 polymerized into multiple oligomeric complexes with a variable number of subunits greater than four. Moreover, these mutant proteins failed to traffic to the plasma membrane, instead of accumulating in the endoplasmic reticulum (ER). Structure-based modeling revealed that these residues are largely involved in interactions between TM helices within monomers. These results suggest that inter-TM interactions occurring both within and between monomers play crucial roles in tetramer formation in the AtPIP2;1 complex. Moreover, the assembly of AtPIP2;1 tetramers is critical for their trafficking from the ER to the plasma membrane, as well as water permeability. PMID:27142778

  1. Transmembrane Photoredox in Model Protocellular Systems. Polycyclic Aromatic Hydrocarbons as Plausible Light-harvesting/Electron Transfer Catalysts

    NASA Astrophysics Data System (ADS)

    Cape, J. L.; Monnard, P.-A.; Ziock, H.-J.; Boncella, J. M.

    2010-04-01

    Mechanistic studies of transmembrane photoredox in a model protocell system indicate a plausible role for membrane solublized PAH species as primitive ‘photosynthetic' energy transduction catalysts.

  2. The Transmembrane Domain C of AMPA Receptors is Critically Involved in Receptor Function and Modulation

    PubMed Central

    Terhag, Jan; Gottschling, Kevin; Hollmann, Michael

    2010-01-01

    Ionotropic glutamate receptors are major players in synaptic transmission and are critically involved in many cognitive events. Although receptors of different subfamilies serve different functions, they all show a conserved domain topology. For most of these domains, structure–function relationships have been established and are well understood. However, up to date the role of the transmembrane domain C in receptor function has been investigated only poorly. We have constructed a series of receptor chimeras and point mutants designed to shed light on the structural and/or functional importance of this domain. We here present evidence that the role of transmembrane domain C exceeds that of a mere scaffolding domain and that several amino acid residues located within the domain are crucial for receptor gating and desensitization. Furthermore, our data suggest that the domain may be involved in receptor interaction with transmembrane AMPA receptor regulatory proteins. PMID:21206529

  3. Interaction of influenza virus haemagglutinin with sphingolipid-cholesterol membrane domains via its transmembrane domain.

    PubMed Central

    Scheiffele, P; Roth, M G; Simons, K

    1997-01-01

    Sphingolipid-cholesterol rafts are microdomains in biological membranes with liquid-ordered phase properties which are implicated in membrane traffic and signalling events. We have used influenza virus haemagglutinin (HA) as a model protein to analyse the interaction of transmembrane proteins with these microdomains. Here we demonstrate that raft association is an intrinsic property encoded in the protein. Mutant HA molecules with foreign transmembrane domain (TMD) sequences lose their ability to associate with the lipid microdomains, and mutations in the HA TMD reveal a requirement for hydrophobic residues in contact with the exoplasmic leaflet of the membrane. We also provide experimental evidence that cholesterol is critically required for association of proteins with lipid rafts. Our data suggest that the binding to specific membrane domains can be encoded in transmembrane proteins and that this information will be used for polarized sorting and signal transduction processes. PMID:9312009

  4. Photosensitized electron transport across lipid vesicle walls: enhancement of quantum yield by ionophores and transmembrane potentials

    SciTech Connect

    Laane, C.; Ford, W.E.; Otvos, J.W.; Calvin, M.

    1981-04-01

    The photosensitized reduction of heptylviologen in the bulk aqueous phase of phosphatidylcholine vesicles containing EDTA inside and a membrane-bound tris(2,2'-bipyridine)ruthenium(2+) derivative is enhanced by a factor of 6.5 by the addition of valinomycin in the presence of K/sup +/. A 3-fold stimulation by gramicidin and carbonyl cyanide m-chlorophenylhydrazone is observed. The results suggest that, under these conditions, the rate of photoinduced electron transfer across vesicle walls in the absence of ion carriers is limited by cotransport of cations. The rate of electron transfer across vesicle walls could be influenced further by generating transmembrane potentials with K/sup +/ gradients inatthe presence of valinomycin. When vesicles are made with transmembrane potentials, interior more negative, the quantum yield of heptylviologen reduction is doubled, and, converseley, when vesicles are made with transmembrane potentials, interior more positive, the quantum yield is decreased and approaches the value found in the absence of valinomycin.

  5. Apoplastic diffusion barriers in Arabidopsis.

    PubMed

    Nawrath, Christiane; Schreiber, Lukas; Franke, Rochus Benni; Geldner, Niko; Reina-Pinto, José J; Kunst, Ljerka

    2013-12-27

    During the development of Arabidopsis and other land plants, diffusion barriers are formed in the apoplast of specialized tissues within a variety of plant organs. While the cuticle of the epidermis is the primary diffusion barrier in the shoot, the Casparian strips and suberin lamellae of the endodermis and the periderm represent the diffusion barriers in the root. Different classes of molecules contribute to the formation of extracellular diffusion barriers in an organ- and tissue-specific manner. Cutin and wax are the major components of the cuticle, lignin forms the early Casparian strip, and suberin is deposited in the stage II endodermis and the periderm. The current status of our understanding of the relationships between the chemical structure, ultrastructure and physiological functions of plant diffusion barriers is discussed. Specific aspects of the synthesis of diffusion barrier components and protocols that can be used for the assessment of barrier function and important barrier properties are also presented.

  6. Apoplastic Diffusion Barriers in Arabidopsis

    PubMed Central

    Schreiber, Lukas; Franke, Rochus Benni; Geldner, Niko; Reina-Pinto, José J.; Kunst, Ljerka

    2013-01-01

    During the development of Arabidopsis and other land plants, diffusion barriers are formed in the apoplast of specialized tissues within a variety of plant organs. While the cuticle of the epidermis is the primary diffusion barrier in the shoot, the Casparian strips and suberin lamellae of the endodermis and the periderm represent the diffusion barriers in the root. Different classes of molecules contribute to the formation of extracellular diffusion barriers in an organ- and tissue-specific manner. Cutin and wax are the major components of the cuticle, lignin forms the early Casparian strip, and suberin is deposited in the stage II endodermis and the periderm. The current status of our understanding of the relationships between the chemical structure, ultrastructure and physiological functions of plant diffusion barriers is discussed. Specific aspects of the synthesis of diffusion barrier components and protocols that can be used for the assessment of barrier function and important barrier properties are also presented. PMID:24465172

  7. Identification of Arabidopsis rat Mutants

    PubMed Central

    Zhu, Yanmin; Nam, Jaesung; Humara, Jaime M.; Mysore, Kirankumar S.; Lee, Lan-Ying; Cao, Hongbin; Valentine, Lisa; Li, Jingling; Kaiser, Anthony D.; Kopecky, Andrea L.; Hwang, Hau-Hsuan; Bhattacharjee, Saikat; Rao, Praveen K.; Tzfira, Tzvi; Rajagopal, Jyothi; Yi, HoChul; Veena; Yadav, Badam S.; Crane, Yan M.; Lin, Kui; Larcher, Yves; Gelvin, Matthew J.K.; Knue, Marnie; Ramos, Cynthia; Zhao, Xiaowen; Davis, Susan J.; Kim, Sang-Ic; Ranjith-Kumar, C.T.; Choi, Yoo-Jin; Hallan, Vipin K.; Chattopadhyay, Sudip; Sui, Xiangzhen; Ziemienowicz, Alicja; Matthysse, Ann G.; Citovsky, Vitaly; Hohn, Barbara; Gelvin, Stanton B.

    2003-01-01

    Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various “reverse genetic” approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants. PMID:12805582

  8. The Arabidopsis metacaspase9 degradome.

    PubMed

    Tsiatsiani, Liana; Timmerman, Evy; De Bock, Pieter-Jan; Vercammen, Dominique; Stael, Simon; van de Cotte, Brigitte; Staes, An; Goethals, Marc; Beunens, Tine; Van Damme, Petra; Gevaert, Kris; Van Breusegem, Frank

    2013-08-01

    Metacaspases are distant relatives of the metazoan caspases, found in plants, fungi, and protists. However, in contrast with caspases, information about the physiological substrates of metacaspases is still scarce. By means of N-terminal combined fractional diagonal chromatography, the physiological substrates of metacaspase9 (MC9; AT5G04200) were identified in young seedlings of Arabidopsis thaliana on the proteome-wide level, providing additional insight into MC9 cleavage specificity and revealing a previously unknown preference for acidic residues at the substrate prime site position P1'. The functionalities of the identified MC9 substrates hinted at metacaspase functions other than those related to cell death. These results allowed us to resolve the substrate specificity of MC9 in more detail and indicated that the activity of phosphoenolpyruvate carboxykinase 1 (AT4G37870), a key enzyme in gluconeogenesis, is enhanced upon MC9-dependent proteolysis.

  9. Tetrapyrrole Metabolism in Arabidopsis thaliana

    PubMed Central

    Tanaka, Ryouichi; Kobayashi, Koichi; Masuda, Tatsuru

    2011-01-01

    Higher plants produce four classes of tetrapyrroles, namely, chlorophyll (Chl), heme, siroheme, and phytochromobilin. In plants, tetrapyrroles play essential roles in a wide range of biological activities including photosynthesis, respiration and the assimilation of nitrogen/sulfur. All four classes of tetrapyrroles are derived from a common biosynthetic pathway that resides in the plastid. In this article, we present an overview of tetrapyrrole metabolism in Arabidopsis and other higher plants, and we describe all identified enzymatic steps involved in this metabolism. We also summarize recent findings on Chl biosynthesis and Chl breakdown. Recent advances in this field, in particular those on the genetic and biochemical analyses of novel enzymes, prompted us to redraw the tetrapyrrole metabolic pathways. In addition, we also summarize our current understanding on the regulatory mechanisms governing tetrapyrrole metabolism. The interactions of tetrapyrrole biosynthesis and other cellular processes including the plastid-to-nucleus signal transduction are discussed. PMID:22303270

  10. Structural and dynamic study of the transmembrane domain of the amyloid precursor protein.

    PubMed

    Nadezhdin, K D; Bocharova, O V; Bocharov, E V; Arseniev, A S

    2011-01-01

    Alzheimer's disease affects people all over the world, regardless of nationality, gender or social status. An adequate study of the disease requires essential understanding of the molecular fundamentals of the pathogenesis. The amyloid β-peptide, which forms amyloid plaques in the brain of people with Alzheimer's disease, is the product of sequential cleavage of a single-span membrane amyloid precursor protein (APP). More than half of the APP mutations found to be associated with familial forms of Alzheimer's disease are located in its transmembrane domain. The pathogenic mutations presumably affect the structural-dynamic properties of the APP transmembrane domain by changing its conformational stability and/or lateral dimerization. In the present study, the structure and dynamics of the recombinant peptide corresponding to the APP fragment, Gln686-Lys726, which comprises the APP transmembrane domain with an adjacent N-terminal juxtamembrane sequence, were determined in the membrane mimetic environment composed of detergent micelles using NMR spectroscopy. The structure obtained in dodecylphosphocholine micelles consists of two α-helices: a short surface-associated juxtamembrane helix (Lys687-Asp694) and a long transmembrane helix (Gly700-Leu723), both connected via a mobile loop region. A minor bend of the transmembrane α-helix is observed near the paired residues Gly708-Gly709. A cholesterol-binding hydrophobic cavity is apparently formed under the loop region, where the juxtamembrane α-helix comes into contact with the membrane surface near the N-terminus of the transmembrane α-helix. PMID:22649674

  11. Hydrophobic Mismatch Drives the Interaction of E5 with the Transmembrane Segment of PDGF Receptor

    PubMed Central

    Windisch, Dirk; Ziegler, Colin; Grage, Stephan L.; Bürck, Jochen; Zeitler, Marcel; Gor’kov, Peter L.; Ulrich, Anne S.

    2015-01-01

    The oncogenic E5 protein from bovine papillomavirus is a short (44 amino acids long) integral membrane protein that forms homodimers. It activates platelet-derived growth factor receptor (PDGFR) β in a ligand-independent manner by transmembrane helix-helix interactions. The nature of this recognition event remains elusive, as numerous mutations are tolerated in the E5 transmembrane segment, with the exception of one hydrogen-bonding residue. Here, we examined the conformation, stability, and alignment of the E5 protein in fluid lipid membranes of substantially varying bilayer thickness, in both the absence and presence of the PDGFR transmembrane segment. Quantitative synchrotron radiation circular dichroism analysis revealed a very long transmembrane helix for E5 of ∼26 amino acids. Oriented circular dichroism and solid-state 15N-NMR showed that the alignment and stability of this unusually long segment depend critically on the membrane thickness. When reconstituted alone in exceptionally thick DNPC lipid bilayers, the E5 helix was found to be inserted almost upright. In moderately thick bilayers (DErPC and DEiPC), it started to tilt and became slightly deformed, and finally it became aggregated in conventional DOPC, POPC, and DMPC membranes due to hydrophobic mismatch. On the other hand, when E5 was co-reconstituted with the transmembrane segment of PDGFR, it was able to tolerate even the most pronounced mismatch and was stabilized by binding to the receptor, which has the same hydrophobic length. As E5 is known to activate PDGFR within the thin membranes of the Golgi compartment, we suggest that the intrinsic hydrophobic mismatch of these two interaction partners drives them together. They seem to recognize each other by forming a closely packed bundle of mutually aligned transmembrane helices, which is further stabilized by a specific pair of hydrogen-bonding residues. PMID:26287626

  12. The Homology Model of PMP22 Suggests Mutations Resulting in Peripheral Neuropathy Disrupt Transmembrane Helix Packing

    PubMed Central

    2015-01-01

    Peripheral myelin protein 22 (PMP22) is a tetraspan membrane protein strongly expressed in myelinating Schwann cells of the peripheral nervous system. Myriad missense mutations in PMP22 result in varying degrees of peripheral neuropathy. We used Rosetta 3.5 to generate a homology model of PMP22 based on the recently published crystal structure of claudin-15. The model suggests that several mutations known to result in neuropathy act by disrupting transmembrane helix packing interactions. Our model also supports suggestions from previous studies that the first transmembrane helix is not tightly associated with the rest of the helical bundle. PMID:25243937

  13. Common Extracellular Sensory Domains in Transmembrane Receptors for Diverse Signal Transduction Pathways in Bacteria and Archaea

    PubMed Central

    Zhulin, Igor B.; Nikolskaya, Anastasia N.; Galperin, Michael Y.

    2003-01-01

    Transmembrane receptors in microorganisms, such as sensory histidine kinases and methyl-accepting chemotaxis proteins, are molecular devices for monitoring environmental changes. We report here that sensory domain sharing is widespread among different classes of transmembrane receptors. We have identified two novel conserved extracellular sensory domains, named CHASE2 and CHASE3, that are found in at least four classes of transmembrane receptors: histidine kinases, adenylate cyclases, predicted diguanylate cyclases, and either serine/threonine protein kinases (CHASE2) or methyl-accepting chemotaxis proteins (CHASE3). Three other extracellular sensory domains were shared by at least two different classes of transmembrane receptors: histidine kinases and either diguanylate cyclases, adenylate cyclases, or phosphodiesterases. These observations suggest that microorganisms use similar conserved domains to sense similar environmental signals and transmit this information via different signal transduction pathways to different regulatory circuits: transcriptional regulation (histidine kinases), chemotaxis (methyl-accepting proteins), catabolite repression (adenylate cyclases), and modulation of enzyme activity (diguanylate cyclases and phosphodiesterases). The variety of signaling pathways using the CHASE-type domains indicates that these domains sense some critically important extracellular signals. PMID:12486065

  14. Molecular basis of transmembrane beta-barrel formation of staphylococcal pore-forming toxins.

    PubMed

    Yamashita, Daichi; Sugawara, Takaki; Takeshita, Miyu; Kaneko, Jun; Kamio, Yoshiyuki; Tanaka, Isao; Tanaka, Yoshikazu; Yao, Min

    2014-01-01

    Pathogenic bacteria secrete pore-forming toxins (PFTs) to attack target cells. PFTs are expressed as water-soluble monomeric proteins, which oligomerize into nonlytic prepore intermediates on the target cell membrane before forming membrane-spanning pores. Despite a wealth of biochemical data, the lack of high-resolution prepore structural information has hampered understanding of the β-barrel formation process. Here, we report crystal structures of staphylococcal γ-haemolysin and leucocidin prepores. The structures reveal a disordered bottom half of the β-barrel corresponding to the transmembrane region, and a rigid upper extramembrane half. Spectroscopic analysis of fluorescently labelled mutants confirmed that the prepore is distinct from the pore within the transmembrane region. Mutational analysis also indicates a pivotal role for the glycine residue located at the lipid-solvent interface as a 'joint' between the two halves of the β-barrel. These observations suggest a two-step transmembrane β-barrel pore formation mechanism in which the upper extramembrane and bottom transmembrane regions are formed independently. PMID:25263813

  15. Detergent properties influence the stability of the glycophorin A transmembrane helix dimer in lysophosphatidylcholine micelles.

    PubMed

    Stangl, Michael; Veerappan, Anbazhagan; Kroeger, Anja; Vogel, Peter; Schneider, Dirk

    2012-12-19

    Detergents might affect membrane protein structures by promoting intramolecular interactions that are different from those found in native membrane bilayers, and fine-tuning detergent properties can be crucial for obtaining structural information of intact and functional transmembrane proteins. To systematically investigate the influence of the detergent concentration and acyl-chain length on the stability of a transmembrane protein structure, the stability of the human glycophorin A transmembrane helix dimer has been analyzed in lyso-phosphatidylcholine micelles of different acyl-chain length. While our results indicate that the transmembrane protein is destabilized in detergents with increasing chain-length, the diameter of the hydrophobic micelle core was found to be less crucial. Thus, hydrophobic mismatch appears to be less important in detergent micelles than in lipid bilayers and individual detergent molecules appear to be able to stretch within a micelle to match the hydrophobic thickness of the peptide. However, the stability of the GpA TM helix dimer linearly depends on the aggregation number of the lyso-PC detergents, indicating that not only is the chemistry of the detergent headgroup and acyl-chain region central for classifying a detergent as harsh or mild, but the detergent aggregation number might also be important.

  16. Comparison between clinical indicators of transmembrane oxygenator thrombosis and multidetector computed tomographic analysis.

    PubMed

    Panigada, Mauro; L'Acqua, Camilla; Passamonti, Serena Maria; Mietto, Cristina; Protti, Alessandro; Riva, Roberto; Gattinoni, Luciano

    2015-04-01

    This study aims to assess whether multidetector computed tomography (MDCT) could accurately confirm the clinical suspicion of transmembrane oxygenator thrombosis (MOT) during extracorporeal membrane oxygenation (ECMO). Twenty-seven oxygenators were examined using MDCT at the end of patient treatment. Transmembrane oxygenator thrombosis was suspected in 15 of them according to the presence of at least 2 of the following clinical indicators: (1) increase in d-dimer, (2) decrease in platelet count, (3) decrease in oxygenator performance, and (4) presence of clots on the surface of the oxygenator. Transmembrane oxygenator thrombosis was confirmed by MDCT in 5 (33%) of them. Transmembrane oxygenator thrombosis was unexpectedly found in 5 (41%) of the remaining 12 oxygenators not suspected for MOT. Eight (80%) of these oxygenators had clots accounting for less than 1% of total volume. Clots were mainly detectable at the apical corner of the oxygenator, most likely due to greater blood stasis. We found a significant increase in d-dimer and in membrane oxygenator shunt and a decrease in platelet count from the start to the discontinuation of ECMO. Hemostatic abnormalities significantly reverted 48 hours after oxygenator removal, suggesting the role of ECMO in activation of the coagulation cascade. Multidetector computed tomographic scan could not accurately confirm the clinical suspicion of MOT.

  17. A mutation in the first transmembrane domain of the lutropin receptor causes male precocious puberty.

    PubMed

    Gromoll, J; Partsch, C J; Simoni, M; Nordhoff, V; Sippell, W G; Nieschlag, E; Saxena, B B

    1998-02-01

    We describe a patient with onset of puberty at the age of 5 yr. characterized by accelerated growth, enlargement of genitalia, pubarche, and serum hormone levels compatible with noncentral precocious puberty. Exon 11 of the LH receptor gene was amplified from genomic DNA by PCR and directly sequenced. We identified a heterozygous C to T base change at nucleotide position 1126, exchanging codon 373 from Ala to Val in the first transmembrane domain. The LH receptor sequence of the parents was normal. The mutated receptor displayed an up to 7.5-fold increase in basal cAMP production compared to that of the wild-type receptor in transiently transfected COS-7 cells. Treatment of the patient with ketoconazole resulted in inconsistent suppression of serum testosterone levels. At the age of 9.1 yr, central activation of the hypothalamic-pituitary-gonadal axis occurred. Additional treatment with a GnRH agonist led to complete suppression of testosterone secretion. This is the first description of constitutive activation of the LH receptor in the first transmembrane segment. It suggests the involvement of the first transmembrane helix in signal transduction and provides further insight into the structural organization of the seven transmembrane domains of the glycoprotein hormone receptor proteins.

  18. Variations in transmembrane Ca2+ gradient and apoptosis of macrophages induced by oxidized low density lipoprotein.

    PubMed

    Yang, X; Zhang, Y; Huang, Y; Yang, F

    2001-10-01

    While Ca2+ has been proposed to be a messenger in OxLDL-induced cell death, few studies have addressed the possibility that it may influence the occurrence of apoptosis and necrosis of macrophages induced by OxLDL in virtue of change of transmembrane Ca2+ gradient including that across plasma membrane and intracellular organelle membranes. In this paper, various lipophilic Ca2+ fluorescent indicators and specific organelle markers were used to study the relationship between the changes of the transmembrane Ca2+ gradients and the OxLDL induced apoptosis of macrophages. Our results showed that following exposure of low dose OxLDL to macrophages, the transmembrane Ca2+ gradient across the plasma membrane, as well as the membrane-proximal Ca2+ gradient, the transnuclear, and the transmitochondrial membrane Ca2+ gradient were all changed significantly. These data suggested that changes in transmembrane Ca2+ gradients might be involved in the apoptosis of macrophages induced by OxLDL.

  19. A quantitative model for using acridine orange as a transmembrane pH gradient probe.

    PubMed

    Clerc, S; Barenholz, Y

    1998-05-15

    Monitoring the acidification of the internal space of membrane vesicles by proton pumps can be achieved easily with optical probes. Transmembrane pH gradients cause a blue-shift in the absorbance spectrum and the quenching of the fluorescence of the cationic dye acridine orange. It has been postulated that these changes are caused by accumulation and aggregation of the dye inside the vesicles. We tested this hypothesis using liposomes with transmembrane concentration gradients of ammonium sulfate as model system. Fluorescence intensity of acridine orange solutions incubated with liposomes was affected by magnitude of the gradient, volume trapped by vesicles, and temperature. These experimental data were compared to a theoretical model describing the accumulation of acridine orange monomers in the vesicles according to the inside-to-outside ratio of proton concentrations, and the intravesicular formation of sandwich-like piles of acridine orange cations. This theoretical model predicted quantitatively the relationship between the transmembrane pH gradients and spectral changes of acridine orange. Therefore, adequate characterization of aggregation of dye in the lumen of biological vesicles provides the theoretical basis for using acridine orange as an optical probe to quantify transmembrane pH gradients.

  20. Theoretical analyses of cellular transmembrane voltage in suspensions induced by high-frequency fields.

    PubMed

    Zou, Yong; Wang, Changzhen; Peng, Ruiyun; Wang, Lifeng; Hu, Xiangjun

    2015-04-01

    A change of the transmembrane voltage is considered to cause biophysical and biochemical responses in cells. The present study focuses on the cellular transmembrane voltage (Δφ) induced by external fields. We detail analytical equations for the transmembrane voltage induced by external high-frequency (above the relaxation frequency of the cell membrane) fields on cells of a spherical shape in suspensions and layers. At direct current (DC) and low frequencies, the cell membrane was assumed to be non-conductive under physiologic conditions. However, with increasing frequency, the permittivity of the cytoplasm/extracellular medium and conductivity of the membrane must be accounted for. Our main work is to extend application of the analytical solution of Δφ to the high-frequency range. We first introduce the transmembrane voltage generated by DC and low-frequency exposures on a single cell. Then, we focus on cell suspensions exposed to high-frequency fields. Using the effective medium theory and the reasonable assumption, the approximate analytical solution of Δφ on cells in suspensions and layers can be derived. Phenomenological effective medium theory equations cannot be used to calculate the local electric field of cell suspensions, so we raised a possible solution based on the Bergman theory.

  1. Terpene Specialized Metabolism in Arabidopsis thaliana

    PubMed Central

    Tholl, Dorothea; Lee, Sungbeom

    2011-01-01

    Terpenes constitute the largest class of plant secondary (or specialized) metabolites, which are compounds of ecological function in plant defense or the attraction of beneficial organisms. Using biochemical and genetic approaches, nearly all Arabidopsis thaliana (Arabidopsis) enzymes of the core biosynthetic pathways producing the 5-carbon building blocks of terpenes have been characterized and closer insight has been gained into the transcriptional and posttranscriptional/translational mechanisms regulating these pathways. The biochemical function of most prenyltransferases, the downstream enzymes that condense the C5-precursors into central 10-, 15-, and 20-carbon prenyldiphosphate intermediates, has been described, although the function of several isoforms of C20-prenyltranferases is not well understood. Prenyl diphosphates are converted to a variety of C10-, C15-, and C20-terpene products by enzymes of the terpene synthase (TPS) family. Genomic organization of the 32 Arabidopsis TPS genes indicates a species-specific divergence of terpene synthases with tissue- and cell-type specific expression profiles that may have emerged under selection pressures by different organisms. Pseudogenization, differential expression, and subcellular segregation of TPS genes and enzymes contribute to the natural variation of terpene biosynthesis among Arabidopsis accessions (ecotypes) and species. Arabidopsis will remain an important model to investigate the metabolic organization and molecular regulatory networks of terpene specialized metabolism in relation to the biological activities of terpenes. PMID:22303268

  2. A fluorescent reporter protein containing AtRMR1 domains is targeted to the storage and central vacuoles in Arabidopsis thaliana and tobacco leaf cells.

    PubMed

    Scabone, Camila María; Frigerio, Lorenzo; Petruccelli, Silvana

    2011-10-01

    To develop a new strategy to target recombinant proteins to the vacuolar storage system in transgenic plants, the ability of the transmembrane and cytosolic domains of Arabidopsis receptor homology-transmembrane-RING H2-1 (AtRMR1) was evaluated. A secreted version of RFP (secRFP) and a fusion of it to the transmembrane and cytosolic domains of AtRMR1 (RFP-TMCT) were produced and studied both in transient and stable expression assays. Transient expression in leaves of Nicotiana tabacum showed that secRFP is secreted to the apoplast while its fusion to TMCT of AtRMR1 is sufficient to prevent secretion of the reporter. In tobacco leaves, RFP-TMCT reporter showed an endoplasmic reticulum pattern in early expression stages while in late expression stages, it was found in the vacuolar lumen. For the first time, the role of TM and CT domains of AtRMR1 in stable expression in Arabidopsis thaliana is presented; the fusion of TMCT to secRFP is sufficient to sort RFP to the lumen of the central vacuoles in leaves and roots and to the lumen of PSV in cotyledons of mature embryos. In addition, biochemical studies performed in extract from transgenic plants showed that RFP-TMCT is an integral membrane protein. Full-length RFP-TMCT was also found in the vacuolar lumen, suggesting internalization into destination vacuole. Not colocalization of RFP-TMCT with tonoplast and plasma membrane markers were observed. This membrane vacuolar determinant sorting signal could be used for future application in molecular pharming as an alternative means to sort proteins of interest to vacuoles.

  3. A fluorescent reporter protein containing AtRMR1 domains is targeted to the storage and central vacuoles in Arabidopsis thaliana and tobacco leaf cells.

    PubMed

    Scabone, Camila María; Frigerio, Lorenzo; Petruccelli, Silvana

    2011-10-01

    To develop a new strategy to target recombinant proteins to the vacuolar storage system in transgenic plants, the ability of the transmembrane and cytosolic domains of Arabidopsis receptor homology-transmembrane-RING H2-1 (AtRMR1) was evaluated. A secreted version of RFP (secRFP) and a fusion of it to the transmembrane and cytosolic domains of AtRMR1 (RFP-TMCT) were produced and studied both in transient and stable expression assays. Transient expression in leaves of Nicotiana tabacum showed that secRFP is secreted to the apoplast while its fusion to TMCT of AtRMR1 is sufficient to prevent secretion of the reporter. In tobacco leaves, RFP-TMCT reporter showed an endoplasmic reticulum pattern in early expression stages while in late expression stages, it was found in the vacuolar lumen. For the first time, the role of TM and CT domains of AtRMR1 in stable expression in Arabidopsis thaliana is presented; the fusion of TMCT to secRFP is sufficient to sort RFP to the lumen of the central vacuoles in leaves and roots and to the lumen of PSV in cotyledons of mature embryos. In addition, biochemical studies performed in extract from transgenic plants showed that RFP-TMCT is an integral membrane protein. Full-length RFP-TMCT was also found in the vacuolar lumen, suggesting internalization into destination vacuole. Not colocalization of RFP-TMCT with tonoplast and plasma membrane markers were observed. This membrane vacuolar determinant sorting signal could be used for future application in molecular pharming as an alternative means to sort proteins of interest to vacuoles. PMID:21611741

  4. Structural and Functional Characterization of the C-terminal Transmembrane Region of NBCe1-A*

    PubMed Central

    Zhu, Quansheng; Kao, Liyo; Azimov, Rustam; Abuladze, Natalia; Newman, Debra; Pushkin, Alexander; Liu, Weixin; Chang, Connie; Kurtz, Ira

    2010-01-01

    NBCe1-A and AE1 both belong to the SLC4 HCO3− transporter family. The two transporters share 40% sequence homology in the C-terminal transmembrane region. In this study, we performed extensive substituted cysteine-scanning mutagenesis analysis of the C-terminal region of NBCe1-A covering amino acids Ala800–Lys967. Location of the introduced cysteines was determined by whole cell labeling with a membrane-permeant biotin maleimide and a membrane-impermeant 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) cysteine-reactive reagent. The results show that the extracellular surface of the NBCe1-A C-terminal transmembrane region is minimally exposed to aqueous media with Met858 accessible to both biotin maleimide and TAMRA and Thr926–Ala929 only to TAMRA labeling. The intracellular surface contains a highly exposed (Met813–Gly828) region and a cryptic (Met887–Arg904) connecting loop. The lipid/aqueous interface of the last transmembrane segment is at Asp960. Our data clearly determined that the C terminus of NBCe1-A contains 5 transmembrane segments with greater average size compared with AE1. Functional assays revealed only two residues in the region of Pro868–Leu967 (a functionally important region in AE1) that are highly sensitive to cysteine substitution. Our findings suggest that the C-terminal transmembrane region of NBCe1-A is tightly folded with unique structural and functional features that differ from AE1. PMID:20837482

  5. BRX promotes Arabidopsis shoot growth.

    PubMed

    Beuchat, Julien; Scacchi, Emanuele; Tarkowska, Danuse; Ragni, Laura; Strnad, Miroslav; Hardtke, Christian S

    2010-10-01

    • BREVIS RADIX (BRX) has been identified through a loss-of-function allele in the Umkirch-1 accession in a natural variation screen for Arabidopsis root growth vigor. Physiological and gene expression analyses have suggested that BRX is rate limiting for auxin-responsive gene expression by mediating cross-talk with the brassinosteroid pathway, as impaired root growth and reduced auxin perception of brx can be (partially) rescued by external brassinosteroid application. • Using genetic tools, we show that brx mutants also display significantly reduced cotyledon and leaf growth. • Similar to the root, the amplitude and penetrance of this phenotype depends on genetic background and shares the physiological features, reduced auxin perception and brassinosteroid rescue. Furthermore, reciprocal grafting experiments between mutant and complemented brx shoot scions and root stocks suggest that the shoot phenotypes are not an indirect consequence of the root phenotype. Finally, BRX gain-of-function lines display epinastic leaf growth and, in the case of dominant negative interference, increased epidermal cell size. Consistent with an impact of BRX on brassinosteroid biosynthesis, this phenotype is accompanied by increased brassinosteroid levels. • In summary, our results demonstrate a ubiquitous, although quantitatively variable role of BRX in modulating the growth rate in both the root and shoot.

  6. Arabidopsis Chitinases: a Genomic Survey

    PubMed Central

    Passarinho, Paul A.; de Vries, Sacco C.

    2002-01-01

    Plant chitinases (EC 3.2.1.14) belong to relatively large gene families subdivided in classes that suggest class-specific functions. They are commonly induced upon the attack of pathogens and by various sources of stress, which led to associating them with plant defense in general. However, it is becoming apparent that most of them display several functions during the plant life cycle, including taking part in developmental processes such as pollination and embryo development. The number of chitinases combined with their multiple functions has been an obstacle to a better understanding of their role in plants. It is therefore important to identify and inventory all chitinase genes of a plant species to be able to dissect their function and understand the relations between the different classes. Complete sequencing of the Arabidopsis genome has made this task feasible and we present here a survey of all putative chitinase-encoding genes accompanied by a detailed analysis of their sequence. Based on their characteristics and on studies on other plant chitinases, we propose an overview of their possible functions as well as modified annotations for some of them. PMID:22303199

  7. Early flower development in Arabidopsis.

    PubMed Central

    Smyth, D R; Bowman, J L; Meyerowitz, E M

    1990-01-01

    The early development of the flower of Arabidopsis thaliana is described from initiation until the opening of the bud. The morphogenesis, growth rate, and surface structure of floral organs were recorded in detail using scanning electron microscopy. Flower development has been divided into 12 stages using a series of landmark events. Stage 1 begins with the initiation of a floral buttress on the flank of the apical meristem. Stage 2 commences when the flower primordium becomes separate from the meristem. Sepal primordia then arise (stage 3) and grow to overlie the primordium (stage 4). Petal and stamen primordia appear next (stage 5) and are soon enclosed by the sepals (stage 6). During stage 6, petal primordia grow slowly, whereas stamen primordia enlarge more rapidly. Stage 7 begins when the medial stamens become stalked. These soon develop locules (stage 8). A long stage 9 then commences with the petal primordia becoming stalked. During this stage all organs lengthen rapidly. This includes the gynoecium, which commences growth as an open-ended tube during stage 6. When the petals reach the length of the lateral stamens, stage 10 begins. Stigmatic papillae appear soon after (stage 11), and the petals rapidly reach the height of the medial stamens (stage 12). This final stage ends when the 1-millimeter-long bud opens. Under our growing conditions 1.9 buds were initiated per day on average, and they took 13.25 days to progress through the 12 stages from initiation until opening. PMID:2152125

  8. Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.

    PubMed

    Wei, Shipeng; Roessler, Bryan C; Chauvet, Sylvain; Guo, Jingyu; Hartman, John L; Kirk, Kevin L

    2014-07-18

    ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs.

  9. Wheat Brassinosteroid-Insensitive1 (TaBRI1) Interacts with Members of TaSERK Gene Family and Cause Early Flowering and Seed Yield Enhancement in Arabidopsis

    PubMed Central

    Singh, Akanksha; Breja, Priyanka; Khurana, Jitendra P.; Khurana, Paramjit

    2016-01-01

    Brassinosteroids (BRs) hormones are important for plant growth, development and immune responses. They are sensed by the transmembrane receptor kinase Brassinosteroid-Insensitive 1 (BRI1) when they bind to its extracellular Leu-rich repeat (LRR) domain. We cloned and characterized the TaBRI1 from T. aestivum and raised overexpression transgenics in Arabidopsis to decipher its functional role. TaBRI1 protein consists of a putative signal peptide followed by 25 leucine rich repeats (LRR), a transmembrane domain and a C-terminal kinase domain. The analysis determined the interaction of TaBRI1 with five members of the wheat Somatic Embryogenesis Receptor Kinase (TaSERKs) gene family (TaSERK1, TaSERK2, TaSERK3, TaSERK4 and TaSERK5), at the plasma membrane. Furthermore, overexpression of TaBRI1 in Arabidopsis leads to the early flowering, increased silique size and seed yield. Root growth analysis of TaBRI1 overexpressing transgenic plants showed hypersensitivity to epi-brassinolide (epi-BL) hormone in a dose-dependent manner. Interestingly, transgenic Arabidopsis plants show thermotolerance phenotype at the seedling stages as revealed by chlorophyll content, photosystem II activity and membrane stability. The transcriptome profiling on the basis of microarray analysis indicates up-regulation of several genes related to brassinosteroid signaling pathway, abiotic stress response, defense response and transcription factors. These studies predict the possible role of TaBRI1 gene in plant growth and development imparting tolerance to thermal stress. PMID:27322749

  10. An Overdose of the Arabidopsis Coreceptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 or Its Ectodomain Causes Autoimmunity in a SUPPRESSOR OF BIR1-1-Dependent Manner.

    PubMed

    Domínguez-Ferreras, Ana; Kiss-Papp, Marta; Jehle, Anna Kristina; Felix, Georg; Chinchilla, Delphine

    2015-07-01

    The membrane-bound Brassinosteroid insensitive1-associated receptor kinase1 (BAK1) is a common coreceptor in plants and regulates distinct cellular programs ranging from growth and development to defense against pathogens. BAK1 functions through binding to ligand-stimulated transmembrane receptors and activating their kinase domains via transphosphorylation. In the absence of microbes, BAK1 activity may be suppressed by different mechanisms, like interaction with the regulatory BIR (for BAK1-interacting receptor-like kinase) proteins. Here, we demonstrated that BAK1 overexpression in Arabidopsis (Arabidopsis thaliana) could cause detrimental effects on plant development, including growth arrest, leaf necrosis, and reduced seed production. Further analysis using an inducible expression system showed that BAK1 accumulation quickly stimulated immune responses, even under axenic conditions, and led to increased resistance to pathogenic Pseudomonas syringae pv tomato DC3000. Intriguingly, our study also revealed that the plasma membrane-associated BAK1 ectodomain was sufficient to induce autoimmunity, indicating a novel mode of action for BAK1 in immunity control. We postulate that an excess of BAK1 or its ectodomain could trigger immune receptor activation in the absence of microbes through unbalancing regulatory interactions, including those with BIRs. Consistently, mutation of suppressor of BIR1-1, which encodes an emerging positive regulator of transmembrane receptors in plants, suppressed the effects of BAK1 overexpression. In conclusion, our findings unravel a new role for the BAK1 ectodomain in the tight regulation of Arabidopsis immune receptors necessary to avoid inappropriate activation of immunity. PMID:25944825

  11. Wheat Brassinosteroid-Insensitive1 (TaBRI1) Interacts with Members of TaSERK Gene Family and Cause Early Flowering and Seed Yield Enhancement in Arabidopsis.

    PubMed

    Singh, Akanksha; Breja, Priyanka; Khurana, Jitendra P; Khurana, Paramjit

    2016-01-01

    Brassinosteroids (BRs) hormones are important for plant growth, development and immune responses. They are sensed by the transmembrane receptor kinase Brassinosteroid-Insensitive 1 (BRI1) when they bind to its extracellular Leu-rich repeat (LRR) domain. We cloned and characterized the TaBRI1 from T. aestivum and raised overexpression transgenics in Arabidopsis to decipher its functional role. TaBRI1 protein consists of a putative signal peptide followed by 25 leucine rich repeats (LRR), a transmembrane domain and a C-terminal kinase domain. The analysis determined the interaction of TaBRI1 with five members of the wheat Somatic Embryogenesis Receptor Kinase (TaSERKs) gene family (TaSERK1, TaSERK2, TaSERK3, TaSERK4 and TaSERK5), at the plasma membrane. Furthermore, overexpression of TaBRI1 in Arabidopsis leads to the early flowering, increased silique size and seed yield. Root growth analysis of TaBRI1 overexpressing transgenic plants showed hypersensitivity to epi-brassinolide (epi-BL) hormone in a dose-dependent manner. Interestingly, transgenic Arabidopsis plants show thermotolerance phenotype at the seedling stages as revealed by chlorophyll content, photosystem II activity and membrane stability. The transcriptome profiling on the basis of microarray analysis indicates up-regulation of several genes related to brassinosteroid signaling pathway, abiotic stress response, defense response and transcription factors. These studies predict the possible role of TaBRI1 gene in plant growth and development imparting tolerance to thermal stress. PMID:27322749

  12. Proteins are polyisoprenylated in Arabidopsis thaliana.

    PubMed

    Gutkowska, Malgorzata; Bieńkowski, Tomasz; Hung, Vo Si; Wanke, Malgorzata; Hertel, Jozefina; Danikiewicz, Witold; Swiezewska, Ewa

    2004-09-24

    Isoprenoid lipids were found to be covalently linked to proteins of Arabidopsis thaliana. Their identity (polyprenols: Prenol-9-11 with Pren-10 dominating and dolichols: Dol-15-17 with Dol-16 dominating) was confirmed by means of HPLC/ESI-MS with application of the multiple reaction monitoring technique as well as metabolic labeling of Arabidopsis plants with [(3)H]mevalonate and other precursors. The occurrence of typical farnesol-, geranylgeraniol-, and phytol-modified proteins was also noted. Radioisotopic labeling allowed detection of several proteins that were covalently bound to mevalonate-derived isoprenoid alcohols. A significant portion of polyisoprenylated proteins was recovered in the cytosolic/light vesicular fraction of Arabidopsis cells upon subfractionation. Taken together our data prove that a subset of plant proteins is polyisoprenylated.

  13. Characterization of StABF1, a stress-responsive bZIP transcription factor from Solanum tuberosum L. that is phosphorylated by StCDPK2 in vitro.

    PubMed

    Muñiz García, María Noelia; Giammaria, Verónica; Grandellis, Carolina; Téllez-Iñón, María Teresa; Ulloa, Rita María; Capiati, Daniela Andrea

    2012-04-01

    ABF/AREB bZIP transcription factors mediate plant abiotic stress responses by regulating the expression of stress-related genes. These proteins bind to the abscisic acid (ABA)-responsive element (ABRE), which is the major cis-acting regulatory sequence in ABA-dependent gene expression. In an effort to understand the molecular mechanisms of abiotic stress resistance in cultivated potato (Solanum tuberosum L.), we have cloned and characterized an ABF/AREB-like transcription factor from potato, named StABF1. The predicted protein shares 45-57% identity with A. thaliana ABFs proteins and 96% identity with the S. lycopersicum SlAREB1 and presents all of the distinctive features of ABF/AREB transcription factors. Furthermore, StABF1 is able to bind to the ABRE in vitro. StABF1 gene is induced in response to ABA, drought, salt stress and cold, suggesting that it might be a key regulator of ABA-dependent stress signaling pathways in cultivated potato. StABF1 is phosphorylated in response to ABA and salt stress in a calcium-dependent manner, and we have identified a potato CDPK isoform (StCDPK2) that phosphorylates StABF1 in vitro. Interestingly, StABF1 expression is increased during tuber development and by tuber-inducing conditions (high sucrose/nitrogen ratio) in leaves. We also found that StABF1 calcium-dependent phosphorylation is stimulated by tuber-inducing conditions and inhibited by gibberellic acid, which inhibits tuberization.

  14. Gibberellins control fruit patterning in Arabidopsis thaliana

    PubMed Central

    Arnaud, Nicolas; Girin, Thomas; Sorefan, Karim; Fuentes, Sara; Wood, Thomas A.; Lawrenson, Tom; Sablowski, Robert; Østergaard, Lars

    2010-01-01

    The Arabidopsis basic helix–loop–helix (bHLH) proteins INDEHISCENT (IND) and ALCATRAZ (ALC) specify tissues required for fruit opening that have major roles in seed dispersal and plant domestication. Here, we show that synthesis of the phytohormone gibberellin is a direct and necessary target of IND, and that ALC interacts directly with DELLA repressors, which antagonize ALC function but are destabilized by gibberellin. Thus, the gibberellin/DELLA pathway has a key role in patterning the Arabidopsis fruit, and the interaction between DELLA and bHLH proteins, previously shown to connect gibberellin and light responses, is a versatile regulatory module also used in tissue patterning. PMID:20889713

  15. Gibberellins control fruit patterning in Arabidopsis thaliana.

    PubMed

    Arnaud, Nicolas; Girin, Thomas; Sorefan, Karim; Fuentes, Sara; Wood, Thomas A; Lawrenson, Tom; Sablowski, Robert; Østergaard, Lars

    2010-10-01

    The Arabidopsis basic helix-loop-helix (bHLH) proteins INDEHISCENT (IND) and ALCATRAZ (ALC) specify tissues required for fruit opening that have major roles in seed dispersal and plant domestication. Here, we show that synthesis of the phytohormone gibberellin is a direct and necessary target of IND, and that ALC interacts directly with DELLA repressors, which antagonize ALC function but are destabilized by gibberellin. Thus, the gibberellin/DELLA pathway has a key role in patterning the Arabidopsis fruit, and the interaction between DELLA and bHLH proteins, previously shown to connect gibberellin and light responses, is a versatile regulatory module also used in tissue patterning. PMID:20889713

  16. The Arabidopsis LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE3 Regulates the Cross Talk between Immunity and Abscisic Acid Responses1[W][OPEN

    PubMed Central

    Paparella, Chiara; Savatin, Daniel Valentin; Marti, Lucia; De Lorenzo, Giulia; Ferrari, Simone

    2014-01-01

    Transmembrane receptor-like kinases characterized by the presence of one or more lysin motif (LysM) domains in the extracytoplasmic portion (LysM-containing receptor-like kinases [LYKs]) mediate recognition of symbiotic and pathogenic microorganisms in plants. The Arabidopsis (Arabidopsis thaliana) genome encodes five putative LYKs; among them, AtLYK1/CHITIN ELICITOR RECEPTOR KINASE1 is required for response to chitin and peptidoglycan, and AtLYK4 contributes to chitin perception. More recently, AtLYK3 has been shown to be required for full repression, mediated by Nod factors, of Arabidopsis innate immune responses. In this work, we show that AtLYK3 also negatively regulates basal expression of defense genes and resistance to Botrytis cinerea and Pectobacterium carotovorum infection. Enhanced resistance of atlyk3 mutants requires PHYTOALEXIN-DEFICIENT3, which is crucial for camalexin biosynthesis. The expression of AtLYK3 is strongly repressed by elicitors and fungal infection and is induced by the hormone abscisic acid (ABA), which has a negative impact on resistance against B. cinerea and P. carotovorum. Plants lacking a functional AtLYK3 also show reduced physiological responses to ABA and are partially resistant to ABA-induced inhibition of PHYTOALEXIN-DEFICIENT3 expression. These results indicate that AtLYK3 is important for the cross talk between signaling pathways activated by ABA and pathogens. PMID:24639336

  17. A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate.

    PubMed

    Kalbina, Irina; Wallin, Anita; Lindh, Ingrid; Engström, Peter; Andersson, Sören; Strid, Ke

    2011-12-01

    The major outer membrane protein (MOMP) of Chlamydia trachomatis is a highly antigenic and hydrophobic transmembrane protein. Our attempts to express the full-length protein in a soluble form in Escherichia coli and in transgenic plants failed. A chimeric gene construct of C. trachomatis serovar E MOMP was designed in order to increase solubility of the MOMP protein but with retained antigenicity. The designed construct was successfully expressed in E. coli, in Arabidopsis thaliana, and in Daucus carota. The chimeric MOMP expressed in and purified from E. coli was used as antigen for production of antibodies in rabbits. The anti-chimeric MOMP antibodies recognized the corresponding protein in both E. coli and in transgenic plants, as well as in inactivated C. trachomatis elementary bodies. Transgenic Arabidopsis and carrots were characterized for the number of MOMP chimeric genetic inserts and for protein expression. Stable integration of the transgene and the corresponding protein expression were demonstrated in Arabidopsis plants over at least six generations. Transgenic carrots showed a high level of expression of the chimeric MOMP - up to 3% of TSP.

  18. O-Acetylation of Arabidopsis Hemicellulose Xyloglucan Requires AXY4 or AXY4L, Proteins with a TBL and DUF231 Domain[W][OA

    PubMed Central

    Gille, Sascha; de Souza, Amancio; Xiong, Guangyan; Benz, Monique; Cheng, Kun; Schultink, Alex; Reca, Ida-Barbara; Pauly, Markus

    2011-01-01

    In an Arabidopsis thaliana forward genetic screen aimed at identifying mutants with altered structures of their hemicellulose xyloglucan (axy mutants) using oligosaccharide mass profiling, two nonallelic mutants (axy4-1 and axy4-2) that have a 20 to 35% reduction in xyloglucan O-acetylation were identified. Mapping of the mutation in axy4-1 identified AXY4, a type II transmembrane protein with a Trichome Birefringence-Like domain and a domain of unknown function (DUF231). Loss of AXY4 transcript results in a complete lack of O-acetyl substituents on xyloglucan in several tissues, except seeds. Seed xyloglucan is instead O-acetylated by the paralog AXY4like, as demonstrated by the analysis of the corresponding T-DNA insertional lines. Wall fractionation analysis of axy4 knockout mutants indicated that only a fraction containing xyloglucan is non-O-acetylated. Hence, AXY4/AXY4L is required for the O-acetylation of xyloglucan, and we propose that these proteins represent xyloglucan-specific O-acetyltransferases, although their donor and acceptor substrates have yet to be identified. An Arabidopsis ecotype, Ty-0, has reduced xyloglucan O-acetylation due to mutations in AXY4, demonstrating that O-acetylation of xyloglucan does not impact the plant’s fitness in its natural environment. The relationship of AXY4 with another previously identified group of Arabidopsis proteins involved in general wall O-acetylation, reduced wall acetylation, is discussed. PMID:22086088

  19. The Arabidopsis LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE3 regulates the cross talk between immunity and abscisic acid responses.

    PubMed

    Paparella, Chiara; Savatin, Daniel Valentin; Marti, Lucia; De Lorenzo, Giulia; Ferrari, Simone

    2014-05-01

    Transmembrane receptor-like kinases characterized by the presence of one or more lysin motif (LysM) domains in the extracytoplasmic portion (LysM-containing receptor-like kinases [LYKs]) mediate recognition of symbiotic and pathogenic microorganisms in plants. The Arabidopsis (Arabidopsis thaliana) genome encodes five putative LYKs; among them, AtLYK1/CHITIN ELICITOR RECEPTOR KINASE1 is required for response to chitin and peptidoglycan, and AtLYK4 contributes to chitin perception. More recently, AtLYK3 has been shown to be required for full repression, mediated by Nod factors, of Arabidopsis innate immune responses. In this work, we show that AtLYK3 also negatively regulates basal expression of defense genes and resistance to Botrytis cinerea and Pectobacterium carotovorum infection. Enhanced resistance of atlyk3 mutants requires PHYTOALEXIN-DEFICIENT3, which is crucial for camalexin biosynthesis. The expression of AtLYK3 is strongly repressed by elicitors and fungal infection and is induced by the hormone abscisic acid (ABA), which has a negative impact on resistance against B. cinerea and P. carotovorum. Plants lacking a functional AtLYK3 also show reduced physiological responses to ABA and are partially resistant to ABA-induced inhibition of PHYTOALEXIN-DEFICIENT3 expression. These results indicate that AtLYK3 is important for the cross talk between signaling pathways activated by ABA and pathogens.

  20. Lipid exposure prediction enhances the inference of rotational angles of transmembrane helices

    PubMed Central

    2013-01-01

    Background Since membrane protein structures are challenging to crystallize, computational approaches are essential for elucidating the sequence-to-structure relationships. Structural modeling of membrane proteins requires a multidimensional approach, and one critical geometric parameter is the rotational angle of transmembrane helices. Rotational angles of transmembrane helices are characterized by their folded structures and could be inferred by the hydrophobic moment; however, the folding mechanism of membrane proteins is not yet fully understood. The rotational angle of a transmembrane helix is related to the exposed surface of a transmembrane helix, since lipid exposure gives the degree of accessibility of each residue in lipid environment. To the best of our knowledge, there have been few advances in investigating whether an environment descriptor of lipid exposure could infer a geometric parameter of rotational angle. Results Here, we present an analysis of the relationship between rotational angles and lipid exposure and a support-vector-machine method, called TMexpo, for predicting both structural features from sequences. First, we observed from the development set of 89 protein chains that the lipid exposure, i.e., the relative accessible surface area (rASA) of residues in the lipid environment, generated from high-resolution protein structures could infer the rotational angles with a mean absolute angular error (MAAE) of 46.32˚. More importantly, the predicted rASA from TMexpo achieved an MAAE of 51.05˚, which is better than 71.47˚ obtained by the best of the compared hydrophobicity scales. Lastly, TMexpo outperformed the compared methods in rASA prediction on the independent test set of 21 protein chains and achieved an overall Matthew’s correlation coefficient, accuracy, sensitivity, specificity, and precision of 0.51, 75.26%, 81.30%, 69.15%, and 72.73%, respectively. TMexpo is publicly available at http

  1. The arabidopsis thaliana AGRAVITROPIC 1 gene encodes a component of the polar-auxin-transport efflux carrier

    NASA Technical Reports Server (NTRS)

    Chen, R.; Hilson, P.; Sedbrook, J.; Rosen, E.; Caspar, T.; Masson, P. H.

    1998-01-01

    Auxins are plant hormones that mediate many aspects of plant growth and development. In higher plants, auxins are polarly transported from sites of synthesis in the shoot apex to their sites of action in the basal regions of shoots and in roots. Polar auxin transport is an important aspect of auxin functions and is mediated by cellular influx and efflux carriers. Little is known about the molecular identity of its regulatory component, the efflux carrier [Estelle, M. (1996) Current Biol. 6, 1589-1591]. Here we show that mutations in the Arabidopsis thaliana AGRAVITROPIC 1 (AGR1) gene involved in root gravitropism confer increased root-growth sensitivity to auxin and decreased sensitivity to ethylene and an auxin transport inhibitor, and cause retention of exogenously added auxin in root tip cells. We used positional cloning to show that AGR1 encodes a putative transmembrane protein whose amino acid sequence shares homologies with bacterial transporters. When expressed in Saccharomyces cerevisiae, AGR1 promotes an increased efflux of radiolabeled IAA from the cells and confers increased resistance to fluoro-IAA, a toxic IAA-derived compound. AGR1 transcripts were localized to the root distal elongation zone, a region undergoing a curvature response upon gravistimulation. We have identified several AGR1-related genes in Arabidopsis, suggesting a global role of this gene family in the control of auxin-regulated growth and developmental processes.

  2. The Arabidopsis thaliana AGRAVITROPIC 1 gene encodes a component of the polar-auxin-transport efflux carrier

    PubMed Central

    Chen, Rujin; Hilson, Pierre; Sedbrook, John; Rosen, Elizabeth; Caspar, Timothy; Masson, Patrick H.

    1998-01-01

    Auxins are plant hormones that mediate many aspects of plant growth and development. In higher plants, auxins are polarly transported from sites of synthesis in the shoot apex to their sites of action in the basal regions of shoots and in roots. Polar auxin transport is an important aspect of auxin functions and is mediated by cellular influx and efflux carriers. Little is known about the molecular identity of its regulatory component, the efflux carrier [Estelle, M. (1996) Current Biol. 6, 1589–1591]. Here we show that mutations in the Arabidopsis thaliana AGRAVITROPIC 1 (AGR1) gene involved in root gravitropism confer increased root-growth sensitivity to auxin and decreased sensitivity to ethylene and an auxin transport inhibitor, and cause retention of exogenously added auxin in root tip cells. We used positional cloning to show that AGR1 encodes a putative transmembrane protein whose amino acid sequence shares homologies with bacterial transporters. When expressed in Saccharomyces cerevisiae, AGR1 promotes an increased efflux of radiolabeled IAA from the cells and confers increased resistance to fluoro-IAA, a toxic IAA-derived compound. AGR1 transcripts were localized to the root distal elongation zone, a region undergoing a curvature response upon gravistimulation. We have identified several AGR1-related genes in Arabidopsis, suggesting a global role of this gene family in the control of auxin-regulated growth and developmental processes. PMID:9844024

  3. The novel protein DELAYED PALE-GREENING1 is required for early chloroplast biogenesis in Arabidopsis thaliana

    PubMed Central

    Liu, Dong; Li, Weichun; Cheng, Jianfeng

    2016-01-01

    Chloroplast biogenesis is one of the most important subjects in plant biology. In this study, an Arabidopsis early chloroplast biogenesis mutant with a delayed pale-greening phenotype (dpg1) was isolated from a T-DNA insertion mutant collection. Both cotyledons and true leaves of dpg1 mutants were initially albino but gradually became pale green as the plant matured. Transmission electron microscopic observations revealed that the mutant displayed a delayed proplastid-to-chloroplast transition. Sequence and transcription analyses showed that AtDPG1 encodes a putatively chloroplast-localized protein containing three predicted transmembrane helices and that its expression depends on both light and developmental status. GUS staining for AtDPG1::GUS transgenic lines showed that this gene was widely expressed throughout the plant and that higher expression levels were predominantly found in green tissues during the early stages of Arabidopsis seedling development. Furthermore, quantitative real-time RT-PCR analyses revealed that a number of chloroplast- and nuclear-encoded genes involved in chlorophyll biosynthesis, photosynthesis and chloroplast development were substantially down-regulated in the dpg1 mutant. These data indicate that AtDPG1 plays an essential role in early chloroplast biogenesis, and its absence triggers chloroplast-to-nucleus retrograde signalling, which ultimately down-regulates the expression of nuclear genes encoding chloroplast-localized proteins. PMID:27160321

  4. The novel protein DELAYED PALE-GREENING1 is required for early chloroplast biogenesis in Arabidopsis thaliana.

    PubMed

    Liu, Dong; Li, Weichun; Cheng, Jianfeng

    2016-01-01

    Chloroplast biogenesis is one of the most important subjects in plant biology. In this study, an Arabidopsis early chloroplast biogenesis mutant with a delayed pale-greening phenotype (dpg1) was isolated from a T-DNA insertion mutant collection. Both cotyledons and true leaves of dpg1 mutants were initially albino but gradually became pale green as the plant matured. Transmission electron microscopic observations revealed that the mutant displayed a delayed proplastid-to-chloroplast transition. Sequence and transcription analyses showed that AtDPG1 encodes a putatively chloroplast-localized protein containing three predicted transmembrane helices and that its expression depends on both light and developmental status. GUS staining for AtDPG1::GUS transgenic lines showed that this gene was widely expressed throughout the plant and that higher expression levels were predominantly found in green tissues during the early stages of Arabidopsis seedling development. Furthermore, quantitative real-time RT-PCR analyses revealed that a number of chloroplast- and nuclear-encoded genes involved in chlorophyll biosynthesis, photosynthesis and chloroplast development were substantially down-regulated in the dpg1 mutant. These data indicate that AtDPG1 plays an essential role in early chloroplast biogenesis, and its absence triggers chloroplast-to-nucleus retrograde signalling, which ultimately down-regulates the expression of nuclear genes encoding chloroplast-localized proteins.

  5. Transmembrane segments form tertiary hairpins in the folding vestibule of the ribosome.

    PubMed

    Tu, Liwei; Khanna, Pooja; Deutsch, Carol

    2014-01-01

    Folding of membrane proteins begins in the ribosome as the peptide is elongated. During this process, the nascent peptide navigates along 100Å of tunnel from the peptidyltransferase center to the exit port. Proximal to the exit port is a "folding vestibule" that permits the nascent peptide to compact and explore conformational space for potential tertiary folding partners. The latter occurs for cytosolic subdomains but has not yet been shown for transmembrane segments. We now demonstrate, using an accessibility assay and an improved intramolecular crosslinking assay, that the helical transmembrane S3b-S4 hairpin ("paddle") of a voltage-gated potassium (Kv) channel, a critical region of the Kv voltage sensor, forms in the vestibule. S3-S4 hairpin interactions are detected at an early stage of Kv biogenesis. Moreover, this vestibule hairpin is consistent with a closed-state conformation of the Kv channel in the plasma membrane.

  6. Transmembrane Segments Form Tertiary Hairpins in the Folding Vestibule of the Ribosome.

    PubMed Central

    Tu, LiWei; Khanna, Pooja; Deutsch, Carol

    2013-01-01

    Folding of membrane proteins begins in the ribosome as the peptide is elongated. During this process, the nascent peptide navigates along 100 Å of tunnel from the peptidyltransferase center to the exit port. Proximal to the exit port is a ‘folding vestibule’ that permits the nascent peptide to compact and explore conformational space for potential tertiary folding partners. The latter occurs for cytosolic subdomains, but has not yet been shown for transmembrane segments. We now demonstrate, using an accessibility assay and an improved, intramolecular crosslinking assay, that the helical transmembrane S3b-S4 hairpin (‘paddle’) of a voltage-gated potassium (Kv) channel, a critical region of the Kv voltage sensor, forms in the vestibule. S3-S4 hairpin interactions are detected at an early stage of Kv biogenesis. Moreover, this vestibule hairpin is consistent with a closed-state conformation of the Kv channel in the plasma membrane. PMID:24055377

  7. Combined approaches of EPR and NMR illustrate only one transmembrane helix in the human IFITM3

    PubMed Central

    Ling, Shenglong; Zhang, Chengwei; Wang, Wei; Cai, Xiaoying; Yu, Lu; Wu, Fangming; Zhang, Longhua; Tian, Changlin

    2016-01-01

    Interferon-inducible transmembrane protein IFITM3 was known to restrict the entry of a wide spectrum of viruses to the cytosol of the host. The mechanism used by the protein to restrict viral entry is unclear given the unavailability of the membrane topology and structures of the IFITM family proteins. Systematic site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) studies of IFITM3 in detergent micelles identified a single, long transmembrane helix in the C-terminus and an intramembrane segment in the N-terminal hydrophobic region. Solution NMR studies of the same sample verified the secondary structure distribution and demonstrated two rigid regions interacting with the micellar surface. The resulting membrane topology of IFITM3 supports the mechanism of an enhanced restricted membrane hemi-fusion. PMID:27046158

  8. Biomimetic Transmembrane Channels with High Stability and Transporting Efficiency from Helically Folded Macromolecules.

    PubMed

    Lang, Chao; Li, Wenfang; Dong, Zeyuan; Zhang, Xin; Yang, Feihu; Yang, Bing; Deng, Xiaoli; Zhang, Chenyang; Xu, Jiayun; Liu, Junqiu

    2016-08-01

    Membrane channels span the cellular lipid bilayers to transport ions and molecules into cells with sophisticated properties including high efficiency and selectivity. It is of particular biological importance in developing biomimetic transmembrane channels with unique functions by means of chemically synthetic strategies. An artificial unimolecular transmembrane channel using pore-containing helical macromolecules is reported. The self-folding, shape-persistent, pore-containing helical macromolecules are able to span the lipid bilayer, and thus result in extraordinary channel stability and high transporting efficiency for protons and cations. The lifetime of this artificial unimolecular channel in the lipid bilayer membrane is impressively long, rivaling those of natural protein channels. Natural channel mimics designed by helically folded polymeric scaffolds will display robust and versatile transport-related properties at single-molecule level.

  9. Transmembrane pH gradients in vivo: measurements using fluorinated vitamin B6 derivatives.

    PubMed

    Mason, R P

    1999-06-01

    It is well recognized that pH plays a significant regulatory role in most cellular processes. Increasingly, there is interest in transmembrane pH gradients, particularly with respect to tumor growth and response to therapy. NMR offers a non-invasive approach to monitoring cellular pH and detecting changes in response to interventions. This review will consider the strengths of various approaches to measuring pH with particular focus on the reporter molecules designed to interrogate the cellular milieu. In particular, fluorinated vitamin B6 derivatives (6-fluoropyridoxol and 6-fluoropyridoxamine) will be described, which for the first time provide a practical non-destructive method to measure simultaneously intra- and extracellular pH, i.e., the transmembrane pH gradient in animals in vivo based on a single reporter molecule.

  10. Cancer Research Advance in CKLF-like MARVEL Transmembrane Domain Containing Member Family (Review).

    PubMed

    Lu, Jia; Wu, Qian-Qian; Zhou, Ya-Bo; Zhang, Kai-Hua; Pang, Bing-Xin; Li, Liang; Sun, Nan; Wang, Heng-Shu; Zhang, Song; Li, Wen-Jian; Zheng, Wei; Liu, Wei

    2016-01-01

    CKLF-like MARVEL transmembrane domain-containing family (CMTM) is a novel family of genes first reported at international level by Peking University Human Disease Gene Research Center. The gene products are between chemokines and the transmembrane-4 superfamily. Loaceted in several human chromosomes, CMTMs, which are unregulated in kinds of tumors, are potential tumor suppressor genes consisting of CKLF and CMTM1 to CMTM8. CMTMs play important roles in immune, male reproductive and hematopoietic systems. Also, it has been approved that CMTM family has strong connection with diseases of autoimmunity, haematopoietic system and haematopoietic system. The in-depth study in recent years found the close relation between CMTMs and umorigenesis, tumor development and metastasis. CMTM family has a significant clinical value in diagnosis and treatment to the diseases linking to tumor and immune system. PMID:27356683

  11. Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active.

    PubMed

    Schönitzer, Veronika; Eichner, Norbert; Clausen-Schaumann, Hauke; Weiss, Ingrid M

    2011-12-01

    Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA(-) cell lines are shown.

  12. First principles design of a core bioenergetic transmembrane electron-transfer protein.

    PubMed

    Goparaju, Geetha; Fry, Bryan A; Chobot, Sarah E; Wiedman, Gregory; Moser, Christopher C; Dutton, P Leslie; Discher, Bohdana M

    2016-05-01

    Here we describe the design, Escherichia coli expression and characterization of a simplified, adaptable and functionally transparent single chain 4-α-helix transmembrane protein frame that binds multiple heme and light activatable porphyrins. Such man-made cofactor-binding oxidoreductases, designed from first principles with minimal reference to natural protein sequences, are known as maquettes. This design is an adaptable frame aiming to uncover core engineering principles governing bioenergetic transmembrane electron-transfer function and recapitulate protein archetypes proposed to represent the origins of photosynthesis. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

  13. Fluorophores, environments and quantification techniques in the analysis of transmembrane helix interaction using FRET

    PubMed Central

    Khadria, Ambalika S.; Senes, Alessandro

    2015-01-01

    FRET has been widely used as a spectroscopic tool in vitro to study the interactions between transmembrane helices in detergent and lipid environments. This technique has been instrumental to many studies that have greatly contributed to quantitative understanding of the physical principles that govern helix-helix interaction in the membrane. These studies have also improved our understanding of the biological role of oligomerization in membrane proteins. In this review, we focus on the combinations of fluorophores used, the membrane mimetic environments and measurement techniques that have been applied to study model systems as well as biological oligomeric complexes in vitro. We highlight the different formalisms used to calculate FRET efficiency, and the challenges associated with accurate quantification. The goal is to provide the reader with a comparative summary of the relevant literature for planning and designing FRET experiments aimed at measuring transmembrane helix-helix association. PMID:25968159

  14. Transmembrane segments form tertiary hairpins in the folding vestibule of the ribosome.

    PubMed

    Tu, Liwei; Khanna, Pooja; Deutsch, Carol

    2014-01-01

    Folding of membrane proteins begins in the ribosome as the peptide is elongated. During this process, the nascent peptide navigates along 100Å of tunnel from the peptidyltransferase center to the exit port. Proximal to the exit port is a "folding vestibule" that permits the nascent peptide to compact and explore conformational space for potential tertiary folding partners. The latter occurs for cytosolic subdomains but has not yet been shown for transmembrane segments. We now demonstrate, using an accessibility assay and an improved intramolecular crosslinking assay, that the helical transmembrane S3b-S4 hairpin ("paddle") of a voltage-gated potassium (Kv) channel, a critical region of the Kv voltage sensor, forms in the vestibule. S3-S4 hairpin interactions are detected at an early stage of Kv biogenesis. Moreover, this vestibule hairpin is consistent with a closed-state conformation of the Kv channel in the plasma membrane. PMID:24055377

  15. First principles design of a core bioenergetic transmembrane electron-transfer protein.

    PubMed

    Goparaju, Geetha; Fry, Bryan A; Chobot, Sarah E; Wiedman, Gregory; Moser, Christopher C; Dutton, P Leslie; Discher, Bohdana M

    2016-05-01

    Here we describe the design, Escherichia coli expression and characterization of a simplified, adaptable and functionally transparent single chain 4-α-helix transmembrane protein frame that binds multiple heme and light activatable porphyrins. Such man-made cofactor-binding oxidoreductases, designed from first principles with minimal reference to natural protein sequences, are known as maquettes. This design is an adaptable frame aiming to uncover core engineering principles governing bioenergetic transmembrane electron-transfer function and recapitulate protein archetypes proposed to represent the origins of photosynthesis. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson. PMID:26672896

  16. PBL13 Is a Serine/Threonine Protein Kinase That Negatively Regulates Arabidopsis Immune Responses.

    PubMed

    Lin, Zuh-Jyh Daniel; Liebrand, Thomas W H; Yadeta, Koste A; Coaker, Gitta

    2015-12-01

    Receptor-like cytoplasmic kinases (RLCKs) are a subset of plant receptor-like kinases lacking both extracellular and transmembrane domains. Some of the 46 members in the Arabidopsis (Arabidopsis thaliana) RLCK subfamily VII have been linked to plant innate immunity; however, most remain uncharacterized. Thus, multiple subfamily VII members are expected to be involved in plant immune signaling. Here, we investigate the role of AvrPphB SUSCEPTIBLE1-LIKE13 (PBL13), a subfamily VII RLCK with unique domain architecture. Unlike other characterized RLCKs, PBL13 transfer DNA insertion lines exhibit enhanced disease resistance after inoculation with virulent Pseudomonas syringae. The pbl13-2 knockout also exhibits elevated basal-level expression of the PATHOGENESIS-RELATED GENE1 defense marker gene, enhanced reactive oxygen species (ROS) burst in response to perception of bacterial microbial patterns, and accelerated flagellin-induced activation of mitogen-activated protein kinases. Recombinant PBL13 is an active kinase, and its primary autophosphorylated sites map to a 15-amino acid repeat motif unique to PBL13. Complementation of pbl13-2 with PBL13-3xFLAG converts the enhanced resistance and elevated ROS phenotypes back to wild-type levels. In contrast, kinase-dead PBL13(K111A)-3xFLAG was unable to rescue pbl13-2 disease phenotypes. Consistent with the enhanced ROS burst in the pbl13-2 knockout, PBL13 is able to associate with the nicotinamide adenine dinucleotide phosphate, reduced oxidase RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN D (RBOHD) by split-luciferase complementation assay, and this association is disrupted by flagellin treatment. We conclude that the PBL13 kinase negatively regulates plant innate immunity to pathogenic bacteria and can associate with RBOHD before pathogen perception. These data are consistent with the hypothesis that PBL13 acts to prevent inappropriate activation of defense responses in the absence of pathogen challenge.

  17. Shotgun Proteomic Analysis of Arabidopsis thaliana Leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two shotgun tandem mass spectrometry proteomics approaches, Multidimensional Protein Identification Technology (MudPIT) and 1D-Gel-LC-MS/MS, were used to identify Arabidopsis thaliana leaf proteins. These methods utilize different protein/peptide separation strategies. Detergents not compatible wit...

  18. Biosynthetic Pathways of Brassinolide in Arabidopsis1

    PubMed Central

    Noguchi, Takahiro; Fujioka, Shozo; Choe, Sunghwa; Takatsuto, Suguru; Tax, Frans E.; Yoshida, Shigeo; Feldmann, Kenneth A.

    2000-01-01

    Our previous studies on the endogenous brassinosteroids (BRs) in Arabidopsis have provided suggestive evidence for the operation of the early C6-oxidation and the late C6-oxidation pathways, leading to brassinolide (BL) in Arabidopsis. However, to date the in vivo operation of these pathways has not been fully confirmed in this species. This paper describes metabolic studies using deuterium-labeled BRs in wild-type and BR-insensitive mutant (bri1) seedlings to establish the intermediates of the biosynthetic pathway of BL in Arabidopsis. The first evidence for the conversion of campestanol to 6-deoxocathasterone and the conversion of 6-deoxocathasterone to 6-deoxoteasterone is provided. The later biosynthetic steps (6-deoxoteasterone → 3-dehydro-6-deoxoteasterone → 6-deoxotyphasterol → 6-deoxocastasterone → 6α-hydroxycastasterone → castasterone → BL) were demonstrated by stepwise metabolic experiments. Therefore, these studies complete the documentation of the late C6-oxidation pathway. The biosynthetic sequence involved in the early C6-oxidation pathway (teasterone → 3-dehydroteasterone → typhasterol → castasterone → BL) was also demonstrated. These results show that both the early and late C6-oxidation pathways are functional in Arabidopsis. In addition we report two new observations: the presence of a new branch in the pathway, C6 oxidation of 6-deoxotyphasterol to typhasterol, and increased metabolic flow in BR-insensitive mutants. PMID:10982435

  19. Arabidopsis gene expression patterns during spaceflight

    NASA Astrophysics Data System (ADS)

    Paul, A.-L.; Ferl, R. J.

    The exposure of Arabidopsis thaliana (Arabidopsis) plants to spaceflight environments resulted in the differential expression of hundreds of genes. A 5 day mission on orbiter Columbia in 1999 (STS-93) carried transgenic Arabidopsis plants engineered with a transgene composed of the alcohol dehydrogenase (Adh) gene promoter linked to the β -Glucuronidase (GUS) reporter gene. The plants were used to evaluate the effects of spaceflight on two fronts. First, expression patterns visualized with the Adh/GUS transgene were used to address specifically the possibility that spaceflight induces a hypoxic stress response, and to assess whether any spaceflight response was similar to control terrestrial hypoxia-induced gene expression patterns. (Paul et al., Plant Physiol. 2001, 126:613). Second, genome-wide patterns of native gene expression were evaluated utilizing the Affymetrix ATH1 GeneChip? array of 8,000 Arabidopsis genes. As a control for the veracity of the array analyses, a selection of genes identified with the arrays was further characterized with quantitative Real-Time RT PCR (ABI - TaqmanTM). Comparison of the patterns of expression for arrays of hybridized with RNA isolated from plants exposed to spaceflight compared to the control arrays revealed hundreds of genes that were differentially expressed in response to spaceflight, yet most genes that are hallmarks of hypoxic stress were unaffected. These results will be discussed in light of current models for plant responses to the spaceflight environment, and with regard to potential future flight opportunities.

  20. Engineering calcium oxalate crystal formation in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many plants accumulate crystals of calcium oxalate. Just how these crystals form remains unknown. To gain insight into the mechanisms regulating calcium oxalate crystal formation, a crystal engineering approach was initiated utilizing the non-crystal accumulating plant, Arabidopsis. The success of t...

  1. Transmembrane potential induced in a spherical cell model under low-frequency magnetic stimulation

    NASA Astrophysics Data System (ADS)

    Ye, Hui; Cotic, Marija; Carlen, Peter L.

    2007-09-01

    Time-varying magnetic fields can induce electric fields in the neuronal tissue, a phenomenon that has been recently explored in clinical applications such as peripheral nerve stimulation and transcranial magnetic stimulation. Although the transmembrane potential induced during direct electric stimulation has already been the subject of a number of theoretical studies, an analytical solution for the magnetically induced transmembrane potential change is still unavailable. In addition, although several studies have analyzed the impact of stimulation parameters, including stimulation intensity and frequency, as well as coil design and position, on the amount of tissue polarization, the effects of tissue non-homogeneity on cell polarization have not been fully elucidated. In this study, we have derived an analytical expression for the transmembrane potential induced by a low-frequency magnetic field in a spherical neuronal structure. This model is representative of a spherical cell body or any neuronal structure of a similar shape. The model cell is located in an extracellular medium and possesses a low-conductive membrane and an internal cytoplasm. These three regions represent the basic tissue non-homogeneity of a neuron at a microscopic level. The sensitivity of the induced transmembrane potential to the coil position and to the geometrical and electrical parameters of the model structure was studied in a broad physiologically relevant range. Our results demonstrate that the structure is regionally polarized, with the pattern of polarization depending on the relative positioning between the model cell and the stimulation coil. In addition, both the geometrical and electrical parameters of the structure affect the amount of polarization. These results may be generalized to other neuronal tissues that possess similar non-homogenous properties, but different shapes, such as an axon. Our results support the idea that aside from coil design and position, tissue non

  2. Transmembrane signaling and assembly of the cytochrome b6f-lipidic charge transfer complex.

    PubMed

    Saif Hasan, S; Yamashita, Eiki; Cramer, William A

    2013-01-01

    Structure-function properties of the cytochrome b6f complex are sufficiently unique compared to those of the cytochrome bc1 complex that b6f should not be considered a trivially modified bc1 complex. A unique property of the dimeric b6f complex is its involvement in transmembrane signaling associated with the p-side oxidation of plastoquinol. Structure analysis of lipid binding sites in the cyanobacterial b6f complex prepared by hydrophobic chromatography shows that the space occupied by the H transmembrane helix in the cytochrome b subunit of the bc1 complex is mostly filled by a lipid in the b6f crystal structure. It is suggested that this space can be filled by the domain of a transmembrane signaling protein. The identification of lipid sites and likely function defines the intra-membrane conserved central core of the b6f complex, consisting of the seven trans-membrane helices of the cytochrome b and subunit IV polypeptides. The other six TM helices, contributed by cytochrome f, the iron-sulfur protein, and the four peripheral single span subunits, define a peripheral less conserved domain of the complex. The distribution of conserved and non-conserved domains of each monomer of the complex, and the position and inferred function of a number of the lipids, suggests a model for the sequential assembly in the membrane of the eight subunits of the b6f complex, in which the assembly is initiated by formation of the cytochrome b6-subunit IV core sub-complex in a monomer unit. Two conformations of the unique lipidic chlorophyll a, defined in crystal structures, are described, and functions of the outlying β-carotene, a possible 'latch' in supercomplex formation, are discussed. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes. PMID:23507619

  3. tincar encodes a novel transmembrane protein expressed in the Tinman-expressing cardioblasts of Drosophila.

    PubMed

    Hirota, Yuki; Sawamoto, Kazunobu; Okano, Hideyuki

    2002-12-01

    We cloned and characterized the Drosophila gene, tincar (tinc), which encodes a novel protein with eight putative transmembrane domains. The tinc mRNA was expressed specifically in four of the six pairs of cardioblasts in each segment, in a pattern identical to that of tinman (tin), a homeobox gene required for the specification of the dorsal vessel. In the non-Tin-expressing pairs of cardioblasts, tinc transcription seemed to be repressed by Seven-up. PMID:14516698

  4. tincar encodes a novel transmembrane protein expressed in the Tinman-expressing cardioblasts of Drosophila.

    PubMed

    Hirota, Yuki; Sawamoto, Kazunobu; Okano, Hideyuki

    2002-12-01

    We cloned and characterized the Drosophila gene, tincar (tinc), which encodes a novel protein with eight putative transmembrane domains. The tinc mRNA was expressed specifically in four of the six pairs of cardioblasts in each segment, in a pattern identical to that of tinman (tin), a homeobox gene required for the specification of the dorsal vessel. In the non-Tin-expressing pairs of cardioblasts, tinc transcription seemed to be repressed by Seven-up. PMID:12617821

  5. Development of a Proteoliposome Model to Probe Transmembrane Electron-Transfer Reactions

    SciTech Connect

    White, Gaye F.; Shi, Zhi; Shi, Liang; Dohnalkova, Alice; Fredrickson, Jim K.; Zachara, John M.; Butt, Julea N.; Richardson, David J.; Clarke, Thomas

    2012-12-01

    The mineral respiring bacterium Shewanella oneidensis uses a protein complex, MtrCAB, composed of two decaheme cytochromes brought together inside a transmembrane porin to transport electrons across the outer membrane to a variety of mineral-based electron acceptors. A proteoliposome system has been developed that contains methyl viologen (MV) as an internalised electron acceptor and valinomycin (V) as a membrane associated cation exchanger. These proteoliposomes can be used as a model system to investigate MtrCAB function.

  6. Transmembrane signaling and assembly of the cytochrome b6f-lipidic charge transfer complex.

    PubMed

    Saif Hasan, S; Yamashita, Eiki; Cramer, William A

    2013-01-01

    Structure-function properties of the cytochrome b6f complex are sufficiently unique compared to those of the cytochrome bc1 complex that b6f should not be considered a trivially modified bc1 complex. A unique property of the dimeric b6f complex is its involvement in transmembrane signaling associated with the p-side oxidation of plastoquinol. Structure analysis of lipid binding sites in the cyanobacterial b6f complex prepared by hydrophobic chromatography shows that the space occupied by the H transmembrane helix in the cytochrome b subunit of the bc1 complex is mostly filled by a lipid in the b6f crystal structure. It is suggested that this space can be filled by the domain of a transmembrane signaling protein. The identification of lipid sites and likely function defines the intra-membrane conserved central core of the b6f complex, consisting of the seven trans-membrane helices of the cytochrome b and subunit IV polypeptides. The other six TM helices, contributed by cytochrome f, the iron-sulfur protein, and the four peripheral single span subunits, define a peripheral less conserved domain of the complex. The distribution of conserved and non-conserved domains of each monomer of the complex, and the position and inferred function of a number of the lipids, suggests a model for the sequential assembly in the membrane of the eight subunits of the b6f complex, in which the assembly is initiated by formation of the cytochrome b6-subunit IV core sub-complex in a monomer unit. Two conformations of the unique lipidic chlorophyll a, defined in crystal structures, are described, and functions of the outlying β-carotene, a possible 'latch' in supercomplex formation, are discussed. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

  7. Combined effect of cortical cytoskeleton and transmembrane proteins on domain formation in biomembranes

    PubMed Central

    Sikder, Md. Kabir Uddin; Stone, Kyle A.; Kumar, P. B. Sunil; Laradji, Mohamed

    2014-01-01

    We investigate the combined effects of transmembrane proteins and the subjacent cytoskeleton on the dynamics of phase separation in multicomponent lipid bilayers using computer simulations of a particle-based implicit solvent model for lipid membranes with soft-core interactions. We find that microphase separation can be achieved by the protein confinement by the cytoskeleton. Our results have relevance to the finite size of lipid rafts in the plasma membrane of mammalian cells. PMID:25106608

  8. Mapping the Homodimer Interface of an Optimized, Artificial, Transmembrane Protein Activator of the Human Erythropoietin Receptor

    PubMed Central

    Bears, Zachary; Barrera, Francisco N.; Alonso, Miriam; Engelman, Donald M.; DiMaio, Daniel

    2014-01-01

    Transmembrane proteins constitute a large fraction of cellular proteins, and specific interactions involving membrane-spanning protein segments play an important role in protein oligomerization, folding, and function. We previously isolated an artificial, dimeric, 44-amino acid transmembrane protein that activates the human erythropoietin receptor (hEPOR) in trans. This artificial protein supports limited erythroid differentiation of primary human hematopoietic progenitor cells in vitro, even though it does not resemble erythropoietin, the natural ligand of this receptor. Here, we used a directed-evolution approach to explore the structural basis for the ability of transmembrane proteins to activate the hEPOR. A library that expresses thousands of mutants of the transmembrane activator was screened for variants that were more active than the original isolate at inducing growth factor independence in mouse cells expressing the hEPOR. The most active mutant, EBC5-16, supports erythroid differentiation in human cells with activity approaching that of EPO, as assessed by cell-surface expression of glycophorin A, a late-stage marker of erythroid differentiation. EBC5-16 contains a single isoleucine to serine substitution at position 25, which increases its ability to form dimers. Genetic studies confirmed the importance of dimerization for activity and identified the residues constituting the homodimer interface of EBC5-16. The interface requires a GxxxG dimer packing motif and a small amino acid at position 25 for maximal activity, implying that tight packing of the EBC5-16 dimer is a crucial determinant of activity. These experiments identified an artificial protein that causes robust activation of its target in a natural host cell, demonstrated the importance of dimerization of this protein for engagement of the hEPOR, and provided the framework for future structure-function studies of this novel mechanism of receptor activation. PMID:24788775

  9. Combined effect of cortical cytoskeleton and transmembrane proteins on domain formation in biomembranes

    NASA Astrophysics Data System (ADS)

    Sikder, Md. Kabir Uddin; Stone, Kyle A.; Kumar, P. B. Sunil; Laradji, Mohamed

    2014-08-01

    We investigate the combined effects of transmembrane proteins and the subjacent cytoskeleton on the dynamics of phase separation in multicomponent lipid bilayers using computer simulations of a particle-based implicit solvent model for lipid membranes with soft-core interactions. We find that microphase separation can be achieved by the protein confinement by the cytoskeleton. Our results have relevance to the finite size of lipid rafts in the plasma membrane of mammalian cells.

  10. Comparative phylogenetic analysis of genome-wide Mlo gene family members from Glycine max and Arabidopsis thaliana.

    PubMed

    Deshmukh, Reena; Singh, V K; Singh, B D

    2014-06-01

    Powdery mildew locus O (Mlo) gene family is one of the largest seven transmembrane protein-encoding gene families. The Mlo proteins act as negative regulators of powdery mildew resistance and a loss-of-function mutation in Mlo is known to confer broad-spectrum resistance to powdery mildew. In addition, the Mlo gene family members are known to participate in various developmental and biotic and abiotic stress response-related pathways. Therefore, a genome-wide similarity search using the characterized Mlo protein sequences of Arabidopsis thaliana was carried out to identify putative Mlo genes in soybean (Glycine max) genome. This search identified 39 Mlo domain containing protein-encoding genes that were distributed on 15 of the 20 G. max chromosomes. The putative promoter regions of these Mlo genes contained response elements for different external stimuli, including different hormones and abiotic stresses. Of the 39 GmMlo proteins, 35 were rich (8.7-13.1 %) in leucine, while five were serine-rich (9.2-11.9 %). Furthermore, all the GmMlo members were localized in the plasma membrane. Phylogenetic analysis of the GmMlo and the AtMlo proteins classified them into three main clusters, and the cluster I comprised two sub-clusters. Multiple sequence alignment visualized the location of seven transmembrane domains, and a conserved CaM-binding domain. Some of the GmMlo proteins (GmMlo10, 20, 22, 23, 32, 36, 37) contained less than seven transmembrane domains. The motif analysis yielded 27 motifs; out of these, motif 2, the only motif present in all the GmMlos, was highly conserved and three amino acid residues were essentially invariant. Five of the GmMlo members were much smaller in size; presumably they originated through deletion following a gene duplication event. The presence of a large number of GmMlo members in the G. max genome may be due to its paleopolyploid nature and the large genome size as compared to that of Arabidopsis. The findings of this study may

  11. UV-Induced DNA Damage Promotes Resistance to the Biotrophic Pathogen Hyaloperonospora parasitica in Arabidopsis1[C][OA

    PubMed Central

    Kunz, Bernard A.; Dando, Paige K.; Grice, Desma M.; Mohr, Peter G.; Schenk, Peer M.; Cahill, David M.

    2008-01-01

    Plant innate immunity to pathogenic microorganisms is activated in response to recognition of extracellular or intracellular pathogen molecules by transmembrane receptors or resistance proteins, respectively. The defense signaling pathways share components with those involved in plant responses to UV radiation, which can induce expression of plant genes important for pathogen resistance. Such intriguing links suggest that UV treatment might activate resistance to pathogens in normally susceptible host plants. Here, we demonstrate that pre-inoculative UV (254 nm) irradiation of Arabidopsis (Arabidopsis thaliana) susceptible to infection by the biotrophic oomycete Hyaloperonospora parasitica, the causative agent of downy mildew, induces dose- and time-dependent resistance to the pathogen detectable up to 7 d after UV exposure. Limiting repair of UV photoproducts by postirradiation incubation in the dark, or mutational inactivation of cyclobutane pyrimidine dimer photolyase, (6-4) photoproduct photolyase, or nucleotide excision repair increased the magnitude of UV-induced pathogen resistance. In the absence of treatment with 254-nm UV, plant nucleotide excision repair mutants also defective for cyclobutane pyrimidine dimer or (6-4) photoproduct photolyase displayed resistance to H. parasitica, partially attributable to short wavelength UV-B (280–320 nm) radiation emitted by incubator lights. These results indicate UV irradiation can initiate the development of resistance to H. parasitica in plants normally susceptible to the pathogen and point to a key role for UV-induced DNA damage. They also suggest UV treatment can circumvent the requirement for recognition of H. parasitica molecules by Arabidopsis proteins to activate an immune response. PMID:18667719

  12. Structural role of the conserved cysteines in the dimerization of the viral transmembrane oncoprotein E5.

    PubMed

    Windisch, Dirk; Hoffmann, Silke; Afonin, Sergii; Vollmer, Stefanie; Benamira, Soraya; Langer, Birgid; Bürck, Jochen; Muhle-Goll, Claudia; Ulrich, Anne S

    2010-09-22

    The E5 oncoprotein is the major transforming protein of bovine papillomavirus type 1. This 44-residue transmembrane protein can interact with the platelet-derived growth factor receptor β, leading to ligand-independent activation and cell transformation. For productive interaction, E5 needs to dimerize via a C-terminal pair of cysteines, though a recent study suggested that its truncated transmembrane segment can dimerize on its own. To analyze the structure of the full protein in a membrane environment and elucidate the role of the Cys-Ser-Cys motif, we produced recombinantly the wild-type protein and four cysteine mutants. Comparison by circular dichroism in detergent micelles and lipid vesicular dispersion and by NMR in trifluoroethanol demonstrates that the absence of one or both cysteines does not influence the highly α-helical secondary structure, nor does it impair the ability of E5 to dimerize, observations that are further supported by sodium dodecylsulfate polyacrylamide gel electrophoresis. We also observed assemblies of higher order. Oriented circular dichroism in lipid bilayers shows that E5 is aligned as a transmembrane helix with a slight tilt angle, and that this membrane alignment is also independent of any cysteines. We conclude that the Cys-containing motif represents a disordered region of the protein that serves as an extra covalent connection for stabilization.

  13. Light induced transmembrane proton gradient in artificial lipid vesicles reconstituted with photosynthetic reaction centers.

    PubMed

    Milano, Francesco; Trotta, Massimo; Dorogi, Márta; Fischer, Béla; Giotta, Livia; Agostiano, Angela; Maróti, Péter; Kálmán, László; Nagy, László

    2012-06-01

    Photosynthetic reaction center (RC) is the minimal nanoscopic photoconverter in the photosynthetic membrane that catalyzes the conversion of solar light to energy readily usable for the metabolism of the living organisms. After electronic excitation the energy of light is converted into chemical potential by the generation of a charge separated state accompanied by intraprotein and ultimately transmembrane proton movements. We designed a system which fulfills the minimum structural and functional requirements to investigate the physico/chemical conditions of the processes: RCs were reconstituted in closed lipid vesicles made of selected lipids entrapping a pH sensitive indicator, and electron donors (cytochrome c₂ and K₄[Fe(CN)₆]) and acceptors (decylubiquinone) were added to sustain the photocycle. Thanks to the low proton permeability of our preparations, we could show the formation of a transmembrane proton gradient under illumination and low buffering conditions directly by measuring proton-related signals simultaneously inside and outside the vesicles. The effect of selected ionophores such as gramicidin, nigericin and valinomycin was used to gain more information on the transmembrane proton gradient driven by the RC photochemistry.

  14. Factors influencing the trans-membrane transport of n-octadecane by Pseudomonas sp. DG17

    PubMed Central

    Hua, Fei; Wang, Hong Qi; Zhao, Yi Cun

    2014-01-01

    In soil bioremediation techniques, the trans-membrane transport of hydrocarbons across the cell membrane is a new and complex point of understanding the process of hydrocarbons biodegradation. In this study, the effect of different environmental factors, including substrate concentration, bacterial inoculums, pH, salinity, substrate analogues and nutrients, on the transport of [14C]n-octadecane by Pseudomonas sp. DG17 was investigated. The results showed that cellular [14C]n-octadecane levels increased along with the increase in the substrate concentration. However, the trans-membrane transport of [14C]n-octadecane was a saturable process in the case of equal amounts of inoculum (biomass). The highest concentration of accumulated [14C]n-octadecane was 0.51 μmol mg−1 ± 0.028 μmol mg−1 after incubation for 20 min. Meanwhile, the cellular n-octadecane concentration decreased along with the biomass increase, and reached a stable level. Acidic/alkaline conditions, high salinity, and supplement of substrate analogues could inhibit the transport of [14C]n-octadecane by Pseudomonas sp. DG17, whereas nitrogen or phosphorus deficiency did not influence this transport. The results suggested that trans-membrane transport of octadecane depends on both the substrate concentration and the microorganism biomass, and extreme environmental conditions could influence the biodegradation ability of microorganisms through inhibiting the transport of extracellular octadecane. PMID:26740764

  15. Structure of FGFR3 transmembrane domain dimer: implications for signaling and human pathologies.

    PubMed

    Bocharov, Eduard V; Lesovoy, Dmitry M; Goncharuk, Sergey A; Goncharuk, Marina V; Hristova, Kalina; Arseniev, Alexander S

    2013-11-01

    Fibroblast growth factor receptor 3 (FGFR3) transduces biochemical signals via lateral dimerization in the plasma membrane, and plays an important role in human development and disease. Eight different pathogenic mutations, implicated in cancers and growth disorders, have been identified in the FGFR3 transmembrane segment. Here, we describe the dimerization of the FGFR3 transmembrane domain in membrane-mimicking DPC/SDS (9/1) micelles. In the solved NMR structure, the two transmembrane helices pack into a symmetric left-handed dimer, with intermolecular stacking interactions occurring in the dimer central region. Some pathogenic mutations fall within the helix-helix interface, whereas others are located within a putative alternative interface. This implies that although the observed dimer structure is important for FGFR3 signaling, the mechanism of FGFR3-mediated transduction across the membrane is complex. We propose an FGFR3 signaling mechanism that is based on the solved structure, available structures of isolated soluble FGFR domains, and published biochemical and biophysical data.

  16. Distribution of the SynDIG4/proline-rich transmembrane protein 1 in rat brain.

    PubMed

    Kirk, Lyndsey M; Ti, Shu W; Bishop, Hannah I; Orozco-Llamas, Mayra; Pham, Michelle; Trimmer, James S; Díaz, Elva

    2016-08-01

    The modulation of AMPA receptor (AMPAR) content at synapses is thought to be an underlying molecular mechanism of memory and learning. AMPAR content at synapses is highly plastic and is regulated by numerous AMPAR accessory transmembrane proteins such as TARPs, cornichons, and CKAMPs. SynDIG (synapse differentiation-induced gene) defines a family of four genes (SynDIG1-4) expressed in distinct and overlapping patterns in the brain. SynDIG1 was previously identified as a novel transmembrane AMPAR-associated protein that regulates synaptic strength. The related protein SynDIG4 [also known as Prrt1 (proline-rich transmembrane protein 1)] has recently been identified as a component of AMPAR complexes. In this study, we show that SynDIG1 and SynDIG4 have distinct yet overlapping patterns of expression in the central nervous system, with SynDIG4 having especially prominent expression in the hippocampus and particularly within CA1. In contrast to SynDIG1 and other traditional AMPAR auxiliary subunits, SynDIG4 is de-enriched at the postsynaptic density and colocalizes with extrasynaptic GluA1 puncta in primary dissociated neuron culture. These results indicate that, although SynDIG4 shares sequence similarity with SynDIG1, it might act through a unique mechanism as an auxiliary factor for extrasynaptic GluA1-containing AMPARs. J. Comp. Neurol. 524:2266-2280, 2016. © 2015 Wiley Periodicals, Inc.

  17. Different Transmembrane Domains Associate with Distinct Endoplasmic Reticulum Components during Membrane Integration of a Polytopic Protein

    PubMed Central

    Meacock, Suzanna L.; Lecomte, Fabienne J.L.; Crawshaw, Samuel G.; High, Stephen

    2002-01-01

    We have been studying the insertion of the seven transmembrane domain (TM) protein opsin to gain insights into how the multiple TMs of polytopic proteins are integrated at the endoplasmic reticulum (ER). We find that the ER components associated with the first and second TMs of the nascent opsin polypeptide chain are clearly distinct. The first TM (TM1) is adjacent to the α and β subunits of the Sec61 complex, and a novel component, a protein associated with the ER translocon of 10 kDa (PAT-10). The most striking characteristic of PAT-10 is that it remains adjacent to TM1 throughout the biogenesis and membrane integration of the full-length opsin polypeptide. TM2 is also found to be adjacent to Sec61α and Sec61β during its membrane integration. However, TM2 does not form any adducts with PAT-10; rather, a transient association with the TRAM protein is observed. We show that the association of PAT-10 with opsin TM1 does not require the N-glycosylation of the nascent chain and occurs irrespective of the amino acid sequence and transmembrane topology of TM1. We conclude that the precise makeup of the ER membrane insertion site can be distinct for the different transmembrane domains of a polytopic protein. We find that the environment of a particular TM can be influenced by both the “stage” of nascent chain biosynthesis reached, and the TM's relative location within the polypeptide. PMID:12475939

  18. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR): CLOSED AND OPEN STATE CHANNEL MODELS.

    PubMed

    Corradi, Valentina; Vergani, Paola; Tieleman, D Peter

    2015-09-18

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily. CFTR controls the flow of anions through the apical membrane of epithelia. Dysfunctional CFTR causes the common lethal genetic disease cystic fibrosis. Transitions between open and closed states of CFTR are regulated by ATP binding and hydrolysis on the cytosolic nucleotide binding domains, which are coupled with the transmembrane (TM) domains forming the pathway for anion permeation. Lack of structural data hampers a global understanding of CFTR and thus the development of "rational" approaches directly targeting defective CFTR. In this work, we explored possible conformational states of the CFTR gating cycle by means of homology modeling. As templates, we used structures of homologous ABC transporters, namely TM(287-288), ABC-B10, McjD, and Sav1866. In the light of published experimental results, structural analysis of the transmembrane cavity suggests that the TM(287-288)-based CFTR model could correspond to a commonly occupied closed state, whereas the McjD-based model could represent an open state. The models capture the important role played by Phe-337 as a filter/gating residue and provide structural information on the conformational transition from closed to open channel.

  19. CFTR anion channel modulates expression of human transmembrane mucin MUC3 through the PDZ protein GOPC.

    PubMed

    Pelaseyed, Thaher; Hansson, Gunnar C

    2011-09-15

    The transmembrane mucins in the enterocyte are type 1 transmembrane proteins with long and rigid mucin domains, rich in proline, threonine and serine residues that carry numerous O-glycans. Three of these mucins, MUC3, MUC12 and MUC17 are unique in harboring C-terminal class I PDZ motifs, making them suitable ligands for PDZ proteins. A screening of 123 different human PDZ domains for binding to MUC3 identified a strong interaction with the PDZ protein GOPC (Golgi-associated PDZ and coiled-coil motif-containing protein). This interaction was mediated by the C-terminal PDZ motif of MUC3, binding to the single GOPC PDZ domain. GOPC is also a binding partner for cystic fibrosis transmembrane conductance regulator (CFTR) that directs CFTR for degradation. Overexpression of GOPC downregulated the total levels of MUC3, an effect that was reversed by introducing CFTR. The results suggest that CFTR and MUC3 compete for binding to GOPC, which in turn can regulate levels of these two proteins. For the first time a direct coupling between mucins and the CFTR channel is demonstrated, a finding that will shed further light on the still poorly understood relationship between cystic fibrosis and the mucus phenotype of this disease.

  20. Localization and Characterization of Rat Transmembrane Protein 225 Specifically Expressed in Testis

    PubMed Central

    Yang, Shirui; Wang, Weiping; Lei, Chen; Liu, Qingmei; Xu, Fengqin; Xing, Xiaowei; Chen, Hao; Liu, Jiajia; Wu, Shiliang

    2011-01-01

    Testis is the one and only location of spermatogenesis and sexual hormone production. Spermatogenesis is a complicated physiological process regulated by many genes specifically and differentially expressed in the testis. In this study, Transmembrane Protein 225 (TMEM225), which is specifically expressed in rat testis, has been identified. TMEM225 was cloned from the testis cDNA library and was mapped to chromosome 8q22 by browsing the University of California Santa Cruz genomic database. It contains an open reading frame with a length of 696 bp, encoding a protein with four putative transmembrane helices. TMEM225 mRNA expression was evaluated by reverse transcription–polymerase chain reaction and in situ hybridization. In addition, the subcellular location of TMEM225 was evaluated. The results obtained highlighted age related specific expression of TMEM225 in testis, specifically during the adult period after age of 13 months. In situ hybridization analysis indicated that TMEM225 mRNA was mainly expressed in spermatocyte cells and round spermatids. Green fluorescence protein localization analysis showed that rat TMEM225 mainly surrounded the nuclear membrane, with a minority distribution in the cytoplasm, and the distribution of TMEM225 was affected by the deletion of N-terminal transmembrane domain. As the expression phase is not related to the first wave of spermatozoon development, our data presented here suggest that TMEM225 may play an important role in sperm degeneration but not in spermatogenesis. PMID:20979528

  1. Cystic fibrosis transmembrane conductance regulator protein expression in the male excretory duct system during development.

    PubMed

    Marcorelles, Pascale; Gillet, Danièle; Friocourt, Gaëlle; Ledé, Françoise; Samaison, Laura; Huguen, Geneviève; Ferec, Claude

    2012-03-01

    Sterility due to bilateral destruction in utero or in early infancy resulting in congenital absence of the vas deferens is the rule in male patients with cystic fibrosis. To understand the developmental pattern of this anomaly, the microscopic morphology of the male excretory system was analyzed during development and the expression of the cystic fibrosis transmembrane conductance regulator protein was explored by immunohistochemistry. We observed that cystic fibrosis fetuses had no excretory ducts agenesis or obstruction until 22 weeks of gestation. However, a focal inflammatory pattern and mucinous plugs in the oldest cystic fibrosis case suggested a disruptive mechanism. Immunolabeling of cytoplasmic epithelial cystic fibrosis transmembrane conductance regulator protein was demonstrated in all cystic fibrosis and control cases with a similar pattern of expression of the protein between age-matched controls and cystic fibrosis cases. At midgestation, an apical intensification appeared in both cystic fibrosis and control cases and was stable during the remainder of fetal life. No gradient of intensity could be detected between the different segments of the excretory tract. These findings are different from those reported in adults. The absence of any morphologic anomaly until 22 weeks of gestation, the focal destruction of the epithelial structures during the second trimester, and the chronological pattern of expression of cystic fibrosis transmembrane conductance regulator are of interest for a better understanding of the pathophysiology of this disease.

  2. Transmembrane semaphorins, forward and reverse signaling: have a look both ways.

    PubMed

    Battistini, Chiara; Tamagnone, Luca

    2016-04-01

    Semaphorins are signaling molecules playing pivotal roles not only as axon guidance cues, but are also involved in the regulation of a range of biological processes, such as immune response, angiogenesis and invasive tumor growth. The main functional receptors for semaphorins are plexins, which are large single-pass transmembrane molecules. Semaphorin signaling through plexins-the "classical" forward signaling-affects cytoskeletal remodeling and integrin-dependent adhesion, consequently influencing cell migration. Intriguingly, semaphorins and plexins can interact not only in trans, but also in cis, leading to differentiated and highly regulated signaling outputs. Moreover, transmembrane semaphorins can also mediate a so-called "reverse" signaling, by acting not as ligands but rather as receptors, and initiate a signaling cascade through their own cytoplasmic domains. Semaphorin reverse signaling has been clearly demonstrated in fruit fly Sema1a, which is required to control motor axon defasciculation and target recognition during neuromuscular development. Sema1a invertebrate semaphorin is most similar to vertebrate class-6 semaphorins, and examples of semaphorin reverse signaling in mammalians have been described for these family members. Reverse signaling is also reported for other vertebrate semaphorin subsets, e.g. class-4 semaphorins, which bear potential PDZ-domain interaction motifs in their cytoplasmic regions. Therefore, thanks to their various signaling abilities, transmembrane semaphorins can play multifaceted roles both in developmental processes and in physiological as well as pathological conditions in the adult. PMID:26794845

  3. The Expanding Social Network of Ionotropic Glutamate Receptors: TARPs and Other Transmembrane Auxiliary Subunits

    PubMed Central

    Jackson, Alexander C.; Nicoll, Roger A.

    2011-01-01

    Ionotropic glutamate receptors (iGluRs) underlie rapid, excitatory synaptic signaling throughout the CNS. After years of intense research, our picture of iGluRs has evolved from imagining them companionless in the postsynaptic membrane to being the hub of dynamic supramolecular signaling complexes, interacting with an ever-expanding litany of other proteins that regulate their trafficking, scaffolding, stability, signaling and turnover. In particular, the discovery that transmembrane AMPA receptor regulatory proteins (TARPs) are auxiliary subunits of AMPA receptors, that are critical determinants of their trafficking, gating and pharmacology, has changed the way we think about iGluR function. Recently, a number of novel transmembrane proteins have been uncovered that may also serve as iGluR auxiliary proteins. Here we review pivotal developments in our understanding of the role of TARPs in AMPA receptor trafficking and gating, as well as an overview of how newly discovered transmembrane proteins expand our view of iGluR function in the CNS. PMID:21521608

  4. Cu**+ Transporting ATPases: Structure of the Two Transmembrane Cu**+ Transport Sites

    SciTech Connect

    Gonzalez-Guerrero, M.; Eren, E.; Rawat, S.; Stemmler, T.L.; Arguello, J.M.

    2009-05-18

    Cu{sup +}-ATPases drive metal efflux from the cell cytoplasm. Paramount to this function is the binding of Cu{sup +} within the transmembrane region and its coupled translocation across the permeability barrier. Here, we describe the two transmembrane Cu{sup +} transport sites present in Archaeoglobus fulgidus CopA. Both sites can be independently loaded with Cu{sup +}. However, their simultaneous occupation is associated with enzyme turnover. Site I is constituted by two Cys in transmembrane segment (TM) 6 and a Tyr in TM7. An Asn in TM7 and Met and Ser in TM8 form Site II. Single site x-ray spectroscopic analysis indicates a trigonal coordination in both sites. This architecture is distinct from that observed in Cu{sup +}-trafficking chaperones and classical cuproproteins. The high affinity of these sites for Cu{sup +} (Site I K{sub {alpha}} = 1.3 fM{sup -1}, Site II K{sub {alpha}} = 1.1 fM{sup -1}), in conjunction with reversible direct Cu{sup +} transfer from chaperones, points to a transport mechanism where backward release of free Cu{sup +} to the cytoplasm is largely prevented.

  5. Trans-membrane transport of n-octadecane by Pseudomonas sp. DG17.

    PubMed

    Hua, Fei; Wang, Hong Qi; Li, Yi; Zhao, Yi Cun

    2013-12-01

    The trans-membrane transport of hydrocarbons is an important and complex aspect of the process of biodegradation of hydrocarbons by microorganisms. The mechanism of transport of (14)C n-octadecane by Pseudomonas sp. DG17, an alkane-degrading bacterium, was studied by the addition of ATP inhibitors and different substrate concentrations. When the concentration of n-octadecane was higher than 4.54 μmol/L, the transport of (14)C n-octadecane was driven by a facilitated passive mechanism following the intra/extra substrate concentration gradient. However, when the cells were grown with a low concentration of the substrate, the cellular accumulation of n-octadecane, an energy-dependent process, was dramatically decreased by the presence of ATP inhibitors, and n-octadecane accumulation continually increased against its concentration gradient. Furthermore, the presence of non-labeled alkanes blocked (14)C n-octadecane transport only in the induced cells, and the trans-membrane transport of n-octadecane was specific with an apparent dissociation constant K t of 11.27 μmol/L and V max of 0.96 μmol/min/mg protein. The results indicated that the trans-membrane transport of n-octadecane by Pseudomonas sp. DG17 was related to the substrate concentration and ATP.

  6. Bioassay-guided Isolation of Constituents of Piper sarmentosum Using a Mitochondrial Transmembrane Potential Assay

    PubMed Central

    Pan, Li; Matthew, Susan; Lantvit, Daniel D.; Zhang, Xiaoli; Ninh, Tran Ngoc; Chai, Heebyung; de Blanco, Esperanza J. Carcache; Soejarto, Djaja D.; Swanson, Steven M.; Kinghorn, A. Douglas

    2011-01-01

    Bioassay-guided fractionation was conducted on a chloroform-soluble extract of the aerial parts of Piper sarmentosum collected in Vietnam, monitored by a mitochondrial transmembrane potential (MTP) assay using HT-29 human colon cancer cells. This led to the isolation of four new C-benzylated dihydroflavones, sarmentosumins A-D (1-4), as well as 14 known compounds. The structures of the new compounds were elucidated on the basis of spectroscopic data interpretation. Among these compounds, 1-4 as well as five known C-benzylated dihydroflavones (5-9), and pipercallosine, a piperamide (11), were found to induce apoptosis in HT-29 cells by moderately reducing the mitochondrial transmembrane potential (ΔΨm), with ED50 values ranging from 1.6 to 13.6 μM. Furthermore, 7-methoxydichamanetin (8) and pinocembrin (10) exhibited proteasome inhibitory activities in a human 20S proteasome bioassay with IC50 values of 3.45 ± 0.18 μM and 2.87 ± 0.26 μM, respectively. This is the first time that C-benzylated dihydroflavones have been reported to demonstrate an apoptotic effect associated with disruption of the mitochondrial transmembrane potential. PMID:21973101

  7. [The effect of the transmembrane potential level on the dynamic extinction of the amplitude of the synaptic reactions in the snail under potential-fixation conditions].

    PubMed

    Gusev, P V

    1995-01-01

    The goal of this work was to describe a contribution of postsynaptic membrane in synaptic plasticity. To solve this problem the influence of the level of transmembrane potential on the dynamics of transmembrane synaptic current amplitude was studied during rhythmical stimulation of the nerve. It was shown that with increase of transmembrane potential value the slope of the function of transmembrane current decrease became steeper. It was also shown that with higher stimulation frequency the influence of transmembrane holding potential upon the dynamics of synaptic reaction increased. The data obtained can be considered as an evidence to the contribution of postsynaptic membrane in synaptic plasticity.

  8. Transmembrane topography of nicotinic acetylcholine receptor: immunochemical tests contradict theoretical predictions based on hydrophobicity profiles.

    PubMed

    Ratnam, M; Nguyen, D L; Rivier, J; Sargent, P B; Lindstrom, J

    1986-05-01

    In our preceding paper [Ratnam, M., Sargent, P. B., Sarin, V., Fox, J. L., Le Nguyen, D., Rivier, J., Criado, M., & Lindstrom, J. (1986) Biochemistry (preceding paper in this issue)], we presented results from peptide mapping studies of purified subunits of the Torpedo acetylcholine receptor which suggested that the sequence beta 429-441 is on the cytoplasmic surface of the receptor. Since this finding contradicts earlier theoretical models of the transmembrane structure of the receptor, which placed this sequence of the beta subunit on the extracellular surface, we investigated the location of the corresponding sequence (389-408) and adjacent sequences of the alpha subunit by a more direct approach. We synthesized peptides including the sequences alpha 330-346, alpha 349-364, alpha 360-378, alpha 379-385, and alpha 389-408 and shorter parts of these peptides. These peptides corresponded to a highly immunogenic region, and by using 125I-labeled peptides as antigens, we were able to detect in our library of monoclonal antibodies to alpha subunits between two and six which bound specifically to each of these peptides, except alpha 389-408. We obtained antibodies specific for alpha 389-408 both from antisera against the denatured alpha subunit and from antisera made against the peptide. These antibodies were specific to alpha 389-396. In binding assays, antibodies specific for all of these five peptides bound to receptor-rich membrane vesicles only after permeabilization of the vesicles to permit access of the antibodies to the cytoplasmic surface of the receptors, suggesting that the receptor sequences which bound these antibodies were located on the intracellular side of the membrane. Electron microscopy using colloidal gold to visualize the bound antibodies was used to conclusively demonstrate that all of these sequences are exposed on the cytoplasmic surface of the receptor. These results, along with our previous demonstration that the C-terminal 10 amino acids of

  9. A Trigger Residue for Transmembrane Signaling in the Escherichia coli Serine Chemoreceptor

    PubMed Central

    Kitanovic, Smiljka; Ames, Peter

    2015-01-01

    ABSTRACT The transmembrane Tsr protein of Escherichia coli mediates chemotactic responses to environmental serine gradients. Serine binds to the periplasmic domain of the homodimeric Tsr molecule, promoting a small inward displacement of one transmembrane helix (TM2). TM2 piston displacements, in turn, modulate the structural stability of the Tsr-HAMP domain on the cytoplasmic side of the membrane to control the autophosphorylation activity of the signaling CheA kinase bound to the membrane-distal cytoplasmic tip of Tsr. A five-residue control cable segment connects TM2 to the AS1 helix of HAMP and transmits stimulus and sensory adaptation signals between them. To explore the possible role of control cable helicity in transmembrane signaling by Tsr, we characterized the signaling properties of mutant receptors with various control cable alterations. An all-alanine control cable shifted Tsr output toward the kinase-on state, whereas an all-glycine control cable prevented Tsr from reaching either a fully on or fully off output state. Restoration of the native isoleucine (I214) in these synthetic control cables largely alleviated their signaling defects. Single amino acid replacements at Tsr-I214 shifted output toward the kinase-off (L, N, H, and R) or kinase-on (A and G) states, whereas other control cable residues tolerated most amino acid replacements with little change in signaling behavior. These findings indicate that changes in control cable helicity might mediate transitions between the kinase-on and kinase-off states during transmembrane signaling by chemoreceptors. Moreover, the Tsr-I214 side chain plays a key role, possibly through interaction with the membrane interfacial environment, in triggering signaling changes in response to TM2 piston displacements. IMPORTANCE The Tsr protein of E. coli mediates chemotactic responses to environmental serine gradients. Stimulus signals from the Tsr periplasmic sensing domain reach its cytoplasmic kinase control

  10. Integrin α1 Has a Long Helix, Extending from the Transmembrane Region to the Cytoplasmic Tail in Detergent Micelles

    PubMed Central

    Lai, Chaohua; Liu, Xiaoxi; Tian, Changlin; Wu, Fangming

    2013-01-01

    Integrin proteins are very important adhesion receptors that mediate cell-cell and cell-extracellular matrix interactions. They play essential roles in cell signaling and the regulation of cellular shape, motility, and the cell cycle. Here, the transmembrane and cytoplasmic (TMC) domains of integrin α1 and β1 were over-expressed and purified in detergent micelles. The structure and backbone relaxations of α1-TMC in LDAO micelles were determined and analyzed using solution NMR. A long helix, extending from the transmembrane region to the cytoplasmic tail, was observed in α1-TMC. Structural comparisons of α1-TMC with reported αIIb-TMC domains indicated different conformations in the transmembrane regions and cytoplasmic tails. An NMR titration experiment indicated weak interactions between α1-TMC and β1-TMC through several α1-TMC residues located at its N-terminal juxta-transmembrane region and C-terminal extended helix region. PMID:23646163

  11. 17th International Conference on Arabidopsis Research

    SciTech Connect

    Judith Bender

    2006-07-01

    The 17th International Conference on Arabidopsis Research was held at the University of Madison, Wisconsin from June 27- July 2, 2006. ICAR-2006 included approximately 625 scientists from across the world. The scientific program was of excellent quality featuring 73 talks, including 30 from invited speakers. There were also 6 community-organized workshops (facilitated by conference staff) featuring additional talks on topics including ‘Submitting data to long-term repositories,’ ‘TAIR introductory workshop,’ ‘Web services and demonstration,’ ‘Public engagement: broadening the impact of your research,’ ‘Systems biology approaches to analysis of metabolic and regulatory networks of Arabidopsis,’ and ‘Mechanotransduction in Arabidopsis.’ Approximately 440 posters were presented in general topic areas including, among others, Development, Modeling/Other Systems, Energy, Environment, and Genetic/Epigenetic mechanisms. Graduate students, postdoctoral researchers, junior faculty, and underrepresented minorities made up a significant portion of the oral presentations thereby promoting the training of young scientists and facilitating important career development opportunities for speakers. Several poster sessions provided an opportunity for younger participants to freely meet with more established scientists. The North American Arabidopsis Steering Committee (NAASC) continued its outreach effort and again sponsored two special luncheons to encourage personal and professional development of young scientists and also underrepresented minorities. The ‘Emerging Scientists Luncheon’ featured 10 graduate students selected on the basis of scientific excellence of their submitted research abstracts. The ‘Minority Funding Luncheon,’ featured 8 awardees selected by the NAASC through a widely-publicized application process. This luncheon was established specifically to provide an opportunity for underrepresented minorities, and/or scientists from

  12. Substituted cysteine accessibility reveals a novel transmembrane 2-3 reentrant loop and functional role for transmembrane domain 2 in the human proton-coupled folate transporter.

    PubMed

    Wilson, Mike R; Hou, Zhanjun; Matherly, Larry H

    2014-09-01

    The proton-coupled folate transporter (PCFT) is a folate-proton symporter highly expressed in solid tumors that can selectively target cytotoxic antifolates to tumors under acidic microenvironment conditions. Predicted topology models for PCFT suggest that the loop domain between transmembrane domains (TMDs) 2 and 3 resides in the cytosol. Mutations involving Asp-109 or Arg-113 in the TMD2-3 loop result in loss of activity. By structural homology to other solute carriers, TMD2 may form part of the PCFT substrate binding domain. In this study we mutated the seven cysteine (Cys) residues of human PCFT to serine, creating Cys-less PCFT. Thirty-three single-Cys mutants spanning TMD2 and the TMD2-3 loop in a Cys-less PCFT background were transfected into PCFT-null HeLa cells. All 33 mutants were detected by Western blotting, and 28 were active for [(3)H]methotrexate uptake at pH 5.5. For the active residues, we performed pulldown assays with membrane-impermeable 2-aminoethyl methanethiosulfonate-biotin and streptavidin beads to determine their aqueous-accessibilities. Multiple residues in TMD2 and the TMD2-3 loop domain reacted with 2-aminoethyl methanethiosulfonate-biotin, establishing aqueous accessibilities. Pemetrexed pretreatment inhibited biotinylation of TMD2 mutants G93C and F94C, and biotinylation of these residues inhibited methotrexate transport activity. Our results suggest that the TMD 2-3 loop domain is aqueous-accessible and forms a novel reentrant loop structure. Residues in TMD2 form an aqueous transmembrane pathway for folate substrates, and Gly-93 and Phe-94 may contribute to a substrate binding domain. Characterization of PCFT structure is essential to understanding the transport mechanism including the critical determinants of substrate binding.

  13. Substituted Cysteine Accessibility Reveals a Novel Transmembrane 2–3 Reentrant Loop and Functional Role for Transmembrane Domain 2 in the Human Proton-coupled Folate Transporter*

    PubMed Central

    Wilson, Mike R.; Hou, Zhanjun; Matherly, Larry H.

    2014-01-01

    The proton-coupled folate transporter (PCFT) is a folate-proton symporter highly expressed in solid tumors that can selectively target cytotoxic antifolates to tumors under acidic microenvironment conditions. Predicted topology models for PCFT suggest that the loop domain between transmembrane domains (TMDs) 2 and 3 resides in the cytosol. Mutations involving Asp-109 or Arg-113 in the TMD2-3 loop result in loss of activity. By structural homology to other solute carriers, TMD2 may form part of the PCFT substrate binding domain. In this study we mutated the seven cysteine (Cys) residues of human PCFT to serine, creating Cys-less PCFT. Thirty-three single-Cys mutants spanning TMD2 and the TMD2-3 loop in a Cys-less PCFT background were transfected into PCFT-null HeLa cells. All 33 mutants were detected by Western blotting, and 28 were active for [3H]methotrexate uptake at pH 5.5. For the active residues, we performed pulldown assays with membrane-impermeable 2-aminoethyl methanethiosulfonate-biotin and streptavidin beads to determine their aqueous-accessibilities. Multiple residues in TMD2 and the TMD2-3 loop domain reacted with 2-aminoethyl methanethiosulfonate-biotin, establishing aqueous accessibilities. Pemetrexed pretreatment inhibited biotinylation of TMD2 mutants G93C and F94C, and biotinylation of these residues inhibited methotrexate transport activity. Our results suggest that the TMD 2–3 loop domain is aqueous-accessible and forms a novel reentrant loop structure. Residues in TMD2 form an aqueous transmembrane pathway for folate substrates, and Gly-93 and Phe-94 may contribute to a substrate binding domain. Characterization of PCFT structure is essential to understanding the transport mechanism including the critical determinants of substrate binding. PMID:25053408

  14. Roles of Arabidopsis PARC6 in Coordination of the Chloroplast Division Complex and Negative Regulation of FtsZ Assembly1[OPEN

    PubMed Central

    Chen, Cheng; Froehlich, John E.; TerBush, Allan D.

    2016-01-01

    Chloroplast division is driven by the simultaneous constriction of the inner FtsZ ring (Z ring) and the outer DRP5B ring. The assembly and constriction of these rings in Arabidopsis (Arabidopsis thaliana) are coordinated partly through the inner envelope membrane protein ACCUMULATION AND REPLICATION OF CHLOROPLASTS6 (ARC6). Previously, we showed that PARC6 (PARALOG OF ARC6), also in the inner envelope membrane, negatively regulates FtsZ assembly and acts downstream of ARC6 to position the outer envelope membrane protein PLASTID DIVISION1 (PDV1), which functions together with its paralog PDV2 to recruit DYNAMIN-RELATED PROTEIN 5B (DRP5B) from a cytosolic pool to the outer envelope membrane. However, whether PARC6, like ARC6, also functions in coordination of the chloroplast division contractile complexes was unknown. Here, we report a detailed topological analysis of Arabidopsis PARC6, which shows that PARC6 has a single transmembrane domain and a topology resembling that of ARC6. The newly identified stromal region of PARC6 interacts not only with ARC3, a direct inhibitor of Z-ring assembly, but also with the Z-ring protein FtsZ2. Overexpression of PARC6 inhibits FtsZ assembly in Arabidopsis but not in a heterologous yeast system (Schizosaccharomyces pombe), suggesting that the negative regulation of FtsZ assembly by PARC6 is a consequence of its interaction with ARC3. A conserved carboxyl-terminal peptide in FtsZ2 mediates FtsZ2 interaction with both PARC6 and ARC6. Consistent with its role in the positioning of PDV1, the intermembrane space regions of PARC6 and PDV1 interact. These findings provide new insights into the functions of PARC6 and suggest that PARC6 coordinates the inner Z ring and outer DRP5B ring through interaction with FtsZ2 and PDV1 during chloroplast division. PMID:26527658

  15. Structural Characterization of the Mechanosensitive Channel Candidate MCA2 from Arabidopsis thaliana

    PubMed Central

    Shigematsu, Hideki; Iida, Kazuko; Nakano, Masataka; Chaudhuri, Pratima; Iida, Hidetoshi; Nagayama, Kuniaki

    2014-01-01

    Mechanosensing in plants is thought to be governed by sensory complexes containing a Ca2+-permeable, mechanosensitive channel. The plasma membrane protein MCA1 and its paralog MCA2 from Arabidopsis thaliana are involved in mechanical stress-induced Ca2+ influx and are thus considered as candidates for such channels or their regulators. Both MCA1 and MCA2 were functionally expressed in Sf9 cells using a baculovirus system in order to elucidate their molecular natures. Because of the abundance of protein in these cells, MCA2 was chosen for purification. Purified MCA2 in a detergent-solubilized state formed a tetramer, which was confirmed by chemical cross-linking. Single-particle analysis of cryo-electron microscope images was performed to depict the overall shape of the purified protein. The three-dimensional structure of MCA2 was reconstructed at a resolution of 26 Å from 5,500 particles and appears to comprise a small transmembrane region and large cytoplasmic region. PMID:24475319

  16. Structure of Voltage-gated Two-pore Channel TPC1 from Arabidopsis thaliana

    PubMed Central

    Guo, Jiangtao; Zeng, Weizhong; Chen, Qingfeng; Lee, Changkeun; Chen, Liping; Yang, Yi; Cang, Chunlei; Ren, Dejian; Jiang, Youxing

    2015-01-01

    Two-pore channels (TPCs) contain two copies of a Shaker-like six-transmembrane (6-TM) domain in each subunit and are ubiquitously expressed in both animals and plants as organellar cation channels. Here, we present the first crystal structure of a vacuolar two-pore channel from Arabidopsis thaliana, AtTPC1, which functions as a homodimer. AtTPC1 activation requires both voltage and cytosolic Ca2+. Ca2+ binding to the cytosolic EF-hand domain triggers conformational changes coupled to the pair of pore-lining inner helices (IS6 helices) from the first 6-TM domains, whereas membrane potential only activates the second voltage-sensing domain (VSD2) whose conformational changes are coupled to the pair of inner helices (IIS6 helices) from the second 6-TM domains. Luminal Ca2+ or Ba2+ can modulate voltage activation by stabilizing VSD2 in the resting state and shifts voltage activation towards more positive potentials. Our Ba2+ bound AtTPC1 structure reveals a voltage sensor in the resting state, providing hitherto unseen structural insight into the general voltage-gating mechanism among voltage-gated channels. PMID:26689363

  17. Structural characterization of the mechanosensitive channel candidate MCA2 from Arabidopsis thaliana.

    PubMed

    Shigematsu, Hideki; Iida, Kazuko; Nakano, Masataka; Chaudhuri, Pratima; Iida, Hidetoshi; Nagayama, Kuniaki

    2014-01-01

    Mechanosensing in plants is thought to be governed by sensory complexes containing a Ca²⁺-permeable, mechanosensitive channel. The plasma membrane protein MCA1 and its paralog MCA2 from Arabidopsis thaliana are involved in mechanical stress-induced Ca²⁺ influx and are thus considered as candidates for such channels or their regulators. Both MCA1 and MCA2 were functionally expressed in Sf9 cells using a baculovirus system in order to elucidate their molecular natures. Because of the abundance of protein in these cells, MCA2 was chosen for purification. Purified MCA2 in a detergent-solubilized state formed a tetramer, which was confirmed by chemical cross-linking. Single-particle analysis of cryo-electron microscope images was performed to depict the overall shape of the purified protein. The three-dimensional structure of MCA2 was reconstructed at a resolution of 26 Å from 5,500 particles and appears to comprise a small transmembrane region and large cytoplasmic region.

  18. Cell-free translation and purification of Arabidopsis thaliana regulator of G signaling 1 protein.

    PubMed

    Li, Bo; Makino, Shin-Ichi; Beebe, Emily T; Urano, Daisuke; Aceti, David J; Misenheimer, Tina M; Peters, Jonathan; Fox, Brian G; Jones, Alan M

    2016-10-01

    Arabidopsis thaliana Regulator of G protein Signalling 1 (AtRGS1) is a protein with a predicted N-terminal 7-transmembrane (7TM) domain and a C-terminal cytosolic RGS1 box domain. The RGS1 box domain exerts GTPase activation (GAP) activity on Gα (AtGPA1), a component of heterotrimeric G protein signaling in plants. AtRGS1 may perceive an exogenous agonist to regulate the steady-state levels of the active form of AtGPA1. It is uncertain if the full-length AtRGS1 protein exerts any atypical effects on Gα, nor has it been established exactly how AtRGS1 contributes to perception of an extracellular signal and transmits this response to a G-protein dependent signaling cascade. Further studies on full-length AtRGS1 have been inhibited due to the extreme low abundance of the endogenous AtRGS1 protein in plants and lack of a suitable heterologous system to express AtRGS1. Here, we describe methods to produce full-length AtRGS1 by cell-free synthesis into unilamellar liposomes and nanodiscs. The cell-free synthesized AtRGS1 exhibits GTPase activating activity on Gα and can be purified to a level suitable for biochemical analyses.

  19. ACA12 Is a Deregulated Isoform of Plasma Membrane Ca2+-ATPase of Arabidopsis thaliana

    PubMed Central

    Limonta, Margherita; Romanowsky, Shawn; Olivari, Claudio; Bonza, Maria Cristina; Luoni, Laura; Rosenberg, Alexa; Harper, Jeffrey F.; De Michelis, Maria Ida

    2014-01-01

    Plant auto-inhibited Ca2+-ATPases (ACA) are crucial in defining the shape of calcium transients and therefore in eliciting plant responses to various stimuli. Arabidopsis thaliana genome encodes ten ACA isoforms that can be divided into four clusters based on gene structure and sequence homology. While isoforms from clusters 1, 2 and 4 have been characterized, virtually nothing is known about members of cluster 3 (ACA12 and ACA13). Here we show that a GFP-tagged ACA12 localizes at the plasma membrane and that expression of ACA12 rescues the phenotype of partial male sterility of a null mutant of the plasma membrane isoform ACA9, thus providing genetic evidence that ACA12 is a functional plasma membrane-resident Ca2+-ATPase. By ACA12 expression in yeast and purification by CaM-affinity chromatography, we show that, unlike other ACAs, the activity of ACA12 is not stimulated by CaM. Moreover, full length ACA12 is able to rescue a yeast mutant deficient in calcium pumps. Analysis of single point ACA12 mutants suggests that ACA12 loss of auto-inhibition can be ascribed to the lack of two acidic residues - highly conserved in other ACA isoforms - localized at the cytoplasmic edge of the second and third transmembrane segments. Together, these results support a model in which the calcium pump activity of ACA12 is primarily regulated by increasing or decreasing mRNA expression and/or protein translation and degradation. PMID:24101142

  20. Analysis of Phosphorylation of the Receptor-Like Protein Kinase HAESA during Arabidopsis Floral Abscission

    PubMed Central

    Taylor, Isaiah; Wang, Ying; Seitz, Kati; Baer, John; Bennewitz, Stefan; Mooney, Brian P.; Walker, John C.

    2016-01-01

    Receptor-like protein kinases (RLKs) are the largest family of plant transmembrane signaling proteins. Here we present functional analysis of HAESA, an RLK that regulates floral organ abscission in Arabidopsis. Through in vitro and in vivo analysis of HAE phosphorylation, we provide evidence that a conserved phosphorylation site on a region of the HAE protein kinase domain known as the activation segment positively regulates HAE activity. Additional analysis has identified another putative activation segment phosphorylation site common to multiple RLKs that potentially modulates HAE activity. Comparative analysis suggests that phosphorylation of this second activation segment residue is an RLK specific adaptation that may regulate protein kinase activity and substrate specificity. A growing number of RLKs have been shown to exhibit biologically relevant dual specificity toward serine/threonine and tyrosine residues, but the mechanisms underlying dual specificity of RLKs are not well understood. We show that a phospho-mimetic mutant of both HAE activation segment residues exhibits enhanced tyrosine auto-phosphorylation in vitro, indicating phosphorylation of this residue may contribute to dual specificity of HAE. These results add to an emerging framework for understanding the mechanisms and evolution of regulation of RLK activity and substrate specificity. PMID:26784444

  1. Role of Dickeya dadantii 3937 chemoreceptors in the entry to Arabidopsis leaves through wounds.

    PubMed

    Río-Álvarez, Isabel; Muñoz-Gómez, Cristina; Navas-Vásquez, Mariela; Martínez-García, Pedro M; Antúnez-Lamas, María; Rodríguez-Palenzuela, Pablo; López-Solanilla, Emilia

    2015-09-01

    Chemotaxis enables bacteria to move towards an optimal environment in response to chemical signals. In the case of plant-pathogenic bacteria, chemotaxis allows pathogens to explore the plant surface for potential entry sites with the ultimate aim to prosper inside plant tissues and to cause disease. Chemoreceptors, which constitute the sensory core of the chemotaxis system, are usually transmembrane proteins which change their conformation when sensing chemicals in the periplasm and transduce the signal through a kinase pathway to the flagellar motor. In the particular case of the soft-rot pathogen Dickeya dadantii 3937, jasmonic acid released in a plant wound has been found to be a strong chemoattractant which drives pathogen entry into the plant apoplast. In order to identify candidate chemoreceptors sensing wound-derived plant compounds, we carried out a bioinformatics search of candidate chemoreceptors in the genome of Dickeya dadantii 3937. The study of the chemotactic response to several compounds and the analysis of the entry process to Arabidopsis leaves of 10 selected mutants in chemoreceptors allowed us to determine the implications of at least two of them (ABF-0020167 and ABF-0046680) in the chemotaxis-driven entry process through plant wounds. Our data suggest that ABF-0020167 and ABF-0046680 may be candidate receptors of jasmonic acid and xylose, respectively.

  2. ROP GTPase-mediated auxin signaling regulates pavement cell interdigitation in Arabidopsis thaliana.

    PubMed

    Lin, Deshu; Ren, Huibo; Fu, Ying

    2015-01-01

    In multicellular plant organs, cell shape formation depends on molecular switches to transduce developmental or environmental signals and to coordinate cell-to-cell communication. Plants have a specific subfamily of the Rho GTPase family, usually called Rho of Plants (ROP), which serve as a critical signal transducer involved in many cellular processes. In the last decade, important advances in the ROP-mediated regulation of plant cell morphogenesis have been made by using Arabidopsis thaliana leaf and cotyledon pavement cells. Especially, the auxin-ROP signaling networks have been demonstrated to control interdigitated growth of pavement cells to form jigsaw-puzzle shapes. Here, we review findings related to the discovery of this novel auxin-signaling mechanism at the cell surface. This signaling pathway is to a large extent independent of the well-known Transport Inhibitor Response (TIR)-Auxin Signaling F-Box (AFB) pathway, and instead requires Auxin Binding Protein 1 (ABP1) interaction with the plasma membrane-localized, transmembrane kinase (TMK) receptor-like kinase to regulate ROP proteins. Once activated, ROP influences cytoskeletal organization and inhibits endocytosis of the auxin transporter PIN1. The present review focuses on ROP signaling and its self-organizing feature allowing ROP proteins to serve as a bustling signal decoder and integrator for plant cell morphogenesis.

  3. Structure of the voltage-gated two-pore channel TPC1 from Arabidopsis thaliana.

    PubMed

    Guo, Jiangtao; Zeng, Weizhong; Chen, Qingfeng; Lee, Changkeun; Chen, Liping; Yang, Yi; Cang, Chunlei; Ren, Dejian; Jiang, Youxing

    2016-03-10

    Two-pore channels (TPCs) contain two copies of a Shaker-like six-transmembrane (6-TM) domain in each subunit and are ubiquitously expressed in both animals and plants as organellar cation channels. Here we present the crystal structure of a vacuolar two-pore channel from Arabidopsis thaliana, AtTPC1, which functions as a homodimer. AtTPC1 activation requires both voltage and cytosolic Ca(2+). Ca(2+) binding to the cytosolic EF-hand domain triggers conformational changes coupled to the pair of pore-lining inner helices from the first 6-TM domains, whereas membrane potential only activates the second voltage-sensing domain, the conformational changes of which are coupled to the pair of inner helices from the second 6-TM domains. Luminal Ca(2+) or Ba(2+) can modulate voltage activation by stabilizing the second voltage-sensing domain in the resting state and shift voltage activation towards more positive potentials. Our Ba(2+)-bound AtTPC1 structure reveals a voltage sensor in the resting state, providing hitherto unseen structural insight into the general voltage-gating mechanism among voltage-gated channels. PMID:26689363

  4. Evolutionary Divergence of Arabidopsis thaliana Classical Peroxidases.

    PubMed

    Kupriyanova, E V; Mamoshina, P O; Ezhova, T A

    2015-10-01

    Polymorphisms of 62 peroxidase genes derived from Arabidopsis thaliana were investigated to evaluate evolutionary dynamics and divergence of peroxidase proteins. By comparing divergence of duplicated genes AtPrx53-AtPrx54 and AtPrx36-AtPrx72 and their products, nucleotide and amino acid substitutions were identified that were apparently targets of positive selection. These substitutions were detected among paralogs of 461 ecotypes from Arabidopsis thaliana. Some of these substitutions are conservative and matched paralogous peroxidases in other Brassicaceae species. These results suggest that after duplication, peroxidase genes evolved under the pressure of positive selection, and amino acid substitutions identified during our study provided divergence of properties and physiological functions in peroxidases. Our predictions regarding functional significance for amino acid residues identified in variable sites of peroxidases may allow further experimental assessment of evolution of peroxidases after gene duplication.

  5. Root gravitropism in maize and Arabidopsis

    NASA Technical Reports Server (NTRS)

    Evans, Michael L.

    1993-01-01

    Research during the period 1 March 1992 to 30 November 1993 focused on improvements in a video digitizer system designed to automate the recording of surface extension in plants responding to gravistimulation. The improvements included modification of software to allow detailed analysis of localized extension patterns in roots of Arabidopsis. We used the system to analyze the role of the postmitotic isodiametric growth zone (a region between the meristem and the elongation zone) in the response of maize roots to auxin, calcium, touch and gravity. We also used the system to analyze short-term auxin and gravitropic responses in mutants of Arabidopsis with reduced auxin sensitivity. In a related project, we studied the relationship between growth rate and surface electrical currents in roots by examining the effects of gravity and thigmostimulation on surface potentials in maize roots.

  6. Unraveling the circadian clock in Arabidopsis

    PubMed Central

    Wang, Xiaoxue; Ma, Ligeng

    2013-01-01

    The circadian clock is an endogenous timing system responsible for coordinating an organism’s biological processes with its environment. Interlocked transcriptional feedback loops constitute the fundamental architecture of the circadian clock. In Arabidopsis, three feedback loops, the core loop, morning loop and evening loop, comprise a network that is the basis of the circadian clock. The components of these three loops are regulated in distinct ways, including transcriptional, post-transcriptional and posttranslational mechanisms. The discovery of the DNA-binding and repressive activities of TOC1 has overturned our initial concept of its function in the circadian clock. The alternative splicing of circadian clock-related genes plays an essential role in normal functioning of the clock and enables organisms to sense environmental changes. In this review, we describe the regulatory mechanisms of the circadian clock that have been identified in Arabidopsis. PMID:23221775

  7. DYn-2 Based Identification of Arabidopsis Sulfenomes*

    PubMed Central

    Akter, Salma; Huang, Jingjing; Bodra, Nandita; De Smet, Barbara; Wahni, Khadija; Rombaut, Debbie; Pauwels, Jarne; Gevaert, Kris; Carroll, Kate; Van Breusegem, Frank; Messens, Joris

    2015-01-01

    Identifying the sulfenylation state of stressed cells is emerging as a strategic approach for the detection of key reactive oxygen species signaling proteins. Here, we optimized an in vivo trapping method for cysteine sulfenic acids in hydrogen peroxide (H2O2) stressed plant cells using a dimedone based DYn-2 probe. We demonstrated that DYn-2 specifically detects sulfenylation events in an H2O2 dose- and time-dependent way. With mass spectrometry, we identified 226 sulfenylated proteins after H2O2 treatment of Arabidopsis cells, residing in the cytoplasm (123); plastid (68); mitochondria (14); nucleus (10); endoplasmic reticulum, Golgi and plasma membrane (7) and peroxisomes (4). Of these, 123 sulfenylated proteins have never been reported before to undergo cysteine oxidative post-translational modifications in plants. All in all, with this DYn-2 approach, we have identified new sulfenylated proteins, and gave a first glance on the locations of the sulfenomes of Arabidopsis thaliana. PMID:25693797

  8. From genome to function: the Arabidopsis aquaporins

    PubMed Central

    Quigley, Francoise; Rosenberg, Joshua M; Shachar-Hill, Yair; Bohnert, Hans J

    2002-01-01

    Background In the post-genomic era newly sequenced genomes can be used to deduce organismal functions from our knowledge of other systems. Here we apply this approach to analyzing the aquaporin gene family in Arabidopsis thaliana. The aquaporins are intrinsic membrane proteins that have been characterized as facilitators of water flux. Originally termed major intrinsic proteins (MIPs), they are now also known as water channels, glycerol facilitators and aqua-glyceroporins, yet recent data suggest that they facilitate the movement of other low-molecular-weight metabolites as well. Results The Arabidopsis genome contains 38 sequences with homology to aquaporin in four subfamilies, termed PIP, TIP, NIP and SIP. We have analyzed aquaporin family structure and expression using the A. thaliana genome sequence, and introduce a new NMR approach for the purpose of analyzing water movement in plant roots in vivo. Conclusions Our preliminary data indicate a strongly transcellular component for the flux of water in roots. PMID:11806824

  9. PHOTOSYSTEM II PROTEIN33, a Protein Conserved in the Plastid Lineage, Is Associated with the Chloroplast Thylakoid Membrane and Provides Stability to Photosystem II Supercomplexes in Arabidopsis1[OPEN

    PubMed Central

    Fristedt, Rikard; Herdean, Andrei; Blaby-Haas, Crysten E.; Mamedov, Fikret; Lundin, Björn

    2015-01-01

    Photosystem II (PSII) is a multiprotein complex that catalyzes the light-driven water-splitting reactions of oxygenic photosynthesis. Light absorption by PSII leads to the production of excited states and reactive oxygen species that can cause damage to this complex. Here, we describe Arabidopsis (Arabidopsis thaliana) At1g71500, which encodes a previously uncharacterized protein that is a PSII auxiliary core protein and hence is named PHOTOSYSTEM II PROTEIN33 (PSB33). We present evidence that PSB33 functions in the maintenance of PSII-light-harvesting complex II (LHCII) supercomplex organization. PSB33 encodes a protein with a chloroplast transit peptide and one transmembrane segment. In silico analysis of PSB33 revealed a light-harvesting complex-binding motif within the transmembrane segment and a large surface-exposed head domain. Biochemical analysis of PSII complexes further indicates that PSB33 is an integral membrane protein located in the vicinity of LHCII and the PSII CP43 reaction center protein. Phenotypic characterization of mutants lacking PSB33 revealed reduced amounts of PSII-LHCII supercomplexes, very low state transition, and a lower capacity for nonphotochemical quenching, leading to increased photosensitivity in the mutant plants under light stress. Taken together, these results suggest a role for PSB33 in regulating and optimizing photosynthesis in response to changing light levels. PMID:25511433

  10. Meiosis in autopolyploid and allopolyploid Arabidopsis.

    PubMed

    Lloyd, Andrew; Bomblies, Kirsten

    2016-04-01

    All newly formed polyploids face a challenge in meiotic chromosome segregation due to the presence of an additional set of chromosomes. Nevertheless, naturally occurring auto and allopolyploids are common and generally show high fertility, showing that evolution can find solutions. Exactly how meiosis is adapted in these cases, however, remains a mystery. The rise of Arabidopsis as a model genus for polyploid and meiosis research has seen several new studies begin to shed light on this long standing question.

  11. Analyzing Synthetic Promoters Using Arabidopsis Protoplasts.

    PubMed

    Stracke, Ralf; Thiedig, Katharina; Kuhlmann, Melanie; Weisshaar, Bernd

    2016-01-01

    This chapter describes a transient protoplast co-transfection method that can be used to quantitatively study in vivo the activity and function of promoters and promoter elements (reporters), and their induction or repression by transcription factors (effectors), stresses, hormones, or metabolites. A detailed protocol for carrying out transient co-transfection assays with Arabidopsis At7 protoplasts and calculating the promoter activity is provided. PMID:27557761

  12. Flavonoid-specific staining of Arabidopsis thaliana.

    PubMed

    Sheahan, J J; Rechnitz, G A

    1992-12-01

    Crop yields may be threatened by increases in UV-B radiation resulting from depletion of the ozone layer. In higher plants, the presence of flavonols provides a protective mechanism, and we report a novel staining procedure for the visualization of such protectants in plant tissue. It is shown that the proposed technique provides sensitive and specific fluorescence of flavonoids in chlorophyll-bleached tissue of Arabidopsis thaliana.

  13. Fluorescence-Activated Nucleolus Sorting in Arabidopsis.

    PubMed

    Pontvianne, Frédéric; Boyer-Clavel, Myriam; Sáez-Vásquez, Julio

    2016-01-01

    Nucleolar isolation allows exhaustive characterization of the nucleolar content. Centrifugation-based protocols are not adapted to isolation of nucleoli directly from a plant tissue because of copurification of cellular debris. We describe here a method that allows the purification of nucleoli using fluorescent-activated cell sorting from Arabidopsis thaliana leaves. This approach requires the expression of a specific nucleolar protein such as fibrillarin fused to green fluorescent protein in planta. PMID:27576720

  14. Resolving the biophysics of axon transmembrane polarization in a single closed-form description

    NASA Astrophysics Data System (ADS)

    Melendy, Robert F.

    2015-12-01

    When a depolarizing event occurs across a cell membrane there is a remarkable change in its electrical properties. A complete depolarization event produces a considerably rapid increase in voltage that propagates longitudinally along the axon and is accompanied by changes in axial conductance. A dynamically changing magnetic field is associated with the passage of the action potential down the axon. Over 75 years of research has gone into the quantification of this phenomenon. To date, no unified model exist that resolves transmembrane polarization in a closed-form description. Here, a simple but formative description of propagated signaling phenomena in the membrane of an axon is presented in closed-form. The focus is on using both biophysics and mathematical methods for elucidating the fundamental mechanisms governing transmembrane polarization. The results presented demonstrate how to resolve electromagnetic and thermodynamic factors that govern transmembrane potential. Computational results are supported by well-established quantitative descriptions of propagated signaling phenomena in the membrane of an axon. The findings demonstrate how intracellular conductance, the thermodynamics of magnetization, and current modulation function together in generating an action potential in a unified closed-form description. The work presented in this paper provides compelling evidence that three basic factors contribute to the propagated signaling in the membrane of an axon. It is anticipated this work will compel those in biophysics, physical biology, and in the computational neurosciences to probe deeper into the classical and quantum features of membrane magnetization and signaling. It is hoped that subsequent investigations of this sort will be advanced by the computational features of this model without having to resort to numerical methods of analysis.

  15. Resolving the biophysics of axon transmembrane polarization in a single closed-form description

    SciTech Connect

    Melendy, Robert F.

    2015-12-28

    When a depolarizing event occurs across a cell membrane there is a remarkable change in its electrical properties. A complete depolarization event produces a considerably rapid increase in voltage that propagates longitudinally along the axon and is accompanied by changes in axial conductance. A dynamically changing magnetic field is associated with the passage of the action potential down the axon. Over 75 years of research has gone into the quantification of this phenomenon. To date, no unified model exist that resolves transmembrane polarization in a closed-form description. Here, a simple but formative description of propagated signaling phenomena in the membrane of an axon is presented in closed-form. The focus is on using both biophysics and mathematical methods for elucidating the fundamental mechanisms governing transmembrane polarization. The results presented demonstrate how to resolve electromagnetic and thermodynamic factors that govern transmembrane potential. Computational results are supported by well-established quantitative descriptions of propagated signaling phenomena in the membrane of an axon. The findings demonstrate how intracellular conductance, the thermodynamics of magnetization, and current modulation function together in generating an action potential in a unified closed-form description. The work presented in this paper provides compelling evidence that three basic factors contribute to the propagated signaling in the membrane of an axon. It is anticipated this work will compel those in biophysics, physical biology, and in the computational neurosciences to probe deeper into the classical and quantum features of membrane magnetization and signaling. It is hoped that subsequent investigations of this sort will be advanced by the computational features of this model without having to resort to numerical methods of analysis.

  16. Interaction of inhibitors of the vacuolar H(+)-ATPase with the transmembrane Vo-sector.

    PubMed

    Páli, Tibor; Whyteside, Graham; Dixon, Neil; Kee, Terence P; Ball, Stephen; Harrison, Michael A; Findlay, John B C; Finbow, Malcolm E; Marsh, Derek

    2004-09-28

    The macrolide antibiotic concanamycin A and a designed derivative of 5-(2-indolyl)-2,4-pentadienamide (INDOL0) are potent inhibitors of vacuolar H(+)-ATPases, with IC(50) values in the low and medium nanomolar range, respectively. Interaction of these V-ATPase inhibitors with spin-labeled subunit c in the transmembrane V(o)-sector of the ATPase was studied by using the transport-active 16-kDa proteolipid analogue of subunit c from the hepatopancreas of Nephrops norvegicus. Analogous experiments were also performed with vacuolar membranes from Saccharomyces cerevisiae. Membranous preparations of the Nephrops 16-kDa proteolipid were spin-labeled either on the unique cysteine C54, with a nitroxyl maleimide, or on the functionally essential glutamate E140, with a nitroxyl analogue of dicyclohexylcarbodiimide (DCCD). These residues were previously demonstrated to be accessible to lipid. Interaction of the inhibitors with these lipid-exposed residues was studied by using both conventional and saturation transfer EPR spectroscopy. Immobilization of the spin-labeled residues by the inhibitors was observed on both the nanosecond and microsecond time scales. The perturbation by INDOL0 was mostly greater than that by concanamycin A. Qualitatively similar but quantitatively greater effects were obtained with the same spin-label reagents and vacuolar membranes in which the Nephrops 16-kDa proteolipid was expressed in place of the native vma3p proteolipid of yeast. The spin-label immobilization corresponds to a direct interaction of the inhibitors with these intramembranous sites on the protein. A mutational analysis on transmembrane segment 4 known to give resistance to concanamycin A also gave partial resistance to INDOL0. The results are consistent with transmembrane segments 2 and 4 of the 16-kDa putative four-helix bundle, and particularly the functionally essential protonation locus, being involved in the inhibitor binding sites. Inhibition of proton transport may also

  17. A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2)

    PubMed Central

    Rossi, Pia; Sterlini, Bruno; Castroflorio, Enrico; Marte, Antonella; Onofri, Franco; Valtorta, Flavia; Maragliano, Luca; Corradi, Anna; Benfenati, Fabio

    2016-01-01

    Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (Ncyt/Cexo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders. PMID:26797119

  18. Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore.

    PubMed

    El Hiani, Yassine; Linsdell, Paul

    2015-06-19

    As an ion channel, the cystic fibrosis transmembrane conductance regulator must form a continuous pathway for the movement of Cl(-) and other anions between the cytoplasm and the extracellular solution. Both the structure and the function of the membrane-spanning part of this pathway are well defined. In contrast, the structure of the pathway that connects the cytoplasm to the membrane-spanning regions is unknown, and functional roles for different parts of the protein forming this pathway have not been described. We used patch clamp recording and substituted cysteine accessibility mutagenesis to identify positively charged amino acid side chains that attract cytoplasmic Cl(-) ions to the inner mouth of the pore. Our results indicate that the side chains of Lys-190, Arg-248, Arg-303, Lys-370, Lys-1041, and Arg-1048, located in different intracellular loops of the protein, play important roles in the electrostatic attraction of Cl(-) ions. Mutation and covalent modification of these residues have charge-dependent effects on the rate of Cl(-) permeation, demonstrating their functional role in maximization of Cl(-) flux. Other nearby positively charged side chains were not involved in electrostatic interactions with Cl(-). The location of these Cl(-)-attractive residues suggests that cytoplasmic Cl(-) ions enter the pore via a lateral portal located between the cytoplasmic extensions to the fourth and sixth transmembrane helices; a secondary, functionally less relevant portal might exist between the extensions to the 10th and 12th transmembrane helices. These results define the cytoplasmic mouth of the pore and show how it attracts Cl(-) ions from the cytoplasm. PMID:25944907

  19. Ethanol Modulation is Quantitatively Determined by the Transmembrane Domain of Human α1 Glycine Receptors

    PubMed Central

    Horani, Suzzane; Stater, Evan P.; Corringer, Pierre-Jean; Trudell, James R.; Harris, R. Adron; Howard, Rebecca J.

    2015-01-01

    Background Mutagenesis and labeling studies have identified amino acids from the human α1 glycine receptor (GlyR) extracellular, transmembrane (TM), and intracellular domains in mediating ethanol potentiation. However, limited high-resolution structural data for physiologically relevant receptors in this Cys-loop receptor superfamily have made pinpointing the critical amino acids difficult. Homologous ion channels from lower organisms provide conserved models for structural and functional properties of Cys-loop receptors. We previously demonstrated that a single amino acid variant of the Gloeobacter violaceus ligand-gated ion channel (GLIC) produced ethanol and anesthetic sensitivity similar to that of GlyRs and provided crystallographic evidence for ethanol binding to GLIC. Methods We directly compared ethanol modulation of the α1 GlyR and GLIC to a chimera containing the transmembrane domain from human α1 GlyRs and the ligand-binding domain of GLIC using two-electrode voltage clamp electrophysiology of receptors expressed in Xenopus laevis oocytes. Results Ethanol potentiated α1 GlyRs in a concentration-dependent manner in the presence of zinc-chelating agents, but did not potentiate GLIC at pharmacologically relevant concentrations. The GLIC/GlyR chimera recapitulated the ethanol potentiation of GlyRs, without apparent sensitivity to zinc chelation. For chimera expression in oocytes, it was essential to suppress leakage current by adding 50 μM picrotoxin to the media, a technique that may have applications in expression of other ion channels. Conclusions Our results are consistent with a transmembrane mechanism of ethanol modulation in Cys-loop receptors. This work highlights the relevance of bacterial homologs as valuable model systems for studying ion channel function of human receptors and demonstrates the modularity of these channels across species. PMID:25973519

  20. Algorithms for incorporating prior topological information in HMMs: application to transmembrane proteins

    PubMed Central

    Bagos, Pantelis G; Liakopoulos, Theodore D; Hamodrakas, Stavros J

    2006-01-01

    Background Hidden Markov Models (HMMs) have been extensively used in computational molecular biology, for modelling protein and nucleic acid sequences. In many applications, such as transmembrane protein topology prediction, the incorporation of limited amount of information regarding the topology, arising from biochemical experiments, has been proved a very useful strategy that increased remarkably the performance of even the top-scoring methods. However, no clear and formal explanation of the algorithms that retains the probabilistic interpretation of the models has been presented so far in the literature. Results We present here, a simple method that allows incorporation of prior topological information concerning the sequences at hand, while at the same time the HMMs retain their full probabilistic interpretation in terms of conditional probabilities. We present modifications to the standard Forward and Backward algorithms of HMMs and we also show explicitly, how reliable predictions may arise by these modifications, using all the algorithms currently available for decoding HMMs. A similar procedure may be used in the training procedure, aiming at optimizing the labels of the HMM's classes, especially in cases such as transmembrane proteins where the labels of the membrane-spanning segments are inherently misplaced. We present an application of this approach developing a method to predict the transmembrane regions of alpha-helical membrane proteins, trained on crystallographically solved data. We show that this method compares well against already established algorithms presented in the literature, and it is extremely useful in practical applications. Conclusion The algorithms presented here, are easily implemented in any kind of a Hidden Markov Model, whereas the prediction method (HMM-TM) is freely available for academic users at , offering the most advanced decoding options currently available. PMID:16597327

  1. Neuregulin 1 Expression and Electrophysiological Abnormalities in the Neuregulin 1 Transmembrane Domain Heterozygous Mutant Mouse

    PubMed Central

    Frank, Elisabeth; Shaw, Alex; Liu, Shijie; Huang, Xu-Feng; Pinault, Didier; Karl, Tim; O’Brien, Terence J.; Shannon Weickert, Cynthia; Jones, Nigel C.

    2015-01-01

    Background The Neuregulin 1 transmembrane domain heterozygous mutant (Nrg1 TM HET) mouse is used to investigate the role of Nrg1 in brain function and schizophrenia-like behavioural phenotypes. However, the molecular alterations in brain Nrg1 expression that underpin the behavioural observations have been assumed, but not directly determined. Here we comprehensively characterise mRNA Nrg1 transcripts throughout development of the Nrg1 TM HET mouse. In addition, we investigate the regulation of high-frequency (gamma) electrophysiological oscillations in this mutant mouse to associate molecular changes in Nrg1 with a schizophrenia-relevant neurophysiological profile. Methods Using exonic probes spanning the cysteine-rich, epidermal growth factor (EGF)-like, transmembrane and intracellular domain encoding regions of Nrg1, mRNA levels were measured using qPCR in hippocampus and frontal cortex from male and female Nrg1 TM HET and wild type-like (WT) mice throughout development. We also performed electrophysiological recordings in adult mice and analysed gamma oscillatory at baseline, in responses to auditory stimuli and to ketamine. Results In both hippocampus and cortex, Nrg1 TM HET mice show significantly reduced expression of the exon encoding the transmembrane domain of Nrg1 compared with WT, but unaltered mRNA expression encoding the extracellular bioactive EGF-like and the cysteine-rich (type III) domains, and development-specific and region-specific reductions in the mRNA encoding the intracellular domain. Hippocampal Nrg1 protein expression was not altered, but NMDA receptor NR2B subunit phosphorylation was lower in Nrg1 TM HET mice. We identified elevated ongoing and reduced sensory-evoked gamma power in Nrg1 TM HET mice. Interpretation We found no evidence to support the claim that the Nrg1 TM HET mouse represents a simple haploinsufficient model. Further research is required to explore the possibility that mutation results in a gain of Nrg1 function. PMID

  2. Structural organization of FtsB, a transmembrane protein of the bacterial divisome

    PubMed Central

    LaPointe, Loren M.; Taylor, Keenan C.; Subramaniam, Sabareesh; Khadria, Ambalika; Rayment, Ivan; Senes, Alessandro

    2013-01-01

    We report the first structural analysis of an integral membrane protein of the bacterial divisome. FtsB is a single-pass membrane protein with a periplasmic coiled coil. Its heterologous association with its partner FtsL represents an essential event for the recruitment of the late components to the division site. Using a combination of mutagenesis, computational modeling and X-ray crystallography, we determined that FtsB self-associates and we investigated its structural organization. We found that the transmembrane domain of FtsB homo-oligomerizes through an evolutionarily conserved interaction interface where a polar residue (Gln 16) plays a critical role through the formation of an inter-helical hydrogen bond. The crystal structure of the periplasmic domain, solved as a fusion with Gp7, shows that 30 juxta-membrane amino acids of FtsB form a canonical coiled coil. The presence of conserved Gly residue in the linker region suggests that flexibility between the transmembrane and coiled coil domains is functionally important. We hypothesize that the transmembrane helices of FtsB form a stable dimeric core for its association with FtsL into a higher-order oligomer, and that FtsL is required to stabilize the periplasmic domain of FtsB, leading to the formation of a complex that is competent for binding to FtsQ, and to their consequent recruitment to the divisome. The study provides an experimentally validated structural model and identifies point mutations that disrupt association, thereby establishing important groundwork for the functional characterization of FtsB in vivo. PMID:23520975

  3. Analysis of the Arabidopsis Mitochondrial Proteome1

    PubMed Central

    Millar, A. Harvey; Sweetlove, Lee J.; Giegé, Philippe; Leaver, Christopher J.

    2001-01-01

    The complete set of nuclear genes that encode proteins targeted to mitochondria in plants is currently undefined and thus the full range of mitochondrial functions in plants is unknown. Analysis of two-dimensional gel separations of Arabidopsis cell culture mitochondrial protein revealed approximately 100 abundant proteins and 250 low-abundance proteins. Comparison of subfractions of mitochondrial protein on two-dimensional gels provided information on the soluble, membrane, or integral membrane locations of this protein set. A total of 170 protein spots were excised, trypsin-digested, and matrix-assisted laser desorption ionization/time of flight mass spectrometry spectra obtained. Using this dataset, 91 of the proteins were identified by searching translated Arabidopsis genomic databases. Of this set, 81 have defined functions based on sequence comparison. These functions include respiratory electron transport, tricarboxylic acid cycle metabolism, amino acid metabolism, protein import, processing, and assembly, transcription, membrane transport, and antioxidant defense. A total of 10 spectra were matched to Arabidopsis putative open reading frames for which no specific function has been determined. A total of 64 spectra did not match to an identified open reading frame. Analysis of full-length putative protein sequences using bioinformatic tools to predict subcellular targeting (TargetP, Psort, and MitoProt) revealed significant variation in predictions, and also a lack of mitochondrial targeting prediction for several characterized mitochondrial proteins. PMID:11743115

  4. Regulatory Proteolysis in Arabidopsis-Pathogen Interactions

    PubMed Central

    Pogány, Miklós; Dankó, Tamás; Kámán-Tóth, Evelin; Schwarczinger, Ildikó; Bozsó, Zoltán

    2015-01-01

    Approximately two and a half percent of protein coding genes in Arabidopsis encode enzymes with known or putative proteolytic activity. Proteases possess not only common housekeeping functions by recycling nonfunctional proteins. By irreversibly cleaving other proteins, they regulate crucial developmental processes and control responses to environmental changes. Regulatory proteolysis is also indispensable in interactions between plants and their microbial pathogens. Proteolytic cleavage is simultaneously used both by plant cells, to recognize and inactivate invading pathogens, and by microbes, to overcome the immune system of the plant and successfully colonize host cells. In this review, we present available results on the group of proteases in the model plant Arabidopsis thaliana whose functions in microbial pathogenesis were confirmed. Pathogen-derived proteolytic factors are also discussed when they are involved in the cleavage of host metabolites. Considering the wealth of review papers available in the field of the ubiquitin-26S proteasome system results on the ubiquitin cascade are not presented. Arabidopsis and its pathogens are conferred with abundant sets of proteases. This review compiles a list of those that are apparently involved in an interaction between the plant and its pathogens, also presenting their molecular partners when available. PMID:26404238

  5. Transcriptional profiling of the Arabidopsis embryo.

    PubMed

    Spencer, Matthew W B; Casson, Stuart A; Lindsey, Keith

    2007-02-01

    We have used laser-capture microdissection to isolate RNA from discrete tissues of globular, heart, and torpedo stage embryos of Arabidopsis (Arabidopsis thaliana). This was amplified and analyzed by DNA microarray using the Affymetrix ATH1 GeneChip, representing approximately 22,800 Arabidopsis genes. Cluster analysis showed that spatial differences in gene expression were less significant than temporal differences. Time course analysis reveals the dynamics and complexity of gene expression in both apical and basal domains of the developing embryo, with several classes of synexpressed genes identifiable. The transition from globular to heart stage is associated in particular with an up-regulation of genes involved in cell cycle control, transcriptional regulation, and energetics and metabolism. The transition from heart to torpedo stage is associated with a repression of cell cycle genes and an up-regulation of genes encoding storage proteins, and pathways of cell growth, energy, and metabolism. The torpedo stage embryo shows strong functional differentiation in the root and cotyledon, as inferred from the classes of genes expressed in these tissues. The time course of expression of the essential EMBRYO-DEFECTIVE genes shows that most are expressed at unchanging levels across all stages of embryogenesis. We show how identified genes can be used to generate cell type-specific markers and promoter activities for future application in cell biology.

  6. Phosphorylation of plastoglobular proteins in Arabidopsis thaliana

    PubMed Central

    Lohscheider, Jens N.; Friso, Giulia; van Wijk, Klaas J.

    2016-01-01

    Plastoglobules (PGs) are plastid lipid–protein particles with a small specialized proteome and metabolome. Among the 30 core PG proteins are six proteins of the ancient ABC1 atypical kinase (ABC1K) family and their locations in an Arabidopsis mRNA-based co-expression network suggested central regulatory roles. To identify candidate ABC1K targets and a possible ABC1K hierarchical phosphorylation network within the chloroplast PG proteome, we searched Arabidopsis phosphoproteomics data from publicly available sources. Evaluation of underlying spectra and/or associated information was challenging for a variety of reasons, but supported pSer sites and a few pThr sites in nine PG proteins, including five FIBRILLINS. PG phosphorylation motifs are discussed in the context of possible responsible kinases. The challenges of collection and evaluation of published Arabidopsis phosphorylation data are discussed, illustrating the importance of deposition of all mass spectrometry data in well-organized repositories such as PRIDE and ProteomeXchange. This study provides a starting point for experimental testing of phosho-sites in PG proteins and also suggests that phosphoproteomics studies specifically designed toward the PG proteome and its ABC1K are needed to understand phosphorylation networks in these specialized particles. PMID:26962209

  7. Computational identification of 69 retroposons in Arabidopsis.

    PubMed

    Zhang, Yujun; Wu, Yongrui; Liu, Yilei; Han, Bin

    2005-06-01

    Retroposition is a shot-gun strategy of the genome to achieve evolutionary diversities by mixing and matching coding sequences with novel regulatory elements. We have identified 69 retroposons in the Arabidopsis (Arabidopsis thaliana) genome by a computational approach. Most of them were derivatives of mature mRNAs, and 20 genes contained relics of the reverse transcription process, such as truncations, deletions, and extra sequence additions. Of them, 22 are processed pseudogenes, and 52 genes are likely to be actively transcribed, especially in tissues from apical meristems (roots and flowers). Functional compositions of these retroposon parental genes imply that not the mRNA itself but its expression in gamete cells defines a suitable template for retroposition. The presence/absence patterns of retroposons can be used as cladistic markers for biogeographic research. Effects of human and the Mediterranean Pleistocene refugia in Arabidopsis biogeographic distributions were revealed based on two recent retroposons (At1g61410 and At5g52090). An evolutionary rate of new gene creation by retroposition was calculated as 0.6 genes per million years. Retroposons can also be used as molecular fossils of the parental gene expressions in ancient time. Extensions of 3' untranslated regions for those expressed parental genes are revealed as a possible trend of plant transcriptome evolution. In addition, we reported the first plant functional chimeric gene that adapts to intercompartmental transport by capturing two additional exons after retroposition. PMID:15923328

  8. Artificial Diels–Alderase based on the transmembrane protein FhuA

    PubMed Central

    Beckerle, Klaus; Arlt, Marcus; Himiyama, Tomoki; Polen, Tino; Onoda, Akira; Schwaneberg, Ulrich; Hayashi, Takashi

    2016-01-01

    Summary Copper(I) and copper(II) complexes were covalently linked to an engineered variant of the transmembrane protein Ferric hydroxamate uptake protein component A (FhuA ΔCVFtev). Copper(I) was incorporated using an N-heterocyclic carbene (NHC) ligand equipped with a maleimide group on the side arm at the imidazole nitrogen. Copper(II) was attached by coordination to a terpyridyl ligand. The spacer length was varied in the back of the ligand framework. These biohybrid catalysts were shown to be active in the Diels–Alder reaction of a chalcone derivative with cyclopentadiene to preferentially give the endo product. PMID:27559380

  9. Site-directed ELISA identifies a highly antigenic region of the simian immunodeficiency virus transmembrane glycoprotein.

    PubMed

    Johnson, P R; Parks, D E; Norrby, E; Lerner, R A; Purcell, R H; Chanock, R M

    1988-06-01

    The transmembrane glycoprotein (gp32) of the simian immunodeficiency virus (SIV) contains a highly antigenic region that includes amino acid residues 606-628. A synthetic peptide representing this region was highly immunoreactive with sera from SIV-infected primates in a site-directed enzyme-linked immunosorbent assay (ELISA). This reactivity extended across four primate species from three genera and identified infection with at least two distinct isolates of SIV. This site-directed ELISA represents a simple, accessible method with broad specificity for screening large numbers of primates for antibodies against SIV.

  10. Seven transmembrane receptors as nature's prototype allosteric protein: de-emphasizing the geography of binding.

    PubMed

    Kenakin, Terry P

    2008-09-01

    The article in this issue by Redka et al. (p. 834) illustrates some interesting interactions between classified orthosteric (bind to the same recognition site as endogenous agonist) and allosteric (bind to a different site) ligands. Of particular interest are the methods used to deal with an obfuscating factor in these kinds of studies, namely the propensity of seven transmembrane receptors to form dimers and thus demonstrate allosteric effects through binding at the orthosteric site. The judicious use of kinetics to detect and quantify allosteric action also is demonstrated. The various unique properties of allosteric modulators are discussed in the context of the increasing prevalence of allosteric ligands as investigational drugs.

  11. Artificial Diels-Alderase based on the transmembrane protein FhuA.

    PubMed

    Osseili, Hassan; Sauer, Daniel F; Beckerle, Klaus; Arlt, Marcus; Himiyama, Tomoki; Polen, Tino; Onoda, Akira; Schwaneberg, Ulrich; Hayashi, Takashi; Okuda, Jun

    2016-01-01

    Copper(I) and copper(II) complexes were covalently linked to an engineered variant of the transmembrane protein Ferric hydroxamate uptake protein component A (FhuA ΔCVF(tev)). Copper(I) was incorporated using an N-heterocyclic carbene (NHC) ligand equipped with a maleimide group on the side arm at the imidazole nitrogen. Copper(II) was attached by coordination to a terpyridyl ligand. The spacer length was varied in the back of the ligand framework. These biohybrid catalysts were shown to be active in the Diels-Alder reaction of a chalcone derivative with cyclopentadiene to preferentially give the endo product. PMID:27559380

  12. Molecular Genetics of Cystic Fibrosis Transmembrane Conductance Regulator: Genotype and Phenotype.

    PubMed

    Sosnay, Patrick R; Raraigh, Karen S; Gibson, Ronald L

    2016-08-01

    The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene encodes an epithelial ion channel. Although one mutation remains the most common cause of CF (F508del), there have been more than 2000 reported variations in CFTR. For the most part, individuals who carry only one mutation (heterozygotes) have no symptoms; individuals who inherit deleterious mutations from both parents have CF. However, growing awareness of CFTR mutations that do not ever or do not always cause CF, and individuals with mild or single-organ system manifestations of CFTR-related disease have made this Mendelian relationship more complex. PMID:27469177

  13. New Therapeutic Approaches to Modulate and Correct Cystic Fibrosis Transmembrane Conductance Regulator.

    PubMed

    Ong, Thida; Ramsey, Bonnie W

    2016-08-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) modulators are clinically available personalized medicines approved for some individuals with cystic fibrosis (CF) to target the underlying defect of disease. This review summarizes strategies used to develop CFTR modulators as therapies that improve function and availability of CFTR protein. Lessons learned from dissemination of ivacaftor across the CF population responsive to this therapy and future approaches to predict and monitor treatment response of CFTR modulators are discussed. The goal remains to expand patient-centered and personalized therapy to all patients with CF, ultimately improving life expectancy and quality of life for this disease. PMID:27469186

  14. Experimental investigation into the transmembrane electrical potential of the forward osmosis membrane process in electrolyte solutions.

    PubMed

    Bian, Lixia; Fang, Yanyan; Wang, Xiaolin

    2014-01-01

    The transmembrane electrical potential (TMEP) in a forward osmosis membrane process with a single electrolyte solution as the draw and feed solutions was investigated by experiments. The effects of membrane orientation, the electrolyte species (KCl, NaCl, MgCl2, and CaCl2), concentration and concentration ratio of solutions at both sides of membrane on water flux and TMEP were investigated. The results showed that the TMEPs at different membrane orientation cannot completely coincide, which confirmed the effect of membrane asymmetry. The ion diffusion coefficients significantly affected the TMEP across the membrane, with different patterns for different electrolytes and concentrations. PMID:24957177

  15. MAPS: an interactive web server for membrane annotation of transmembrane protein structures.

    PubMed

    Cheema, Jitender; Basu, Gautam

    2011-04-01

    The exact positioning of the membrane in transmembrane (TM) proteins plays important functional roles. Yet, the structures of TM proteins in protein data bank (pdb) have no information about the explicit position of the membrane. Using a simple hydrophobic lipid-protein mismatch energy function and a flexible lipid/water boundary, the position of lipid bilayer for representative TM proteins in pdb have been annotated. A web server called MAPS (Membrane Annotation of Protein Structures; available at: http://www.boseinst.ernet.in/gautam/maps) has been set up that allows the user to interactively analyze membrane-protein orientations of any uploaded pdb structure with user-defined membrane flexibility parameters.

  16. Experimental Investigation into the Transmembrane Electrical Potential of the Forward Osmosis Membrane Process in Electrolyte Solutions

    PubMed Central

    Bian, Lixia; Fang, Yanyan; Wang, Xiaolin

    2014-01-01

    The transmembrane electrical potential (TMEP) in a forward osmosis membrane process with a single electrolyte solution as the draw and feed solutions was investigated by experiments. The effects of membrane orientation, the electrolyte species (KCl, NaCl, MgCl2, and CaCl2), concentration and concentration ratio of solutions at both sides of membrane on water flux and TMEP were investigated. The results showed that the TMEPs at different membrane orientation cannot completely coincide, which confirmed the effect of membrane asymmetry. The ion diffusion coefficients significantly affected the TMEP across the membrane, with different patterns for different electrolytes and concentrations. PMID:24957177

  17. Structure and dynamics of one-dimensional ionic solutions in biological transmembrane channels.

    PubMed Central

    Skerra, A; Brickmann, J

    1987-01-01

    The structure and dynamics of solvated alkali metal cations in transmembrane channels are treated using the molecular dynamics simulation technique. The simulations are based on a modified Fischer-Brickmann model (Fischer, W., and J. Brickmann, 1983, Biophys. Chem., 18:323-337) for gramicidin A-type channels. The trajectories of all particles in the channel as well as two-dimensional pair correlation functions are analyzed. It is found from the analysis of the stationary simulation state that one-dimensional solvation complexes are formed and that the number of water molecules in the channel varies for different alkali metal cations. PMID:2440485

  18. TMB-Hunt: a web server to screen sequence sets for transmembrane beta-barrel proteins.

    PubMed

    Garrow, Andrew G; Agnew, Alison; Westhead, David R

    2005-07-01

    TMB-Hunt is a program that uses a modified k-nearest neighbour (k-NN) algorithm to classify protein sequences as transmembrane beta-barrel (TMB) or non-TMB on the basis of whole sequence amino acid composition. By including differentially weighted amino acids, evolutionary information and by calibrating the scoring, a discrimination accuracy of 92.5% was achieved, as tested using a rigorous cross-validation procedure. The TMB-Hunt web server, available at www.bioinformatics.leeds.ac.uk/betaBarrel, allows screening of up to 10,000 sequences in a single query and provides results and key statistics in a simple colour coded format.

  19. AtHIPM, an ortholog of the apple HrpN-interacting protein, is a negative regulator of plant growth and mediates the growth-enhancing effect of HrpN in Arabidopsis.

    PubMed

    Oh, Chang-Sik; Beer, Steven V

    2007-10-01

    HrpN (harpin) protein is critical to the virulence of the fire blight pathogen Erwinia amylovora in host plants like apple (Malus x domestica). Moreover, exogenous treatment of Arabidopsis (Arabidopsis thaliana), a nonhost plant, with partially purified HrpN enhances growth. To address the bases of the effects of HrpN in disease, we sought a HrpN-interacting protein(s) in apple, using a yeast two-hybrid assay. A single positive clone, designated HIPM (HrpN-interacting protein from Malus), was found. HIPM, a 6.5-kD protein, interacted with HrpN in yeast and in vitro. Deletion analysis showed that the N-terminal 198 of 403 amino acids of HrpN are required for interaction with HIPM. HIPM orthologs were found in Arabidopsis (AtHIPM) and rice (Oryza sativa; OsHIPM). HrpN also interacted with AtHIPM in yeast and in vitro. In silico analyses revealed that the three plant proteins contain putative signal peptides and putative transmembrane domains. We showed that both HIPM and AtHIPM have functional signal peptides, and green fluorescent protein-tagged HIPM and AtHIPM associated, in clusters, with plasma membranes. Both HIPM and AtHIPM are expressed constitutively; however, they are expressed more strongly in apple and Arabidopsis flowers than in leaves and stems. The size of AtHIPM knockout mutant plants of Arabidopsis was slightly larger than the wild-type plants. Interestingly, the knockout mutant did not exhibit enhanced plant growth in response to treatment with HrpN. Overexpression of AtHIPM conversely resulted in smaller plants. These results indicate that AtHIPM functions as a negative regulator of plant growth and mediates enhanced growth that results from treatment with HrpN. PMID:17704235

  20. The Arabidopsis TAC Position Viewer: a high-resolution map of transformation-competent artificial chromosome (TAC) clones aligned with the Arabidopsis thaliana Columbia-0 genome.

    PubMed

    Hirose, Yoshitsugu; Suda, Kunihiro; Liu, Yao-Guang; Sato, Shusei; Nakamura, Yukino; Yokoyama, Koji; Yamamoto, Naoki; Hanano, Shigeru; Takita, Eiji; Sakurai, Nozomu; Suzuki, Hideyuki; Nakamura, Yasukazu; Kaneko, Takakazu; Yano, Kentaro; Tabata, Satoshi; Shibata, Daisuke

    2015-09-01

    We present a high-resolution map of genomic transformation-competent artificial chromosome (TAC) clones extending over all Arabidopsis thaliana (Arabidopsis) chromosomes. The Arabidopsis genomic TAC clones have been valuable genetic tools. Previously, we constructed an Arabidopsis genomic TAC library consisting of more than 10,000 TAC clones harboring large genomic DNA fragments extending over the whole Arabidopsis genome. Here, we determined 13,577 end sequences from 6987 Arabidopsis TAC clones and mapped 5937 TAC clones to precise locations, covering approximately 90% of the Arabidopsis chromosomes. We present the large-scale data set of TAC clones with high-resolution mapping information as a Java application tool, the Arabidopsis TAC Position Viewer, which provides ready-to-go transformable genomic DNA clones corresponding to certain loci on Arabidopsis chromosomes. The TAC clone resources will accelerate genomic DNA cloning, positional walking, complementation of mutants and DNA transformation for heterologous gene expression. PMID:26227242

  1. Comparative genomics of Arabidopsis and maize: prospects and limitations

    PubMed Central

    Brendel, Volker; Kurtz, Stefan; Walbot, Virginia

    2002-01-01

    The completed Arabidopsis genome seems to be of limited value as a model for maize genomics. In addition to the expansion of repetitive sequences in maize and the lack of genomic micro-colinearity, maize-specific or highly-diverged proteins contribute to a predicted maize proteome of about 50,000 proteins, twice the size of that of Arabidopsis. PMID:11897028

  2. Signal transmission through the CXC chemokine receptor 4 (CXCR4) transmembrane helices.

    PubMed

    Wescott, Melanie P; Kufareva, Irina; Paes, Cheryl; Goodman, Jason R; Thaker, Yana; Puffer, Bridget A; Berdougo, Eli; Rucker, Joseph B; Handel, Tracy M; Doranz, Benjamin J

    2016-08-30

    The atomic-level mechanisms by which G protein-coupled receptors (GPCRs) transmit extracellular ligand binding events through their transmembrane helices to activate intracellular G proteins remain unclear. Using a comprehensive library of mutations covering all 352 residues of the GPCR CXC chemokine receptor 4 (CXCR4), we identified 41 amino acids that are required for signaling induced by the chemokine ligand CXCL12 (stromal cell-derived factor 1). CXCR4 variants with each of these mutations do not signal properly but remain folded, based on receptor surface trafficking, reactivity to conformationally sensitive monoclonal antibodies, and ligand binding. When visualized on the structure of CXCR4, the majority of these residues form a continuous intramolecular signaling chain through the transmembrane helices; this chain connects chemokine binding residues on the extracellular side of CXCR4 to G protein-coupling residues on its intracellular side. Integrated into a cohesive model of signal transmission, these CXCR4 residues cluster into five functional groups that mediate (i) chemokine engagement, (ii) signal initiation, (iii) signal propagation, (iv) microswitch activation, and (v) G protein coupling. Propagation of the signal passes through a "hydrophobic bridge" on helix VI that coordinates with nearly every known GPCR signaling motif. Our results agree with known conserved mechanisms of GPCR activation and significantly expand on understanding the structural principles of CXCR4 signaling. PMID:27543332

  3. Cystic fibrosis transmembrane conductance regulator (CFTR) anion binding as a probe of the pore.

    PubMed Central

    Mansoura, M K; Smith, S S; Choi, A D; Richards, N W; Strong, T V; Drumm, M L; Collins, F S; Dawson, D C

    1998-01-01

    We compared the effects of mutations in transmembrane segments (TMs) TM1, TM5, and TM6 on the conduction and activation properties of the cystic fibrosis transmembrane conductance regulator (CFTR) to determine which functional property was most sensitive to mutations and, thereby, to develop a criterion for measuring the importance of a particular residue or TM for anion conduction or activation. Anion substitution studies provided strong evidence for the binding of permeant anions in the pore. Anion binding was highly sensitive to point mutations in TM5 and TM6. Permeability ratios, in contrast, were relatively unaffected by the same mutations, so that anion binding emerged as the conduction property most sensitive to structural changes in CFTR. The relative insensitivity of permeability ratios to CFTR mutations was in accord with the notion that anion-water interactions are important determinants of permeability selectivity. By the criterion of anion binding, TM5 and TM6 were judged to be likely to contribute to the structure of the anion-selective pore, whereas TM1 was judged to be less important. Mutations in TM5 and TM6 also dramatically reduced the sensitivity of CFTR to activation by 3-isobutyl 1-methyl xanthine (IBMX), as expected if these TMs are intimately involved in the physical process that opens and closes the channel. PMID:9512029

  4. Rate-related changes of the transmembrane potential in the rabbit atrial myocardium.

    PubMed

    Nilius, B; Boldt, W

    1980-01-01

    We studied changes of the transmembrane resting and steady state action potentials in the rabbit atrial trabecular preparations (length 2-4 mm, diameter 150-800 micrometers, standard microelectrode techniques). Following cessation of rapid drive the transmembrane potential shows an initial depolarization and a terminal hyperpolarization. Increasing the stimulation frequencies before the cessation of driving the initial depolarization and the terminal hyperpolarization are more accentuated. When the stimulation frequency is abruptly reduced, the enddiastolic ("resting") potential increases (steady state resting potential - 82 +/- 3 mV at 1.0 s, -73 +/- 6 mV at 0.25 s driving interval). Decreasing the rate of stimulation the duration of the steady state action potential shortens at 25% and prolongs at 90% repolarization. A calculated background conductivity is diminished using a slower drive. From the experimental findings ensue some considerations about a K+ accumulation, the rate dependent sodium pump and the rate related shifts of the steady state outward current-voltage relationships.

  5. Transmembrane Helices Tilt, Bend, Slide, Torque, and Unwind between Functional States of Rhodopsin

    PubMed Central

    Ren, Zhong; Ren, Peter X.; Balusu, Rohith; Yang, Xiaojing

    2016-01-01

    The seven-helical bundle of rhodopsin and other G-protein coupled receptors undergoes structural rearrangements as the transmembrane receptor protein is activated. These structural changes are known to involve tilting and bending of various transmembrane helices. However, the cause and effect relationship among structural events leading to a cytoplasmic crevasse for G-protein binding is less well defined. Here we present a mathematical model of the protein helix and a simple procedure to determine multiple parameters that offer precise depiction of a helical conformation. A comprehensive survey of bovine rhodopsin structures shows that the helical rearrangements during the activation of rhodopsin involve a variety of angular and linear motions such as torsion, unwinding, and sliding in addition to the previously reported tilting and bending. These hitherto undefined motion components unify the results obtained from different experimental approaches, and demonstrate conformational similarity between the active opsin structure and the photoactivated structures in crystallo near the retinal anchor despite their marked differences. PMID:27658480

  6. Order Parameters of a Transmembrane Helix in a Fluid Bilayer: Case Study of a WALP Peptide

    PubMed Central

    Holt, Andrea; Rougier, Léa; Réat, Valérie; Jolibois, Franck; Saurel, Olivier; Czaplicki, Jerzy; Killian, J. Antoinette; Milon, Alain

    2010-01-01

    Abstract A new solid-state NMR-based strategy is established for the precise and efficient analysis of orientation and dynamics of transmembrane peptides in fluid bilayers. For this purpose, several dynamically averaged anisotropic constraints, including 13C and 15N chemical shift anisotropies and 13C-15N dipolar couplings, were determined from two different triple-isotope-labeled WALP23 peptides (2H, 13C, and 15N) and combined with previously published quadrupolar splittings of the same peptide. Chemical shift anisotropy tensor orientations were determined with quantum chemistry. The complete set of experimental constraints was analyzed using a generalized, four-parameter dynamic model of the peptide motion, including tilt and rotation angle and two associated order parameters. A tilt angle of 21° was determined for WALP23 in dimyristoylphosphatidylcholine, which is much larger than the tilt angle of 5.5° previously determined from 2H NMR experiments. This approach provided a realistic value for the tilt angle of WALP23 peptide in the presence of hydrophobic mismatch, and can be applied to any transmembrane helical peptide. The influence of the experimental data set on the solution space is discussed, as are potential sources of error. PMID:20441750

  7. TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation

    PubMed Central

    Batrakou, Dzmitry G.; de las Heras, Jose I.; Czapiewski, Rafal; Mouras, Rabah; Schirmer, Eric C.

    2015-01-01

    Recent work indicates that the nuclear envelope is a major signaling node for the cell that can influence tissue differentiation processes. Here we present two nuclear envelope trans-membrane proteins TMEM120A and TMEM120B that are paralogs encoded by the Tmem120A and Tmem120B genes. The TMEM120 proteins are expressed preferentially in fat and both are induced during 3T3-L1 adipocyte differentiation. Knockdown of one or the other protein altered expression of several genes required for adipocyte differentiation, Gata3, Fasn, Glut4, while knockdown of both together additionally affected Pparg and Adipoq. The double knockdown also increased the strength of effects, reducing for example Glut4 levels by 95% compared to control 3T3-L1 cells upon pharmacologically induced differentiation. Accordingly, TMEM120A and B knockdown individually and together impacted on adipocyte differentiation/metabolism as measured by lipid accumulation through binding of Oil Red O and coherent anti-Stokes Raman scattering microscopy (CARS). The nuclear envelope is linked to several lipodystrophies through mutations in lamin A; however, lamin A is widely expressed. Thus it is possible that the TMEM120A and B fat-specific nuclear envelope transmembrane proteins may play a contributory role in the tissue-specific pathology of this disorder or in the wider problem of obesity. PMID:26024229

  8. Nano-pipette directed transport of nanotube transmembrane channels and hybrid vesicles

    NASA Astrophysics Data System (ADS)

    Dutt, Meenakshi; Kuksenok, Olga; Balazs, Anna C.

    2013-09-01

    Using computational modeling, we simulate the interactions between a nanopipette and transmembrane, end-functionalized nanotubes that are localized within flat bilayers or nanoscopic vesicles. The functional groups (hairs) provide a ``handle'' for the moving pipette to controllably pick up and move the nanotubes to specific locations in the flat membrane, or the hybrid vesicle to specified regions on a surface. The ability to localize these hybrid vesicles on surfaces paves the way for creating nanoreactor arrays in fluidic devices.Using computational modeling, we simulate the interactions between a nanopipette and transmembrane, end-functionalized nanotubes that are localized within flat bilayers or nanoscopic vesicles. The functional groups (hairs) provide a ``handle'' for the moving pipette to controllably pick up and move the nanotubes to specific locations in the flat membrane, or the hybrid vesicle to specified regions on a surface. The ability to localize these hybrid vesicles on surfaces paves the way for creating nanoreactor arrays in fluidic devices. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr33991b

  9. Transmembrane Protein Diffusion in Gel-Supported Dual-Leaflet Membranes

    PubMed Central

    Wang, Chih-Ying; Hill, Reghan J.

    2014-01-01

    Tools to measure transmembrane-protein diffusion in lipid bilayer membranes have advanced in recent decades, providing a need for predictive theoretical models that account for interleaflet leaflet friction on tracer mobility. Here we address the fully three-dimensional flows driven by a (nonprotruding) transmembrane protein embedded in a dual-leaflet membrane that is supported above and below by soft porous supports (e.g., hydrogel or extracellular matrix), each of which has a prescribed permeability and solvent viscosity. For asymmetric configurations, i.e., supports with contrasting permeability, as realized for cells in contact with hydrogel scaffolds or culture media, the diffusion coefficient can reflect interleaflet friction. Reasonable approximations, for sufficiently large tracers on low-permeability supports, are furnished by a recent phenomenological theory from the literature. Interpreting literature data, albeit for hard-supported membranes, provides a theoretical basis for the phenomenological Stokes drag law as well as strengthening assertions that nonhydrodynamic interactions are important in supported bilayer systems, possibly leading to overestimates of the membrane/leaflet viscosity. Our theory provides a theoretical foundation for future experimental studies of tracer diffusion in gel-supported membranes. PMID:25418298

  10. A pump-pore model for transmembrane transport of hydrophilic solutes.

    PubMed

    Roberts, E

    1993-08-15

    Transmembrane transport of a hydrophilic solute is presumed to begin when hydrated ligand adheres in Velcro-like fashion to hydrated membrane surface. Asymmetric physical forces cause rolling movements of ligand over membrane surface until contact occurs with appropriate transport machinery, consisting of a pump (Pu) to which is tethered a ligand (Li)-specific perm-selective pore (Po). The Po is in the open form when the Li is attached to an external high-affinity allosteric site on it. The active form of the Pu is stabilized by attachment of the Li to high-affinity internal or low-affinity external allosteric sites. The active form of the Pu induces closure of the Po, even when ligand is bound to it; the inactive conformation of the Pu permits Po opening. Attachment of Li to either one of two binding sites on the active Pu and irreversible envelopment by it in Venus fly-trap fashion trigger transmembrane transport of Li. Multistep attachment of Li is rate-limiting in the transport process. Application of a simple equation derived from relevant kinetic considerations relating velocity of transport (V) to concentration of Li (L), V = k1(L)1/2, gives V-L curves approximating transport data obtained in a variety of biological systems. This model is congruent with the ability of cells to concentrate substances from extremely dilute solutions and with the adaptive informational value to cells of rates of transport.

  11. Analysis of Light-Induced Transmembrane Ion Gradients and Membrane Potential in Photosystem I Proteoliposomes

    SciTech Connect

    Pennisi, Cristian P.; Greenbaum, Elias; Yoshida, Ken

    2010-01-01

    Photosystem I (PSI) complexes can support a light-driven electrochemical gradient for protons, which is the driving force for energy-conserving reactions across biological membranes. In this work, a computational model that enables a quantitative description of the light-induced proton gradients across the membrane of PSI proteoliposomes is presented. Using a set of electrodiffusion equations, a compartmental model of a vesicle suspended in aqueous medium was studied. The light-mediated proton movement was modeled as a single proton pumping step with backpressure of the electric potential. The model fits determinations of pH obtained from PSI proteoliposomes illuminated in the presence of mediators of cyclic electron transport. The model also allows analysis of the proton gradients in relation to the transmembrane ion fluxes and electric potential. Sensitivity analysis enabled a determination of the parameters that have greater influence on steady-state levels and onset/decay rates of transmembrane pH and electric potential. This model could be used as a tool for optimizing PSI proteoliposomes for photo-electrochemical applications.

  12. Transmembrane domain II of the human bile acid transporter SLC10A2 coordinates sodium translocation.

    PubMed

    Sabit, Hairat; Mallajosyula, Sairam S; MacKerell, Alexander D; Swaan, Peter W

    2013-11-01

    Human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) is responsible for intestinal reabsorption of bile acids and plays a key role in cholesterol homeostasis. We used a targeted and systematic approach to delineate the role of highly conserved transmembrane helix 2 on the expression and function of hASBT. Cysteine mutation significantly depressed transport activity for >60% of mutants without affecting cell surface localization of the transporter. All mutants were inaccessible toward chemical modification by membrane-impermeant MTSET reagent, strongly suggesting that transmembrane 2 (TM2) plays an indirect role in bile acid substrate translocation. Both bile acid uptake and sodium dependence of TM2 mutants revealed a distinct α-helical periodicity. Kinetic studies with conservative and non-conservative mutants of sodium sensitive residues further underscored the importance of Gln(75), Phe(76), Met(79), Gly(83), Leu(86), Phe(90), and Asp(91) in hASBT function. Computational analysis indicated that Asp(91) may coordinate with sodium during the transport cycle. Combined, our data propose that a consortium of sodium-sensitive residues along with previously reported residues (Thr(134), Leu(138), and Thr(149)) from TM3 may form the sodium binding and translocation pathway. Notably, residues Gln(75), Met(79), Thr(82), and Leu(86) from TM2 are highly conserved in TM3 of a putative remote bacterial homologue (ASBTNM), suggesting a universal mechanism for the SLC10A transporter family.

  13. Samp1, a RanGTP binding transmembrane protein in the inner nuclear membrane.

    PubMed

    Vijayaraghavan, Balaje; Jafferali, Mohammed Hakim; Figueroa, Ricardo A; Hallberg, Einar

    2016-07-01

    Samp1 is a transmembrane protein of the inner nuclear membrane (INM), which interacts with the nuclear lamina and the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex in interphase and during mitosis, it localizes to the mitotic spindle. Samp1 was recently found to coprecipitate a protein complex containing Ran, a GTPase with fundamental regulatory functions both in interphase and in mitosis. To investigate the interaction between Samp1 and Ran in further detail, we have designed and expressed recombinant fusion proteins of the Chaetomium thermophilum homolog of Samp1 (Ct.Samp1) and human Ran. Pulldown experiments show that Samp1 binds directly to Ran and that Samp1 binds better to RanGTP compared to RanGDP. Samp1 also preferred RanGTP over RanGDP in living tsBN2 cells. We also show that the Ran binding domain is located between amino acids 75-135 in the nucleoplasmically exposed N-terminal tail of Samp1. This domain is unique for Samp1, without homology in any other proteins in fungi or metazoa. Samp1 is the first known transmembrane protein that binds to Ran and could provide a unique local binding site for RanGTP in the INM. Samp1 overexpression resulted in increased Ran concentrations in the nuclear periphery supporting this idea. PMID:27541860

  14. In silico partitioning and transmembrane insertion of hydrophobic peptides under equilibrium conditions.

    PubMed

    Ulmschneider, Jakob P; Smith, Jeremy C; White, Stephen H; Ulmschneider, Martin B

    2011-10-01

    Nascent transmembrane (TM) polypeptide segments are recognized and inserted into the lipid bilayer by the cellular translocon machinery. The recognition rules, described by a biological hydrophobicity scale, correlate strongly with physical hydrophobicity scales that describe the free energy of insertion of TM helices from water. However, the exact relationship between the physical and biological scales is unknown, because solubility problems limit our ability to measure experimentally the direct partitioning of hydrophobic peptides across lipid membranes. Here we use microsecond molecular dynamics (MD) simulations in which monomeric polyleucine segments of different lengths are allowed to partition spontaneously into and out of lipid bilayers. This approach directly reveals all states populated at equilibrium. For the hydrophobic peptides studied here, only surface-bound and transmembrane-inserted helices are found. The free energy of insertion is directly obtained from the relative occupancy of these states. A water-soluble state was not observed, consistent with the general insolubility of hydrophobic peptides. The approach further allows determination of the partitioning pathways and kinetics. Surprisingly, the transfer free energy appears to be independent of temperature, which implies that surface-to-bilayer peptide insertion is a zero-entropy process. We find that the partitioning free energy of the polyleucine segments correlates strongly with values from translocon experiments but reveals a systematic shift favoring shorter peptides, suggesting that translocon-to-bilayer partitioning is not equivalent but related to spontaneous surface-to-bilayer partitioning. PMID:21861483

  15. The electrophysiology of the beta-cell based on single transmembrane protein characteristics.

    PubMed

    Meyer-Hermann, Michael E

    2007-10-15

    The electrophysiology of beta-cells is at the origin of insulin secretion. beta-Cells exhibit a complex behavior upon stimulation with glucose including repeated bursts and continuous spiking. Mathematical modeling is most suitable to improve knowledge about the function of various transmembrane currents provided the model is based on reliable data. This is the first attempt to build a mathematical model for the beta-cell electrophysiology in a bottom-up approach that relies on single protein conductance data. The results of previous whole-cell-based models are reconsidered. The full simulation including all prominent transmembrane proteins in beta-cells is used to provide a functional interpretation of their role in beta-cell bursting and an updated vantage point of beta-cell electrophysiology. As a result of a number of in silico knock-out and block experiments the novel model makes some unexpected predictions: single-channel conductance data imply that large-conductance calcium-gated potassium currents acquire the potential of driving oscillations at supralarge glucose levels. A more complex burst interruption model is presented. It also turns out that, depending on the species, sodium currents may be more relevant than considered so far. Experiments are proposed to verify these predictions. PMID:17573431

  16. Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Ganglia of Human Gastrointestinal Tract.

    PubMed

    Xue, Ruiqi; Gu, Huan; Qiu, Yamei; Guo, Yong; Korteweg, Christine; Huang, Jin; Gu, Jiang

    2016-01-01

    CF is caused by mutations of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) which is an anion selective transmembrane ion channel that mainly regulates chloride transport, expressed in the epithelia of various organs. Recently, we have demonstrated CFTR expression in the brain, the spinal cord and the sympathetic ganglia. This study aims to investigate the expression and distribution of CFTR in the ganglia of the human gastrointestinal tract. Fresh tissue and formalin-fixed paraffin-embedded normal gastrointestinal tract samples were collected from eleven surgical patients and five autopsy cases. Immunohistochemistry, in situ hybridization, laser-assisted microdissection and nested reverse transcriptase polymerase chain reaction were performed. Expression of CFTR protein and mRNA was detected in neurons of the ganglia of all segments of the human gastrointestinal tract examined, including the stomach, duodenum, jejunum, ileum, cecum, appendix, colon and rectum. The extensive expression of CFTR in the enteric ganglia suggests that CFTR may play a role in the physiology of the innervation of the gastro-intestinal tract. The presence of dysfunctional CFTRs in enteric ganglia could, to a certain extent, explain the gastrointestinal symptoms frequently experienced by CF patients. PMID:27491544

  17. Real-time transmembrane translocation of penetratin driven by light-generated proton pumping.

    PubMed

    Björklund, Jörgen; Biverståhl, Henrik; Gräslund, Astrid; Mäler, Lena; Brzezinski, Peter

    2006-08-15

    Cell penetrating peptides (CPPs) are small peptides that are able to penetrate the plasma membrane of mammalian cells. Because these peptides can also carry large hydrophilic cargos such as proteins, they could potentially be used to transport biologically active drugs across cell membranes to modulate in vivo biology. One characteristic feature of the CPPs is that they typically have a net positive charge. Therefore, a key issue associated with the transport mechanism is the role of the transmembrane electrochemical potential in driving the peptides across the membrane. In this study, we have reconstituted bacteriorhodopsin (bR) in large unilamellar vesicles (LUVs) with fluorescein-labeled CPP penetratin enclosed within the LUVs under conditions when the fluorescence is quenched. Illumination of the bacteriorhodopsin-containing LUVs resulted in creation of a transmembrane proton electrochemical gradient (positive on the inside). Upon generation of this gradient, an increase in fluorescence was observed, which shows that the proton gradient drives the translocation of penetratin. The mechanism most likely can be generalized to other CPPs.

  18. Role of transmembrane pH gradient and membrane binding in nisin pore formation.

    PubMed

    Moll, G N; Clark, J; Chan, W C; Bycroft, B W; Roberts, G C; Konings, W N; Driessen, A J

    1997-01-01

    Nisin is a cationic antimicrobial peptide that belongs to the group of lantibiotics. It is thought to form oligomeric pores in the target membrane by a mechanism that requires the transmembrane electrical potential delta psi and that involves local pertubation of the lipid bilayer structure. Here we show that nisin does not form exclusively voltage-dependent pores: even in the absence of a delta psi, nisin is able to dissipate the transmembrane pH gradient (delta pH) in sensitive Lactococcus lactis cells and proteoliposomes. The rate of dissipation increases with the magnitude of the delta pH. Nisin forms pores only when the delta pH is inside alkaline. The efficiency of delta psi-induced pore formation is strongly affected by the external pH, whereas delta pH-induced pore formation is rather insensitive to the external pH. Nisin(1-12), an amino-terminal fragment of nisin, and (des-deltaAla5)-(nisin(1-32) amide have a strongly reduced capacity to dissipate the delta psi and delta pH in cytochrome c oxidase proteoliposomes and L. lactis cells. Both variants bind with reduced efficiency to liposomes containing negatively charged phospholipids, suggesting that both ring A and rings C to E play a role in membrane binding. Nisin(1-12) competes with nisin for membrane binding and antagonizes pore formation. These findings are consistent with the wedge model of nisin-induced pore formation.

  19. Analysis of light-induced transmembrane ion gradients and membrane potential in Photosystem I proteoliposomes.

    PubMed

    Pennisi, Cristian Pablo; Greenbaum, Elias; Yoshida, Ken

    2010-01-01

    Photosystem I (PSI) complexes can support a light-driven electrochemical gradient for protons, which is the driving force for energy-conserving reactions across biological membranes. In this work, a computational model that enables a quantitative description of the light-induced proton gradients across the membrane of PSI proteoliposomes is presented. Using a set of electrodiffusion equations, a compartmental model of a vesicle suspended in aqueous medium was studied. The light-mediated proton movement was modeled as a single proton pumping step with backpressure of the electric potential. The model fits determinations of pH obtained from PSI proteoliposomes illuminated in the presence of mediators of cyclic electron transport. The model also allows analysis of the proton gradients in relation to the transmembrane ion fluxes and electric potential. Sensitivity analysis enabled a determination of the parameters that have greater influence on steady-state levels and onset/decay rates of transmembrane pH and electric potential. This model could be used as a tool for optimizing PSI proteoliposomes for photo-electrochemical applications.

  20. Channel Gating Regulation by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) First Cytosolic Loop.

    PubMed

    Ehrhardt, Annette; Chung, W Joon; Pyle, Louise C; Wang, Wei; Nowotarski, Krzysztof; Mulvihill, Cory M; Ramjeesingh, Mohabir; Hong, Jeong; Velu, Sadanandan E; Lewis, Hal A; Atwell, Shane; Aller, Steve; Bear, Christine E; Lukacs, Gergely L; Kirk, Kevin L; Sorscher, Eric J

    2016-01-22

    In this study, we present data indicating a robust and specific domain interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) first cytosolic loop (CL1) and nucleotide binding domain 1 (NBD1) that allows ion transport to proceed in a regulated fashion. We used co-precipitation and ELISA to establish the molecular contact and showed that binding kinetics were not altered by the common clinical mutation F508del. Both intrinsic ATPase activity and CFTR channel gating were inhibited severely by CL1 peptide, suggesting that NBD1/CL1 binding is a crucial requirement for ATP hydrolysis and channel function. In addition to cystic fibrosis, CFTR dysregulation has been implicated in the pathogenesis of prevalent diseases such as chronic obstructive pulmonary disease, acquired rhinosinusitis, pancreatitis, and lethal secretory diarrhea (e.g. cholera). On the basis of clinical relevance of the CFTR as a therapeutic target, a cell-free drug screen was established to identify modulators of NBD1/CL1 channel activity independent of F508del CFTR and pharmacologic rescue. Our findings support a targetable mechanism of CFTR regulation in which conformational changes in the NBDs cause reorientation of transmembrane domains via interactions with CL1 and result in channel gating.

  1. Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Ganglia of Human Gastrointestinal Tract

    PubMed Central

    Xue, Ruiqi; Gu, Huan; Qiu, Yamei; Guo, Yong; Korteweg, Christine; Huang, Jin; Gu, Jiang

    2016-01-01

    CF is caused by mutations of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) which is an anion selective transmembrane ion channel that mainly regulates chloride transport, expressed in the epithelia of various organs. Recently, we have demonstrated CFTR expression in the brain, the spinal cord and the sympathetic ganglia. This study aims to investigate the expression and distribution of CFTR in the ganglia of the human gastrointestinal tract. Fresh tissue and formalin-fixed paraffin-embedded normal gastrointestinal tract samples were collected from eleven surgical patients and five autopsy cases. Immunohistochemistry, in situ hybridization, laser-assisted microdissection and nested reverse transcriptase polymerase chain reaction were performed. Expression of CFTR protein and mRNA was detected in neurons of the ganglia of all segments of the human gastrointestinal tract examined, including the stomach, duodenum, jejunum, ileum, cecum, appendix, colon and rectum. The extensive expression of CFTR in the enteric ganglia suggests that CFTR may play a role in the physiology of the innervation of the gastro-intestinal tract. The presence of dysfunctional CFTRs in enteric ganglia could, to a certain extent, explain the gastrointestinal symptoms frequently experienced by CF patients. PMID:27491544

  2. BAliBASE (Benchmark Alignment dataBASE): enhancements for repeats, transmembrane sequences and circular permutations.

    PubMed

    Bahr, A; Thompson, J D; Thierry, J C; Poch, O

    2001-01-01

    BAliBASE is specifically designed to serve as an evaluation resource to address all the problems encountered when aligning complete sequences. The database contains high quality, manually constructed multiple sequence alignments together with detailed annotations. The alignments are all based on three-dimensional structural superpositions, with the exception of the transmembrane sequences. The first release provided sets of reference alignments dealing with the problems of high variability, unequal repartition and large N/C-terminal extensions and internal insertions. Here we describe version 2.0 of the database, which incorporates three new reference sets of alignments containing structural repeats, trans-membrane sequences and circular permutations to evaluate the accuracy of detection/prediction and alignment of these complex sequences. BAliBASE can be viewed at the web site http://www-igbmc.u-strasbg. fr/BioInfo/BAliBASE2/index.html or can be downloaded from ftp://ftp-igbmc.u-strasbg.fr/pub/BAliBASE2 /.

  3. Phospholipid Flip-flop Modulated by Transmembrane Peptides WALP and Melittin

    PubMed Central

    Anglin, Timothy C.; Brown, Krystal L.; Conboy, John C.

    2010-01-01

    Select transmembrane proteins found in biogenic membranes are known to facilitate rapid bidirectional flip-flop of lipids between the membrane leaflets, while others have no little or no effect. The particular characteristics which determine the extent to which a protein will facilitate flip-flop are still unknown. To determine if the relative polarity of the transmembrane protein segment influences its capacity for facilitation of flip-flop, we have studied lipid flip-flop dynamics for bilayers containing the peptides WALP23 and melittin. WALP23 is used as a model hydrophobic peptide, while melittin consists of both hydrophobic and hydrophilic residues. Sum-frequency vibrational spectroscopy (SFVS) was used to characterize the bilayers and determine the kinetics of flip-flop for the lipid component, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), within the mixed bilayers. The kinetics data were utilized to determine the activation thermodynamics for DSPC flip-flop in the presence of the peptides. Melittin was found to significantly reduce the free energy barrier to DSPC flip-flop when incorporated into the bilayer at 1 mol%, while incorporation of WALP23 at the same concentration led to a more modest reduction of the free energy barrier. The possible mechanisms by which these peptides facilitate flip-flop are analyzed and discussed in terms of the observed activation thermodynamics. PMID:19508895

  4. Channel Gating Regulation by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) First Cytosolic Loop.

    PubMed

    Ehrhardt, Annette; Chung, W Joon; Pyle, Louise C; Wang, Wei; Nowotarski, Krzysztof; Mulvihill, Cory M; Ramjeesingh, Mohabir; Hong, Jeong; Velu, Sadanandan E; Lewis, Hal A; Atwell, Shane; Aller, Steve; Bear, Christine E; Lukacs, Gergely L; Kirk, Kevin L; Sorscher, Eric J

    2016-01-22

    In this study, we present data indicating a robust and specific domain interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) first cytosolic loop (CL1) and nucleotide binding domain 1 (NBD1) that allows ion transport to proceed in a regulated fashion. We used co-precipitation and ELISA to establish the molecular contact and showed that binding kinetics were not altered by the common clinical mutation F508del. Both intrinsic ATPase activity and CFTR channel gating were inhibited severely by CL1 peptide, suggesting that NBD1/CL1 binding is a crucial requirement for ATP hydrolysis and channel function. In addition to cystic fibrosis, CFTR dysregulation has been implicated in the pathogenesis of prevalent diseases such as chronic obstructive pulmonary disease, acquired rhinosinusitis, pancreatitis, and lethal secretory diarrhea (e.g. cholera). On the basis of clinical relevance of the CFTR as a therapeutic target, a cell-free drug screen was established to identify modulators of NBD1/CL1 channel activity independent of F508del CFTR and pharmacologic rescue. Our findings support a targetable mechanism of CFTR regulation in which conformational changes in the NBDs cause reorientation of transmembrane domains via interactions with CL1 and result in channel gating. PMID:26627831

  5. Regulation of the cystic fibrosis transmembrane conductance regulator anion channel by tyrosine phosphorylation

    PubMed Central

    Billet, Arnaud; Jia, Yanlin; Jensen, Tim; Riordan, John R.; Hanrahan, John W.

    2015-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) channel is activated by PKA phosphorylation of a regulatory domain that interacts dynamically with multiple CFTR domains and with other proteins. The large number of consensus sequences for phosphorylation by PKA has naturally focused most attention on regulation by this kinase. We report here that human CFTR is also phosphorylated by the tyrosine kinases p60c-Src (proto-oncogene tyrosine-protein kinase) and the proline-rich tyrosine kinase 2 (Pyk2), and they can also cause robust activation of quiescent CFTR channels. In excised patch-clamp experiments, CFTR activity during exposure to Src or Pyk2 reached ∼80% of that stimulated by PKA. Exposure to PKA after Src or Pyk2 caused a further increase to the level induced by PKA alone, implying a common limiting step. Channels became spontaneously active when v-Src or the catalytic domain of Pyk2 was coexpressed with CFTR and were further stimulated by the tyrosine phosphatase inhibitor dephostatin. Exogenous Src also activated 15SA-CFTR, a variant that lacks 15 potential PKA sites and has little response to PKA. PKA-independent activation by tyrosine phosphorylation has implications for the mechanism of regulation by the R domain and for the physiologic functions of CFTR.—Billet, A., Jia, Y., Jensen, T., Riordan, J. R., Hanrahan, J. W. Regulation of the cystic fibrosis transmembrane conductance regulator anion channel by tyrosine phosphorylation. PMID:26062600

  6. Quantitative estimation of transmembrane ion transport in rat renal collecting duct principal cells.

    PubMed

    Ilyaskin, Alexander V; Karpov, Denis I; Medvedev, Dmitriy A; Ershov, Alexander P; Baturina, Galina S; Katkova, Liubov E; Solenov, Evgeniy I

    2014-01-01

    Kidney collecting duct principal cells play a key role in regulated tubular reabsorption of water and sodium and secretion of potassium. The importance of this function for the maintenance of the osmotic homeostasis of the whole organism motivates extensive study of the ion transport properties of collecting duct principal cells. We performed experimental measurements of cell volume and intracellular sodium concentration in rat renal collecting duct principal cells from the outer medulla (OMCD) and used a mathematical model describing transmembrane ion fluxes to analyze the experimental data. The sodium and chloride concentrations ([Na+]in = 37.3 ± 3.3 mM, [Cl-]in = 32.2 ± 4.0 mM) in OMCD cells were quantitatively estimated. Correspondence between the experimentally measured cell physiological characteristics and the values of model permeability parameters was established. Plasma membrane permeabilities and the rates of transmembrane fluxes for sodium, potassium and chloride ions were estimated on the basis of ion substitution experiments and model predictions. In particular, calculated sodium (PNa), potassium (PK) and chloride (PCl) permeabilities were equal to 3.2 × 10-6 cm/s, 1.0 × 10-5 cm/s and 3.0 × 10-6 cm/s, respectively. This approach sets grounds for utilization of experimental measurements of intracellular sodium concentration and cell volume to quantify the ion permeabilities of OMCD principal cells and aids us in understanding the physiology of the adjustment of renal sodium and potassium excretion.

  7. Trans-membrane transport of fluoranthene by Rhodococcus sp. BAP-1 and optimization of uptake process.

    PubMed

    Li, Yi; Wang, Hongqi; Hua, Fei; Su, Mengyuan; Zhao, Yicun

    2014-03-01

    The mechanism of transport of (14)C-fluoranthene by Rhodococcus sp. BAP-1, a Gram-positive bacterium isolated from crude oil-polluted soil, was examined. Our finding demonstrated that the mechanism for fluoranthene travel across the cell membrane in Rhodococcus sp. BAP-1 requires energy. Meanwhile, the transport of fluoranthene involves concurrent catabolism of (14)C, that leading to the generation of significant amount of (14)CO2. Combined with trans-membrane transport dynamic and response surface methodology, a significant influence of temperature, pH and salinity on cellular uptake rate was screened by Plackett-Burman design. Then, Box-Behnken design was employed to optimize and enhanced the trans-membrane transport process. The results predicted by Box-Behnken design indicated that the maximum cellular uptake rate of fluoranthene could be achieve to 0.308μmolmin(-1)mg(-1)·protein (observed) and 0.304μmolmin(-1)mg(-1)·protein (predicted) when the initial temperature, pH and salinity were set at 20°C, 9% and 1%, respectively.

  8. Analysis of transmembrane helix integration in the endoplasmic reticulum in S. cerevisiae.

    PubMed

    Hessa, Tara; Reithinger, Johannes H; von Heijne, Gunnar; Kim, Hyun

    2009-03-13

    What sequence features in integral membrane proteins determine which parts of the polypeptide chain will form transmembrane alpha-helices and which parts will be located outside the lipid bilayer? Previous studies on the integration of model transmembrane segments into the mammalian endoplasmic reticulum (ER) have provided a rather detailed quantitative picture of the relation between amino acid sequence and membrane-integration propensity for proteins targeted to the Sec61 translocon. We have now carried out a comparative study of the integration of N out-C in-orientated 19-residue-long polypeptide segments into the ER of the yeast Saccharomyces cerevisiae. We find that the 'threshold hydrophobicity' required for insertion into the ER membrane is very similar in S. cerevisiae and in mammalian cells. Further, when comparing the contributions to the apparent free energy of membrane insertion of the 20 natural amino acids between the S. cerevisiae and the mammalian ER, we find that the two scales are strongly correlated but that the absolute difference between the most hydrophobic and most hydrophilic residues is approximately 2-fold smaller in S. cerevisiae.

  9. Transmembrane signal transduction by peptide hormones via family B G protein-coupled receptors

    PubMed Central

    Culhane, Kelly J.; Liu, Yuting; Cai, Yingying; Yan, Elsa C. Y.

    2015-01-01

    Although family B G protein-coupled receptors (GPCRs) contain only 15 members, they play key roles in transmembrane signal transduction of hormones. Family B GPCRs are drug targets for developing therapeutics for diseases ranging from metabolic to neurological disorders. Despite their importance, the molecular mechanism of activation of family B GPCRs remains largely unexplored due to the challenges in expression and purification of functional receptors to the quantity for biophysical characterization. Currently, there is no crystal structure available of a full-length family B GPCR. However, structures of key domains, including the extracellular ligand binding regions and seven-helical transmembrane regions, have been solved by X-ray crystallography and NMR, providing insights into the mechanisms of ligand recognition and selectivity, and helical arrangements within the cell membrane. Moreover, biophysical and biochemical methods have been used to explore functions, key residues for signaling, and the kinetics and dynamics of signaling processes. This review summarizes the current knowledge of the signal transduction mechanism of family B GPCRs at the molecular level and comments on the challenges and outlook for mechanistic studies of family B GPCRs. PMID:26594176

  10. Dynamic behaviors and transport properties of ethanol molecules in transmembrane cyclic peptide nanotubes

    NASA Astrophysics Data System (ADS)

    Li, Rui; Fan, Jianfen; Li, Hui; Yan, Xiliang; Yu, Yi

    2015-07-01

    Classical molecular dynamics simulations have been performed to investigate the dynamic behaviors and transport properties of ethanol molecules in transmembrane cyclic peptide nanotubes (CPNTs) with various radii, i.e., 8 × ( W L ¯ ) n = 3 , 4 , 5 / POPE . The results show that ethanol molecules spontaneously fill the octa- and deca-CPNTs, but not the hexa-CPNT. In the octa-CPNT, ethanol molecules are trapped at individual gaps with their carbon skeletons perpendicular to the tube axis and hydroxyl groups towards the tube wall, forming a broken single-file chain. As the channel radius increases, ethanol molecules inside the deca-CPNT tend to form a tubular layer and the hydroxyl groups mainly stretch towards the tube axis. Computations of diffusion coefficients indicate that ethanol molecules in the octa-CPNT nearly lost their diffusion abilities, while those in the deca-CPNT diffuse as 4.5 times as in a (8, 8) carbon nanotube with a similar tube diameter. The osmotic and diffusion permeabilities (pf and pd, respectively) of the octa- and deca-CPNTs transporting ethanol were deduced for the first time. The distributions of the gauche and trans conformers of ethanol molecules in two CPNTs are quite similar, both with approximately 57% gauche conformers. The non-bonded interactions of channel ethanol with a CPNT wall and surrounding ethanol were explored. The potential of mean force elucidates the mechanism underlying the transporting characteristics of channel ethanol in a transmembrane CPNT.

  11. Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active

    SciTech Connect

    Schoenitzer, Veronika; Eichner, Norbert; Clausen-Schaumann, Hauke; Weiss, Ingrid M.

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Dictyostelium produces the 264 kDa myosin chitin synthase of bivalve mollusc Atrina. Black-Right-Pointing-Pointer Chitin synthase activity releases chitin, partly associated with the cell surface. Black-Right-Pointing-Pointer Membrane extracts of transgenic slime molds produce radiolabeled chitin in vitro. Black-Right-Pointing-Pointer Chitin producing Dictyostelium cells can be characterized by atomic force microscopy. Black-Right-Pointing-Pointer This model system enables us to study initial processes of chitin biomineralization. -- Abstract: Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA{sup -} cell lines are shown.

  12. Dynamic behaviors and transport properties of ethanol molecules in transmembrane cyclic peptide nanotubes.

    PubMed

    Li, Rui; Fan, Jianfen; Li, Hui; Yan, Xiliang; Yu, Yi

    2015-07-01

    Classical molecular dynamics simulations have been performed to investigate the dynamic behaviors and transport properties of ethanol molecules in transmembrane cyclic peptide nanotubes (CPNTs) with various radii, i.e., 8×(WL¯)n=3,4,5/POPE. The results show that ethanol molecules spontaneously fill the octa- and deca-CPNTs, but not the hexa-CPNT. In the octa-CPNT, ethanol molecules are trapped at individual gaps with their carbon skeletons perpendicular to the tube axis and hydroxyl groups towards the tube wall, forming a broken single-file chain. As the channel radius increases, ethanol molecules inside the deca-CPNT tend to form a tubular layer and the hydroxyl groups mainly stretch towards the tube axis. Computations of diffusion coefficients indicate that ethanol molecules in the octa-CPNT nearly lost their diffusion abilities, while those in the deca-CPNT diffuse as 4.5 times as in a (8, 8) carbon nanotube with a similar tube diameter. The osmotic and diffusion permeabilities (pf and pd, respectively) of the octa- and deca-CPNTs transporting ethanol were deduced for the first time. The distributions of the gauche and trans conformers of ethanol molecules in two CPNTs are quite similar, both with approximately 57% gauche conformers. The non-bonded interactions of channel ethanol with a CPNT wall and surrounding ethanol were explored. The potential of mean force elucidates the mechanism underlying the transporting characteristics of channel ethanol in a transmembrane CPNT. PMID:26156492

  13. Samp1, a RanGTP binding transmembrane protein in the inner nuclear membrane

    PubMed Central

    Vijayaraghavan, Balaje; Jafferali, Mohammed Hakim; Figueroa, Ricardo A.; Hallberg, Einar

    2016-01-01

    ABSTRACT Samp1 is a transmembrane protein of the inner nuclear membrane (INM), which interacts with the nuclear lamina and the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex in interphase and during mitosis, it localizes to the mitotic spindle. Samp1 was recently found to coprecipitate a protein complex containing Ran, a GTPase with fundamental regulatory functions both in interphase and in mitosis. To investigate the interaction between Samp1 and Ran in further detail, we have designed and expressed recombinant fusion proteins of the Chaetomium thermophilum homolog of Samp1 (Ct.Samp1) and human Ran. Pulldown experiments show that Samp1 binds directly to Ran and that Samp1 binds better to RanGTP compared to RanGDP. Samp1 also preferred RanGTP over RanGDP in living tsBN2 cells. We also show that the Ran binding domain is located between amino acids 75–135 in the nucleoplasmically exposed N-terminal tail of Samp1. This domain is unique for Samp1, without homology in any other proteins in fungi or metazoa. Samp1 is the first known transmembrane protein that binds to Ran and could provide a unique local binding site for RanGTP in the INM. Samp1 overexpression resulted in increased Ran concentrations in the nuclear periphery supporting this idea. PMID:27541860

  14. Influence of Lithium Ions on the Transmembrane Potential and Cation Content of Cardiac Cells

    PubMed Central

    Carmeliet, E. E.

    1964-01-01

    The effect of lithium ions on cardiac cells was investigated by recording the changes in transmembrane potential and by following the movement of Li, Na, and K across the cell membrane. Isolated preparations of calf Purkinje fibers and cat ventricular muscles were used. Potentials were measured by intracellular microelectrodes; ion transport was estimated by flame photometric analysis and by using the radioactive isotopes of Na and K. It was shown (a) that Li ions can replace Na ions in the mechanism generating the cardiac action potential but that they also cause a marked depolarization and pronounced changes in action potential configuration; (b) that the resting permeability to Li ions is high and that these ions accumulate in the cell interior as if they were not actively pumped outwards. In Li-Tyrode [K]i decreases markedly while the K permeability seems to be increased. In a kinetic study of net K and Na fluxes, the outward movement of each ion was found to be proportional to the second power of its intracellular concentration. The effect on the transmembrane potential is explained in terms of changes in ion movement and intracellular ion concentration. PMID:14100967

  15. Tris-thiourea tripodal-based molecules as chloride transmembrane transporters: insights from molecular dynamics simulations.

    PubMed

    Marques, Igor; Colaço, Ana R; Costa, Paulo J; Busschaert, Nathalie; Gale, Philip A; Félix, Vítor

    2014-05-28

    The interaction of six tripodal synthetic chloride transmembrane transporters with a POPC bilayer was investigated by means of molecular dynamics simulations using the general Amber force field (GAFF) for the transporters and the LIPID11 force field for phospholipids. These transporters are structurally simple molecules, based on the tris(2-aminoethyl)amine scaffold, containing three thiourea binding units coupled with three n-butyl (1), phenyl (2), fluorophenyl (3), pentafluorophenyl (4), trifluoromethylphenyl (5), or bis(trifluoromethyl)phenyl (6) substituents. The passive diffusion of 1-6⊃ Cl(-) was evaluated with the complexes initially positioned either in the water phase or inside the bilayer. In the first scenario the chloride is released in the water solution before the synthetic molecules achieve the water-lipid interface and permeate the membrane. In the latter one, only when the chloride complex reaches the interface is the anion released to the water phase, with the transporter losing the initial ggg tripodal shape. Independently of the transporter used in the membrane system, the bilayer structure is preserved and the synthetic molecules interact with the POPC molecules at the phosphate headgroup level, via N-H···O hydrogen bonds. Overall, the molecular dynamics simulations' results indicate that the small tripodal molecules in this series have a low impact on the bilayer and are able to diffuse with chloride inside the lipid environment. Indeed, these are essential conditions for these molecules to promote the transmembrane transport as anion carriers, in agreement with experimental efflux data. PMID:24663079

  16. Endogenous transmembrane protein UT2 inhibits pSTAT3 and suppresses hematological malignancy

    PubMed Central

    Wang, Ying-Hua; Kalaitzidis, Demetrios; Ramachandran, Janani; Sykes, David B.; Raje, Noopur; Scadden, David T.

    2016-01-01

    Regulation of STAT3 activation is critical for normal and malignant hematopoietic cell proliferation. Here, we have reported that the endogenous transmembrane protein upstream-of-mTORC2 (UT2) negatively regulates activation of STAT3. Specifically, we determined that UT2 interacts directly with GP130 and inhibits phosphorylation of STAT3 on tyrosine 705 (STAT3Y705). This reduces cytokine signaling including IL6 that is implicated in multiple myeloma and other hematopoietic malignancies. Modulation of UT2 resulted in inverse effects on animal survival in myeloma models. Samples from multiple myeloma patients also revealed a decreased copy number of UT2 and decreased expression of UT2 in genomic and transcriptomic analyses, respectively. Together, these studies identify a transmembrane protein that functions to negatively regulate cytokine signaling through GP130 and pSTAT3Y705 and is molecularly and mechanistically distinct from the suppressors of cytokine signaling (SOCS) family of genes. Moreover, this work provides evidence that perturbations of this activation-dampening molecule participate in hematologic malignancies and may serve as a key determinant of multiple myeloma pathophysiology. UT2 is a negative regulator shared across STAT3 and mTORC2 signaling cascades, functioning as a tumor suppressor in hematologic malignancies driven by those pathways. PMID:26927669

  17. Nanoporous microbead supported bilayers: stability, physical characterization, and incorporation of functional transmembrane proteins.

    SciTech Connect

    Davis, Ryan W. (University of New Mexico, Albuquerque, NM); Brozik, James A. (University of New Mexico, Albuquerque, NM); Brozik, Susan Marie; Cox, Jason M.; Lopez, Gabriel P.; Barrick, Todd A.; Flores, Adrean

    2007-03-01

    The introduction of functional transmembrane proteins into supported bilayer-based biomimetic systems presents a significant challenge for biophysics. Among the various methods for producing supported bilayers, liposomal fusion offers a versatile method for the introduction of membrane proteins into supported bilayers on a variety of substrates. In this study, the properties of protein containing unilamellar phosphocholine lipid bilayers on nanoporous silica microspheres are investigated. The effects of the silica substrate, pore structure, and the substrate curvature on the stability of the membrane and the functionality of the membrane protein are determined. Supported bilayers on porous silica microspheres show a significant increase in surface area on surfaces with structures in excess of 10 nm as well as an overall decrease in stability resulting from increasing pore size and curvature. Comparison of the liposomal and detergent-mediated introduction of purified bacteriorhodopsin (bR) and the human type 3 serotonin receptor (5HT3R) are investigated focusing on the resulting protein function, diffusion, orientation, and incorporation efficiency. In both cases, functional proteins are observed; however, the reconstitution efficiency and orientation selectivity are significantly enhanced through detergent-mediated protein reconstitution. The results of these experiments provide a basis for bulk ionic and fluorescent dye-based compartmentalization assays as well as single-molecule optical and single-channel electrochemical interrogation of transmembrane proteins in a biomimetic platform.

  18. Solvation of transmembrane proteins by isotropic membrane mimetics: a molecular dynamics study.

    PubMed

    Mottamal, Madhusoodanan; Shen, Sui; Guembe, Cristina; Krilov, Goran

    2007-09-27

    Mixtures of organic solvents are often used as membrane mimetics in structure determination of transmembrane proteins by solution NMR; however, the mechanism through which these isotropic solvents mimic the anisotropic environment of cell membranes is not known. Here, we use molecular dynamics simulations to study the solvation thermodynamics of the c-subunit of Escherichia coli F1F0 ATP synthase in membrane mimetic mixtures of methanol, chloroform, and water with varying fractions of components as well as in lipid bilayers. We show that the protein induces a local phase separation of the solvent components into hydrophobic and hydrophilic layers, which provides the anisotropic solvation environment to stabilize the amphiphilic peptide. The extent of this effect varies with solvent composition and is most pronounced in the ternary methanol-chloroform-water mixtures. Analysis of the solvent structure, including the local mole fraction, density profiles, and pair distribution functions, reveals considerable variation among solvent mixtures in the solvation environment surrounding the hydrophobic transmembrane region of the protein. Hydrogen bond analysis indicates that this is primarily driven by the hydrogen-bonding propensity of the essential Asp(61) residue. The impact of the latter on the conformational stability of the solvated protein is discussed. Comparison with the simulations in explicit all-atom models of lipid bilayer indicates a higher flexibility and reduced structural integrity of the membrane mimetic solvated c-subunit. This was particularly true for the deprotonated form of the protein and found to be linked to solvent stabilization of the charged Asp(61).

  19. Structural Organization of a Full-Length Gp130/LIF-R Cytokine Receptor Transmembrane Complex

    SciTech Connect

    Skiniotis, G.; Lupardus, P.J.; Martick, M.; Walz, T.; Garcia, K.C.

    2009-05-26

    gp130 is a shared receptor for at least nine cytokines, and can signal either as a homodimer, or as a heterodimer with Leukemia Inhibitory Factor Receptor (LIF-R). Here we biophysically and structurally characterize the full-length, transmembrane form of a quaternary cytokine receptor complex consisting of gp130, LIF-R, the cytokine Ciliary Neurotrophic Factor (CNTF), and its alpha receptor (CNTF-R{alpha}). Thermodynamic analysis indicates that, unlike the cooperative assembly of the symmetric gp130/Interleukin-6/IL-6R{alpha} hexameric complex, CNTF/CNTF-R{alpha} heterodimerizes gp130 and LIF-R via non-cooperative energetics to form an asymmetric 1:1:1:1 complex. Single particle electron microscopic (EM) analysis of the full-length gp130/LIF-R/CNTF-R{alpha}/CNTF quaternary complex elucidates an asymmetric structural arrangement, in which the receptor extracellular and transmembrane segments join as a continuous, rigid unit, poised to sensitively transduce ligand engagement to the membrane-proximal intracellular signaling regions. These studies also enumerate the organizing principles for assembly of the 'tall' class of gp130-family cytokine receptor complexes including LIF, IL-27, IL-12, and others.

  20. Regulation of apoptosis by p53-inducible transmembrane protein containing sushi domain.

    PubMed

    Cui, Hongyan; Kamino, Hiroki; Nakamura, Yasuyuki; Kitamura, Noriaki; Miyamoto, Takafumi; Shinogi, Daisuke; Goda, Olga; Arakara, Hirofumi; Futamura, Manabu

    2010-11-01

    The tumor suppressor p53 is a transcription factor that induces the transcription of various target genes in response to DNA damage and it protects the cells from malignant transformation. In this study, we performed cDNA microarray analysis and found that the transmembrane protein containing sushi domain (TMPS) gene, which encodes a putative type I transmembrane protein, is a novel p53-target gene. TMPS contains a sushi domain in the extracellular region, which is associated with protein-protein interaction. TMPS expression is induced by endogenous p53 under genotoxic stress in several cancer cell lines. Reporter assay revealed p53-dependent transactivation of the p53 binding-sites (BSs) located in the intron 1 of TMPS. Chromatin immunoprecipitation (ChIP) assay showed that p53 binds to these BSs in vivo. Overexpression of TMPS induced apoptosis through the activation of caspase-3, 8, and 9 in various cancer cell lines. Moreover, γ-irradiation induced the expression of TMPS mRNA in the spleen and colon of p53+/+ mice but not in those of p53-/- mice. These data indicate that TMPS may play a role in p53-dependent apoptosis under DNA damage condition.

  1. Recognition of a Single Transmembrane Degron by Sequential Quality Control Checkpoints

    PubMed Central

    Fayadat, Laurence; Kopito, Ron R.

    2003-01-01

    To understand the relationship between conformational maturation and quality control–mediated proteolysis in the secretory pathway, we engineered the well-characterized degron from the α-subunit of the T-cell antigen receptor (TCRα) into the α-helical transmembrane domain of homotrimeric type I integral membrane protein, influenza hemagglutinin (HA). Although the membrane degron does not appear to interfere with acquisition of native secondary structure, as assessed by the formation of native intrachain disulfide bonds, only ∼50% of nascent mutant HA chains (HA++) become membrane-integrated and acquire complex N-linked glycans indicative of transit to a post-ER compartment. The remaining ∼50% of nascent HA++ chains fail to integrate into the lipid bilayer and are subject to proteasome-dependent degradation. Site-specific cleavage by extracellular trypsin and reactivity with conformation-specific monoclonal antibodies indicate that membrane-integrated HA++ molecules are able to mature to the plasma membrane with a conformation indistinguishable from that of HAwt. These apparently native HA++ molecules are, nevertheless, rapidly degraded by a process that is insensitive to proteasome inhibitors but blocked by lysosomotropic amines. These data suggest the existence in the secretory pathway of at least two sequential quality control checkpoints that recognize the same transmembrane degron, thereby ensuring the fidelity of protein deployment to the plasma membrane. PMID:12631739

  2. Evolution of a computer program for classifying protein segments as transmembrane domains using genetic programming.

    PubMed

    Koza, J R

    1994-01-01

    The recently-developed genetic programming paradigm is used to evolve a computer program to classify a given protein segment as being a transmembrane domain or non-transmembrane area of the protein. Genetic programming starts with a primordial ooze of randomly generated computer programs composed of available programmatic ingredients and then genetically breeds the population of programs using the Darwinian principle of survival of the fittest and an analog of the naturally occurring genetic operation of crossover (sexual recombination). Automatic function definition enables genetic programming to dynamically create subroutines dynamically during the run. Genetic programming is given a training set of differently-sized protein segments and their correct classification (but no biochemical knowledge, such as hydrophobicity values). Correlation is used as the fitness measure to drive the evolutionary process. The best genetically-evolved program achieves an out-of-sample correlation of 0.968 and an out-of-sample error rate of 1.6%. This error rate is better than that reported for four other algorithms reported at the First International Conference on Intelligent Systems for Molecular Biology. Our genetically evolved program is an instance of an algorithm discovered by an automated learning paradigm that is superior to that written by human investigators.

  3. Multifunctional Transmembrane Protein Ligands for Cell-Specific Targeting of Plasma Membrane-Derived Vesicles.

    PubMed

    Zhao, Chi; Busch, David J; Vershel, Connor P; Stachowiak, Jeanne C

    2016-07-01

    Liposomes and nanoparticles that bind selectively to cell-surface receptors can target specific populations of cells. However, chemical conjugation of ligands to these particles is difficult to control, frequently limiting ligand uniformity and complexity. In contrast, the surfaces of living cells are decorated with highly uniform populations of sophisticated transmembrane proteins. Toward harnessing cellular capabilities, here it is demonstrated that plasma membrane vesicles (PMVs) derived from donor cells can display engineered transmembrane protein ligands that precisely target cells on the basis of receptor expression. These multifunctional targeting proteins incorporate (i) a protein ligand, (ii) an intrinsically disordered protein spacer to make the ligand sterically accessible, and (iii) a fluorescent protein domain that enables quantification of the ligand density on the PMV surface. PMVs that display targeting proteins with affinity for the epidermal growth factor receptor (EGFR) bind at increasing concentrations to breast cancer cells that express increasing levels of EGFR. Further, as an example of the generality of this approach, PMVs expressing a single-domain antibody against green fluorescence protein (eGFP) bind to cells expressing eGFP-tagged receptors with a selectivity of ≈50:1. The results demonstrate the versatility of PMVs as cell targeting systems, suggesting diverse applications from drug delivery to tissue engineering. PMID:27294846

  4. Using experimental information to produce a model of the transmembrane domain of the ion channel phospholamban.

    PubMed Central

    Herzyk, P; Hubbard, R E

    1998-01-01

    Molecular models of the transmembrane domain of the phospholamban pentamer have been generated by a computational method that uses the experimentally measured effects of systematic single-site mutations as a guiding force in the modeling procedure. This method makes the assumptions that 1) the phospholamban transmembrane domain is a parallel five-helix bundle, and 2) nondisruptive mutation positions are lipid exposed, whereas 3) disruptive or partially disruptive mutations are not. Our procedure requires substantially less computer time than systematic search methods, allowing rapid assessment of the effects of different experimental results on the helix arrangement. The effectiveness of the approach is investigated in test calculations on two helix-dimer systems of known structure. Two independently derived sets of mutagenesis data were used to define the restraints for generating models of phospholamban. Both resulting models are left-handed, highly symmetrical pentamers. Although the overall bundle geometry is very similar in the two models, the orientation of individual helices differs by approximately 50 degrees, resulting in different sets of residues facing the pore. This demonstrates how differences in restraints can have an effect on the model structures generated, and how the violation of these restraints can identify inconsistent experimental data. PMID:9512019

  5. Monolayer freeze-fracture autoradiography: quantitative analysis of the transmembrane distribution of radioiodinated concanavalin A

    SciTech Connect

    Fisher, K.A.

    1982-04-01

    The technique of monolayer freeze-fracture autoradiography (MONOFARG) has been developed and the principles, quantitation, and application of the method are described. Cell monolayers attached to polylysine-treated glass were freeze-fractured, shadowed, and coated with dry, Parlodion-supported Ilford L4 photographic emulsion at room temperature. Quantitative aspects of MONOFARG were examined using radioiodinated test systems. Background was routinely <2.5 x 10/sup -4/ grains/..mu..m/sup 2//day, the highest overall efficiency was between 25% and 45%, and grain density and efficiency were dependent on radiation dose for iodine-125 and D-19 development. Corrected grain densities were linearly proportional to iodine-125 concentration. The method was applied to an examination of the transmembrane distribution of radioiodinated and fluoresceinated concanavalin A (/sup 125/I-FITC-Con-A). Human erythrocytes were labeled, column-purified, freeze-dried or freeze-fractured, autoradiographed, and examined by electron microscopy. The number of silver grains per square micrometer of unsplit single membrane was essentially identical to that of split extracellular membrane halves. These data demonstrate that /sup 125/I-FITC-Con-A partitions exclusively with the extracellular half of the membrane upon freeze-fracturing and can be used as a quantitative marker for the fraction of extracellular split membrane halves. This method should be able to provide new information about certain transmembrane properties of biological membrane molecules and probes, as well as about the process of freeze-fracture per se.

  6. Noninvasive reconstruction of cardiac transmembrane potentials using a kernelized extreme learning method

    NASA Astrophysics Data System (ADS)

    Jiang, Mingfeng; Zhang, Heng; Zhu, Lingyan; Cao, Li; Wang, Yaming; Xia, Ling; Gong, Yinglan

    2015-04-01

    Non-invasively reconstructing the cardiac transmembrane potentials (TMPs) from body surface potentials can act as a regression problem. The support vector regression (SVR) method is often used to solve the regression problem, however the computational complexity of the SVR training algorithm is usually intensive. In this paper, another learning algorithm, termed as extreme learning machine (ELM), is proposed to reconstruct the cardiac transmembrane potentials. Moreover, ELM can be extended to single-hidden layer feed forward neural networks with kernel matrix (kernelized ELM), which can achieve a good generalization performance at a fast learning speed. Based on the realistic heart-torso models, a normal and two abnormal ventricular activation cases are applied for training and testing the regression model. The experimental results show that the ELM method can perform a better regression ability than the single SVR method in terms of the TMPs reconstruction accuracy and reconstruction speed. Moreover, compared with the ELM method, the kernelized ELM method features a good approximation and generalization ability when reconstructing the TMPs.

  7. Transmembrane sodium and potassium gradients modulate histamine secretion induced by ionophore A23187.

    PubMed Central

    Amellal, M.; Bronner, C.; Landry, Y.

    1985-01-01

    Histamine secretion was induced from rat peritoneal mast cells by calcium ionophore A23187 in the presence of various extracellular calcium concentrations. Transmembrane sodium and potassium gradients were altered by cold pretreatment of mast cells or through the inhibition of sodium-potassium ATPase by the use of ouabain or potassium-deprivation. Such pretreatments led to a parallel shift to the left of the extracellular calcium concentration-histamine secretion curve, i.e. to an apparent decrease of extracellular calcium requirement for the ionophore-induced histamine release. These effects were fully reversed by warming mast cells, by washing out ouabain or by adding potassium. Metabolic inhibition of mast cells prevented the ionophore-induced secretion in all the experimental conditions described. Secretion observed in the absence of added calcium was inhibited by short term treatment of cells with 5 X 10(-6) M EGTA or EDTA provided magnesium was absent from the assay medium. Data show that ionophore A23187 was able to induce secretion in the presence of micromolar concentrations of extracellular calcium, when the efficiency of the ionophore was not decreased by extracellular magnesium and when transmembrane sodium and potassium gradients were altered. PMID:2412623

  8. Transport properties of simple organic molecules in a transmembrane cyclic peptide nanotube.

    PubMed

    Xu, Jian; Fan, Jian Fen; Zhang, Ming Ming; Weng, Pei Pei; Lin, Hui Fang

    2016-05-01

    Multiple molecular dynamics simulations have been performed to explore the transport properties of single methane, methanol, and ethanol molecules through the water-filled transmembrane cyclic peptide nanotube (CPNT) of 8 × (WL)₄-POPE, as well as the potential application of this CPNT in the separation of an alcohol/water mixture. Molecular size and hydrophilicity/hydrophobicity were found to significantly influence molecular diffusion behavior in the channel. Methane and ethanol display more explicit distributions in midplane regions, while methanol mainly occurs in α-plane zones. Methane and ethanol drift faster near an α-plane zone, whereas methanol diffuses uniformly throughout the whole transmembrane region. The dipole orientation of channel methanol is significantly affected by the bare carbonyl groups at the tube mouths and flips mainly in gap 4, whereas the rotation of ethanol is blocked. Ball-shaped hydrophobic methane experiences more flips in gap 4. The PMF (potential of mean force) profiles of the three organic molecules disclose their different diffusion behaviors in the CPNT. Amphiphilic alcohols are able to form direct H-bonds with channel water and the tube. Both single and double water bridges with the tube were observed in the methanol and ethanol systems. The different adsorption behaviors of the alcohols and water in the dehydrated CPNT may lead to the potential application of the CPNT as a means of separating alcohols from water. PMID:27083567

  9. Prediction of transmembrane regions of beta-barrel proteins using ANN- and SVM-based methods.

    PubMed

    Natt, Navjyot K; Kaur, Harpreet; Raghava, G P S

    2004-07-01

    This article describes a method developed for predicting transmembrane beta-barrel regions in membrane proteins using machine learning techniques: artificial neural network (ANN) and support vector machine (SVM). The ANN used in this study is a feed-forward neural network with a standard back-propagation training algorithm. The accuracy of the ANN-based method improved significantly, from 70.4% to 80.5%, when evolutionary information was added to a single sequence as a multiple sequence alignment obtained from PSI-BLAST. We have also developed an SVM-based method using a primary sequence as input and achieved an accuracy of 77.4%. The SVM model was modified by adding 36 physicochemical parameters to the amino acid sequence information. Finally, ANN- and SVM-based methods were combined to utilize the full potential of both techniques. The accuracy and Matthews correlation coefficient (MCC) value of SVM, ANN, and combined method are 78.5%, 80.5%, and 81.8%, and 0.55, 0.63, and 0.64, respectively. These methods were trained and tested on a nonredundant data set of 16 proteins, and performance was evaluated using "leave one out cross-validation" (LOOCV). Based on this study, we have developed a Web server, TBBPred, for predicting transmembrane beta-barrel regions in proteins (available at http://www.imtech.res.in/raghava/tbbpred).

  10. Different transport behaviors of NH4 (+) and NH3 in transmembrane cyclic peptide nanotubes.

    PubMed

    Zhang, Mingming; Fan, Jianfen; Xu, Jian; Weng, Peipei; Lin, Huifang

    2016-10-01

    Two water-filled transmembrane cyclic peptide nanotubes (CPNTs) of 8×cyclo-(WL)n=4,5/POPE were chosen to investigate the dependences of the transport properties of the positive NH4 (+) and neutral NH3 on the channel radius. Molecular dynamic simulations revealed that molecular charge, size, ability to form H-bonds and channel radius all significantly influence the behaviors of NH4 (+) and NH3 in a CPNT. Higher electrostatic interactions, more H-bonds, and water-bridges were found in the NH4 (+) system, resulting in NH4 (+) meeting higher energy barriers, while NH3 can enter, exit and permeate the channels effortlessly. This work sheds a first light on the differences between the mechanisms of NH4 (+) and NH3 moving in a CPNT at an atomic level. Graphical Abstract Snapshot of the simulation system of NH4 (+)_octa-CPNT with an NH4 (+) initially positioned at one mouth of the tube, PMF profiles for single NH4 (+) ion and NH3 molecule moving through water-filled transmembrane CPNTs of 8×cyclo-(WL)n=4,5/POPE and sketch graphs of the possible H-bond forms of NH3 and NH4 (+) with the neighboring water. PMID:27600817

  11. Structural classification and prediction of reentrant regions in alpha-helical transmembrane proteins: application to complete genomes.

    PubMed

    Viklund, Håkan; Granseth, Erik; Elofsson, Arne

    2006-08-18

    Alongside the well-studied membrane spanning helices, alpha-helical transmembrane (TM) proteins contain several functionally and structurally important types of substructures. Here, existing 3D structures of transmembrane proteins have been used to define and study the concept of reentrant regions, i.e. membrane penetrating regions that enter and exit the membrane on the same side. We find that these regions can be divided into three distinct categories based on secondary structure motifs, namely long regions with a helix-coil-helix motif, regions of medium length with the structure helix-coil or coil-helix and regions of short to medium length consisting entirely of irregular secondary structure. The residues situated in reentrant regions are significantly smaller on average compared to other regions and reentrant regions can be detected in the inter-transmembrane loops with an accuracy of approximately 70% based on their amino acid composition. Using TOP-MOD, a novel method for predicting reentrant regions, we have scanned the genomes of Escherichia coli, Saccharomyces cerevisiae and Homo sapiens. The results suggest that more than 10% of transmembrane proteins contain reentrant regions and that the occurrence of reentrant regions increases linearly with the number of transmembrane regions. Reentrant regions seem to be most commonly found in channel proteins and least commonly in signal receptors.

  12. Role of the vaccinia virus O3 protein in cell entry can be fulfilled by its Sequence flexible transmembrane domain

    SciTech Connect

    Satheshkumar, P.S.; Chavre, James; Moss, Bernard

    2013-09-15

    The vaccinia virus O3 protein, a component of the entry–fusion complex, is encoded by all chordopoxviruses. We constructed truncation mutants and demonstrated that the transmembrane domain, which comprises two-thirds of this 35 amino acid protein, is necessary and sufficient for interaction with the entry–fusion complex and function in cell entry. Nevertheless, neither single amino acid substitutions nor alanine scanning mutagenesis revealed essential amino acids within the transmembrane domain. Moreover, replication-competent mutant viruses were generated by randomization of 10 amino acids of the transmembrane domain. Of eight unique viruses, two contained only two amino acids in common with wild type and the remainder contained one or none within the randomized sequence. Although these mutant viruses formed normal size plaques, the entry–fusion complex did not co-purify with the mutant O3 proteins suggesting a less stable interaction. Thus, despite low specific sequence requirements, the transmembrane domain is sufficient for function in entry. - Highlights: • The 35 amino acid O3 protein is required for efficient vaccinia virus entry. • The transmembrane domain of O3 is necessary and sufficient for entry. • Mutagenesis demonstrated extreme sequence flexibility compatible with function.

  13. How to grow transgenic Arabidopsis in the field.

    PubMed

    Jänkänpää, Hanna Johansson; Jansson, Stefan

    2012-01-01

    Arabidopsis is naturally adapted to habitats in which both biotic variables (e.g., light, wind, and humidity) and abiotic variables (e.g., competition, herbivory, and pathogen densities) strongly fluctuate. Hence, conditions in controlled growth chambers (in which Arabidopsis is typically grown for scientific experiments) differ substantially from those in natural environments. In order to mimic more closely natural conditions, we grow Arabidopsis outdoors under "semi-natural" field conditions. Performing experiments on transgenic Arabidopsis grown in the field that are sufficiently reliable for publication is challenging. In this chapter, we present some of our experiences based on 10 years of field experimentation, which may be of use to researchers seeking to perform field experiments using transgenic Arabidopsis.

  14. An Overdose of the Arabidopsis Coreceptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 or Its Ectodomain Causes Autoimmunity in a SUPPRESSOR OF BIR1-1-Dependent Manner1

    PubMed Central

    Domínguez-Ferreras, Ana; Kiss-Papp, Marta; Jehle, Anna Kristina; Felix, Georg; Chinchilla, Delphine

    2015-01-01

    The membrane-bound BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1) is a common coreceptor in plants and regulates distinct cellular programs ranging from growth and development to defense against pathogens. BAK1 functions through binding to ligand-stimulated transmembrane receptors and activating their kinase domains via transphosphorylation. In the absence of microbes, BAK1 activity may be suppressed by different mechanisms, like interaction with the regulatory BIR (for BAK1-INTERACTING RECEPTOR-LIKE KINASE) proteins. Here, we demonstrated that BAK1 overexpression in Arabidopsis (Arabidopsis thaliana) could cause detrimental effects on plant development, including growth arrest, leaf necrosis, and reduced seed production. Further analysis using an inducible expression system showed that BAK1 accumulation quickly stimulated immune responses, even under axenic conditions, and led to increased resistance to pathogenic Pseudomonas syringae pv tomato DC3000. Intriguingly, our study also revealed that the plasma membrane-associated BAK1 ectodomain was sufficient to induce autoimmunity, indicating a novel mode of action for BAK1 in immunity control. We postulate that an excess of BAK1 or its ectodomain could trigger immune receptor activation in the absence of microbes through unbalancing regulatory interactions, including those with BIRs. Consistently, mutation of SUPPRESSOR OF BIR1-1, which encodes an emerging positive regulator of transmembrane receptors in plants, suppressed the effects of BAK1 overexpression. In conclusion, our findings unravel a new role for the BAK1 ectodomain in the tight regulation of Arabidopsis immune receptors necessary to avoid inappropriate activation of immunity. PMID:25944825

  15. Conformational rearrangements in the transmembrane domain of CNGA1 channels revealed by single-molecule force spectroscopy

    NASA Astrophysics Data System (ADS)

    Maity, Sourav; Mazzolini, Monica; Arcangeletti, Manuel; Valbuena, Alejandro; Fabris, Paolo; Lazzarino, Marco; Torre, Vincent

    2015-05-01

    Cyclic nucleotide-gated (CNG) channels are activated by binding of cyclic nucleotides. Although structural studies have identified the channel pore and selectivity filter, conformation changes associated with gating remain poorly understood. Here we combine single-molecule force spectroscopy (SMFS) with mutagenesis, bioinformatics and electrophysiology to study conformational changes associated with gating. By expressing functional channels with SMFS fingerprints in Xenopus laevis oocytes, we were able to investigate gating of CNGA1 in a physiological-like membrane. Force spectra determined that the S4 transmembrane domain is mechanically coupled to S5 in the closed state, but S3 in the open state. We also show there are multiple pathways for the unfolding of the transmembrane domains, probably caused by a different degree of α-helix folding. This approach demonstrates that CNG transmembrane domains have dynamic structure and establishes SMFS as a tool for probing conformational change in ion channels.

  16. Conformational rearrangements in the transmembrane domain of CNGA1 channels revealed by single-molecule force spectroscopy

    PubMed Central

    Maity, Sourav; Mazzolini, Monica; Arcangeletti, Manuel; Valbuena, Alejandro; Fabris, Paolo; Lazzarino, Marco; Torre, Vincent

    2015-01-01

    Cyclic nucleotide-gated (CNG) channels are activated by binding of cyclic nucleotides. Although structural studies have identified the channel pore and selectivity filter, conformation changes associated with gating remain poorly understood. Here we combine single-molecule force spectroscopy (SMFS) with mutagenesis, bioinformatics and electrophysiology to study conformational changes associated with gating. By expressing functional channels with SMFS fingerprints in Xenopus laevis oocytes, we were able to investigate gating of CNGA1 in a physiological-like membrane. Force spectra determined that the S4 transmembrane domain is mechanically coupled to S5 in the closed state, but S3 in the open state. We also show there are multiple pathways for the unfolding of the transmembrane domains, probably caused by a different degree of α-helix folding. This approach demonstrates that CNG transmembrane domains have dynamic structure and establishes SMFS as a tool for probing conformational change in ion channels. PMID:25963832

  17. Functional investigation of the plant-specific long coiled-coil proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL) in Arabidopsis thaliana.

    PubMed

    Venkatakrishnan, Sowmya; Mackey, David; Meier, Iris

    2013-01-01

    We have identified and characterized two Arabidopsis long coiled-coil proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL). PICC (147 kDa) and PICL (87 kDa) are paralogs that consist predominantly of a long coiled-coil domain (expanded in PICC), with a predicted transmembrane domain at the immediate C-terminus. Orthologs of PICC and PICL were found exclusively in vascular plants. PICC and PICL GFP fusion proteins are anchored to the cytoplasmic surface of the endoplasmic reticulum (ER) membrane by a C-terminal transmembrane domain and a short tail domain, via a tail-anchoring mechanism. T-DNA-insertion mutants of PICC and PICL as well as the double mutant show an increased sensitivity to the plant abiotic stress hormone abscisic acid (ABA) in a post-germination growth response. PICC, but not PICL gene expression is induced by the bacterial pathogen-associated molecular pattern (PAMP) flg22. T-DNA insertion alleles of PICC, but not PICL, show increased susceptibility to the non-virulent strain P. syringae pv. tomato DC3000 hrcC, but not to the virulent strain P. syringae pv. tomato DC3000. This suggests that PICC mutants are compromised in PAMP-triggered immunity (PTI). The data presented here provide first evidence for the involvement of a plant long coiled-coil protein in a plant defense response.

  18. Arabidopsis snc2-1D Activates Receptor-Like Protein-Mediated Immunity Transduced through WRKY70[C][W

    PubMed Central

    Zhang, Yaxi; Yang, Yuanai; Fang, Bin; Gannon, Patrick; Ding, Pingtao; Li, Xin; Zhang, Yuelin

    2010-01-01

    Plant immune receptors belonging to the receptor-like protein (RLP) family contain extracellular leucine-rich repeats (LRRs) and a short cytoplasmic tail linked by a single transmembrane motif. Here, we report the identification of snc2-1D (for suppressor of npr1-1, constitutive 2), a semidominant Arabidopsis thaliana mutant with constitutively activated defense responses. Map-based cloning of snc2-1D showed that it encodes an RLP. The point mutation in snc2-1D leads to substitution of the second Gly for Arg in the conserved GXXXG motif of the transmembrane helix, suggesting that this residue is important for negative regulation of the protein. Epistasis analysis revealed that the snc2-1D mutant phenotype is not affected by mutations in genes known to be required for the nucleotide binding (NB)-LRR Resistance (R) protein signaling. A suppressor screen of snc2-1D was performed, and map-based cloning of one suppressor revealed that mutations in WRKY70 suppress the constitutive defense responses in snc2-1D, suggesting that WRKY70 functions downstream of snc2-1D. The identification of snc2-1D provides us with a unique system for genetic analysis of resistance pathways downstream of RLPs, which may be distinct from those downstream of NB-LRR type R proteins. PMID:20841424

  19. Functional Investigation of the Plant-Specific Long Coiled-Coil Proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL) in Arabidopsis thaliana

    PubMed Central

    Venkatakrishnan, Sowmya; Mackey, David; Meier, Iris

    2013-01-01

    We have identified and characterized two Arabidopsis long coiled-coil proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL). PICC (147 kDa) and PICL (87 kDa) are paralogs that consist predominantly of a long coiled-coil domain (expanded in PICC), with a predicted transmembrane domain at the immediate C-terminus. Orthologs of PICC and PICL were found exclusively in vascular plants. PICC and PICL GFP fusion proteins are anchored to the cytoplasmic surface of the endoplasmic reticulum (ER) membrane by a C-terminal transmembrane domain and a short tail domain, via a tail-anchoring mechanism. T-DNA-insertion mutants of PICC and PICL as well as the double mutant show an increased sensitivity to the plant abiotic stress hormone abscisic acid (ABA) in a post-germination growth response. PICC, but not PICL gene expression is induced by the bacterial pathogen-associated molecular pattern (PAMP) flg22. T-DNA insertion alleles of PICC, but not PICL, show increased susceptibility to the non-virulent strain P. syringae pv. tomato DC3000 hrcC, but not to the virulent strain P. syringae pv. tomato DC3000. This suggests that PICC mutants are compromised in PAMP-triggered immunity (PTI). The data presented here provide first evidence for the involvement of a plant long coiled-coil protein in a plant defense response. PMID:23451199

  20. Functional investigation of the plant-specific long coiled-coil proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL) in Arabidopsis thaliana.

    PubMed

    Venkatakrishnan, Sowmya; Mackey, David; Meier, Iris

    2013-01-01

    We have identified and characterized two Arabidopsis long coiled-coil proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL). PICC (147 kDa) and PICL (87 kDa) are paralogs that consist predominantly of a long coiled-coil domain (expanded in PICC), with a predicted transmembrane domain at the immediate C-terminus. Orthologs of PICC and PICL were found exclusively in vascular plants. PICC and PICL GFP fusion proteins are anchored to the cytoplasmic surface of the endoplasmic reticulum (ER) membrane by a C-terminal transmembrane domain and a short tail domain, via a tail-anchoring mechanism. T-DNA-insertion mutants of PICC and PICL as well as the double mutant show an increased sensitivity to the plant abiotic stress hormone abscisic acid (ABA) in a post-germination growth response. PICC, but not PICL gene expression is induced by the bacterial pathogen-associated molecular pattern (PAMP) flg22. T-DNA insertion alleles of PICC, but not PICL, show increased susceptibility to the non-virulent strain P. syringae pv. tomato DC3000 hrcC, but not to the virulent strain P. syringae pv. tomato DC3000. This suggests that PICC mutants are compromised in PAMP-triggered immunity (PTI). The data presented here provide first evidence for the involvement of a plant long coiled-coil protein in a plant defense response. PMID:23451199

  1. Active conformation of the erythropoietin receptor: random and cysteine-scanning mutagenesis of the extracellular juxtamembrane and transmembrane domains.

    PubMed

    Lu, Xiaohui; Gross, Alec W; Lodish, Harvey F

    2006-03-17

    In the absence of erythropoietin (Epo) cell surface Epo receptors (EpoR) are dimeric; dimerization is mediated mainly by the transmembrane domain. Binding of Epo changes the orientation of the two receptor subunits. This conformational change is transmitted through the juxtamembrane and transmembrane domains, leading to activation of JAK2 kinase and induction of proliferation and survival signals. To define the active EpoR conformation(s) we screened libraries of EpoRs with random mutations in the transmembrane domain and identified several point mutations that activate the EpoR in the absence of ligand, including changes of either of the first two transmembrane domain residues (Leu(226) and Ile(227)) to cysteine. Following this discovery, we performed cysteine-scanning mutagenesis in the EpoR juxtamembrane and transmembrane domains. Many mutants formed disulfide-linked receptor dimers, but only EpoR dimers linked by cysteines at positions 223, 226, or 227 activated EpoR signal transduction pathways and supported proliferation of Ba/F3 cells in the absence of cytokines. These data suggest that activation of dimeric EpoR by Epo binding is achieved by reorienting the EpoR transmembrane and the connected cytosolic domains and that certain disulfide-bonded dimers represent the activated dimeric conformation of the EpoR, constitutively activating downstream signaling. Based on our data and the previously determined structure of Epo bound to a dimer of the EpoR extracellular domain, we present a model of the active and inactive conformations of the Epo receptor.

  2. All-Atom Structural Models of the Transmembrane Domains of Insulin and Type 1 Insulin-Like Growth Factor Receptors

    PubMed Central

    Mohammadiarani, Hossein; Vashisth, Harish

    2016-01-01

    The receptor tyrosine kinase superfamily comprises many cell-surface receptors including the insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF1R) that are constitutively homodimeric transmembrane glycoproteins. Therefore, these receptors require ligand-triggered domain rearrangements rather than receptor dimerization for activation. Specifically, binding of peptide ligands to receptor ectodomains transduces signals across the transmembrane domains for trans-autophosphorylation in cytoplasmic kinase domains. The molecular details of these processes are poorly understood in part due to the absence of structures of full-length receptors. Using MD simulations and enhanced conformational sampling algorithms, we present all-atom structural models of peptides containing 51 residues from the transmembrane and juxtamembrane regions of IR and IGF1R. In our models, the transmembrane regions of both receptors adopt helical conformations with kinks at Pro961 (IR) and Pro941 (IGF1R), but the C-terminal residues corresponding to the juxtamembrane region of each receptor adopt unfolded and flexible conformations in IR as opposed to a helix in IGF1R. We also observe that the N-terminal residues in IR form a kinked-helix sitting at the membrane–solvent interface, while homologous residues in IGF1R are unfolded and flexible. These conformational differences result in a larger tilt-angle of the membrane-embedded helix in IGF1R in comparison to IR to compensate for interactions with water molecules at the membrane–solvent interfaces. Our metastable/stable states for the transmembrane domain of IR, observed in a lipid bilayer, are consistent with a known NMR structure of this domain determined in detergent micelles, and similar states in IGF1R are consistent with a previously reported model of the dimerized transmembrane domains of IGF1R. Our all-atom structural models suggest potentially unique structural organization of kinase domains in each receptor. PMID

  3. Generation and identification of Arabidopsis EMS mutants.

    PubMed

    Qu, Li-Jia; Qin, Genji

    2014-01-01

    EMS mutant analysis is a routine experiment to identify new players in a specific biological process or signaling pathway using forward genetics. It begins with the generation of mutants by treating Arabidopsis seeds with EMS. A mutant with a phenotype of interest (mpi) is obtained by screening plants of the M2 generation under a specific condition. Once the phenotype of the mpi is confirmed in the next generation, map-based cloning is performed to locate the mpi mutation. During the map-based cloning, mpi plants (Arabidopsis Columbia-0 (Col-0) ecotype background) are first crossed with Arabidopsis Landsberg erecta (Ler) ecotype, and the presence or absence of the phenotype in the F1 hybrids indicates whether the mpi is recessive or dominant. F2 plants with phenotypes similar to the mpi, if the mpi is recessive, or those without the phenotype, if the mpi is dominant, are used as the mapping population. As few as 24 such plants are selected for rough mapping. After finding one marker (MA) linked to the mpi locus or mutant phenotype, more markers near MA are tested to identify recombinants. The recombinants indicate the interval in which the mpi is located. Additional recombinants and molecular markers are then required to narrow down the interval. This is an iterative process of narrowing down the mapping interval until no further recombinants or molecular markers are available. The genes in the mapping interval are then sequenced to look for the mutation. In the last step, the wild-type or mutated gene is cloned to generate binary constructs. Complementation or recapitulation provides the most convincing evidence in determining the mutation that causes the phenotype of the mpi. Here, we describe the procedures for generating mutants with EMS and analyzing EMS mutations by map-based cloning.

  4. Near-IR resonance Raman spectroscopy of archaerhodopsin 3: effects of transmembrane potential.

    PubMed

    Saint Clair, Erica C; Ogren, John I; Mamaev, Sergey; Russano, Daniel; Kralj, Joel M; Rothschild, Kenneth J

    2012-12-20

    Archaerhodopsin 3 (AR3) is a light driven proton pump from Halorubrum sodomense that has been used as a genetically targetable neuronal silencer and an effective fluorescent sensor of transmembrane potential. Unlike the more extensively studied bacteriorhodopsin (BR) from Halobacterium salinarum, AR3 readily incorporates into the plasma membrane of both E. coli and mammalian cells. Here, we used near-IR resonance Raman confocal microscopy to study the effects of pH and membrane potential on the AR3 retinal chromophore structure. Measurements were performed both on AR3 reconstituted into E. coli polar lipids and in vivo in E. coli expressing AR3 in the absence and presence of a negative transmembrane potential. The retinal chromophore structure of AR3 is in an all-trans configuration almost identical to BR over the entire pH range from 3 to 11. Small changes are detected in the retinal ethylenic stretching frequency and Schiff Base (SB) hydrogen bonding strength relative to BR which may be related to a different water structure near the SB. In the case of the AR3 mutant D95N, at neutral pH an all-trans retinal O-like species (O(all-trans)) is found. At higher pH a second 13-cis retinal N-like species (N(13-cis)) is detected which is attributed to a slowly decaying intermediate in the red-light photocycle of D95N. However, the amount of N(13-cis) detected is less in E. coli cells but is restored upon addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or sonication, both of which dissipate the normal negative membrane potential. We postulate that these changes are due to the effect of membrane potential on the N(13-cis) to M(13-cis) levels accumulated in the D95N red-light photocycle and on a molecular level by the effects of the electric field on the protonation/deprotonation of the cytoplasmic accessible SB. This mechanism also provides a possible explanation for the observed fluorescence dependence of AR3 and other microbial rhodopsins on transmembrane

  5. LsbB Bacteriocin Interacts with the Third Transmembrane Domain of the YvjB Receptor.

    PubMed

    Miljkovic, Marija; Uzelac, Gordana; Mirkovic, Nemanja; Devescovi, Giulia; Diep, Dzung B; Venturi, Vittorio; Kojic, Milan

    2016-09-01

    The Zn-dependent membrane-located protease YvjB has previously been shown to serve as a target receptor for LsbB, a class II leaderless lactococcal bacteriocin. Although yvjB is highly conserved in the genus Lactococcus, the bacteriocin appears to be active only against the subspecies L. lactis subsp. lactis Comparative analysis of the YvjB proteins of a sensitive strain (YvjBMN) and a resistant strain (YvjBMG) showed that they differ from each other in 31 positions. In this study, we applied site-directed mutagenesis and performed directed binding studies to provide biochemical evidence that LsbB interacts with the third transmembrane helix of YvjB in susceptible cells. The site-directed mutagenesis of LsbB and YvjB proteins showed that certain amino acids and the length of LsbB are responsible for the bacteriocin activity, most probably through adequate interaction of these two proteins; the essential amino acids in LsbB responsible for the activity are tryptophan (Trp(25)) and terminal alanine (Ala(30)). It was also shown that the distance between Trp(25) and terminal alanine is crucial for LsbB activity. The crucial region in YvjB for the interaction with LsbB is the beginning of the third transmembrane helix, particularly amino acids tyrosine (Tyr(356)) and alanine (Ala(353)). In vitro experiments showed that LsbB could interact with both YvjBMN and YvjBMG, but the strength of interaction is significantly less with YvjBMG In vivo experiments with immunofluorescently labeled antibody demonstrated that LsbB specifically interacts only with cells carrying YvjBMN IMPORTANCE: The antimicrobial activity of LsbB bacteriocin depends on the correct interaction with the corresponding receptor in the bacterial membrane of sensitive cells. Membrane-located bacteriocin receptors have essential primary functions, such as cell wall synthesis or sugar transport, and it seems that interaction with bacteriocins is suicidal for cells. This study showed that the C-terminal part

  6. Transmembrane delivery of anticancer drugs through self-assembly of cyclic peptide nanotubes.

    PubMed

    Chen, Jian; Zhang, Bei; Xia, Fei; Xie, Yunchang; Jiang, Sifan; Su, Rui; Lu, Yi; Wu, Wei

    2016-04-01

    Breaking the natural barriers of cell membranes achieves fast entry of therapeutics, which leads to enhanced efficacy and helps overcome multiple drug resistance. Herein, transmembrane delivery of a series of small molecule anticancer drugs was achieved by the construction of artificial transmembrane nanochannels formed by self-assembly of cyclic peptide (cyclo[Gln-(d-Leu-Trp)4-d-Leu], CP) nanotubes (CPNTs) in the lipid bilayers. Our in vitro study in liposomes indicated that the transport of molecules with sizes smaller than 1.0 nm, which is the internal diameter of the CPNTs, could be significantly enhanced by CPNTs in a size-selective and dose-dependent manner. Facilitated uptake of 5-fluorouracil (5-FU) was also confirmed in the BEL7402 cell line. On the contrary, CPs could facilitate neither the transport across liposomal membranes nor the uptake by cell lines of cytarabine, a counterevidence drug with a size of 1.1 nm. CPs had a very weak anticancer efficacy, but could significantly reduce the IC50 of 5-FU in BEL7402, HeLa and S180 cell lines. Analysis by a q test revealed that a combination of 5-FU and CP had a synergistic effect in BEL7402 at all CP levels, in S180 at CP levels higher than 64 μg mL(-1), but not in HeLa, where an additive effect was observed. Temporarily, intratumoral injection is believed to be the best way for CP administration. In vivo imaging using (125)I radio-labelled CP confirmed that CPNPTs were completely localized in the tumor tissues, and translocation to other tissues was negligible. In vivo anticancer efficacy was studied in the grafted S180 solid tumor model in mice, and the results indicated that tumor growth was greatly inhibited by the combinatory use of 5-FU and CP, and a synergistic effect was observed at CP doses of 0.25 mg per kg bw. It is concluded that facilitated transmembrane delivery of anticancer drugs with sizes smaller than 1.0 nm was achieved, and the synergistic anticancer effect was confirmed both in cell

  7. Transmembrane helix prediction using amino acid property features and latent semantic analysis

    PubMed Central

    Ganapathiraju, Madhavi; Balakrishnan, N; Reddy, Raj; Klein-Seetharaman, Judith

    2008-01-01

    Background Prediction of transmembrane (TM) helices by statistical methods suffers from lack of sufficient training data. Current best methods use hundreds or even thousands of free parameters in their models which are tuned to fit the little data available for training. Further, they are often restricted to the generally accepted topology "cytoplasmic-transmembrane-extracellular" and cannot adapt to membrane proteins that do not conform to this topology. Recent crystal structures of channel proteins have revealed novel architectures showing that the above topology may not be as universal as previously believed. Thus, there is a need for methods that can better predict TM helices even in novel topologies and families. Results Here, we describe a new method "TMpro" to predict TM helices with high accuracy. To avoid overfitting to existing topologies, we have collapsed cytoplasmic and extracellular labels to a single state, non-TM. TMpro is a binary classifier which predicts TM or non-TM using multiple amino acid properties (charge, polarity, aromaticity, size and electronic properties) as features. The features are extracted from sequence information by applying the framework used for latent semantic analysis of text documents and are input to neural networks that learn the distinction between TM and non-TM segments. The model uses only 25 free parameters. In benchmark analysis TMpro achieves 95% segment F-score corresponding to 50% reduction in error rate compared to the best methods not requiring an evolutionary profile of a protein to be known. Performance is also improved when applied to more recent and larger high resolution datasets PDBTM and MPtopo. TMpro predictions in membrane proteins with unusual or disputed TM structure (K+ channel, aquaporin and HIV envelope glycoprotein) are discussed. Conclusion TMpro uses very few free parameters in modeling TM segments as opposed to the very large number of free parameters used in state-of-the-art membrane

  8. Functional Analysis of Transcription Factors in Arabidopsis

    PubMed Central

    Mitsuda, Nobutaka; Ohme-Takagi, Masaru

    2009-01-01

    Transcription factors (TFs) regulate the expression of genes at the transcriptional level. Modification of TF activity dynamically alters the transcriptome, which leads to metabolic and phenotypic changes. Thus, functional analysis of TFs using ‘omics-based’ methodologies is one of the most important areas of the post-genome era. In this mini-review, we present an overview of Arabidopsis TFs and introduce strategies for the functional analysis of plant TFs, which include both traditional and recently developed technologies. These strategies can be assigned to five categories: bioinformatic analysis; analysis of molecular function; expression analysis; phenotype analysis; and network analysis for the description of entire transcriptional regulatory networks. PMID:19478073

  9. Transmembrane signaling in the sensor kinase DcuS of Escherichia coli: A long-range piston-type displacement of transmembrane helix 2

    PubMed Central

    Monzel, Christian; Unden, Gottfried

    2015-01-01

    The C4-dicarboxylate sensor kinase DcuS is membrane integral because of the transmembrane (TM) helices TM1 and TM2. Fumarate-induced movement of the helices was probed in vivo by Cys accessibility scanning at the membrane–water interfaces after activation of DcuS by fumarate at the periplasmic binding site. TM1 was inserted with amino acid residues 21–41 in the membrane in both the fumarate-activated (ON) and inactive (OFF) states. In contrast, TM2 was inserted with residues 181–201 in the OFF state and residues 185–205 in the ON state. Replacement of Trp 185 by an Arg residue caused displacement of TM2 toward the outside of the membrane and a concomitant induction of the ON state. Results from Cys cross-linking of TM2/TM2′ in the DcuS homodimer excluded rotation; thus, data from accessibility changes of TM2 upon activation, either by ligand binding or by mutation of TM2, and cross-linking of TM2 and the connected region in the periplasm suggest a piston-type shift of TM2 by four residues to the periplasm upon activation (or fumarate binding). This mode of function is supported by the suggestion from energetic calculations of two preferred positions for TM2 insertion in the membrane. The shift of TM2 by four residues (or 4–6 Å) toward the periplasm upon activation is complementary to the periplasmic displacement of 3–4 Å of the C-terminal part of the periplasmic ligand-binding domain upon ligand occupancy in the citrate-binding domain in the homologous CitA sensor kinase. PMID:26283365

  10. Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis.

    PubMed

    Gao, Qi-Fei; Gu, Li-Li; Wang, Hui-Qin; Fei, Cui-Fang; Fang, Xiang; Hussain, Jamshaid; Sun, Shu-Jing; Dong, Jing-Yun; Liu, Hongtao; Wang, Yong-Fei

    2016-03-15

    In flowering plants, pollen tubes are guided into ovules by multiple attractants from female gametophytes to release paired sperm cells for double fertilization. It has been well-established that Ca(2+) gradients in the pollen tube tips are essential for pollen tube guidance and that plasma membrane Ca(2+) channels in pollen tube tips are core components that regulate Ca(2+) gradients by mediating and regulating external Ca(2+) influx. Therefore, Ca(2+) channels are the core components for pollen tube guidance. However, there is still no genetic evidence for the identification of the putative Ca(2+) channels essential for pollen tube guidance. Here, we report that the point mutations R491Q or R578K in cyclic nucleotide-gated channel 18 (CNGC18) resulted in abnormal Ca(2+) gradients and strong pollen tube guidance defects by impairing the activation of CNGC18 in Arabidopsis. The pollen tube guidance defects of cngc18-17 (R491Q) and of the transfer DNA (T-DNA) insertion mutant cngc18-1 (+/-) were completely rescued by CNGC18. Furthermore, domain-swapping experiments showed that CNGC18's transmembrane domains are indispensable for pollen tube guidance. Additionally, we found that, among eight Ca(2+) channels (including six CNGCs and two glutamate receptor-like channels), CNGC18 was the only one essential for pollen tube guidance. Thus, CNGC18 is the long-sought essential Ca(2+) channel for pollen tube guidance in Arabidopsis.

  11. Overexpression of BrMORN, a novel 'membrane occupation and recognition nexus' motif protein gene from Chinese cabbage, promotes vegetative growth and seed production in Arabidopsis.

    PubMed

    Lee, Jeongyeo; Han, Ching-Tack; Hur, Yoonkang

    2010-02-28

    Proteins that contain membrane occupation and recognition nexus (MORN) motifs regulate various aspects of cellular metabolism by localizing proteins in different cellular organelles. The full-length Brassica rapa MORN motif protein (BrMORN) cDNA consists of 1,510 bp encoding 502 deduced amino acids with a predicted molecular mass of 55.8 kDa and an isoelectric point of 9.72. BrMORN is a novel protein composed of two N-terminal transmembrane helices and seven C-terminal MORN motifs and it appears to be localized on the plastid envelope. BrMORN expression was relatively high in actively-growing tissues, but low in mature tissues and under some abiotic stresses. Arabidopsis thaliana plants overexpressing BrMORN showed an enhanced rate of growth, hypocotyl elongation, and increases in the size of vegetative organs and seed productivity under normal growth conditions. In addition, cell size in Arabidopsis plants overexpressing BrMORN was 24% larger than that of wild-type plants, implying that the increase in the size of vegetative organs is due to cell enlargement. The increased size of the vegetative organs also led to increased seed production. Our data suggest that the MORN motif of BrMORN may act at the plastid envelope and facilitate plant growth via cell enlargement.

  12. MzPIP2;1: An Aquaporin Involved in Radial Water Movement in Both Water Uptake and Transportation, Altered the Drought and Salt Tolerance of Transgenic Arabidopsis

    PubMed Central

    Lei, Qiong; Feng, Chao; Gao, Yinan; Zheng, Xiaodong; Zhao, Yu; Wang, Zhi; Kong, Jin

    2015-01-01

    Background Plants are unavoidably subjected to various abiotic stressors, including high salinity, drought and low temperature, which results in water deficit and even death. Water uptake and transportation play a critical role in response to these stresses. Many aquaporin proteins, localized at different tissues, function in various transmembrane water movements. We targeted at the key aquaporin in charge of both water uptake in roots and radial water transportation from vascular tissues through the whole plant. Results The MzPIP2;1 gene encoding a plasma membrane intrinsic protein was cloned from salt-tolerant apple rootstock Malus zumi Mats. The GUS gene was driven by MzPIP2;1 promoter in transgenic Arabidopsis. It indicated that MzPIP2;1 might function in the epidermal and vascular cells of roots, parenchyma cells around vessels through the stems and vascular tissues of leaves. The ectopically expressed MzPIP2;1 conferred the transgenic Arabidopsis plants enhanced tolerance to slight salt and drought stresses, but sensitive to moderate salt stress, which was indicated by root length, lateral root number, fresh weight and K+/Na+ ratio. In addition, the possible key cis-elements in response to salt, drought and cold stresses were isolated by the promoter deletion experiment. Conclusion The MzPIP2;1 protein, as a PIP2 aquaporins subgroup member, involved in radial water movement, controls water absorption and usage efficiency and alters transgenic plants drought and salt tolerance. PMID:26562158

  13. The Membrane-Bound NAC Transcription Factor ANAC013 Functions in Mitochondrial Retrograde Regulation of the Oxidative Stress Response in Arabidopsis[C][W

    PubMed Central

    De Clercq, Inge; Vermeirssen, Vanessa; Van Aken, Olivier; Vandepoele, Klaas; Murcha, Monika W.; Law, Simon R.; Inzé, Annelies; Ng, Sophia; Ivanova, Aneta; Rombaut, Debbie; van de Cotte, Brigitte; Jaspers, Pinja; Van de Peer, Yves; Kangasjärvi, Jaakko; Whelan, James; Van Breusegem, Frank

    2013-01-01

    Upon disturbance of their function by stress, mitochondria can signal to the nucleus to steer the expression of responsive genes. This mitochondria-to-nucleus communication is often referred to as mitochondrial retrograde regulation (MRR). Although reactive oxygen species and calcium are likely candidate signaling molecules for MRR, the protein signaling components in plants remain largely unknown. Through meta-analysis of transcriptome data, we detected a set of genes that are common and robust targets of MRR and used them as a bait to identify its transcriptional regulators. In the upstream regions of these mitochondrial dysfunction stimulon (MDS) genes, we found a cis-regulatory element, the mitochondrial dysfunction motif (MDM), which is necessary and sufficient for gene expression under various mitochondrial perturbation conditions. Yeast one-hybrid analysis and electrophoretic mobility shift assays revealed that the transmembrane domain–containing NO APICAL MERISTEM/ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR/CUP-SHAPED COTYLEDON transcription factors (ANAC013, ANAC016, ANAC017, ANAC053, and ANAC078) bound to the MDM cis-regulatory element. We demonstrate that ANAC013 mediates MRR-induced expression of the MDS genes by direct interaction with the MDM cis-regulatory element and triggers increased oxidative stress tolerance. In conclusion, we characterized ANAC013 as a regulator of MRR upon stress in Arabidopsis thaliana. PMID:24045019

  14. Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis

    PubMed Central

    Gao, Qi-Fei; Gu, Li-Li; Wang, Hui-Qin; Fei, Cui-Fang; Fang, Xiang; Hussain, Jamshaid; Sun, Shu-Jing; Dong, Jing-Yun; Liu, Hongtao; Wang, Yong-Fei

    2016-01-01

    In flowering plants, pollen tubes are guided into ovules by multiple attractants from female gametophytes to release paired sperm cells for double fertilization. It has been well-established that Ca2+ gradients in the pollen tube tips are essential for pollen tube guidance and that plasma membrane Ca2+ channels in pollen tube tips are core components that regulate Ca2+ gradients by mediating and regulating external Ca2+ influx. Therefore, Ca2+ channels are the core components for pollen tube guidance. However, there is still no genetic evidence for the identification of the putative Ca2+ channels essential for pollen tube guidance. Here, we report that the point mutations R491Q or R578K in cyclic nucleotide-gated channel 18 (CNGC18) resulted in abnormal Ca2+ gradients and strong pollen tube guidance defects by impairing the activation of CNGC18 in Arabidopsis. The pollen tube guidance defects of cngc18-17 (R491Q) and of the transfer DNA (T-DNA) insertion mutant cngc18-1 (+/−) were completely rescued by CNGC18. Furthermore, domain-swapping experiments showed that CNGC18’s transmembrane domains are indispensable for pollen tube guidance. Additionally, we found that, among eight Ca2+ channels (including six CNGCs and two glutamate receptor-like channels), CNGC18 was the only one essential for pollen tube guidance. Thus, CNGC18 is the long-sought essential Ca2+ channel for pollen tube guidance in Arabidopsis. PMID:26929345

  15. Genomic structure of SAS, a member of the transmembrane 4 superfamily amplified in human sarcomas

    SciTech Connect

    Jankowski, S.A.; De Jong, P.; Meltzer, P.S.

    1995-01-20

    SAS is a recently identified member of the transmembrane 4 superfamily (TM4SF) that is frequently amplified in human sarcomas. To further its characterization and to confirm its classification, the genomic structure of the SAS gene was determined. The SAS gene covers approximately 3.2 kb of DNA. It contains six exons within its translated region, three of which are highly conserved in the TM4SF. 5{prime} to the translation start site are two putative transcription start sites, two CCAAT consensus sequences, and potential binding sites for both Sp1 and ATF transcription factors. Comparison of SAS organization to human ME491, CD9, and CD53 and murine CD53 and TAPA-1 confirms that SAS is a member of this family of genes and is consistent with the theory that these genes arose through duplication and divergent evolution. 44 refs., 4 figs., 2 tabs.

  16. The transmembrane protein, Tincar, is involved in the development of the compound eye in Drosophila melanogaster.

    PubMed

    Hirota, Yuki; Sawamoto, Kazunobu; Takahashi, Kuniaki; Ueda, Ryu; Okano, Hideyuki

    2005-02-01

    We previously cloned and characterized the Drosophila gene, tincar (tinc), which encodes a novel protein with eight putative transmembrane domains. Here, we have studied the expression pattern and functions of tinc during developmental processes. tinc mRNA is expressed in the central and peripheral nervous systems, and midgut during embryogenesis. In the third-instar larval eye disc, tinc mRNA is strongly expressed in all the differentiating ommatidial cells within and in the vicinity of the morphogenetic furrow. Loss-of-function analysis using the RNA-interference method revealed severe defects of eye morphogenesis during the late developmental stages. Our results suggested that tinc may have an indispensable role in the normal differentiation of ommatidial cells. PMID:15654626

  17. Interferon-Induced Transmembrane Protein-Mediated Inhibition of Host Cell Entry of Ebolaviruses.

    PubMed

    Wrensch, Florian; Karsten, Christina B; Gnirß, Kerstin; Hoffmann, Markus; Lu, Kai; Takada, Ayato; Winkler, Michael; Simmons, Graham; Pöhlmann, Stefan

    2015-10-01

    Ebolaviruses are highly pathogenic in humans and nonhuman primates and pose a severe threat to public health. The interferon-induced transmembrane (IFITM) proteins can restrict entry of ebolaviruses, influenza A viruses, and other enveloped viruses. However, the breadth and mechanism of the antiviral activity of IFITM proteins are incompletely understood. Here, we employed ebolavirus glycoprotein-pseudotyped vectors and ebolavirus-like particles to address this question. We show that IFITM proteins inhibit the cellular entry of diverse ebolaviruses and demonstrate that type I interferon induces IFITM protein expression in macrophages, major viral targets. Moreover, we show that IFITM proteins block entry of influenza A viruses and ebolaviruses by different mechanisms and provide evidence that antibodies and IFITM proteins can synergistically inhibit cellular entry of ebolaviruses. These results provide insights into the role of IFITM proteins in infection by ebolaviruses and suggest a mechanism by which antibodies, though poorly neutralizing in vitro, might contribute to viral control in vivo.

  18. [The activation effect of nobiletin on cystic fibrosis transmembrane conductance regulator chloride channel].

    PubMed

    Yang, Shuang; Yu, Bo; Zhang, Yao-Fang; Wang, Xue; Yang, Hong

    2013-06-01

    Aim of the present study is to investigate activation effect of nobiletin on cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity. CFTR-mediated iodide influx assay and patch-clamp tests were done on FRT cells stably co-transfected with human CFTR and EYFP/H148Q. Nobiletin potently activated CFTR chloride channel activity in a dose- and time-dependent manner. The CFTR blocker CFTR(inh)-172 could completely reverse the effect. Preliminary mechanism study indicated that nobiletin activated CFTR chloride channel through a direct binding way. In addition, ex vivo tests done on mice trachea showed that nobiletin time-dependently stimulated submucosal gland fluid secretion. Nobiletin may be a therapeutic lead compound in treating CFTR-related diseases including disseminated bronchiectasis.

  19. Single-molecule visualization of dynamic transitions of pore-forming peptides among multiple transmembrane positions

    PubMed Central

    Li, Ying; Qian, Zhenyu; Ma, Li; Hu, Shuxin; Nong, Daguan; Xu, Chunhua; Ye, Fangfu; Lu, Ying; Wei, Guanghong; Li, Ming

    2016-01-01

    Research on the dynamics of single-membrane proteins remains underdeveloped due to the lack of proper approaches that can probe in real time the protein's insertion depth in lipid bilayers. Here we report a single-molecule visualization method to track both vertical insertion and lateral diffusion of membrane proteins in supported lipid bilayers by exploiting the surface-induced fluorescence attenuation (SIFA) of fluorophores. The attenuation follows a d−4 dependency, where d is the fluorophore-to-surface distance. The method is validated by observing the antimicrobial peptide LL-37 to transfer among five transmembrane positions: the surface, the upper leaflet, the centre, the lower leaflet and the bottom of the lipid bilayer. These results demonstrate the power of SIFA to study protein-membrane interactions and provide unprecedented in-depth understanding of molecular mechanisms of the insertion and translocation of membrane proteins. PMID:27686409

  20. Energetics of side-chain snorkeling in transmembrane helices probed by nonproteinogenic amino acids

    PubMed Central

    Öjemalm, Karin; Higuchi, Takashi; Lara, Patricia; Lindahl, Erik; Suga, Hiroaki

    2016-01-01

    Cotranslational translocon-mediated insertion of membrane proteins into the endoplasmic reticulum is a key process in membrane protein biogenesis. Although the mechanism is understood in outline, quantitative data on the energetics of the process is scarce. Here, we have measured the effect on membrane integration efficiency of nonproteinogenic analogs of the positively charged amino acids arginine and lysine incorporated into model transmembrane segments. We provide estimates of the influence on the apparent free energy of membrane integration (ΔGapp) of “snorkeling” of charged amino acids toward the lipid–water interface, and of charge neutralization. We further determine the effect of fluorine atoms and backbone hydrogen bonds (H-bonds) on ΔGapp. These results help establish a quantitative basis for our understanding of membrane protein assembly in eukaryotic cells. PMID:27601675

  1. Cystic Fibrosis Transmembrane Conductance Regulator. Implications in Cystic Fibrosis and Chronic Obstructive Pulmonary Disease.

    PubMed

    Cantin, André M

    2016-04-01

    Cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) have traditionally been viewed as two distinct entities of unrelated origins. However, molecular, cellular, and clinical studies have revealed that cystic fibrosis transmembrane conductance regulator (CFTR) protein dysfunction is common to both conditions, one (CF) being defined genetically and the other (COPD) as an acquired CFTR deficiency. Multiple molecular mechanisms of cigarette smoke-induced CFTR dysfunction have been reported. More importantly, considerable evidence of cigarette smoke-induced CFTR dysfunction in several respiratory and nonrespiratory tissues have been confirmed, making CFTR a target that cannot be overlooked in our quest to understand COPD and improve therapies for individuals affected by this disease. This review summarizes the molecular, cellular, and clinical evidence that CFTR dysfunction is induced by cigarette smoke exposure both in vitro and in vivo, and explores how this may contribute to the development of COPD. PMID:27115950

  2. Failure of the Cystic Fibrosis Transmembrane Conductance Regulator to Conduct ATP

    NASA Astrophysics Data System (ADS)

    Reddy, M. M.; Quinton, P. M.; Haws, C.; Wine, J. J.; Grygorczyk, R.; Tabcharani, J. A.; Hanrahan, J. W.; Gunderson, K. L.; Kopito, R. R.

    1996-03-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is chloride ion channel regulated by protein kinase A and adenosine triphosphate (ATP). Loss of CFTR-mediated chloride ion conductance from the apical plasma membrane of epithelial cells is a primary physiological lesion in cystic fibrosis. CFTR has also been suggested to function as an ATP channel, although the size of the ATP anion is much larger than the estimated size of the CFTR pore. ATP was not conducted through CFTR in intact organs, polarized human lung cell lines, stably transfected mammalian cell lines, or planar lipid bilayers reconstituted with CFTR protein. These findings suggest that ATP permeation through the CFTR is unlikely to contribute to the normal function of CFTR or to the pathogenesis of cystic fibrosis.

  3. Transmembrane Potential Modeling: Comparison between Methods of Constant Electric Field and Ion Imbalance.

    PubMed

    Melcr, Josef; Bonhenry, Daniel; Timr, Štěpán; Jungwirth, Pavel

    2016-05-10

    Two approaches for modeling of the transmembrane potential, as present in all eukaryotic cells, are examined in detail and compared with each other. One approach uses an externally applied electric field, whereas the other maintains an imbalance of ions on the two sides of a membrane. We demonstrate that both methods provide converged results concerning structural parameters of the membrane which are practically indistinguishable from each other, at least for monovalent ions. Effects of the electric field on the detailed molecular structure of the phospholipid bilayer are also presented and discussed. In addition, we achieve a considerable speed-up of the underlying molecular dynamics simulations by implementing the virtual interaction sites method for the Slipids force field.

  4. Type II transmembrane serine proteases as potential targets for cancer therapy

    PubMed Central

    Murray, Andrew S.; Varela, Fausto A.

    2016-01-01

    Carcinogenesis is accompanied by increased protein and activity levels of extracellular cell-surface proteases that are capable of modifying the tumor micro-environment by directly cleaving the extracellular matrix, as well as activating growth factors and proinflammatory mediators involved in proliferation and invasion of cancer cells, and recruitment of inflammatory cells. These complex processes ultimately potentiate neoplastic progression leading to local tumor cell invasion, entry into the vasculature, and metastasis to distal sites. Several members of the type II transmembrane serine protease (TTSP) family have been shown to play critical roles in cancer progression. In this review the knowledge collected over the past two decades about the molecular mechanisms underlying the pro-cancerous properties of selected TTSPs will be summarized. Furthermore, we will discuss how these insights may facilitate the translation into clinical settings in the future by specifically targeting TTSPs as part of novel cancer treatment regimens. PMID:27078673

  5. Visualization and quantification of transmembrane ion transport into giant unilamellar vesicles.

    PubMed

    Valkenier, Hennie; López Mora, Néstor; Kros, Alexander; Davis, Anthony P

    2015-02-01

    Transmembrane ion transporters (ionophores) are widely investigated as supramolecular agents with potential for biological activity. Tests are usually performed in synthetic membranes that are assembled into large unilamellar vesicles (LUVs). However transport must be followed through bulk properties of the vesicle suspension, because LUVs are too small for individual study. An alternative approach is described whereby ion transport can be revealed and quantified through direct observation. The method employs giant unilamellar vesicles (GUVs), which are 20-60 μm in diameter and readily imaged by light microscopy. This allows characterization of individual GUVs containing transporter molecules, followed by studies of transport through fluorescence emission from encapsulated indicators. The method provides new levels of certainty and relevance, given that the GUVs are similar in size to living cells. It has been demonstrated using a highly active anion carrier, and should aid the development of compounds for treating channelopathies such as cystic fibrosis.

  6. TIM23-mediated insertion of transmembrane α-helices into the mitochondrial inner membrane.

    PubMed

    Botelho, Salomé Calado; Osterberg, Marie; Reichert, Andreas S; Yamano, Koji; Björkholm, Patrik; Endo, Toshiya; von Heijne, Gunnar; Kim, Hyun

    2011-03-16

    While overall hydrophobicity is generally recognized as the main characteristic of transmembrane (TM) α-helices, the only membrane system for which there are detailed quantitative data on how different amino acids contribute to the overall efficiency of membrane insertion is the endoplasmic reticulum (ER) of eukaryotic cells. Here, we provide comparable data for TIM23-mediated membrane protein insertion into the inner mitochondrial membrane of yeast cells. We find that hydrophobicity and the location of polar and aromatic residues are strong determinants of membrane insertion. These results parallel what has been found previously for the ER. However, we see striking differences between the effects elicited by charged residues flanking the TM segments when comparing the mitochondrial inner membrane and the ER, pointing to an unanticipated difference between the two insertion systems.

  7. Measuring Transmembrane Helix Interaction Strengths in Lipid Bilayers Using Steric Trapping

    PubMed Central

    Hong, Heedeok; Chang, Yu-Chu; Bowie, James U.

    2016-01-01

    We have developed a method to measure strong transmembrane (TM) helix interaction affinities in lipid bilayers that are difficult to measure by traditional dilution methods. The method, called steric trapping, couples dissociation of biotinylated TM helices to a competitive binding by monovalent streptavidin (mSA), so that dissociation is driven by the affinity of mSA for biotin and mSA concentration. By adjusting the binding affinity of mSA through mutation, the method can obtain dissociation constants of TM helix dimers (Kd,dimer) over a range of six orders of magnitudes. The Kd,dimer limit of measurable target interaction is extended 3–4 orders of magnitude lower than possible by dilution methods. Thus, steric trapping opens up new opportunities to study the folding and assembly of α-helical membrane proteins in lipid bilayer environments. Here we provide detailed methods for applying steric trapping to a TM helix dimer. PMID:23975771

  8. Characterizing diverse orthologues of the cystic fibrosis transmembrane conductance regulator protein for structural studies.

    PubMed

    Pollock, Naomi L; Rimington, Tracy L; Ford, Robert C

    2015-10-01

    As an ion channel, the cystic fibrosis transmembrane conductance regulator (CFTR) protein occupies a unique niche within the ABC family. Orthologues of CFTR are extant throughout the animal kingdom from sharks to platypods to sheep, where the osmoregulatory function of the protein has been applied to differing lifestyles and diverse organ systems. In humans, loss-of-function mutations to CFTR cause the disease cystic fibrosis, which is a significant health burden in populations of white European descent. Orthologue screening has proved fruitful in the pursuit of high-resolution structural data for several membrane proteins, and we have applied some of the princples developed in previous studies to the expression and purification of CFTR. We have overexpressed this protein, along with evolutionarily diverse orthologues, in Saccharomyces cerevisiae and developed a purification to isolate it in quantities sufficient for structural and functional studies. PMID:26517900

  9. Emerging role of cystic fibrosis transmembrane conductance regulator - an epithelial chloride channel in gastrointestinal cancers.

    PubMed

    Hou, Yuning; Guan, Xiaoqing; Yang, Zhe; Li, Chunying

    2016-03-15

    Cystic fibrosis transmembrane conductance regulator (CFTR), a glycoprotein with 1480 amino acids, has been well established as a chloride channel mainly expressed in the epithelial cells of various tissues and organs such as lungs, sweat glands, gastrointestinal system, and reproductive organs. Although defective CFTR leads to cystic fibrosis, a common genetic disorder in the Caucasian population, there is accumulating evidence that suggests a novel role of CFTR in various cancers, especially in gastroenterological cancers, such as pancreatic cancer and colon cancer. In this review, we summarize the emerging findings that link CFTR with various cancers, with focus on the association between CFTR defects and gastrointestinal cancers as well as the underlying mechanisms. Further study of CFTR in cancer biology may help pave a new way for the diagnosis and treatment of gastrointestinal cancers. PMID:26989463

  10. Energetics of side-chain snorkeling in transmembrane helices probed by nonproteinogenic amino acids.

    PubMed

    Öjemalm, Karin; Higuchi, Takashi; Lara, Patricia; Lindahl, Erik; Suga, Hiroaki; von Heijne, Gunnar

    2016-09-20

    Cotranslational translocon-mediated insertion of membrane proteins into the endoplasmic reticulum is a key process in membrane protein biogenesis. Although the mechanism is understood in outline, quantitative data on the energetics of the process is scarce. Here, we have measured the effect on membrane integration efficiency of nonproteinogenic analogs of the positively charged amino acids arginine and lysine incorporated into model transmembrane segments. We provide estimates of the influence on the apparent free energy of membrane integration (ΔGapp) of "snorkeling" of charged amino acids toward the lipid-water interface, and of charge neutralization. We further determine the effect of fluorine atoms and backbone hydrogen bonds (H-bonds) on ΔGapp These results help establish a quantitative basis for our understanding of membrane protein assembly in eukaryotic cells. PMID:27601675

  11. Coarse Grained Molecular Dynamics Simulations of Transmembrane Protein-Lipid Systems

    PubMed Central

    Spijker, Peter; van Hoof, Bram; Debertrand, Michel; Markvoort, Albert J.; Vaidehi, Nagarajan; Hilbers, Peter A.J.

    2010-01-01

    Many biological cellular processes occur at the micro- or millisecond time scale. With traditional all-atom molecular modeling techniques it is difficult to investigate the dynamics of long time scales or large systems, such as protein aggregation or activation. Coarse graining (CG) can be used to reduce the number of degrees of freedom in such a system, and reduce the computational complexity. In this paper the first version of a coarse grained model for transmembrane proteins is presented. This model differs from other coarse grained protein models due to the introduction of a novel angle potential as well as a hydrogen bonding potential. These new potentials are used to stabilize the backbone. The model has been validated by investigating the adaptation of the hydrophobic mismatch induced by the insertion of WALP-peptides into a lipid membrane, showing that the first step in the adaptation is an increase in the membrane thickness, followed by a tilting of the peptide. PMID:20640160

  12. Engineering a Transmembrane Nanopore Ion Channel from a Membrane Breaker Peptide.

    PubMed

    Lella, Muralikrishna; Mahalakshmi, Radhakrishnan

    2016-07-01

    Re-engineering nature's molecules is an ideal strategy to obtain explicit functionality such as synthetic molecular machines, yet novel strategies for producing engineered molecular channels are few. Here we report a peptide engineering strategy through sequence reversal, which we applied on the first transmembrane peptide of the mycobacteriophage membranoporin protein holin. We have successfully redesigned the membrane rupture property of this peptide to form specific nanopore ion channels. We report the structural characterization and electrophysiology measurements of a library of 28-residue engineered membrane peptides, with remarkable ion channel behavior. We further identify that key residues at the peptide terminus, the central proline, charge distribution, and hydropathy index of the peptide together contribute to the channel properties that we measure. Our sequence reversal strategy for peptide engineering to successfully obtain nanopore channels can pave the way for better biobased design of controlled nanopores, using only natural amino acids. PMID:27257735

  13. Transformation optics for efficient calculation of transmembrane voltage induced on cells

    NASA Astrophysics Data System (ADS)

    Liao, Yin-Hong; Zhu, Hua-Cheng; Tang, Zheng-Ming; Huang, Ka-Ma

    2015-10-01

    We present a novel efficient approach in calculating induced transmembrane voltage (ITV) on cells based on transformation optics. As cell membrane is much thinner than the dimension of a typical cell, discretizing the membrane needs numerous meshes. Using an anisotropic medium based on transformation optics, the thickness of the membrane can be exaggerated by at least one order, which eliminates rigorous mesh refinement and reduces unknowns greatly. The accuracy and efficiency of the proposed method are verified by a cylindrical cell model. Moreover, the influence on ITV with bound water (BW) layers is also studied. The results show that when cells are exposed to nanosecond electric field, BW layers should be rigorously considered in calculating ITV. Project supported by the National Key Basic Research Program of China (Grant Nos. 2013CB328900 and 2013CB328905).

  14. v-SNARE transmembrane domains function as catalysts for vesicle fusion.

    PubMed

    Dhara, Madhurima; Yarzagaray, Antonio; Makke, Mazen; Schindeldecker, Barbara; Schwarz, Yvonne; Shaaban, Ahmed; Sharma, Satyan; Böckmann, Rainer A; Lindau, Manfred; Mohrmann, Ralf; Bruns, Dieter

    2016-01-01

    Vesicle fusion is mediated by an assembly of SNARE proteins between opposing membranes, but it is unknown whether transmembrane domains (TMDs) of SNARE proteins serve mechanistic functions that go beyond passive anchoring of the force-generating SNAREpin to the fusing membranes. Here, we show that conformational flexibility of synaptobrevin-2 TMD is essential for efficient Ca(2+)-triggered exocytosis and actively promotes membrane fusion as well as fusion pore expansion. Specifically, the introduction of helix-stabilizing leucine residues within the TMD region spanning the vesicle's outer leaflet strongly impairs exocytosis and decelerates fusion pore dilation. In contrast, increasing the number of helix-destabilizing, ß-branched valine or isoleucine residues within the TMD restores normal secretion but accelerates fusion pore expansion beyond the rate found for the wildtype protein. These observations provide evidence that the synaptobrevin-2 TMD catalyzes the fusion process by its structural flexibility, actively setting the pace of fusion pore expansion.

  15. Interferon-Induced Transmembrane Protein-Mediated Inhibition of Host Cell Entry of Ebolaviruses.

    PubMed

    Wrensch, Florian; Karsten, Christina B; Gnirß, Kerstin; Hoffmann, Markus; Lu, Kai; Takada, Ayato; Winkler, Michael; Simmons, Graham; Pöhlmann, Stefan

    2015-10-01

    Ebolaviruses are highly pathogenic in humans and nonhuman primates and pose a severe threat to public health. The interferon-induced transmembrane (IFITM) proteins can restrict entry of ebolaviruses, influenza A viruses, and other enveloped viruses. However, the breadth and mechanism of the antiviral activity of IFITM proteins are incompletely understood. Here, we employed ebolavirus glycoprotein-pseudotyped vectors and ebolavirus-like particles to address this question. We show that IFITM proteins inhibit the cellular entry of diverse ebolaviruses and demonstrate that type I interferon induces IFITM protein expression in macrophages, major viral targets. Moreover, we show that IFITM proteins block entry of influenza A viruses and ebolaviruses by different mechanisms and provide evidence that antibodies and IFITM proteins can synergistically inhibit cellular entry of ebolaviruses. These results provide insights into the role of IFITM proteins in infection by ebolaviruses and suggest a mechanism by which antibodies, though poorly neutralizing in vitro, might contribute to viral control in vivo. PMID:26034199