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Sample records for acid selenocysteine sec

  1. Fmoc-Sec(Xan)-OH: synthesis and utility of Fmoc selenocysteine SPPS derivatives with acid-labile sidechain protection.

    PubMed

    Flemer, Stevenson

    2015-01-01

    We report here the synthesis of the first selenocysteine SPPS derivatives which bear TFA-labile sidechain protecting groups. New compounds Fmoc-Sec(Xan)-OH and Fmoc-Sec(Trt)-OH are presented as useful and practical alternatives to the traditional Fmoc-Sec-OH derivatives currently available to the peptide chemist. From a bis Fmoc-protected selenocystine precursor, multiple avenues of diselenide reduction were attempted to determine the most effective method for subsequent attachment of the protecting group electrophiles. Our previously reported one-pot reduction methodology was ultimately chosen as the optimal approach toward the synthesis of these novel building blocks, and both were easily obtained in high yield and purity. Fmoc-Sec(Xan)-OH was discovered to be bench-stable for extended timeframes while the corresponding Fmoc-Sec(Trt)-OH derivative appeared to detritylate slowly when not stored at -20 °C. Both Sec derivatives were incorporated into single- and multiple-Sec-containing test peptides in order to ascertain the peptides' deprotection behavior and final form upon TFA cleavage. Single-Sec-containing test peptides were always isolated as their corresponding diselenide dimers, while dual-Sec-containing peptide sequences were afforded exclusively as their intramolecular diselenides. PMID:25504629

  2. The Human SepSecS-tRNA[superscript Sec] Complex Reveals the Mechanism of Selenocysteine Formation

    SciTech Connect

    Palioura, Sotiria; Sherrer, R. Lynn; Steitz, Thomas A.; Söll, Dieter; Simonovic, Miljan

    2009-08-13

    Selenocysteine is the only genetically encoded amino acid in humans whose biosynthesis occurs on its cognate transfer RNA (tRNA). O-Phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS) catalyzes the final step of selenocysteine formation by a poorly understood tRNA-dependent mechanism. The crystal structure of human tRNA{sup Sec} in complex with SepSecS, phosphoserine, and thiophosphate, together with in vivo and in vitro enzyme assays, supports a pyridoxal phosphate-dependent mechanism of Sec-tRNA{sup Sec} formation. Two tRNA{sup Sec} molecules, with a fold distinct from other canonical tRNAs, bind to each SepSecS tetramer through their 13-base pair acceptor-T{Upsilon}C arm (where {Upsilon} indicates pseudouridine). The tRNA binding is likely to induce a conformational change in the enzyme's active site that allows a phosphoserine covalently attached to tRNA{sup Sec}, but not free phosphoserine, to be oriented properly for the reaction to occur.

  3. Decameric SelA•tRNA(Sec) ring structure reveals mechanism of bacterial selenocysteine formation.

    PubMed

    Itoh, Yuzuru; Bröcker, Markus J; Sekine, Shun-ichi; Hammond, Gifty; Suetsugu, Shiro; Söll, Dieter; Yokoyama, Shigeyuki

    2013-04-01

    The 21st amino acid, selenocysteine (Sec), is synthesized on its cognate transfer RNA (tRNA(Sec)). In bacteria, SelA synthesizes Sec from Ser-tRNA(Sec), whereas in archaea and eukaryotes SepSecS forms Sec from phosphoserine (Sep) acylated to tRNA(Sec). We determined the crystal structures of Aquifex aeolicus SelA complexes, which revealed a ring-shaped homodecamer that binds 10 tRNA(Sec) molecules, each interacting with four SelA subunits. The SelA N-terminal domain binds the tRNA(Sec)-specific D-arm structure, thereby discriminating Ser-tRNA(Sec) from Ser-tRNA(Ser). A large cleft is created between two subunits and accommodates the 3'-terminal region of Ser-tRNA(Sec). The SelA structures together with in vivo and in vitro enzyme assays show decamerization to be essential for SelA function. SelA catalyzes pyridoxal 5'-phosphate-dependent Sec formation involving Arg residues nonhomologous to those in SepSecS. Different protein architecture and substrate coordination of the bacterial enzyme provide structural evidence for independent evolution of the two Sec synthesis systems present in nature. PMID:23559248

  4. Understanding selenocysteine through conformational analysis, proton affinities, acidities and bond dissociation energies

    NASA Astrophysics Data System (ADS)

    Kaur, Damanjit; Sharma, Punita; Bharatam, Prasad V.; Kaur, Mondeep

    Density functional methods have been employed to characterize the gas phase conformations of selenocysteine. The 33 stable conformers of selenocysteine have been located on the potential energy surface using density functional B3LYP/6-31+G* method. The conformers are analyzed in terms of intramolecular hydrogen bonding interactions. The proton affinity, gas phase acidities, and bond dissociation energies have also been evaluated for different reactive sites of selenocysteine for the five lowest energy conformers at B3LYP/6-311++G*//B3LYP/6-31+G* level. Evaluation of these intrinsic properties reflects the antioxidant activity of selenium in selenocysteine.0

  5. [Expanding genetic code: amino acids 21 and 22--selenocysteine and pyrrolysine].

    PubMed

    Lukashenko, N P

    2010-08-01

    The discovery of two nonstandard amino acids, selenocysteine and pyrrolysine, in the genetic code is discussed. These findings have expanded our understanding of the genetic code, since the repertoire of amino acids in the genetic code was supplemented by two novel ones, in addition of the standard 20 amino acids. Current views on specific mechanisms of selenocysteine insertion in forming selenoproteins are considered, as well as the results of studies of new translational components involved in biosynthesis and incorporation of selenocysteine at different stages of translation. Similarity in the strategies of decoding UGA and UAG as codons for respectively selenocysteine and pyrrolysine is discussed. The review also presents evidence on the medical and biological role of selenium and selenoproteins containing selenocysteine as the main biological form of selenium. PMID:20873198

  6. Selenocysteine Lyase.

    PubMed

    Stadtman, Thressa C

    2004-12-01

    Selenocysteine is a naturally occurring analog of cysteine in which the sulfur atom of the latter is replaced with selenium. This seleno-amino acid occurs as a specific component of various selenoproteins and selenium-dependent enzymes. Incorporation of selenocysteine into these proteins occurs cotranslationally as directed by the UGA codon. For this process, a special tRNA having an anticodon complimentary to UGA, tRNASec, is utilized. In Escherichia coli and related bacteria, this tRNA first is amino acylated with serine, and the seryl-tRNASec is converted to selenocysteyl-tRNASec. The specific incorporation of selenocysteine into proteins directed by the UGA codon depends on the synthesis of selenocysteyl-tRNASec. Included in the selenium delivery protein category are rhodaneses that mobilize selenium from inorganic sources and NIFS-like proteins that liberate elemental selenium from selenocysteine. The NIFS protein from Azotobacter vinelandii was found to serve as an efficient catalyst in vitro for delivery of selenium from free selenocysteine to Escherichia coli selenophosphate synthetase for selenophosphate formation. The widespread distribution of selenocysteine lyase in numerous bacterial species was reported and the bacterial enzymes, like the pig liver enzyme, required pyridoxal phosphate as cofactor. Three NIFS-like genes were isolated from E. coli by Esaki and coworkers and the expressed gene products were isolated and characterized. One of these NIFS-like proteins also exhibited a high preference for selenocysteine over cysteine. M. vannielii, an anaerobic methane-producing organism, that grows in a mineral medium containing formate as sole organic carbon source, synthesizes several specific selenoenzymes required for growth and energy production under these conditions. PMID:26443359

  7. [Biochemical selenocysteine synthesis and the phylogenic study].

    PubMed

    Mizutani, Takaharu; Osaka, Takashi; Fujiwara, Toshinobu; Shahidzzman, M

    2008-07-01

    Selenium (Se) is an essential trace element. Se is found as selenocysteine (Sec) in Se-proteins. Sec is the 21(st) amino acid, because Sec has its tRNA, the codon UGA and those components in its translational machinery. Sec UGA codon shares with major stop codon UGA. We purified Sec synthesizing enzymes, such as seryl-tRNA synthetase (SerRS), Sec synthetase (SecS) and selenophosphate synthetase (SePS). I described the procedures to prepare Sec tRNA, SerRS, SecS, SePS and [(75)Se]H(2)Se in detail. We clarified that SecS composed of two proteins, SecSalpha and SecSbeta. Sec synthesizing and incorporating systems present in Monela, Animalia and Protoctista but not in Plantae and Fungi. We showed that protozoa had Sec tRNA on which Sec was synthesized from Ser-tRNA by bovine and protozoa SecS. Some worms, such as Caenorhabditis elegans and Fasiola gigantica, also had Sec tRNA on which Sec was synthesized by bovine liver SecS or C. elegans enzymes. We showed recognition sites of mammalian Sec tRNA by SecS. The identity units of Sec tRNA are 9 bp aminoacyl- and 6 bp D-stems. This recognition is not the base-specific manner but the length-specific manner. From comparison of the phylogeny trees of Sec synthesizing system and translation system, we concluded that the evolution of Sec synthesizing system is older than that of the translation system. PMID:18591866

  8. [Methods to Biosynthesize Mammalian Selenocysteine-Containing Proteins in vitro].

    PubMed

    Varlamova, E G; Novoselov, S V

    2016-01-01

    The main problem in studying mammalian selenocysteine-containing proteins is that the proteins are difficult to obtain in a recombinant form because the amino acid selenocysteine (Sec), which is their component, is encoded by TGA, which is one of the stop codons. When only the open reading frame of a target protein is cloned in a plasmid, translation is prematurely terminated at the TGA codon. An intricate natural mechanism allows the codon to be recognized as a selenocysteine codon and involves various cis- and trans-acting factors, such as the selenocysteine insertion sequence (SECIS), mRNA secondary structure, selenocysteine tRNA Sec-tRNA^( [Ser]Sec), SECIS-binding protein 2 (SBP2), selenocysteine-specific elongation factor EFsec, and others. Generation of recombinant selenoproteins in preparative amounts directly depends on the expression levels of the cis- and trans-acting transcription and translation factors to further complicate the problem, and cysteine homologs of selenoproteins are consequently used in many studies. Several methods designed to express mammalian selenoproteins in vitro are considered in the review. PMID:27028810

  9. Selenocysteine: Wherefore Art Thou?

    PubMed

    Fenyö, David; Beavis, Ronald C

    2016-02-01

    Selenocysteine is a naturally occurring proteogenic amino acid that is encoded in the genomic sequence of relatively abundant proteins in many of the model species commonly used for biomedical research. On the basis of an analysis of publicly available proteomics information, it was discovered that peptides containing selenocysteine were not being identified in tandem mass spectrometry proteomics data. Once the chemical basis for this exclusion was understood, a simple alteration in search parameters led to the confident identification of selenocysteine containing peptides from existing proteomics data, with no change in experimental protocols required. PMID:26680273

  10. High content of proteins containing 21st and 22nd amino acids, selenocysteine and pyrrolysine, in a symbiotic deltaproteobacterium of gutless worm Olavius algarvensis

    PubMed Central

    Zhang, Yan; Gladyshev, Vadim N.

    2007-01-01

    Selenocysteine (Sec) and pyrrolysine (Pyl) are rare amino acids that are cotranslationally inserted into proteins and known as the 21st and 22nd amino acids in the genetic code. Sec and Pyl are encoded by UGA and UAG codons, respectively, which normally serve as stop signals. Herein, we report on unusually large selenoproteomes and pyrroproteomes in a symbiont metagenomic dataset of a marine gutless worm, Olavius algarvensis. We identified 99 selenoprotein genes that clustered into 30 families, including 17 new selenoprotein genes that belong to six families. In addition, several Pyl-containing proteins were identified in this dataset. Most selenoproteins and Pyl-containing proteins were present in a single deltaproteobacterium, δ1 symbiont, which contained the largest number of both selenoproteins and Pyl-containing proteins of any organism reported to date. Our data contrast with the previous observations that symbionts and host-associated bacteria either lose Sec utilization or possess a limited number of selenoproteins, and suggest that the environment in the gutless worm promotes Sec and Pyl utilization. Anaerobic conditions and consistent selenium supply might be the factors that support the use of amino acids that extend the genetic code. PMID:17626042

  11. Biosynthesis of Selenocysteine on Its tRNA in Eukaryotes

    PubMed Central

    Mix, Heiko; Zhang, Yan; Saira, Kazima; Glass, Richard S; Berry, Marla J; Gladyshev, Vadim N; Hatfield, Dolph L

    2007-01-01

    Selenocysteine (Sec) is cotranslationally inserted into protein in response to UGA codons and is the 21st amino acid in the genetic code. However, the means by which Sec is synthesized in eukaryotes is not known. Herein, comparative genomics and experimental analyses revealed that the mammalian Sec synthase (SecS) is the previously identified pyridoxal phosphate-containing protein known as the soluble liver antigen. SecS required selenophosphate and O-phosphoseryl-tRNA[Ser]Sec as substrates to generate selenocysteyl-tRNA[Ser]Sec. Moreover, it was found that Sec was synthesized on the tRNA scaffold from selenide, ATP, and serine using tRNA[Ser]Sec, seryl-tRNA synthetase, O-phosphoseryl-tRNA[Ser]Sec kinase, selenophosphate synthetase, and SecS. By identifying the pathway of Sec biosynthesis in mammals, this study not only functionally characterized SecS but also assigned the function of the O-phosphoseryl-tRNA[Ser]Sec kinase. In addition, we found that selenophosphate synthetase 2 could synthesize monoselenophosphate in vitro but selenophosphate synthetase 1 could not. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that synthesize selenoproteins. PMID:17194211

  12. Selenocysteine incorporation: A trump card in the game of mRNA decay.

    PubMed

    Shetty, Sumangala P; Copeland, Paul R

    2015-07-01

    The incorporation of the 21st amino acid, selenocysteine (Sec), occurs on mRNAs that harbor in-frame stop codons because the Sec-tRNA(Sec) recognizes a UGA codon. This sets up an intriguing interplay between translation elongation, translation termination and the complex machinery that marks mRNAs that contain premature termination codons for degradation, leading to nonsense mediated mRNA decay (NMD). In this review we discuss the intricate and complex relationship between this key quality control mechanism and the process of Sec incorporation in mammals. PMID:25622574

  13. Oxidative Deselenization of Selenocysteine: Applications for Programmed Ligation at Serine.

    PubMed

    Malins, Lara R; Mitchell, Nicholas J; McGowan, Sheena; Payne, Richard J

    2015-10-19

    Despite the unique chemical properties of selenocysteine (Sec), ligation at Sec is an under-utilized methodology for protein synthesis. We describe herein an unprecedented protocol for the conversion of Sec to serine (Ser) in a single, high-yielding step. When coupled with ligation at Sec, this transformation provides a new approach to programmed ligations at Ser residues. This new reaction is compatible with a wide range of functionality, including the presence of unprotected amino acid side chains and appended glycans. The utility of the methodology is demonstrated in the rapid synthesis of complex glycopeptide fragments of the epithelial glycoproteins MUC5AC and MUC4 and through the total synthesis of the structured, cysteine (Cys)-free protein eglin C. PMID:26384718

  14. Selenocysteine incorporation: A trump card in the game of mRNA decay

    PubMed Central

    Shetty, Sumangala P.; Copeland, Paul R.

    2015-01-01

    The incorporation of the 21st amino acid, selenocysteine (Sec), occurs on mRNAs that harbor in-frame stop codons because the Sec-tRNASec recognizes a UGA codon. This sets up an intriguing interplay between translation elongation, translation termination and the complex machinery that marks mRNAs that contain premature termination codons for degradation, leading to nonsense mediated mRNA decay (NMD). In this review we discuss the intricate and complex relationship between this key quality control mechanism and the process of Sec incorporation in mammals. PMID:25622574

  15. [cDNA cloning, expression and determination of substrate specificity of mice selenocysteine-containing protein SelV (Selenoprotein V)].

    PubMed

    Varlamova, E G; Novoselov, S V; Novoselov, V I

    2015-01-01

    To date various bioinformatics tools allowed to identify 25 selenocysteine-containing mammalian proteins. The name of these proteins assumes that they contain the amino acid selenocysteine (Sec). Functionally characterized selenocysteine-containing proteins are oxidoreductases with various functions, including glutathione peroxidases, thioredoxin reductases, deiodinases etc. However, the functions of more than half of identified proteins are still unclear, and mammalian selenoprotein SeIV is among them. We studied the selV in all stages of postnatal development with the maximum level of mRNA expression during puberty, whereas in adult mice (8-18 months) we observed a gradual decrease of expression. In order to get closer to the functional role of Selenoprotein V, we have carried out experiments on the substrate specificity and enzymatic activity measurement of this selenocysteine-containing protein. It was shown that SelV posseses glutathionperoxidase and thioredoxinreductase activities. PMID:26510596

  16. Synthesis and decoding of selenocysteine and human health

    PubMed Central

    Schmidt, Rachel L.; Simonović, Miljan

    2012-01-01

    Selenocysteine, the 21st amino acid, has been found in 25 human selenoproteins and selenoenzymes important for fundamental cellular processes ranging from selenium homeostasis maintenance to the regulation of the overall metabolic rate. In all organisms that contain selenocysteine, both the synthesis of selenocysteine and its incorporation into a selenoprotein requires an elaborate synthetic and translational apparatus, which does not resemble the canonical enzymatic system employed for the 20 standard amino acids. In humans, three synthetic enzymes, a specialized elongation factor, an accessory protein factor, two catabolic enzymes, a tRNA, and a stem-loop structure in the selenoprotein mRNA are critical for ensuring that only selenocysteine is attached to selenocysteine tRNA and that only selenocysteine is inserted into the nascent polypeptide in response to a context-dependent UGA codon. The abnormal selenium homeostasis and mutations in selenoprotein genes have been causatively linked to a variety of human diseases, which, in turn, sparked a renewed interest in utilizing selenium as the dietary supplement to either prevent or remedy pathologic conditions. In contrast, the importance of the components of the selenocysteine-synthetic machinery for human health is less clear. Emerging evidence suggests that enzymes responsible for selenocysteine formation and decoding the selenocysteine UGA codon, which by extension are critical for synthesis of the entire selenoproteome, are essential for the development and health of the human organism. PMID:23275319

  17. RNA-dependent conversion of phosphoserine forms selenocysteine in eukaryotes and archaea

    PubMed Central

    Yuan, Jing; Palioura, Sotiria; Salazar, Juan Carlos; Su, Dan; O'Donoghue, Patrick; Hohn, Michael J.; Cardoso, Alexander Machado; Whitman, William B.; Söll, Dieter

    2006-01-01

    The trace element selenium is found in proteins as selenocysteine (Sec), the 21st amino acid to participate in ribosome-mediated translation. The substrate for ribosomal protein synthesis is selenocysteinyl-tRNASec. Its biosynthesis from seryl-tRNASec has been established for bacteria, but the mechanism of conversion from Ser-tRNASec remained unresolved for archaea and eukarya. Here, we provide evidence for a different route present in these domains of life that requires the tRNASec-dependent conversion of O-phosphoserine (Sep) to Sec. In this two-step pathway, O-phosphoseryl-tRNASec kinase (PSTK) converts Ser-tRNASec to Sep-tRNASec. This misacylated tRNA is the obligatory precursor for a Sep-tRNA:Sec-tRNA synthase (SepSecS); this protein was previously annotated as SLA/LP. The human and archaeal SepSecS genes complement in vivo an Escherichia coli Sec synthase (SelA) deletion strain. Furthermore, purified recombinant SepSecS converts Sep-tRNASec into Sec-tRNASec in vitro in the presence of sodium selenite and purified recombinant E. coli selenophosphate synthetase (SelD). Phylogenetic arguments suggest that Sec decoding was present in the last universal common ancestor. SepSecS and PSTK coevolved with the archaeal and eukaryotic lineages, but the history of PSTK is marked by several horizontal gene transfer events, including transfer to non-Sec-decoding Cyanobacteria and fungi. PMID:17142313

  18. Identification of a novel putative non-selenocysteine containing phospholipid hydroperoxide glutathione peroxidase (NPGPx) essential for alleviating oxidative stress generated from polyunsaturated fatty acids in breast cancer cells.

    PubMed

    Utomo, Ahmad; Jiang, Xianzhi; Furuta, Saori; Yun, Jeanho; Levin, David S; Wang, Yi-Chun J; Desai, Kartiki V; Green, Jeffrey E; Chen, Phang-Lang; Lee, Wen-Hwa

    2004-10-15

    A dramatic reduction in the expression of a novel phospholipid hydroperoxide glutathione peroxidase (PHGPx), which incorporates cysteine instead of selenocysteine in the conserved catalytic motif was observed in a microarray analysis using cDNAs amplified from mRNA of Brca1-null mouse embryonic fibroblasts. This non-selenocysteine PHGPx named NPGPx is a cytoplasmic protein with molecular mass of approximately 22 kDa and has little detectable glutathione peroxidase activity in vitro. Ectopic expression of NPGPx in Brca1-null cells that were sensitive to oxidative stress induced by hydrogen peroxide conferred a similar resistance level to that of the wild-type cells, suggesting the importance of this protein in reducing oxidative stress. Expression of NPGPx was found in many tissues, including developing mammary gland. However, the majority of breast cancer cell lines studied (11 of 12) expressed very low or undetectable levels of NPGPx irrespective of BRCA1 status. Re-expression of NPGPx in breast cancer lines, MCF-7 and HCC1937, which have very little or no endogenous NPGPx, induced resistance to eicosapentaenoic acid (an omega-3 type of polyunsaturated fatty acid)-mediated cell death. Conversely, inhibition of the expression of NPGPx by the specific small interfering RNA in HS578T breast cancer cells that originally express substantial amounts of endogenous NPGPx increased their sensitivity to eicosapentaenoic acid-mediated cell death. Thus, NPGPx plays an essential role in breast cancer cells in alleviating oxidative stress generated from polyunsaturated fatty acid metabolism. PMID:15294905

  19. Crystal structure of the full-length bacterial selenocysteine-specific elongation factor SelB.

    PubMed

    Itoh, Yuzuru; Sekine, Shun-Ichi; Yokoyama, Shigeyuki

    2015-10-15

    Selenocysteine (Sec), the 21(st) amino acid in translation, uses its specific tRNA (tRNA(Sec)) to recognize the UGA codon. The Sec-specific elongation factor SelB brings the selenocysteinyl-tRNA(Sec) (Sec-tRNA(Sec)) to the ribosome, dependent on both an in-frame UGA and a Sec-insertion sequence (SECIS) in the mRNA. The bacterial SelB binds mRNA through its C-terminal region, for which crystal structures have been reported. In this study, we determined the crystal structure of the full-length SelB from the bacterium Aquifex aeolicus, in complex with a GTP analog, at 3.2-Å resolution. SelB consists of three EF-Tu-like domains (D1-3), followed by four winged-helix domains (WHD1-4). The spacer region, connecting the N- and C-terminal halves, fixes the position of WHD1 relative to D3. The binding site for the Sec moiety of Sec-tRNA(Sec) is located on the interface between D1 and D2, where a cysteine molecule from the crystallization solution is coordinated by Arg residues, which may mimic Sec binding. The Sec-binding site is smaller and more exposed than the corresponding site of EF-Tu. Complex models of Sec-tRNA(Sec), SECIS RNA, and the 70S ribosome suggest that the unique secondary structure of tRNA(Sec) allows SelB to specifically recognize tRNA(Sec) and characteristically place it at the ribosomal A-site. PMID:26304550

  20. Selenium utilization in thioredoxin and catalytic advantage provided by selenocysteine

    SciTech Connect

    Kim, Moon-Jung; Lee, Byung Cheon; Hwang, Kwang Yeon; Gladyshev, Vadim N.; Kim, Hwa-Young

    2015-06-12

    Thioredoxin (Trx) is a major thiol-disulfide reductase that plays a role in many biological processes, including DNA replication and redox signaling. Although selenocysteine (Sec)-containing Trxs have been identified in certain bacteria, their enzymatic properties have not been characterized. In this study, we expressed a selenoprotein Trx from Treponema denticola, an oral spirochete, in Escherichia coli and characterized this selenoenzyme and its natural cysteine (Cys) homologue using E. coli Trx1 as a positive control. {sup 75}Se metabolic labeling and mutation analyses showed that the SECIS (Sec insertion sequence) of T. denticola selenoprotein Trx is functional in the E. coli Sec insertion system with specific selenium incorporation into the Sec residue. The selenoprotein Trx exhibited approximately 10-fold higher catalytic activity than the Sec-to-Cys version and natural Cys homologue and E. coli Trx1, suggesting that Sec confers higher catalytic activity on this thiol-disulfide reductase. Kinetic analysis also showed that the selenoprotein Trx had a 30-fold higher K{sub m} than Cys-containing homologues, suggesting that this selenoenzyme is adapted to work efficiently with high concentrations of substrate. Collectively, the results of this study support the hypothesis that selenium utilization in oxidoreductase systems is primarily due to the catalytic advantage provided by the rare amino acid, Sec. - Highlights: • The first characterization of a selenoprotein Trx is presented. • The selenoenzyme Trx exhibits 10-fold higher catalytic activity than Cys homologues. • Se utilization in Trx is primarily due to the catalytic advantage provided by Sec residue.

  1. Regulation of Selenocysteine Incorporation into the Selenium Transport Protein, Selenoprotein P*

    PubMed Central

    Shetty, Sumangala P.; Shah, Ravi; Copeland, Paul R.

    2014-01-01

    Selenoproteins are unique as they contain selenium in their active site in the form of the 21st amino acid selenocysteine (Sec), which is encoded by an in-frame UGA stop codon. Sec incorporation requires both cis- and trans-acting factors, which are known to be sufficient for Sec incorporation in vitro, albeit with low efficiency. However, the abundance of the naturally occurring selenoprotein that contains 10 Sec residues (SEPP1) suggests that processive and efficient Sec incorporation occurs in vivo. Here, we set out to study native SEPP1 synthesis in vitro to identify factors that regulate processivity and efficiency. Deletion analysis of the long and conserved 3′-UTR has revealed that the incorporation of multiple Sec residues is inherently processive requiring only the SECIS elements but surprisingly responsive to the selenium concentration. We provide evidence that processive Sec incorporation is linked to selenium utilization and that reconstitution of known Sec incorporation factors in a wheat germ lysate does not permit multiple Sec incorporation events, thus suggesting a role for yet unidentified mammalian-specific processes or factors. The relationship between our findings and the channeling theory of translational efficiency is discussed. PMID:25063811

  2. Lokiarchaeota Marks the Transition between the Archaeal and Eukaryotic Selenocysteine Encoding Systems

    PubMed Central

    Mariotti, Marco; Lobanov, Alexei V.; Manta, Bruno; Santesmasses, Didac; Bofill, Andreu; Guigó, Roderic; Gabaldón, Toni; Gladyshev, Vadim N.

    2016-01-01

    Selenocysteine (Sec) is the 21st amino acid in the genetic code, inserted in response to UGA codons with the help of RNA structures, the SEC Insertion Sequence (SECIS) elements. The three domains of life feature distinct strategies for Sec insertion in proteins and its utilization. While bacteria and archaea possess similar sets of selenoproteins, Sec biosynthesis is more similar among archaea and eukaryotes. However, SECIS elements are completely different in the three domains of life. Here, we analyze the archaeon Lokiarchaeota that resolves the relationships among Sec insertion systems. This organism has selenoproteins representing five protein families, three of which have multiple Sec residues. Remarkably, these archaeal selenoprotein genes possess conserved RNA structures that strongly resemble the eukaryotic SECIS element, including key eukaryotic protein-binding sites. These structures also share similarity with the SECIS element in archaeal selenoprotein VhuD, suggesting a relation of direct descent. These results identify Lokiarchaeota as an intermediate form between the archaeal and eukaryotic Sec-encoding systems and clarify the evolution of the Sec insertion system. PMID:27413050

  3. SerRS-tRNASec complex structures reveal mechanism of the first step in selenocysteine biosynthesis.

    PubMed

    Wang, Caiyan; Guo, Yu; Tian, Qingnan; Jia, Qian; Gao, Yuanzhu; Zhang, Qinfen; Zhou, Chun; Xie, Wei

    2015-12-01

    Selenocysteine (Sec) is found in the catalytic centers of many selenoproteins and plays important roles in living organisms. Malfunctions of selenoproteins lead to various human disorders including cancer. Known as the 21st amino acid, the biosynthesis of Sec involves unusual pathways consisting of several stages. While the later stages of the pathways are well elucidated, the molecular basis of the first stage-the serylation of Sec-specific tRNA (tRNA(Sec)) catalyzed by seryl-tRNA synthetase (SerRS)-is unclear. Here we present two cocrystal structures of human SerRS bound with tRNA(Sec) in different stoichiometry and confirm the formation of both complexes in solution by various characterization techniques. We discovered that the enzyme mainly recognizes the backbone of the long variable arm of tRNA(Sec) with few base-specific contacts. The N-terminal coiled-coil region works like a long-range lever to precisely direct tRNA 3' end to the other protein subunit for aminoacylation in a conformation-dependent manner. Restraints of the flexibility of the coiled-coil greatly reduce serylation efficiencies. Lastly, modeling studies suggest that the local differences present in the D- and T-regions as well as the characteristic U20:G19:C56 base triple in tRNA(Sec) may allow SerRS to distinguish tRNA(Sec) from closely related tRNA(Ser) substrate. PMID:26433229

  4. Lokiarchaeota Marks the Transition between the Archaeal and Eukaryotic Selenocysteine Encoding Systems.

    PubMed

    Mariotti, Marco; Lobanov, Alexei V; Manta, Bruno; Santesmasses, Didac; Bofill, Andreu; Guigó, Roderic; Gabaldón, Toni; Gladyshev, Vadim N

    2016-09-01

    Selenocysteine (Sec) is the 21st amino acid in the genetic code, inserted in response to UGA codons with the help of RNA structures, the SEC Insertion Sequence (SECIS) elements. The three domains of life feature distinct strategies for Sec insertion in proteins and its utilization. While bacteria and archaea possess similar sets of selenoproteins, Sec biosynthesis is more similar among archaea and eukaryotes. However, SECIS elements are completely different in the three domains of life. Here, we analyze the archaeon Lokiarchaeota that resolves the relationships among Sec insertion systems. This organism has selenoproteins representing five protein families, three of which have multiple Sec residues. Remarkably, these archaeal selenoprotein genes possess conserved RNA structures that strongly resemble the eukaryotic SECIS element, including key eukaryotic protein-binding sites. These structures also share similarity with the SECIS element in archaeal selenoprotein VhuD, suggesting a relation of direct descent. These results identify Lokiarchaeota as an intermediate form between the archaeal and eukaryotic Sec-encoding systems and clarify the evolution of the Sec insertion system. PMID:27413050

  5. CHARACTERIZATION OF HUMIC ACID SIZE FRACTIONS BY SEC AND MALS (R822832)

    EPA Science Inventory

    Latahco silt-loam humic acid was separated on a preparatory scale by size exclusion chromatography (SEC) on a gravity-fed Sepharose column. Four fractions from this separation were collected and further analyzed, along with whole humic acid, by high-performance SEC coupled with a...

  6. Formation of a Ternary Complex for Selenocysteine Biosynthesis in Bacteria.

    PubMed

    Silva, Ivan R; Serrão, Vitor H B; Manzine, Livia R; Faim, Lívia M; da Silva, Marco T A; Makki, Raphaela; Saidemberg, Daniel M; Cornélio, Marinônio L; Palma, Mário S; Thiemann, Otavio H

    2015-12-01

    The synthesis of selenocysteine-containing proteins (selenoproteins) involves the interaction of selenocysteine synthase (SelA), tRNA (tRNA(Sec)), selenophosphate synthetase (SelD, SPS), a specific elongation factor (SelB), and a specific mRNA sequence known as selenocysteine insertion sequence (SECIS). Because selenium compounds are highly toxic in the cellular environment, the association of selenium with proteins throughout its metabolism is essential for cell survival. In this study, we demonstrate the interaction of SPS with the SelA-tRNA(Sec) complex, resulting in a 1.3-MDa ternary complex of 27.0 ± 0.5 nm in diameter and 4.02 ± 0.05 nm in height. To assemble the ternary complex, SPS undergoes a conformational change. We demonstrated that the glycine-rich N-terminal region of SPS is crucial for the SelA-tRNA(Sec)-SPS interaction and selenoprotein biosynthesis, as revealed by functional complementation experiments. Taken together, our results provide new insights into selenoprotein biosynthesis, demonstrating for the first time the formation of the functional ternary SelA-tRNA(Sec)-SPS complex. We propose that this complex is necessary for proper selenocysteine synthesis and may be involved in avoiding the cellular toxicity of selenium compounds. PMID:26378233

  7. Microbial distribution of selenocysteine lyase.

    PubMed Central

    Chocat, P; Esaki, N; Nakamura, T; Tanaka, H; Soda, K

    1983-01-01

    We studied the distribution of selenocysteine lyase, a novel enzyme catalyzing the conversion of selenocysteine into alanine and H2Se, which we first demonstrated in various mammalian tissues (Esaki et al., J. Biol. Chem. 257:4386-4391, 1982). Enzyme activity was found in various bacteria such as Alcaligenes viscolactis and Pseudomonas alkanolytica. No significant activity was found in yeasts and fungi. Selenocysteine lyases from A. viscolactis and P. alkanolytica acted specifically on L-selenocysteine and required pyridoxal 5'-phosphate as a cofactor. PMID:6225771

  8. The Selenocysteine tRNA STAF-Binding Region is Essential for Adequate Selenocysteine tRNA Status, Selenoprotein Expression and Early Age Survival of Mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    STAF is a transcription activating factor for a number of RNA Pol III-and RNA Pol II-dependent genes including the selenocysteine (Sec) tRNA gene. Here, the role of STAF in regulating expression of Sec tRNA and selenoproteins was examined in an invivo model. Heterozygous inactivation of the Staf gen...

  9. Facile Recoding of Selenocysteine in Nature.

    PubMed

    Mukai, Takahito; Englert, Markus; Tripp, H James; Miller, Corwin; Ivanova, Natalia N; Rubin, Edward M; Kyrpides, Nikos C; Söll, Dieter

    2016-04-18

    Selenocysteine (Sec or U) is encoded by UGA, a stop codon reassigned by a Sec-specific elongation factor and a distinctive RNA structure. To discover possible code variations in extant organisms we analyzed 6.4 trillion base pairs of metagenomic sequences and 24 903 microbial genomes for tRNA(Sec) species. As expected, UGA is the predominant Sec codon in use. We also found tRNA(Sec) species that recognize the stop codons UAG and UAA, and ten sense codons. Selenoprotein synthesis programmed by UAG in Geodermatophilus and Blastococcus, and by the Cys codon UGU in Aeromonas salmonicida was confirmed by metabolic labeling with (75) Se or mass spectrometry. Other tRNA(Sec) species with different anticodons enabled E. coli to synthesize active formate dehydrogenase H, a selenoenzyme. This illustrates the ease by which the genetic code may evolve new coding schemes, possibly aiding organisms to adapt to changing environments, and show the genetic code is much more flexible than previously thought. PMID:26991476

  10. Crystal structure of the full-length bacterial selenocysteine-specific elongation factor SelB

    PubMed Central

    Itoh, Yuzuru; Sekine, Shun-ichi; Yokoyama, Shigeyuki

    2015-01-01

    Selenocysteine (Sec), the 21st amino acid in translation, uses its specific tRNA (tRNASec) to recognize the UGA codon. The Sec-specific elongation factor SelB brings the selenocysteinyl-tRNASec (Sec-tRNASec) to the ribosome, dependent on both an in-frame UGA and a Sec-insertion sequence (SECIS) in the mRNA. The bacterial SelB binds mRNA through its C-terminal region, for which crystal structures have been reported. In this study, we determined the crystal structure of the full-length SelB from the bacterium Aquifex aeolicus, in complex with a GTP analog, at 3.2-Å resolution. SelB consists of three EF-Tu-like domains (D1–3), followed by four winged-helix domains (WHD1–4). The spacer region, connecting the N- and C-terminal halves, fixes the position of WHD1 relative to D3. The binding site for the Sec moiety of Sec-tRNASec is located on the interface between D1 and D2, where a cysteine molecule from the crystallization solution is coordinated by Arg residues, which may mimic Sec binding. The Sec-binding site is smaller and more exposed than the corresponding site of EF-Tu. Complex models of Sec-tRNASec, SECIS RNA, and the 70S ribosome suggest that the unique secondary structure of tRNASec allows SelB to specifically recognize tRNASec and characteristically place it at the ribosomal A-site. PMID:26304550

  11. Transcription of the Xenopus laevis selenocysteine tRNA(Ser)Sec gene: a system that combines an internal B box and upstream elements also found in U6 snRNA genes.

    PubMed Central

    Carbon, P; Krol, A

    1991-01-01

    The transcription mode of the Xenopus tRNA(Ser)Sec gene by RNA polymerase III was deciphered by injection of mutant templates into Xenopus oocyte nuclei. tRNA(Ser)Sec represents the paradigm of a new class of RNA polymerase III genes combining tRNA and U snRNA gene regulatory elements. Its promoter is tripartite, constituted by two upstream elements, a PSE and a TATA motif that are interchangeable with those of U6 snRNA genes and an internal box B as in other tRNAs. The B box enables the transcription level dependent on the upstream promoter to be increased. Data obtained indicate that U1 snRNA (Pol II) and tRNA(Ser)Sec (Pol III) genes share at least one transcription factor, implying that the border between transcription systems is less tight than expected. Images PMID:2001675

  12. Selenoproteins-What unique properties can arise with selenocysteine in place of cysteine?

    SciTech Connect

    Arner, Elias S.J.

    2010-05-01

    The defining entity of a selenoprotein is the inclusion of at least one selenocysteine (Sec) residue in its sequence. Sec, the 21st naturally occurring genetically encoded amino acid, differs from its significantly more common structural analog cysteine (Cys) by the identity of a single atom: Sec contains selenium instead of the sulfur found in Cys. Selenium clearly has unique chemical properties that differ from sulfur, but more striking are perhaps the similarities between the two elements. Selenium was discovered by Joens Jacob Berzelius, a renowned Swedish scientist instrumental in establishing the institution that would become Karolinska Institutet. Written at the occasion of the bicentennial anniversary of Karolinska Institutet, this mini review focuses on the unique selenium-derived properties that may potentially arise in a protein upon the inclusion of Sec in place of Cys. With 25 human genes encoding selenoproteins and in total several thousand selenoproteins yet described in nature, it seems likely that the presence of that single selenium atom of Sec should convey some specific feature, thereby explaining the existence of selenoproteins in spite of demanding and energetically costly Sec-specific synthesis machineries. Nonetheless, most, if not all, of the currently known selenoproteins are also found as Cys-containing non-selenoprotein orthologues in other organisms, wherefore any potentially unique properties of selenoproteins are yet a matter of debate. The pK{sub a} of free Sec (approximately 5.2) being significantly lower than that of free Cys (approximately 8.5) has often been proposed as one of the unique features of Sec. However, as discussed herein, this pK{sub a} difference between Sec and Cys can hardly provide an evolutionary pressure for maintenance of selenoproteins. Moreover, the typically 10- to 100-fold lower enzymatic efficiencies of Sec-to-Cys mutants of selenoprotein oxidoreductases, are also weak arguments for the overall existence

  13. Selenoproteins-What unique properties can arise with selenocysteine in place of cysteine?

    PubMed

    Arnér, Elias S J

    2010-05-01

    The defining entity of a selenoprotein is the inclusion of at least one selenocysteine (Sec) residue in its sequence. Sec, the 21st naturally occurring genetically encoded amino acid, differs from its significantly more common structural analog cysteine (Cys) by the identity of a single atom: Sec contains selenium instead of the sulfur found in Cys. Selenium clearly has unique chemical properties that differ from sulfur, but more striking are perhaps the similarities between the two elements. Selenium was discovered by Jöns Jacob Berzelius, a renowned Swedish scientist instrumental in establishing the institution that would become Karolinska Institutet. Written at the occasion of the bicentennial anniversary of Karolinska Institutet, this mini review focuses on the unique selenium-derived properties that may potentially arise in a protein upon the inclusion of Sec in place of Cys. With 25 human genes encoding selenoproteins and in total several thousand selenoproteins yet described in nature, it seems likely that the presence of that single selenium atom of Sec should convey some specific feature, thereby explaining the existence of selenoproteins in spite of demanding and energetically costly Sec-specific synthesis machineries. Nonetheless, most, if not all, of the currently known selenoproteins are also found as Cys-containing non-selenoprotein orthologues in other organisms, wherefore any potentially unique properties of selenoproteins are yet a matter of debate. The pK(a) of free Sec (approximately 5.2) being significantly lower than that of free Cys (approximately 8.5) has often been proposed as one of the unique features of Sec. However, as discussed herein, this pK(a) difference between Sec and Cys can hardly provide an evolutionary pressure for maintenance of selenoproteins. Moreover, the typically 10- to 100-fold lower enzymatic efficiencies of Sec-to-Cys mutants of selenoprotein oxidoreductases, are also weak arguments for the overall existence of

  14. Reconstitution of selenocysteine incorporation reveals intrinsic regulation by SECIS elements

    PubMed Central

    Gupta, Nirupama; DeMong, Louise W.; Banda, Sowmya; Copeland, Paul R.

    2013-01-01

    Selenoproteins are present in all three domains of life and are responsible for a major part of a cell’s antioxidant defense against reactive oxygen species. Synthesis of selenoproteins requires the decoding of a UGA codon as selenocysteine (Sec) instead of translation termination. Sec is incorporated into the growing polypeptide chain during translation elongation and is known to require a set of highly specific factors: The Sec insertion sequence (SECIS) element in the 3′ untranslated region (3′ UTR), Sec-tRNASec, the Sec-specific elongation factor eEFSec, and SECIS binding protein 2 (SBP2). Since reconstitution has not been reported, whether these factors are sufficient is unknown. Here we report a novel in vitro translation system in which Sec incorporation has been reconstituted from purified components introduced into a Sec naive system. In addition, we developed a novel method to purify Sec-tRNASec and active eEFSec/GTP/tRNA ternary complex. We found that the known basal factors are sufficient for Sec incorporation in vitro. Using this highly manipulable system, we have also found that ribosomes from non-Sec utilizing organisms cannot support Sec incorporation and that some SECIS elements are intrinsically less efficient than others. Having identified the essential set of factors, this work removes a significant barrier to our understanding of the mechanism of Sec incorporation. PMID:23624110

  15. Regulation of Selenocysteine Content of Human Selenoprotein P by Dietary Selenium and Insertion of Cysteine in Place of Selenocysteine

    PubMed Central

    Turanov, Anton A.; Everley, Robert A.; Hybsier, Sandra; Renko, Kostja; Schomburg, Lutz; Gygi, Steven P.; Hatfield, Dolph L.; Gladyshev, Vadim N.

    2015-01-01

    Selenoproteins are a unique group of proteins that contain selenium in the form of selenocysteine (Sec) co-translationally inserted in response to a UGA codon with the help of cis- and trans-acting factors. Mammalian selenoproteins contain single Sec residues, with the exception of selenoprotein P (SelP) that has 7–15 Sec residues depending on species. Assessing an individual’s selenium status is important under various pathological conditions, which requires a reliable selenium biomarker. Due to a key role in organismal selenium homeostasis, high Sec content, regulation by dietary selenium, and availability of robust assays in human plasma, SelP has emerged as a major biomarker of selenium status. Here, we found that Cys is present in various Sec positions in human SelP. Treatment of cells expressing SelP with thiophosphate, an analog of the selenium donor for Sec synthesis, led to a nearly complete replacement of Sec with Cys, whereas supplementation of cells with selenium supported Sec insertion. SelP isolated directly from human plasma had up to 8% Cys inserted in place of Sec, depending on the Sec position. These findings suggest that a change in selenium status may be reflected in both SelP concentration and its Sec content, and that availability of the SelP-derived selenium for selenoprotein synthesis may be overestimated under conditions of low selenium status due to replacement of Sec with Cys. PMID:26452064

  16. Regulation of Selenocysteine Content of Human Selenoprotein P by Dietary Selenium and Insertion of Cysteine in Place of Selenocysteine.

    PubMed

    Turanov, Anton A; Everley, Robert A; Hybsier, Sandra; Renko, Kostja; Schomburg, Lutz; Gygi, Steven P; Hatfield, Dolph L; Gladyshev, Vadim N

    2015-01-01

    Selenoproteins are a unique group of proteins that contain selenium in the form of selenocysteine (Sec) co-translationally inserted in response to a UGA codon with the help of cis- and trans-acting factors. Mammalian selenoproteins contain single Sec residues, with the exception of selenoprotein P (SelP) that has 7-15 Sec residues depending on species. Assessing an individual's selenium status is important under various pathological conditions, which requires a reliable selenium biomarker. Due to a key role in organismal selenium homeostasis, high Sec content, regulation by dietary selenium, and availability of robust assays in human plasma, SelP has emerged as a major biomarker of selenium status. Here, we found that Cys is present in various Sec positions in human SelP. Treatment of cells expressing SelP with thiophosphate, an analog of the selenium donor for Sec synthesis, led to a nearly complete replacement of Sec with Cys, whereas supplementation of cells with selenium supported Sec insertion. SelP isolated directly from human plasma had up to 8% Cys inserted in place of Sec, depending on the Sec position. These findings suggest that a change in selenium status may be reflected in both SelP concentration and its Sec content, and that availability of the SelP-derived selenium for selenoprotein synthesis may be overestimated under conditions of low selenium status due to replacement of Sec with Cys. PMID:26452064

  17. Dimer-dimer interaction of the bacterial selenocysteine synthase SelA promotes functional active-site formation and catalytic specificity.

    PubMed

    Itoh, Yuzuru; Bröcker, Markus J; Sekine, Shun-ichi; Söll, Dieter; Yokoyama, Shigeyuki

    2014-04-17

    The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins and is synthesized on its specific tRNA (tRNA(Sec)). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNA(Sec), formed by seryl-tRNA synthetase, to Sec-tRNA(Sec). SelA, a member of the fold-type-I pyridoxal 5'-phosphate-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNA(Sec) revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNA(Sec). The SelA catalytic site is close to the dimer-dimer interface, but the significance of the dimer pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of "depentamerized" SelA variants with mutations at the dimer-dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I pyridoxal 5'-phosphate-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures. PMID:24456689

  18. An engineered selenocysteine defines a unique class of antibody derivatives

    PubMed Central

    Hofer, Thomas; Thomas, Joshua D.; Burke, Terrence R.; Rader, Christoph

    2008-01-01

    Selenocysteine is cotranslationally inserted into proteins by recoding the stop codon UGA from termination to selenocysteine insertion. The nucleophilic selenol group of selenocysteine endows this rare amino acid with unique chemical reactivity that allows regiospecific covalent conjugation in the presence of the other natural amino acids. Using a mammalian expression system, we generated an IgG1-derived Fc fragment with a C-terminal selenocysteine in yields comparable to conventional monoclonal antibodies and conjugated it to an electrophilic derivative of a peptidomimetic that binds with high affinity and specificity to integrin α4β1. Through this conjugation, both the biological and chemical components are endowed with pharmacological advantages. We demonstrate that whereas the Fc protein increases the circulatory half-life from minutes to days and mediates transcytosis through binding to the neonatal Fc receptor, the peptidomimetic introduces cross-species binding to cell surface integrin α4β1 and blocks its interaction with vascular cell adhesion molecule-1. Compared with conventional monoclonal antibodies, our technology benefits economically from combining a generic biological component with a variable chemical component. PMID:18719095

  19. Semisynthesis and initial characterization of sortase A mutants containing selenocysteine and homocysteine.

    PubMed

    Schmohl, Lena; Wagner, Felix Roman; Schümann, Michael; Krause, Eberhard; Schwarzer, Dirk

    2015-06-15

    The bacterial transpeptidase sortase A is a well-established tool in protein chemistry and catalyzes the chemoselective ligation of peptides and proteins. During catalysis sortase A cleaves the conserved Leu-Pro-X-Thr-Gly sorting motif at the Thr residue under concomitant thioester formation at active site Cys184. We have used expressed protein ligation (EPL) to generate sortase mutants with Cys184 replaced by selenocysteine (Sec) and homocysteine (Hcy). Sec-sortase showed a moderate 2-3-fold reduction in catalytic activity in contrast to Hcy-sortase which is a poor catalyst with less than 1% of wild-type activity. The sensitivity of the active site nucleophiles towards an alkylation reagent correlated with the pKa values of the mutated residues. Furthermore, the pH-profile of Sec-sortase was shifted to more acidic conditions when compared to the wild-type enzyme. These observations provide information on sortase catalysis and the semisynthetic enzymes might represent useful tools for further biochemical investigations and engineering approaches of sortases A. PMID:25900629

  20. Crystallization and preliminary X-ray crystallographic analysis of Aquifex aeolicus SelA, a bacterial selenocysteine synthase.

    PubMed

    Itoh, Yuzuru; Sekine, Shun-ichi; Yokoyama, Shigeyuki

    2012-09-01

    Selenocysteine (Sec), the 21st amino acid, is synthesized on its specific tRNA (tRNA(Sec)) via a multi-step process. In bacteria, tRNA(Sec) is ligated first with serine by seryl-tRNA synthetase, which is followed by Ser-to-Sec conversion by Sec synthase (SelA). To elucidate its structure and catalytic mechanism, Aquifex aeolicus SelA was crystallized. Although wild-type SelA crystals diffracted X-rays poorly (to up to 8 Å resolution), the resolution was improved by introducing a quadruple point mutation targeting the loop regions and by methylating the lysine residues, which yielded 3.9 Å resolution diffraction data from a full-length SelA crystal. Truncation of the N-terminal region (ΔN) also improved the resolution. A 3.3 Å resolution data set for phase determination was obtained from a crystal of selenomethionine-substituted Lys-methylated SelA-ΔN. PMID:22949212

  1. The selenocysteine-specific elongation factor contains a novel and multi-functional domain.

    PubMed

    Gonzalez-Flores, Jonathan N; Gupta, Nirupama; DeMong, Louise W; Copeland, Paul R

    2012-11-01

    The selenocysteine (Sec)-specific eukaryotic elongation factor (eEFSec) delivers the aminoacylated selenocysteine-tRNA (Sec-tRNA(Sec)) to the ribosome and suppresses UGA codons that are upstream of Sec insertion sequence (SECIS) elements bound by SECIS-binding protein 2 (SBP2). Multiple studies have highlighted the importance of SBP2 forming a complex with the SECIS element, but it is not clear how this regulates eEFSec during Sec incorporation. Compared with the canonical elongation factor eEF1A, eEFSec has a unique C-terminal extension called Domain IV. To understand the role of Domain IV in Sec incorporation, we examined a series of mutant proteins for all of the known molecular functions for eEFSec: GTP hydrolysis, Sec-tRNA(Sec) binding, and SBP2/SECIS binding. In addition, wild-type and mutant versions of eEFSec were analyzed for Sec incorporation activity in a novel eEFSec-dependent translation extract. We have found that Domain IV is essential for both tRNA and SBP2 binding as well as regulating GTPase activity. We propose a model where the SBP2/SECIS complex activates eEFSec by directing functional interactions between Domain IV and the ribosome to promote Sec-tRNA(Sec) binding and accommodation into the ribosomal A-site. PMID:22992746

  2. Size matters: a view of selenocysteine incorporation from the ribosome.

    PubMed

    Caban, K; Copeland, P R

    2006-01-01

    This review focuses on the known factors required for selenocysteine (Sec) incorporation in eukaryotes and highlights recent findings that have compelled us to propose a new model for the mechanism of Sec incorporation. In light of this data we also review the controversial aspects of the previous model specifically regarding the proposed interaction between SBP2 and eEFSec. In addition, the relevance of two recently discovered factors in the recoding of Sec are reviewed. The role of the ribosome in this process is emphasized along with a detailed analysis of kinkturn structures present in the ribosome and the L7Ae RNA-binding motif present in SBP2 and other proteins. PMID:16416259

  3. Autoantibodies against a serine tRNA-protein complex implicated in cotranslational selenocysteine insertion.

    PubMed Central

    Gelpi, C; Sontheimer, E J; Rodriguez-Sanchez, J L

    1992-01-01

    We describe an autoantibody specificity present in a subgroup of patients with a severe form of autoimmune chronic active hepatitis. These antibodies precipitate a 90-nucleotide RNA from human whole cell extracts and recognize a 48-kDa polypeptide in immunoblotting assays. The RNA is a UGA suppressor serine tRNA that carries selenocysteine (tRNA[Ser]Sec)), as shown by sequence analysis. The protein does not appear to be seryl-tRNA synthetase; rather, it is an excellent candidate for a factor involved in cotranslational selenocysteine incorporation in human cells. Images PMID:1409691

  4. Partitioning between recoding and termination at a stop codon-selenocysteine insertion sequence.

    PubMed

    Kotini, Suresh Babu; Peske, Frank; Rodnina, Marina V

    2015-07-27

    Selenocysteine (Sec) is inserted into proteins by recoding a UGA stop codon followed by a selenocysteine insertion sequence (SECIS). UGA recoding by the Sec machinery is believed to be very inefficient owing to RF2-mediated termination at UGA. Here we show that recoding efficiency in vivo is 30-40% independently of the cell growth rate. Efficient recoding requires sufficient selenium concentrations in the medium. RF2 is an unexpectedly poor competitor of Sec. We recapitulate the major characteristics of SECIS-dependent UGA recoding in vitro using a fragment of fdhF-mRNA encoding a natural bacterial selenoprotein. Only 40% of actively translating ribosomes that reach the UGA codon insert Sec, even in the absence of RF2, suggesting that the capacity to insert Sec into proteins is inherently limited. RF2 does not compete with the Sec incorporation machinery; rather, it terminates translation on those ribosomes that failed to incorporate Sec. The data suggest a model in which early recruitment of Sec-tRNA(Sec)-SelB-GTP to the SECIS blocks the access of RF2 to the stop codon, thereby prioritizing recoding over termination at Sec-dedicated stop codons. PMID:26040702

  5. Tertiary structure of bacterial selenocysteine tRNA.

    PubMed

    Itoh, Yuzuru; Sekine, Shun-ichi; Suetsugu, Shiro; Yokoyama, Shigeyuki

    2013-07-01

    Selenocysteine (Sec) is translationally incorporated into proteins in response to the UGA codon. The tRNA specific to Sec (tRNA(Sec)) is first ligated with serine by seryl-tRNA synthetase (SerRS). In the present study, we determined the 3.1 Å crystal structure of the tRNA(Sec) from the bacterium Aquifex aeolicus, in complex with the heterologous SerRS from the archaeon Methanopyrus kandleri. The bacterial tRNA(Sec) assumes the L-shaped structure, from which the long extra arm protrudes. Although the D-arm conformation and the extra-arm orientation are similar to those of eukaryal/archaeal tRNA(Sec)s, A. aeolicus tRNA(Sec) has unique base triples, G14:C21:U8 and C15:G20a:G48, which occupy the positions corresponding to the U8:A14 and R15:Y48 tertiary base pairs of canonical tRNAs. Methanopyrus kandleri SerRS exhibited serine ligation activity toward A. aeolicus tRNA(Sec) in vitro. The SerRS N-terminal domain interacts with the extra-arm stem and the outer corner of tRNA(Sec). Similar interactions exist in the reported tRNA(Ser) and SerRS complex structure from the bacterium Thermus thermophilus. Although the catalytic C-terminal domain of M. kandleri SerRS lacks interactions with A. aeolicus tRNA(Sec) in the present complex structure, the conformational flexibility of SerRS is likely to allow the CCA terminal region of tRNA(Sec) to enter the SerRS catalytic site. PMID:23649835

  6. Partitioning between recoding and termination at a stop codon–selenocysteine insertion sequence

    PubMed Central

    Kotini, Suresh Babu; Peske, Frank; Rodnina, Marina V.

    2015-01-01

    Selenocysteine (Sec) is inserted into proteins by recoding a UGA stop codon followed by a selenocysteine insertion sequence (SECIS). UGA recoding by the Sec machinery is believed to be very inefficient owing to RF2-mediated termination at UGA. Here we show that recoding efficiency in vivo is 30–40% independently of the cell growth rate. Efficient recoding requires sufficient selenium concentrations in the medium. RF2 is an unexpectedly poor competitor of Sec. We recapitulate the major characteristics of SECIS-dependent UGA recoding in vitro using a fragment of fdhF-mRNA encoding a natural bacterial selenoprotein. Only 40% of actively translating ribosomes that reach the UGA codon insert Sec, even in the absence of RF2, suggesting that the capacity to insert Sec into proteins is inherently limited. RF2 does not compete with the Sec incorporation machinery; rather, it terminates translation on those ribosomes that failed to incorporate Sec. The data suggest a model in which early recruitment of Sec-tRNASec–SelB–GTP to the SECIS blocks the access of RF2 to the stop codon, thereby prioritizing recoding over termination at Sec-dedicated stop codons. PMID:26040702

  7. Ex vivo correction of selenoprotein N deficiency in rigid spine muscular dystrophy caused by a mutation in the selenocysteine codon

    PubMed Central

    Rederstorff, M.; Allamand, V.; Guicheney, P.; Gartioux, C.; Richard, P.; Chaigne, D.; Krol, A.; Lescure, A.

    2008-01-01

    Premature termination of translation due to nonsense mutations is a frequent cause of inherited diseases. Therefore, many efforts were invested in the development of strategies or compounds to selectively suppress this default. Selenoproteins are interesting candidates considering the idiosyncrasy of the amino acid selenocysteine (Sec) insertion mechanism. Here, we focused our studies on SEPN1, a selenoprotein gene whose mutations entail genetic disorders resulting in different forms of muscular diseases. Selective correction of a nonsense mutation at the Sec codon (UGA to UAA) was undertaken with a corrector tRNASec that was engineered to harbor a compensatory mutation in the anticodon. We demonstrated that its expression restored synthesis of a full-length selenoprotein N both in HeLa cells and in skin fibroblasts from a patient carrying the mutated Sec codon. Readthrough of the UAA codon was effectively dependent on the Sec insertion machinery, therefore being highly selective for this gene and unlikely to generate off-target effects. In addition, we observed that expression of the corrector tRNASec stabilized the mutated SEPN1 transcript that was otherwise more subject to degradation. In conclusion, our data provide interesting evidence that premature termination of translation due to nonsense mutations is amenable to correction, in the context of the specialized selenoprotein synthesis mechanism. PMID:18025044

  8. Mutation in human selenocysteine transfer RNA selectively disrupts selenoprotein synthesis.

    PubMed

    Schoenmakers, Erik; Carlson, Bradley; Agostini, Maura; Moran, Carla; Rajanayagam, Odelia; Bochukova, Elena; Tobe, Ryuta; Peat, Rachel; Gevers, Evelien; Muntoni, Francesco; Guicheney, Pascale; Schoenmakers, Nadia; Farooqi, Sadaf; Lyons, Greta; Hatfield, Dolph; Chatterjee, Krishna

    2016-03-01

    Selenium is a trace element that is essential for human health and is incorporated into more than 25 human selenocysteine-containing (Sec-containing) proteins via unique Sec-insertion machinery that includes a specific, nuclear genome-encoded, transfer RNA (tRNA[Ser]Sec). Here, we have identified a human tRNA[Ser]Sec mutation in a proband who presented with a variety of symptoms, including abdominal pain, fatigue, muscle weakness, and low plasma levels of selenium. This mutation resulted in a marked reduction in expression of stress-related, but not housekeeping, selenoproteins. Evaluation of primary cells from the homozygous proband and a heterozygous parent indicated that the observed deficit in stress-related selenoprotein production is likely mediated by reduced expression and diminished 2'-O-methylribosylation at uridine 34 in mutant tRNA[Ser]Sec. Moreover, this methylribosylation defect was restored by cellular complementation with normal tRNA[Ser]Sec. This study identifies a tRNA mutation that selectively impairs synthesis of stress-related selenoproteins and demonstrates the importance of tRNA modification for normal selenoprotein synthesis. PMID:26854926

  9. Mutation in human selenocysteine transfer RNA selectively disrupts selenoprotein synthesis

    PubMed Central

    Schoenmakers, Erik; Carlson, Bradley; Agostini, Maura; Moran, Carla; Rajanayagam, Odelia; Bochukova, Elena; Tobe, Ryuta; Peat, Rachel; Gevers, Evelien; Muntoni, Francesco; Guicheney, Pascale; Schoenmakers, Nadia; Farooqi, Sadaf; Lyons, Greta; Hatfield, Dolph; Chatterjee, Krishna

    2016-01-01

    Selenium is a trace element that is essential for human health and is incorporated into more than 25 human selenocysteine-containing (Sec-containing) proteins via unique Sec-insertion machinery that includes a specific, nuclear genome–encoded, transfer RNA (tRNA[Ser]Sec). Here, we have identified a human tRNA[Ser]Sec mutation in a proband who presented with a variety of symptoms, including abdominal pain, fatigue, muscle weakness, and low plasma levels of selenium. This mutation resulted in a marked reduction in expression of stress-related, but not housekeeping, selenoproteins. Evaluation of primary cells from the homozygous proband and a heterozygous parent indicated that the observed deficit in stress-related selenoprotein production is likely mediated by reduced expression and diminished 2′-O-methylribosylation at uridine 34 in mutant tRNA[Ser]Sec. Moreover, this methylribosylation defect was restored by cellular complementation with normal tRNA[Ser]Sec. This study identifies a tRNA mutation that selectively impairs synthesis of stress-related selenoproteins and demonstrates the importance of tRNA modification for normal selenoprotein synthesis. PMID:26854926

  10. Adjustments, extinction, and remains of selenocysteine incorporation machinery in the nematode lineage

    PubMed Central

    Otero, Lucía; Romanelli-Cedrez, Laura; Turanov, Anton A.; Gladyshev, Vadim N.; Miranda-Vizuete, Antonio; Salinas, Gustavo

    2014-01-01

    Selenocysteine (Sec) is encoded by an UGA codon with the help of a SECIS element present in selenoprotein mRNAs. SECIS-binding protein (SBP2/SCBP-2) mediates Sec insertion, but the roles of its domains and the impact of its deficiency on Sec insertion are not fully understood. We used Caenorhabditis elegans to examine SBP2 function since it possesses a single selenoprotein, thioredoxin reductase-1 (TRXR-1). All SBP2 described so far have an RNA-binding domain (RBD) and a Sec-incorporation domain (SID). Surprisingly, C. elegans SBP2 lacks SID and consists only of an RBD. An sbp2 deletion mutant strain ablated Sec incorporation demonstrating SBP2 essentiality for Sec incorporation. Further in silico analyses of nematode genomes revealed conservation of SBP2 lacking SID and maintenance of Sec incorporation linked to TRXR-1. Remarkably, parasitic plant nematodes lost the ability to incorporate Sec, but retained SecP43, a gene associated with Sec incorporation. Interestingly, both selenophosphate synthetase (SPS) genes are absent in plant parasitic nematodes, while only Cys-containing SPS2 is present in Sec-incorporating nematodes. Our results indicate that C. elegans and the nematode lineage provide key insights into Sec incorporation and the evolution of Sec utilization trait, selenoproteomes, selenoproteins, and Sec residues. Finally, our study provides evidence of noncanonical translation initiation in C. elegans, not previously known for this well-established animal model. PMID:24817701

  11. Dimer-dimer interaction of the bacterial selenocysteine synthase SelA promotes functional active site formation and catalytic specificity

    PubMed Central

    Itoh, Yuzuru; Bröcker, Markus J.; Sekine, Shun-ichi; Söll, Dieter; Yokoyama, Shigeyuki

    2015-01-01

    The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins, and is synthesized on its specific tRNA (tRNASec). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNASec, formed by seryl-tRNA synthetase, to Sec-tRNASec. SelA, a member of the fold-type-I pyridoxal 5′-phosphate (PLP)-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500 kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNASec revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNASec. The SelA catalytic site is close to the dimer-dimer interface, but the significance of the dimer-pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions, and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of “depentamerized” SelA variants with mutations at the dimer-dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site, and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I PLP-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures. PMID:24456689

  12. Crystallization and preliminary X-ray crystallographic analysis of Aquifex aeolicus SelA, a bacterial selenocysteine synthase

    PubMed Central

    Itoh, Yuzuru; Sekine, Shun-ichi; Yokoyama, Shigeyuki

    2012-01-01

    Selenocysteine (Sec), the 21st amino acid, is synthesized on its specific tRNA (tRNASec) via a multi-step process. In bacteria, tRNASec is ligated first with serine by seryl-tRNA synthetase, which is followed by Ser-to-Sec conversion by Sec synthase (SelA). To elucidate its structure and catalytic mechanism, Aquifex aeolicus SelA was crystallized. Although wild-type SelA crystals diffracted X-­rays poorly (to up to 8 Å resolution), the resolution was improved by introducing a quadruple point mutation targeting the loop regions and by methylating the lysine residues, which yielded 3.9 Å resolution diffraction data from a full-length SelA crystal. Truncation of the N-terminal region (ΔN) also improved the resolution. A 3.3 Å resolution data set for phase determination was obtained from a crystal of selenomethionine-substituted Lys-methylated SelA-ΔN. PMID:22949212

  13. Removal of the 5-nitro-2-pyridine-sulfenyl protecting group from selenocysteine and cysteine by ascorbolysis.

    PubMed

    Ste Marie, Emma J; Ruggles, Erik L; Hondal, Robert J

    2016-09-01

    We previously reported on a method for the facile removal of 4-methoxybenzyl and acetamidomethyl protecting groups from cysteine (Cys) and selenocysteine (Sec) using 2,2'-dithiobis-5-nitropyridine dissolved in trifluoroacetic acid, with or without thioanisole. The use of this reaction mixture removes the protecting group and replaces it with a 2-thio(5-nitropyridyl) (5-Npys) group. This results in either a mixed selenosulfide bond or disulfide bond (depending on the use of Sec or Cys), which can subsequently be reduced by thiolysis. A major disadvantage of thiolysis is that excess thiol must be used to drive the reaction to completion and then removed before using the Cys-containing or Sec-containing peptide in further applications. Here, we report a further advancement of this method as we have found that ascorbate at pH 4.5 and 25 °C will reduce the selenosulfide to the selenol. Ascorbolysis of the mixed disulfide between Cys and 5-Npys is much less efficient but can be accomplished at higher concentrations of ascorbate at pH 7 and 37 °C with extended reaction times. We envision that our improved method will allow for in situ reactions with alkylating agents and electrophiles without the need for further purification, as well as a number of other applications. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27480992

  14. Determination of modification degree in BDDE-modified hyaluronic acid hydrogel by SEC/MS.

    PubMed

    Yang, Biao; Guo, Xueping; Zang, Hengchang; Liu, Jianjian

    2015-10-20

    Determination of modification degree in BDDE-modified hyaluronic acid (HA) hydrogel is of particular interest. In this paper, three crosslinking parameters (degree of total modification, t-MOD; degree of cross-link modification, c-MOD; degree of pendent modification, p-MOD) are defined and determined by quantification of the modified fragments in hydrogel digestion by size exclusion chromatography combined with mass spectrometry (SEC-MS). The digestion products of a novel hyaluronidase HAase-B produced by Bacillus sp. A50 are studied and only a few modified fragments are identified by (1)H NMR and MS. As a result, Three HA hydrogels prepared in lab have different t-MOD, c-MOD and p-MOD, but the ratio of c-MOD to p-MOD result in the almost same value of 75%. Hydrogel products from Q-Med have nearly same t-MOD about 0.8% and c-MOD about 0.1%, the ratio of c-MOD to p-MOD is about 13%. Hydrogels from ANTEIS S.A all have much higher t-MOD values, the ratio of c-MOD and p-MOD is about 8%. PMID:26256180

  15. Identification of methionine as a possible precursor to the selenocysteine catalytic site of glutathione peroxidase

    SciTech Connect

    Chung, C.K.

    1985-01-01

    The selenium (Se) moiety of glutathione peroxidase (GSHPx) occurs as selenocysteine and is present at the catalytic active site of the enzyme which catalyzes the reduction of hydrogen peroxides and lipid peroxides. The presence of this unusual amino acid at the active site raises the question as to the origin of the carbon skeleton of Se-cysteine. ICR Swiss mice were fed a Se deficient diet for 50 days and then were fed a Se adequate diet (1 ppm Se as SeO/sub 3/). Mice were i.p. injected with either (U-/sup 14/C) methionine, serine, or alanine (0.5 ..mu..Ci/0.1 ml/mouse/day) for 25 days. Recovered GSHPx activity in liver and blood was carboxymethylated (CM) with iodoacetic acid. CM-GSHPx was partially purified by column chromatography. /sup 14/C-GSHPx fractions were collected, lyophilized, and hydrolyzed. /sup 14/C-amino acids were separated by TLC and ion-exchange chromatography. TLC (phenol, cyclohexane, acetic acid, and water (90;6.5;3.5;8)) revealed a GSHPx /sup 14/C-amino acid derived from U-/sup 14/C-methionine, but not from serine or alanine corresponding to CM-selenocysteine (R/sub f/; 0.16). Ion-exchange chromatography of U-/sup 14/C-methionine labeled GSHPx hydrolyzate revealed two radio carbon ninhydrin positive peaks corresponding to /sup 14/C-CM-selenocysteine and /sup 14/C-methionine. No corresponding /sup 14/C-labeled peaks were observed for CM-selenocysteine derived from U-/sup 14/C serine or alanine. The results suggest that methionine may contribute a portion of the carbon skeleton to selenocysteine which may include an alternative metabolic pathway. Animal studies demonstrated that GSHPx activity is increased by methionine supplementation may be due to its contribution of carbon source to the catalytic site of the enzyme.

  16. Structural basis for early-onset neurological disorders caused by mutations in human selenocysteine synthase

    PubMed Central

    Puppala, Anupama K.; French, Rachel L.; Matthies, Doreen; Baxa, Ulrich; Subramaniam, Sriram; Simonović, Miljan

    2016-01-01

    Selenocysteine synthase (SepSecS) catalyzes the terminal reaction of selenocysteine, and is vital for human selenoproteome integrity. Autosomal recessive inheritance of mutations in SepSecS–Ala239Thr, Thr325Ser, Tyr334Cys and Tyr429*–induced severe, early-onset, neurological disorders in distinct human populations. Although harboring different mutant alleles, patients presented remarkably similar phenotypes typified by cerebellar and cerebral atrophy, seizures, irritability, ataxia, and extreme spasticity. However, it has remained unclear how these genetic alterations affected the structure of SepSecS and subsequently elicited the development of a neurological pathology. Herein, our biophysical and structural characterization demonstrates that, with the exception of Tyr429*, pathogenic mutations decrease protein stability and trigger protein misfolding. We propose that the reduced stability and increased propensity towards misfolding are the main causes for the loss of SepSecS activity in afflicted patients, and that these factors contribute to disease progression. We also suggest that misfolding of enzymes regulating protein synthesis should be considered in the diagnosis and study of childhood neurological disorders. PMID:27576344

  17. Structural basis for early-onset neurological disorders caused by mutations in human selenocysteine synthase.

    PubMed

    Puppala, Anupama K; French, Rachel L; Matthies, Doreen; Baxa, Ulrich; Subramaniam, Sriram; Simonović, Miljan

    2016-01-01

    Selenocysteine synthase (SepSecS) catalyzes the terminal reaction of selenocysteine, and is vital for human selenoproteome integrity. Autosomal recessive inheritance of mutations in SepSecS-Ala239Thr, Thr325Ser, Tyr334Cys and Tyr429*-induced severe, early-onset, neurological disorders in distinct human populations. Although harboring different mutant alleles, patients presented remarkably similar phenotypes typified by cerebellar and cerebral atrophy, seizures, irritability, ataxia, and extreme spasticity. However, it has remained unclear how these genetic alterations affected the structure of SepSecS and subsequently elicited the development of a neurological pathology. Herein, our biophysical and structural characterization demonstrates that, with the exception of Tyr429*, pathogenic mutations decrease protein stability and trigger protein misfolding. We propose that the reduced stability and increased propensity towards misfolding are the main causes for the loss of SepSecS activity in afflicted patients, and that these factors contribute to disease progression. We also suggest that misfolding of enzymes regulating protein synthesis should be considered in the diagnosis and study of childhood neurological disorders. PMID:27576344

  18. Molecularly defined antibody conjugation through a selenocysteine interface†

    PubMed Central

    Hofer, Thomas; Skeffington, Lauren R.; Chapman, Colby M.; Rader, Christoph

    2009-01-01

    Antibody conjugates have broad utility in basic, preclinical, and clinical applications. Conventional antibody conjugation through the amine group of lysine or the thiol group of cysteine residues yields heterogeneous products of undefined stoichiometry and considerable batch-to-batch variability. To preserve the two hallmarks of the antibody molecule, precision and predictability, methods that enable site-specific antibody conjugation are in high demand. Based on a mammalian cell expression system, we describe the utilization of the 21st natural amino acid selenocysteine for the generation of IgG and Fab molecules with unique nucleophilic reactivity that affords site-specific conjugation to electrophilic derivatives of biotin, fluorescein, and poly(ethylene glycol). The resulting antibody conjugates were found to fully retain their antigen binding capability and, in case of IgG, the ability to mediate effector functions. Gain-of-function was demonstrated in vitro and in vivo. While these antibody conjugates are relevant for a variety of proteomic, diagnostic, and therapeutic applications, they also constitute a proof-of-principle for the generation of molecularly defined antibody-drug conjugates and radioimmunoconjugates. Compared to other site-specific antibody conjugation methods, selenocysteine interface technology (i) only involves a minor modification at the C-terminus that does not interfere with disulfide bridges, (ii) does not require activation, and (iii) generates unique 1:1 stoichiometries of biological and chemical component. Collectively, our method affords the generation of highly defined antibody conjugates with broad utility from proteomic applications to therapeutic intervention. PMID:19894757

  19. Induction of S-Phase Arrest in Human Glioma Cells by Selenocysteine, a Natural Selenium-Containing Agent Via Triggering Reactive Oxygen Species-Mediated DNA Damage and Modulating MAPKs and AKT Pathways.

    PubMed

    Wang, Kun; Fu, Xiao-Ting; Li, Yuan; Hou, Ya-Jun; Yang, Ming-Feng; Sun, Jing-Yi; Yi, Shu-Ying; Fan, Cun-Dong; Fu, Xiao-Yan; Zhai, Jing; Sun, Bao-Liang

    2016-06-01

    Selenocysteine (SeC) a natural available selenoamino acid exhibits novel anticancer activities against human cancer cell lines. However, the growth inhibitory effect and mechanism of SeC in human glioma cells remain unclear. The present study reveals that SeC time- and dose-dependently inhibited U251 and U87 human glioma cells growth by induction of S-phase cell cycle arrest, followed by the marked decrease of cyclin A. SeC-induced S-phase arrest was achieved by inducing DNA damage through triggering generation of reactive oxygen species (ROS) and superoxide anion, with concomitant increase of TUNEL-positive cells and induction of p21waf1/Cip1 and p53. SeC treatment also caused the activation of p38MAPK, JNK and ERK, and inactivation of AKT. Four inhibitors of MAPKs and AKT pathways further confirmed their roles in SeC-induced S-phase arrest in human glioma cells. Our findings advance the understanding on the molecular mechanisms of SeC in human glioma management. PMID:26846141

  20. Evolutionary history of selenocysteine incorporation from the perspective of SECIS binding proteins

    PubMed Central

    Donovan, Jesse; Copeland, Paul R

    2009-01-01

    Background The co-translational incorporation of selenocysteine into nascent polypeptides by recoding the UGA stop codon occurs in all domains of life. In eukaryotes, this event requires at least three specific factors: SECIS binding protein 2 (SBP2), a specific translation elongation factor (eEFSec), selenocysteinyl tRNA, and a cis-acting selenocysteine insertion sequence (SECIS) element in selenoprotein mRNAs. While the phylogenetic relationships of selenoprotein families and the evolution of selenocysteine usage are well documented, the evolutionary history of SECIS binding proteins has not been explored. Results In this report we present a phylogeny of the eukaryotic SECIS binding protein family which includes SBP2 and a related protein we herein term SBP2L. Here we show that SBP2L is an SBP2 paralogue in vertebrates and is the only form of SECIS binding protein in invertebrate deuterostomes, suggesting a key role in Sec incorporation in these organisms, but an SBP2/SBP2L fusion protein is unable to support Sec incorporation in vitro. An in-depth phylogenetic analysis of the conserved L7Ae RNA binding domain suggests an ancestral relationship with ribosomal protein L30. In addition, we describe the emergence of a motif upstream of the SBP2 RNA binding domain that shares significant similarity with a motif within the pseudouridine synthase Cbf5. Conclusion Our analysis suggests that SECIS binding proteins arose once in evolution but diverged significantly in multiple lineages. In addition, likely due to a gene duplication event in the early vertebrate lineage, SBP2 and SBP2L are paralogous in vertebrates. PMID:19744324

  1. Compensating for the Absence of Selenocysteine in High-Molecular Weight Thioredoxin Reductases: The Electrophilic Activation Hypothesis

    PubMed Central

    2015-01-01

    Mammalian thioredoxin reductase (TR) is a pyridine disulfide oxidoreductase that uses the rare amino acid selenocysteine (Sec) in place of the more commonly used amino acid cysteine (Cys). Selenium is a Janus-faced element because it is both highly nucleophilic and highly electrophilic. Cys orthologs of Sec-containing enzymes may compensate for the absence of a Sec residue by making the active site Cys residue more (i) nucleophilic, (ii) electrophilic, or (iii) reactive by increasing both S-nucleophilicity and S-electrophilicity. It has already been shown that the Cys ortholog TR from Drosophila melanogaster (DmTR) has increased S-nucleophilicity [Gromer, S., Johansson, L., Bauer, H., Arscott, L. D., Rauch, S., Ballou, D. P., Williams, C. H., Jr., Schrimer, R. H., and Arnér, E. S (2003) Active sites of thioredoxin reductases: Why selenoproteins? Proc. Natl. Acad. Sci. U.S.A. 100, 12618–12623]. Here we present evidence that DmTR also enhances the electrophilicity of Cys490 through the use of an “electrophilic activation” mechanism. This mechanism is proposed to work by polarizing the disulfide bond that occurs between Cys489 and Cys490 in the C-terminal redox center by the placement of a positive charge near Cys489. This polarization renders the sulfur atom of Cys490 electron deficient and enhances the rate of thiol/disulfide exchange that occurs between the N- and C-terminal redox centers. Our hypothesis was developed by using a strategy of homocysteine (hCys) for Cys substitution in the Cys-Cys redox dyad of DmTR to differentiate the function of each Cys residue. The results show that hCys could substitute for Cys490 with little loss of thioredoxin reductase activity, but that substitution of hCys for Cys489 resulted in a 238-fold reduction in activity. We hypothesize that replacement of Cys489 with hCys destroys an interaction between the sulfur atom of Cys489 and His464 crucial for the proposed electrophilic activation mechanism. This electrophilic

  2. Compensating for the absence of selenocysteine in high-molecular weight thioredoxin reductases: the electrophilic activation hypothesis.

    PubMed

    Lothrop, Adam P; Snider, Gregg W; Flemer, Stevenson; Ruggles, Erik L; Davidson, Ronald S; Lamb, Audrey L; Hondal, Robert J

    2014-02-01

    Mammalian thioredoxin reductase (TR) is a pyridine disulfide oxidoreductase that uses the rare amino acid selenocysteine (Sec) in place of the more commonly used amino acid cysteine (Cys). Selenium is a Janus-faced element because it is both highly nucleophilic and highly electrophilic. Cys orthologs of Sec-containing enzymes may compensate for the absence of a Sec residue by making the active site Cys residue more (i) nucleophilic, (ii) electrophilic, or (iii) reactive by increasing both S-nucleophilicity and S-electrophilicity. It has already been shown that the Cys ortholog TR from Drosophila melanogaster (DmTR) has increased S-nucleophilicity [Gromer, S., Johansson, L., Bauer, H., Arscott, L. D., Rauch, S., Ballou, D. P., Williams, C. H., Jr., Schrimer, R. H., and Arnér, E. S (2003) Active sites of thioredoxin reductases: Why selenoproteins? Proc. Natl. Acad. Sci. U.S.A. 100, 12618-12623]. Here we present evidence that DmTR also enhances the electrophilicity of Cys490 through the use of an "electrophilic activation" mechanism. This mechanism is proposed to work by polarizing the disulfide bond that occurs between Cys489 and Cys490 in the C-terminal redox center by the placement of a positive charge near Cys489. This polarization renders the sulfur atom of Cys490 electron deficient and enhances the rate of thiol/disulfide exchange that occurs between the N- and C-terminal redox centers. Our hypothesis was developed by using a strategy of homocysteine (hCys) for Cys substitution in the Cys-Cys redox dyad of DmTR to differentiate the function of each Cys residue. The results show that hCys could substitute for Cys490 with little loss of thioredoxin reductase activity, but that substitution of hCys for Cys489 resulted in a 238-fold reduction in activity. We hypothesize that replacement of Cys489 with hCys destroys an interaction between the sulfur atom of Cys489 and His464 crucial for the proposed electrophilic activation mechanism. This electrophilic activation

  3. Assessment of reagents for selenocysteine conjugation and the stability of selenocysteine adducts.

    PubMed

    Pedzisa, Lee; Li, Xiuling; Rader, Christoph; Roush, William R

    2016-06-14

    Conventional antibody-drug conjugates (ADCs) are heterogeneous mixtures that have poor pharmacokinetic properties and decreased efficacy relative to homogenous ADCs. Furthermore, ADCs that are maleimide-based often have inadequate circulatory stability, which can result in premature drug release with consequent off-target toxicities. Selenocysteine-modified antibodies have been developed that allow site-specific antibody conjugation, yielding homogeneous ADCs. Herein, we survey several electrophilic functional groups that react with selenocystine with high efficiency. Several of these result in conjugates with stabilities that are superior to maleimide conjugates. Among these, the allenamide functional group reacts with notably high efficiency, leads to conjugates with remarkable stability, and shows exquisite selectivity for selenocysteine conjugation. PMID:27184239

  4. Purification and characterization of selenocysteine beta-lyase from Citrobacter freundii.

    PubMed Central

    Chocat, P; Esaki, N; Tanizawa, K; Nakamura, K; Tanaka, H; Soda, K

    1985-01-01

    The purification and characterization of bacterial selenocysteine beta-lyase, an enzyme which specifically catalyzes the cleavage of L-selenocysteine to L-alanine and Se0, are presented. The enzyme, purified to near homogeneity from Citrobacter freundii, is monomeric with a molecular weight of ca. 64,000 and contains 1 mol of pyridoxal 5'-phosphate as a cofactor per mol of enzyme. L-Selenocysteine is the sole substrate (Km, 0.95 mM). L-Cysteine is a competitive inhibitor of the enzyme (Ki, 0.65 mM). The enzyme also catalyzes the alpha, beta elimination of beta-chloro-L-alanine to form NH3, pyruvate, and Cl- and is irreversibly inactivated during the reaction. The physicochemical properties, e.g., amino acid composition and subunit structure, of the bacterial enzyme are fairly different from those of the pig liver enzyme (Esaki et al., J. Biol. Chem. 257:4386-4391, 1982). However, the catalytic properties of both enzymes, e.g., substrate specificity and inactivation by the substrate or a mechanism-based inactivator, beta-chloro-L-alanine, are very similar. PMID:2991201

  5. Selenocysteine containing analogues of Atx1-based peptides protect cells from copper ion toxicity.

    PubMed

    Shoshan, Michal S; Lehman, Yonat; Goch, Wojciech; Bal, Wojciech; Tshuva, Edit Y; Metanis, Norman

    2016-08-01

    Seleno-substituted model peptides of copper metallochaperone proteins were analyzed for the metal affinity and in vitro anti-oxidative reactivity. An acyclic MTCXXC (X is any amino acid) reference peptide previously analyzed as a potent inhibitor of ROS production underwent substitution of the cysteine residues with selenocysteine to give two singly substituted derivatives C3U and C6U and the doubly substituted analogue C3U/C6U. Presumably due to the softer nature of Se vs. S, all selenocysteine containing peptides demonstrated high affinity to Cu(i), higher than that of the reference peptide, and in the same order of magnitude as that measured for the native protein, Atox1. A stronger impact of residue 3 confirmed previous findings on its more dominant role in metal coordination. In vitro studies on the HT-29 human colon cancer cell line, MEF mice embryonic fibroblasts, and MEF with the knocked-out Atox1 gene (Atox1-/-) consistently identified C3U/C6U as the most potent inhibitor of ROS cellular production based on the 2',7'-dichlorodihydrofluorescin diacetate (H2DCF-DA) assay, also in comparison with known drugs employed in the clinic for Wilson's disease. The selenocysteine containing peptides are thus promising drug candidates for chelation therapy of Wilson's disease and related conditions relevant to excessive copper levels. PMID:27349676

  6. Purification and characterization of selenocysteine beta-lyase from Citrobacter freundii

    SciTech Connect

    Chocat, P.; Esaki, N.; Tanizawa, K.; Nakamura, K.; Tanaka, H.; Soda, K.

    1985-08-01

    The purification and characterization of bacterial selenocysteine beta-lyase, an enzyme which specifically catalyzes the cleavage of L-selenocysteine to L-alanine and Se0, are presented. The enzyme, purified to near homogeneity from Citrobacter freundii, is monomeric with a molecular weight of ca. 64,000 and contains 1 mol of pyridoxal 5'-phosphate as a cofactor per mol of enzyme. L-Selenocysteine is the sole substrate. L-Cysteine is a competitive inhibitor of the enzyme. The enzyme also catalyzes the alpha, beta elimination of beta-chloro-L-alanine to form NH3, pyruvate, and Cl- and is irreversibly inactivated during the reaction. The physicochemical properties, e.g., amino acid composition and subunit structure, of the bacterial enzyme are fairly different from those of the pig liver enzyme. However, the catalytic properties of both enzymes, e.g., substrate specificity and inactivation by the substrate or a mechanism-based inactivator, beta-chloro-L-alanine, are very similar.

  7. Serine incorporation into the selenocysteine moiety of glutathione peroxidase

    SciTech Connect

    Sunde, R.A.; Evenson, J.K.

    1987-01-15

    The selenium in mammalian glutathione peroxidase is present as a selenocysteine ((Se)Cys) moiety incorporated into the peptide backbone 41-47 residues from the N-terminal end. To study the origin of the skeleton of the (Se)Cys moiety, we perfused isolated rat liver with /sup 14/C- or /sup 3/H-labeled amino acids for 4 h, purified the GSH peroxidase, derivatized the (Se)Cys in GSH peroxidase to carboxymethylselenocysteine ((Se)Cys(Cm)), and determined the amino acid specific activity. Perfusion with (/sup 14/C)cystine resulted in (/sup 14/C)cystine incorporation into GSH peroxidase without labeling (Se)Cys(Cm), indicating that cysteine is not a direct precursor for (Se)Cys. (/sup 14/C)Serine perfusion labeled serine, glycine (the serine hydroxymethyltransferase product), and (Se)Cys(Cm) in purified GSH peroxidase, whereas (3-3H)serine perfusion only labeled serine and (Se)Cys(Cm), thus demonstrating that the (Se)Cys in GSH peroxidase is derived from serine. The similar specific activities of serine and (Se)Cys(Cm) strongly suggest that the precursor pool of serine used for (Se) Cys synthesis is the same or similar to the serine pool used for acylation of seryl-tRNAs.

  8. The Redox State of SECIS Binding Protein 2 Controls Its Localization and Selenocysteine Incorporation Function

    PubMed Central

    Papp, Laura V.; Lu, Jun; Striebel, Frank; Kennedy, Derek; Holmgren, Arne; Khanna, Kum Kum

    2006-01-01

    Selenoproteins are central controllers of cellular redox homeostasis. Incorporation of selenocysteine (Sec) into selenoproteins employs a unique mechanism to decode the UGA stop codon. The process requires the Sec insertion sequence (SECIS) element, tRNASec, and protein factors including the SECIS binding protein 2 (SBP2). Here, we report the characterization of motifs within SBP2 that regulate its subcellular localization and function. We show that SBP2 shuttles between the nucleus and the cytoplasm via intrinsic, functional nuclear localization signal and nuclear export signal motifs and that its nuclear export is dependent on the CRM1 pathway. Oxidative stress induces nuclear accumulation of SBP2 via oxidation of cysteine residues within a redox-sensitive cysteine-rich domain. These modifications are efficiently reversed in vitro by human thioredoxin and glutaredoxin, suggesting that these antioxidant systems might regulate redox status of SBP2 in vivo. Depletion of SBP2 in cell lines using small interfering RNA results in a decrease in Sec incorporation, providing direct evidence for its requirement for selenoprotein synthesis. Furthermore, Sec incorporation is reduced substantially after treatment of cells with agents that cause oxidative stress, suggesting that nuclear sequestration of SBP2 under such conditions may represent a mechanism to regulate the expression of selenoproteins. PMID:16782878

  9. Selective selC-Independent Selenocysteine Incorporation into Formate Dehydrogenases

    PubMed Central

    Zorn, Michael; Ihling, Christian H.; Golbik, Ralph; Sawers, R. Gary; Sinz, Andrea

    2013-01-01

    The formate dehydrogenases (Fdh) Fdh-O, Fdh-N, and Fdh-H, are the only proteins in Escherichia coli that incorporate selenocysteine at a specific position by decoding a UGA codon. However, an excess of selenium can lead to toxicity through misincorporation of selenocysteine into proteins. To determine whether selenocysteine substitutes for cysteine, we grew Escherichia coli in the presence of excess sodium selenite. The respiratory Fdh-N and Fdh-O enzymes, along with nitrate reductase (Nar) were co-purified from wild type strain MC4100 after anaerobic growth with nitrate and either 2 µM or 100 µM selenite. Mass spectrometric analysis of the catalytic subunits of both Fdhs identified the UGA-specified selenocysteine residue and revealed incorporation of additional, ‘non-specific’ selenocysteinyl residues, which always replaced particular cysteinyl residues. Although variable, their incorporation was not random and was independent of the selenite concentration used. Notably, these cysteines are likely to be non-essential for catalysis and they do not coordinate the iron-sulfur cluster. The remaining cysteinyl residues that could be identified were never substituted by selenocysteine. Selenomethionine was never observed in our analyses. Non-random substitution of particular cysteinyl residues was also noted in the electron-transferring subunit of both Fdhs as well as in the subunits of the Nar enzyme. Nar isolated from an E. coli selC mutant also showed a similar selenocysteine incorporation pattern to the wild-type indicating that non-specific selenocysteine incorporation was independent of the specific selenocysteine pathway. Thus, selenide replaces sulfide in the biosynthesis of cysteine and misacylated selenocysteyl-tRNACys decodes either UGU or UGC codons, which usually specify cysteine. Nevertheless, not every UGU or UGC codon was decoded as selenocysteine. Together, our results suggest that a degree of misincorporation of selenocysteine into enzymes

  10. Selenocysteine, Pyrrolysine, and the Unique Energy Metabolism of Methanogenic Archaea

    DOE PAGESBeta

    Rother, Michael; Krzycki, Joseph A.

    2010-01-01

    Methanogenic archaea are a group of strictly anaerobic microorganisms characterized by their strict dependence on the process of methanogenesis for energy conservation. Among the archaea, they are also the only known group synthesizing proteins containing selenocysteine or pyrrolysine. All but one of the known archaeal pyrrolysine-containing and all but two of the confirmed archaeal selenocysteine-containing protein are involved in methanogenesis. Synthesis of these proteins proceeds through suppression of translational stop codons but otherwise the two systems are fundamentally different. This paper highlights these differences and summarizes the recent developments in selenocysteine- and pyrrolysine-related research on archaea and aims to putmore » this knowledge into the context of their unique energy metabolism.« less

  11. Kinetics of the interaction of translation factor SelB from Escherichia coli with guanosine nucleotides and selenocysteine insertion sequence RNA.

    PubMed

    Thanbichler, M; Bock, A; Goody, R S

    2000-07-01

    The kinetics of the interaction of GTP and GDP with SelB, the specific translation factor for the incorporation of selenocysteine into proteins, have been investigated using the stopped-flow method. Useful signals were obtained using intrinsic (i.e. tryptophan) fluorescence, the fluorescence of methylanthraniloyl derivatives of nucleotides, or fluorescence resonance energy transfer from tryptophan to the methylanthraniloyl group. The affinities of SelB for GTP (K(d) = 0.74 micrometer) and GDP (K(d) = 13.4 micrometer) were considerably lower than those of other translation factors. Of functional significance is the fact that the rate constant for GDP release from its complex with SelB (15 s(-)(1)) is many orders of magnitude larger than for elongation factor Tu, explaining why a GDP/GTP exchange factor is not required for the action of SelB. In contrast, the rate of release of GTP is 2 orders of magnitude slower and not significantly faster than for elongation factor Tu. Using a fluorescently labeled 17-nucleotide RNA minihelix that represents a binding site for the protein and that is part of the fdhF selenocysteine insertion sequence element positioned immediately downstream of the UGA triplet coding for selenocysteine incorporation, the kinetics of the interaction were studied. The high affinity of the interaction (K(d) approximately 1 nm) appeared to be increased even further when selenocysteyl-tRNA(Sec) was bound to SelB, but to be independent of the presence or nature of the guanosine nucleotide at the active site. These results suggest that the affinity of SelB for its RNA binding site is maximized when charged tRNA is bound and decreases to allow dissociation and reading of codons downstream of the selenocysteine codon after selenocysteine peptide bond formation. PMID:10781605

  12. Absence of Selenoprotein P but not Selenocysteine Lyase Results in Severe Neurological Dysfunction

    PubMed Central

    Raman, Arjun V.; Pitts, Matthew W.; Seyedali, Ali; Hashimoto, Ann C.; Seale, Lucia A.; Bellinger, Frederick P.; Berry, Marla J.

    2012-01-01

    Dietary selenium restriction in mammals causes bodily selenium to be preferentially retained in the brain relative to other organs. Almost all of the known selenoproteins are found in brain, where expression is facilitated by selenocysteine-laden selenoprotein P. The brain also expresses selenocysteine lyase, an enzyme that putatively salvages selenocysteine and recycles the selenium for selenoprotein translation. We compared mice with a genetic deletion of selenocysteine lyase to selenoprotein P knockout mice for similarity of neurological impairments, and whether dietary selenium modulates these parameters. We report that selenocysteine lyase knockout mice do not display neurological dysfunction comparable to selenoprotein P knockout mice. Feeding a low-selenium diet to selenocysteine lyase knockout mice revealed a mild spatial learning deficit without disrupting motor coordination. Additionally, we report that the neurological phenotype caused by the absence of selenoprotein P is exacerbated in male versus female mice. These findings indicate that selenocysteine recycling via selenocysteine lyase becomes limiting under selenium deficiency, and suggest the presence of a complementary mechanism for processing selenocysteine. Our studies illuminate the interaction between selenoprotein P and selenocysteine lyase in the distribution and turnover of body and brain selenium, and emphasize the consideration of sex differences when studying selenium and selenoproteins in vertebrate biology. PMID:22487427

  13. Complete doping in solid-state by silica-supported perchloric acid as dopant solid acid: Synthesis and characterization of the novel chiral composite of poly [(±)-2-(sec-butyl) aniline

    NASA Astrophysics Data System (ADS)

    Farrokhzadeh, Abdolkarim; Modarresi-Alam, Ali Reza

    2016-05-01

    Poly [(±)-2-(sec-butyl) aniline]/silica-supported perchloric acid composites were synthesized by combination of poly[(±)-2-sec-butylaniline] base (PSBA) and the silica-supported perchloric acid (SSPA) as dopant solid acid in solid-state. The X-ray photoelectron spectroscopy (XPS) and CHNS results confirm nigraniline oxidation state and complete doping for composites (about 75%) and non-complete for the PSBA·HCl salt (about 49%). The conductivity of samples was (≈0.07 S/cm) in agreement with the percent of doping obtained of the XPS analysis. Also, contact resistance was determined by circular-TLM measurement. The morphology of samples by the scanning electron microscopy (SEM) and their coating were investigated by XPS, SEM-map and energy-dispersive X-ray spectroscopy (EDX). The key benefits of this work are the preparation of conductive chiral composite with the delocalized polaron structure under green chemistry and solid-state condition, the improvement of the processability by inclusion of the 2-sec-butyl group and the use of dopant solid acid (SSPA) as dopant.

  14. ERK7 is a negative regulator of protein secretion in response to amino-acid starvation by modulating Sec16 membrane association

    PubMed Central

    Zacharogianni, Margarita; Kondylis, Vangelis; Tang, Yang; Farhan, Hesso; Xanthakis, Despina; Fuchs, Florian; Boutros, Michael; Rabouille, Catherine

    2011-01-01

    RNAi screening for kinases regulating the functional organization of the early secretory pathway in Drosophila S2 cells has identified the atypical Mitotic-Associated Protein Kinase (MAPK) Extracellularly regulated kinase 7 (ERK7) as a new modulator. We found that ERK7 negatively regulates secretion in response to serum and amino-acid starvation, in both Drosophila and human cells. Under these conditions, ERK7 turnover through the proteasome is inhibited, and the resulting higher levels of this kinase lead to a modification in a site within the C-terminus of Sec16, a key ER exit site component. This post-translational modification elicits the cytoplasmic dispersion of Sec16 and the consequent disassembly of the ER exit sites, which in turn results in protein secretion inhibition. We found that ER exit site disassembly upon starvation is TOR complex 1 (TORC1) independent, showing that under nutrient stress conditions, cell growth is not only inhibited at the transcriptional and translational levels, but also independently at the level of secretion by inhibiting the membrane flow through the early secretory pathway. These results reveal the existence of new signalling circuits participating in the complex regulation of cell growth. PMID:21847093

  15. Mutations in the selenocysteine insertion sequence–binding protein 2 gene lead to a multisystem selenoprotein deficiency disorder in humans

    PubMed Central

    Schoenmakers, Erik; Agostini, Maura; Mitchell, Catherine; Schoenmakers, Nadia; Papp, Laura; Rajanayagam, Odelia; Padidela, Raja; Ceron-Gutierrez, Lourdes; Doffinger, Rainer; Prevosto, Claudia; Luan, Jian’an; Montano, Sergio; Lu, Jun; Castanet, Mireille; Clemons, Nick; Groeneveld, Matthijs; Castets, Perrine; Karbaschi, Mahsa; Aitken, Sri; Dixon, Adrian; Williams, Jane; Campi, Irene; Blount, Margaret; Burton, Hannah; Muntoni, Francesco; O’Donovan, Dominic; Dean, Andrew; Warren, Anne; Brierley, Charlotte; Baguley, David; Guicheney, Pascale; Fitzgerald, Rebecca; Coles, Alasdair; Gaston, Hill; Todd, Pamela; Holmgren, Arne; Khanna, Kum Kum; Cooke, Marcus; Semple, Robert; Halsall, David; Wareham, Nicholas; Schwabe, John; Grasso, Lucia; Beck-Peccoz, Paolo; Ogunko, Arthur; Dattani, Mehul; Gurnell, Mark; Chatterjee, Krishna

    2010-01-01

    Selenium, a trace element that is fundamental to human health, is incorporated into some proteins as selenocysteine (Sec), generating a family of selenoproteins. Sec incorporation is mediated by a multiprotein complex that includes Sec insertion sequence–binding protein 2 (SECISBP2; also known as SBP2). Here, we describe subjects with compound heterozygous defects in the SECISBP2 gene. These individuals have reduced synthesis of most of the 25 known human selenoproteins, resulting in a complex phenotype. Azoospermia, with failure of the latter stages of spermatogenesis, was associated with a lack of testis-enriched selenoproteins. An axial muscular dystrophy was also present, with features similar to myopathies caused by mutations in selenoprotein N (SEPN1). Cutaneous deficiencies of antioxidant selenoenzymes, increased cellular ROS, and susceptibility to ultraviolet radiation–induced oxidative damage may mediate the observed photosensitivity. Reduced levels of selenoproteins in peripheral blood cells were associated with impaired T lymphocyte proliferation, abnormal mononuclear cell cytokine secretion, and telomere shortening. Paradoxically, raised ROS in affected subjects was associated with enhanced systemic and cellular insulin sensitivity, similar to findings in mice lacking the antioxidant selenoenzyme glutathione peroxidase 1 (GPx1). Thus, mutation of SECISBP2 is associated with a multisystem disorder with defective biosynthesis of many selenoproteins, highlighting their role in diverse biological processes. PMID:21084748

  16. Pancreatic SEC23B deficiency is sufficient to explain the perinatal lethality of germline SEC23B deficiency in mice.

    PubMed

    Khoriaty, Rami; Everett, Lesley; Chase, Jennifer; Zhu, Guojing; Hoenerhoff, Mark; McKnight, Brooke; Vasievich, Matthew P; Zhang, Bin; Tomberg, Kärt; Williams, John; Maillard, Ivan; Ginsburg, David

    2016-01-01

    In humans, loss of function mutations in SEC23B result in Congenital Dyserythropoietic Anemia type II (CDAII), a disease limited to defective erythroid development. Patients with two nonsense SEC23B mutations have not been reported, suggesting that complete SEC23B deficiency might be lethal. We previously reported that SEC23B-deficient mice die perinatally, exhibiting massive pancreatic degeneration and that mice with hematopoietic SEC23B deficiency do not exhibit CDAII. We now show that SEC23B deficiency restricted to the pancreas is sufficient to explain the lethality observed in mice with global SEC23B-deficiency. Immunohistochemical stains demonstrate an acinar cell defect but normal islet cells. Mammalian genomes contain two Sec23 paralogs, Sec23A and Sec23B. The encoded proteins share ~85% amino acid sequence identity. We generate mice with pancreatic SEC23A deficiency and demonstrate that these mice survive normally, exhibiting normal pancreatic weights and histology. Taken together, these data demonstrate that SEC23B but not SEC23A is essential for murine pancreatic development. We also demonstrate that two BAC transgenes spanning Sec23b rescue the lethality of mice homozygous for a Sec23b gene trap allele, excluding a passenger gene mutation as the cause of the pancreatic lethality, and indicating that the regulatory elements critical for Sec23b pancreatic function reside within the BAC transgenes. PMID:27297878

  17. Pancreatic SEC23B deficiency is sufficient to explain the perinatal lethality of germline SEC23B deficiency in mice

    PubMed Central

    Khoriaty, Rami; Everett, Lesley; Chase, Jennifer; Zhu, Guojing; Hoenerhoff, Mark; McKnight, Brooke; Vasievich, Matthew P.; Zhang, Bin; Tomberg, Kärt; Williams, John; Maillard, Ivan; Ginsburg, David

    2016-01-01

    In humans, loss of function mutations in SEC23B result in Congenital Dyserythropoietic Anemia type II (CDAII), a disease limited to defective erythroid development. Patients with two nonsense SEC23B mutations have not been reported, suggesting that complete SEC23B deficiency might be lethal. We previously reported that SEC23B-deficient mice die perinatally, exhibiting massive pancreatic degeneration and that mice with hematopoietic SEC23B deficiency do not exhibit CDAII. We now show that SEC23B deficiency restricted to the pancreas is sufficient to explain the lethality observed in mice with global SEC23B-deficiency. Immunohistochemical stains demonstrate an acinar cell defect but normal islet cells. Mammalian genomes contain two Sec23 paralogs, Sec23A and Sec23B. The encoded proteins share ~85% amino acid sequence identity. We generate mice with pancreatic SEC23A deficiency and demonstrate that these mice survive normally, exhibiting normal pancreatic weights and histology. Taken together, these data demonstrate that SEC23B but not SEC23A is essential for murine pancreatic development. We also demonstrate that two BAC transgenes spanning Sec23b rescue the lethality of mice homozygous for a Sec23b gene trap allele, excluding a passenger gene mutation as the cause of the pancreatic lethality, and indicating that the regulatory elements critical for Sec23b pancreatic function reside within the BAC transgenes. PMID:27297878

  18. Knockdown of Selenocysteine-Specific Elongation Factor in Amblyomma maculatum Alters the Pathogen Burden of Rickettsia parkeri with Epigenetic Control by the Sin3 Histone Deacetylase Corepressor Complex

    PubMed Central

    Adamson, Steven W.; Browning, Rebecca E.; Budachetri, Khemraj; Ribeiro, José M. C.; Karim, Shahid

    2013-01-01

    Selenocysteine is the 21st naturally-occurring amino acid. Selenoproteins have diverse functions and many remain uncharacterized, but they are typically associated with antioxidant activity. The incorporation of selenocysteine into the nascent polypeptide chain recodes the TGA stop codon and this process depends upon a number of essential factors including the selenocysteine elongation factor (SEF). The transcriptional expression of SEF did not change significantly in tick midguts throughout the blood meal, but decreased in salivary glands to 20% at the end of the fast feeding phase. Since selenoprotein translation requires this specialized elongation factor, we targeted this gene for knockdown by RNAi to gain a global view of the role selenoproteins play in tick physiology. We found no significant differences in tick engorgement and embryogenesis but detected no antioxidant capacity in tick saliva. The transcriptional profile of selenoproteins in R. parkeri-infected Amblyomma maculatum revealed declined activity of selenoprotein M and catalase and increased activity of selenoprotein O, selenoprotein S, and selenoprotein T. Furthermore, the pathogen burden was significantly altered in SEF-knockdowns. We then determined the global impact of SEF-knockdown by RNA-seq, and mapped huge shifts in secretory gene expression that could be the result of downregulation of the Sin3 histone deacetylase corepressor complex. PMID:24282621

  19. Diet-Induced Obesity in the Selenocysteine Lyase Knockout Mouse

    PubMed Central

    Gilman, Christy L.; Hashimoto, Ann C.; Ogawa-Wong, Ashley N.; Berry, Marla J.

    2015-01-01

    Abstract Aims: Selenocysteine lyase (Scly) mediates selenocysteine decomposition. It was previously demonstrated that, upon adequate caloric intake (12% kcal fat) and selenium deficiency, disruption of Scly in mice leads to development of metabolic syndrome. In this study, we investigate the effect of a high-fat (45% kcal) selenium-adequate diet in Scly knockout (KO) mice on development of metabolic syndrome. Involvement of selenoproteins in energy metabolism after Scly disruption was also examined in vitro in the murine hepatoma cell line, Hepa1-6, following palmitate treatment. Results: Scly KO mice were more susceptible to diet-induced obesity than their wild-type counterparts after feeding a high-fat selenium-adequate diet. Scly KO mice had aggravated hyperinsulinemia, hypercholesterolemia, glucose, and insulin intolerance, but unchanged inflammatory cytokines and expression of most selenoproteins, except increased serum selenoprotein P (Sepp1). Scly KO mice also exhibited enhanced hepatic levels of pyruvate and enzymes involved in the regulation of pyruvate cycling, such as pyruvate carboxylase (Pcx) and pyruvate dehydrogenase (Pdh). However, in vitro silencing of Scly in Hepa1-6 cells led to diminished Sepp1 expression, and concomitant palmitate treatment decreased Pdh expression. Innovation: The role of selenium in lipid metabolism is recognized, but specific selenium-dependent mechanisms leading to obesity are unclear. This study uncovers that Scly has a remarkable effect on obesity and metabolic syndrome development triggered by high-fat exposure, independent of the expression of most selenoproteins. Conclusion: Diet-induced obesity in Scly KO mice is aggravated, with effects on pyruvate levels and consequent activation of energy metabolism independent of selenoprotein levels. Antioxid. Redox Signal. 23, 761–774. PMID:26192035

  20. Crystallization and preliminary X-ray crystallographic analysis of bacterial tRNA(Sec) in complex with seryl-tRNA synthetase.

    PubMed

    Itoh, Yuzuru; Sekine, Shun Ichi; Yokoyama, Shigeyuki

    2012-06-01

    Selenocysteine (Sec) is translationally incorporated into proteins in response to the UGA codon. The tRNA specific to Sec (tRNA(Sec)) is first ligated with serine by seryl-tRNA synthetase (SerRS). To elucidate the tertiary structure of bacterial tRNA(Sec) and its specific interaction with SerRS, the bacterial tRNA(Sec) from Aquifex aeolicus was crystallized as the heterologous complex with the archaeal SerRS from Methanopyrus kandleri. Although X-ray diffraction by crystals of tRNA(Sec) in complex with wild-type SerRS was rather poor (to 5.7 Å resolution), the resolution was improved by introducing point mutations targeting the crystal-packing interface. Heavy-atom labelling also contributed to resolution improvement. A 3.2 Å resolution diffraction data set for phase determination was obtained from a K(2)Pt(CN)(4)-soaked crystal. PMID:22684069

  1. Crystal structure of mammalian selenocysteine-dependent iodothyronine deiodinase suggests a peroxiredoxin-like catalytic mechanism

    PubMed Central

    Schweizer, Ulrich; Schlicker, Christine; Braun, Doreen; Köhrle, Josef; Steegborn, Clemens

    2014-01-01

    Local levels of active thyroid hormone (3,3′,5-triiodothyronine) are controlled by the action of activating and inactivating iodothyronine deiodinase enzymes. Deiodinases are selenocysteine-dependent membrane proteins catalyzing the reductive elimination of iodide from iodothyronines through a poorly understood mechanism. We solved the crystal structure of the catalytic domain of mouse deiodinase 3 (Dio3), which reveals a close structural similarity to atypical 2-Cys peroxiredoxin(s) (Prx). The structure suggests a route for proton transfer to the substrate during deiodination and a Prx-related mechanism for subsequent recycling of the transiently oxidized enzyme. The proposed mechanism is supported by biochemical experiments and is consistent with the effects of mutations of conserved amino acids on Dio3 activity. Thioredoxin and glutaredoxin reduce the oxidized Dio3 at physiological concentrations, and dimerization appears to activate the enzyme by displacing an autoinhibitory loop from the iodothyronine binding site. Deiodinases apparently evolved from the ubiquitous Prx scaffold, and their structure and catalytic mechanism reconcile a plethora of partly conflicting data reported for these enzymes. PMID:25002520

  2. Cotranslational insertion of selenocysteine into formate dehydrogenase from Escherichia coli directed by a UGA codon

    SciTech Connect

    Zinoni, F.; Birkmann, A.; Leinfelder, W.; Boeck, A.

    1987-05-01

    The structural gene (fdhF) for the 80-kDa selenopolypeptide of formate dehydrogenase from Escherichia coli contains an in-frame UGA codon at amino acid position 140 that is translated. Translation of gene fusions between N-terminal parts of fdhF with lacZ depends on the availability of selenium in the medium when the hybrid gene contains the UGA codon; it is independent of the presence of selenium when an fdhF portion upstream of the UGA position is fused to lacZ. Transcription does not require the presence of selenium in either case. By localized mutagenesis, the UGA codon was converted into serine (UCA) and cysteine (UGC and UGU) codons. Each mutagion relieved the selenium dependency of fdhF mRNA translation. Selenium incorporation was completely abolished in the case of the UCA insertion and was reduced to about 10% when the UGA was replaced by a cysteine codon. Insertion of UCA yielded an inactive fdhF gene product, while insertion of UGC and UGU resulted in polypeptides with lowered activities as components in the system formerly known as formate hydrogenlyase. Altogether the results indicate that the UGA codon at position 140 directs the cotranslational insertion of selenocysteine into the fdhF polypeptide chain.

  3. Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine.

    PubMed

    Aldag, Caroline; Gromov, Igor A; García-Rubio, Inés; von Koenig, Konstanze; Schlichting, Ilme; Jaun, Bernhard; Hilvert, Donald

    2009-04-01

    The unique monooxygenase activity of cytochrome P450cam has been attributed to coordination of a cysteine thiolate to the heme cofactor. To investigate this interaction, we replaced cysteine with the more electron-donating selenocysteine. Good yields of the selenoenzyme were obtained by bacterial expression of an engineered gene containing the requisite UGA codon for selenocysteine and a simplified yet functional selenocysteine insertion sequence (SECIS). The sulfur-to-selenium substitution subtly modulates the structural, electronic, and catalytic properties of the enzyme. Catalytic activity decreases only 2-fold, whereas substrate oxidation becomes partially uncoupled from electron transfer, implying a more complex role for the axial ligand than generally assumed. PMID:19293375

  4. 46 CFR Sec. 9 - Communications.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... CHANGE IN USE OR IN TERMS GOVERNING UTILIZATION OF PORT FACILITIES Sec. 9 Communications. Communications concerning this part should refer to 32A CFR part 1901 and should be addressed to the Maritime Administrator... 46 Shipping 8 2011-10-01 2011-10-01 false Communications. Sec. 9 Section 9 Shipping...

  5. 46 CFR Sec. 3 - Specifications.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

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  6. 46 CFR Sec. 9 - Payment.

    Code of Federal Regulations, 2010 CFR

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  7. 46 CFR Sec. 9 - Payment.

    Code of Federal Regulations, 2012 CFR

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  8. 46 CFR Sec. 3 - Specifications.

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  9. 46 CFR Sec. 3 - Specifications.

    Code of Federal Regulations, 2010 CFR

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  10. 46 CFR Sec. 9 - Payment.

    Code of Federal Regulations, 2011 CFR

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    ... 46 Shipping 8 2011-10-01 2011-10-01 false Payment. Sec. 9 Section 9 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURE FOR ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 9...

  11. 46 CFR Sec. 9 - Payment.

    Code of Federal Regulations, 2014 CFR

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  12. 46 CFR Sec. 9 - Payment.

    Code of Federal Regulations, 2013 CFR

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  13. 46 CFR Sec. 3 - Specifications.

    Code of Federal Regulations, 2013 CFR

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  14. 46 CFR Sec. 3 - Specifications.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

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  15. 46 CFR Sec. 13 - Insurance.

    Code of Federal Regulations, 2011 CFR

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  16. 46 CFR Sec. 13 - Insurance.

    Code of Federal Regulations, 2010 CFR

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  17. 46 CFR Sec. 5 - Accounting.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Accounting. Sec. 5 Section 5 Shipping MARITIME... Sec. 5 Accounting. The General Agent shall record the amounts of compensation paid from the NSA... Accounting Office, at which time the Maritime Administration will take custody of the records....

  18. 46 CFR Sec. 5 - Accounting.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 8 2012-10-01 2012-10-01 false Accounting. Sec. 5 Section 5 Shipping MARITIME... Sec. 5 Accounting. The General Agent shall record the amounts of compensation paid from the NSA... Accounting Office, at which time the Maritime Administration will take custody of the records....

  19. 46 CFR Sec. 5 - Accounting.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 8 2014-10-01 2014-10-01 false Accounting. Sec. 5 Section 5 Shipping MARITIME... Sec. 5 Accounting. The General Agent shall record the amounts of compensation paid from the NSA... Accounting Office, at which time the Maritime Administration will take custody of the records....

  20. 46 CFR Sec. 5 - Accounting.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Accounting. Sec. 5 Section 5 Shipping MARITIME... Sec. 5 Accounting. The General Agent shall record the amounts of compensation paid from the NSA... Accounting Office, at which time the Maritime Administration will take custody of the records....

  1. 46 CFR Sec. 5 - Accounting.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 8 2013-10-01 2013-10-01 false Accounting. Sec. 5 Section 5 Shipping MARITIME... Sec. 5 Accounting. The General Agent shall record the amounts of compensation paid from the NSA... Accounting Office, at which time the Maritime Administration will take custody of the records....

  2. 46 CFR Sec. 9 - Communications.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... CHANGE IN USE OR IN TERMS GOVERNING UTILIZATION OF PORT FACILITIES Sec. 9 Communications. Communications concerning this part should refer to 32A CFR part 1901 and should be addressed to the Maritime Administrator... 46 Shipping 8 2012-10-01 2012-10-01 false Communications. Sec. 9 Section 9 Shipping...

  3. 46 CFR Sec. 9 - Communications.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CHANGE IN USE OR IN TERMS GOVERNING UTILIZATION OF PORT FACILITIES Sec. 9 Communications. Communications concerning this part should refer to 32A CFR part 1901 and should be addressed to the Maritime Administrator... 46 Shipping 8 2014-10-01 2014-10-01 false Communications. Sec. 9 Section 9 Shipping...

  4. 46 CFR Sec. 12 - Audit.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Audit. Sec. 12 Section 12 Shipping MARITIME... TRANSACTIONS UNDER AGENCY AGREEMENTS Reports and Audit Sec. 12 Audit. (a) The owner will audit as currently as possible subsequent to audit by the agent, all documents relating to the activities, maintenance...

  5. Protection of rat liver against hepatic ischemia-reperfusion injury by a novel selenocysteine-containing 7-mer peptide

    PubMed Central

    Jiang, Qianqian; Pan, Yu; Cheng, Yupeng; Li, Huiling; Li, Hui

    2016-01-01

    Hepatic ischemia-reperfusion (I-R) injury causes acute organ damage or dysfunction, and remains a problem for liver transplantation. In the I-R phase, the generation of reactive oxygen species aggravates the injury. In the current study, a novel selenocysteine-containing 7-mer peptide (H-Arg-Sec-Gly-Arg-Asn-Ala-Gln-OH) was constructed to imitate the active site of an antioxidant enzyme, glutathione peroxidase (GPX). The 7-mer peptide which has a lower molecular weight, and improved water-solubility, higher stability and improved cell membrane permeability compared with other GPX mimics. Its GPX activity reached 13 U/µmol, which was 13 times that of ebselen (a representative GPX mimic). The effect of this GPX mimic on I-R injury of the liver was assessed in rats. The 7-mer peptide significantly inhibited the increase in serum hepatic amino-transferases, tissue malondialdehyde, nitric oxide contents, myeloperoxidase activity and decrease of GPX activity compared with I-R tissue. Following treatment with the 7-mer peptide, the expression of B-cell CLL/lymphoma-2 (Bcl-2) was significantly upregulated at the mRNA and protein level compared with the I-R group, as determined by reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. By contrast, Bcl-2 associated X protein (Bax) was downregulated by the 7-mer peptide compared the I-R group. Histological and ultrastructural changes of the rat liver tissue were also compared among the experimental groups. The results of the current study suggest that the 7-mer peptide protected the liver against hepatic I-R injury via suppression of oxygen-derived free radicals and regulation of Bcl-2 and Bax expression, which are involved in the apoptosis of liver cells. The findings of the present study will further the investigation of the 7-mer peptide as an effective therapeutic agent in hepatic I-R injury. PMID:27431272

  6. Covalent heme attachment to the protein in human heme oxygenase-1 with selenocysteine replacing the His25 proximal iron ligand.

    PubMed

    Jiang, Yongying; Trnka, Michael J; Medzihradszky, Katalin F; Ouellet, Hugues; Wang, Yongqiang; Ortiz de Montellano, Paul R

    2009-03-01

    To characterize heme oxygenase with a selenocysteine (SeCys) as the proximal iron ligand, we have expressed truncated human heme oxygenase-1 (hHO-1) His25Cys, in which Cys-25 is the only cysteine, in the Escherichia coli cysteine auxotroph strain BL21(DE3)cys. Selenocysteine incorporation into the protein was demonstrated by both intact protein mass measurement and mass spectrometric identification of the selenocysteine-containing tryptic peptide. One selenocysteine was incorporated into approximately 95% of the expressed protein. Formation of an adduct with Ellman's reagent (DTNB) indicated that the selenocysteine in the expressed protein was in the reduced state. The heme-His25SeCys hHO-1 complex could be prepared by either (a) supplementing the overexpression medium with heme, or (b) reconstituting the purified apoprotein with heme. Under reducing conditions in the presence of imidazole, a covalent bond is formed by addition of the selenocysteine residue to one of the heme vinyl groups. No covalent bond is formed when the heme is replaced by mesoheme, in which the vinyls are replaced by ethyl groups. These results, together with our earlier demonstration that external selenolate ligands can transfer an electron to the iron [Y. Jiang, P.R. Ortiz de Montellano, Inorg. Chem. 47 (2008) 3480-3482 ], indicate that a selenyl radical is formed in the hHO-1 His25SeCys mutant that adds to a heme vinyl group. PMID:19135260

  7. 46 CFR Sec. 13 - Insurance.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 13 Insurance. Article 9 of the NSA-LUMPSUMREP Contract sets forth the Contractor's liabilities and obligations...

  8. 46 CFR Sec. 2 - Terms.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 2 Terms. The terms employed in this order shall have the same meaning as those contained in NSA Order No. 47....

  9. 46 CFR Sec. 2 - Terms.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 2 Terms. The terms employed in this order shall have the same meaning as those contained in NSA Order No. 47....

  10. 46 CFR Sec. 13 - Insurance.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 13 Insurance. Article 9 of the NSA-LUMPSUMREP Contract sets forth the Contractor's liabilities and obligations...

  11. 46 CFR Sec. 13 - Insurance.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 13 Insurance. Article 9 of the NSA-LUMPSUMREP Contract sets forth the Contractor's liabilities and obligations...

  12. 46 CFR Sec. 2 - Terms.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 2 Terms. The terms employed in this order shall have the same meaning as those contained in NSA Order No. 47....

  13. 46 CFR Sec. 2 - Terms.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 2 Terms. The terms employed in this order shall have the same meaning as those contained in NSA Order No. 47....

  14. 46 CFR Sec. 2 - Terms.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 2 Terms. The terms employed in this order shall have the same meaning as those contained in NSA Order No. 47....

  15. Expression of Selenoproteins Is Maintained in Mice Carrying Mutations in SECp43, the tRNA Selenocysteine 1 Associated Protein (Trnau1ap)

    PubMed Central

    Carlson, Bradley A.; Fradejas, Noelia; Günter, Paul; Braun, Doreen; Southon, Eileen; Tessarollo, Lino; Hatfield, Dolph L.; Schweizer, Ulrich

    2015-01-01

    Selenocysteine tRNA 1 associated protein (Trnau1ap) has been characterized as a tRNA[Ser]Sec-binding protein of 43 kDa, hence initially named SECp43. Previous studies reported its presence in complexes containing tRNA[Ser]Sec implying a role of SECp43 as a co-factor in selenoprotein expression. We generated two conditionally mutant mouse models targeting exons 3+4 and exons 7+8 eliminating parts of the first RNA recognition motif or of the tyrosine-rich domain, respectively. Constitutive inactivation of exons 3+4 of SECp43 apparently did not affect the mice or selenoprotein expression in several organs. Constitutive deletion of exons 7+8 was embryonic lethal. We therefore generated hepatocyte-specific Secp43 knockout mice and characterized selenoprotein expression in livers of mutant mice. We found no significant changes in the levels of 75Se-labelled hepatic proteins, selenoprotein levels as determined by Western blot analysis, enzymatic activity or selenoprotein mRNA abundance. The methylation pattern of tRNA[Ser]Sec remained unchanged. Truncated Secp43 Δ7,8mRNA increased in Secp43-mutant livers suggesting auto-regulation of Secp43 mRNA abundance. We found no signs of liver damage in Secp433-mutant mice, but neuron-specific deletion of exons 7+8 impaired motor performance, while not affecting cerebral selenoprotein expression or cerebellar development. These findings suggest that the targeted domains in the SECp43 protein are not essential for selenoprotein biosynthesis in hepatocytes and neurons. Whether the remaining second RNA recognition motif plays a role in selenoprotein biosynthesis and which other cellular process depends on SECp43 remains to be determined. PMID:26043259

  16. Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation.

    PubMed

    Allen, William John; Corey, Robin Adam; Oatley, Peter; Sessions, Richard Barry; Baldwin, Steve A; Radford, Sheena E; Tuma, Roman; Collinson, Ian

    2016-01-01

    The essential process of protein secretion is achieved by the ubiquitous Sec machinery. In prokaryotes, the drive for translocation comes from ATP hydrolysis by the cytosolic motor-protein SecA, in concert with the proton motive force (PMF). However, the mechanism through which ATP hydrolysis by SecA is coupled to directional movement through SecYEG is unclear. Here, we combine all-atom molecular dynamics (MD) simulations with single molecule FRET and biochemical assays. We show that ATP binding by SecA causes opening of the SecY-channel at long range, while substrates at the SecY-channel entrance feed back to regulate nucleotide exchange by SecA. This two-way communication suggests a new, unifying 'Brownian ratchet' mechanism, whereby ATP binding and hydrolysis bias the direction of polypeptide diffusion. The model represents a solution to the problem of transporting inherently variable substrates such as polypeptides, and may underlie mechanisms of other motors that translocate proteins and nucleic acids. PMID:27183269

  17. Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation

    PubMed Central

    Allen, William John; Corey, Robin Adam; Oatley, Peter; Sessions, Richard Barry; Radford, Sheena E; Tuma, Roman; Collinson, Ian

    2016-01-01

    The essential process of protein secretion is achieved by the ubiquitous Sec machinery. In prokaryotes, the drive for translocation comes from ATP hydrolysis by the cytosolic motor-protein SecA, in concert with the proton motive force (PMF). However, the mechanism through which ATP hydrolysis by SecA is coupled to directional movement through SecYEG is unclear. Here, we combine all-atom molecular dynamics (MD) simulations with single molecule FRET and biochemical assays. We show that ATP binding by SecA causes opening of the SecY-channel at long range, while substrates at the SecY-channel entrance feed back to regulate nucleotide exchange by SecA. This two-way communication suggests a new, unifying 'Brownian ratchet' mechanism, whereby ATP binding and hydrolysis bias the direction of polypeptide diffusion. The model represents a solution to the problem of transporting inherently variable substrates such as polypeptides, and may underlie mechanisms of other motors that translocate proteins and nucleic acids. DOI: http://dx.doi.org/10.7554/eLife.15598.001 PMID:27183269

  18. Symmetry scheme for amino acid codons

    NASA Astrophysics Data System (ADS)

    Balakrishnan, J.

    2002-02-01

    Group theoretical concepts are invoked in a specific model to explain how only twenty amino acids occur in nature out of a possible sixty four. The methods we use enable us to justify the occurrence of the recently discovered 21st amino acid selenocysteine, and also enables us to predict the possible existence of two more, as yet undiscovered amino acids.

  19. KlSEC53 is an essential Kluyveromyces lactis gene and is homologous with the SEC53 gene of Saccharomyces cerevisiae.

    PubMed

    Staneva, Dessislava; Uccelletti, Daniela; Farina, Francesca; Venkov, Pencho; Palleschi, Claudio

    2004-01-15

    Phosphomannomutase (PMM) is a key enzyme, which catalyses one of the first steps in the glycosylation pathway, the conversion of D-mannose-6-phosphate to D-mannose-1-phosphate. The latter is the substrate for the synthesis of GDP-mannose, which serves as the mannosyl donor for the glycosylation reactions in eukaryotic cells. In the yeast Saccharomyces cerevisiae PMM is encoded by the gene SEC53 (ScSEC53) and the deficiency of PMM activity leads to severe defects in both protein glycosylation and secretion. We report here on the isolation of the Kluyveromyces lactis SEC53 (KlSEC53) gene from a genomic library by virtue of its ability to complement a Saccharomyces cerevisiae sec53 mutation. The sequenced DNA fragment contained an open reading frame of 765 bp, coding for a predicted polypeptide, KlSec53p, of 254 amino acids. The KlSec53p displays a high degree of homology with phosphomannomutases from other yeast species, protozoans, plants and humans. Our results have demonstrated that KlSEC53 is the functional homologue of the ScSEC53 gene. Like ScSEC53, the KlSEC53 gene is essential for K. lactis cell viability. Phenotypic analysis of a K. lactis strain overexpressing the KlSEC53 gene revealed defects expected for impaired cell wall integrity. PMID:14745781

  20. Sec1p and Mso1p C-terminal tails cooperate with the SNAREs and Sec4p in polarized exocytosis

    PubMed Central

    Weber-Boyvat, Marion; Aro, Nina; Chernov, Konstantin G.; Nyman, Tuula; Jäntti, Jussi

    2011-01-01

    The Sec1/Munc18 protein family members perform an essential, albeit poorly understood, function in association with soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes in membrane fusion. The Saccharomyces cerevisiae Sec1p has a C-terminal tail that is missing in its mammalian homologues. Here we show that deletion of the Sec1p tail (amino acids 658–724) renders cells temperature sensitive for growth, reduces sporulation efficiency, causes a secretion defect, and abolishes Sec1p-SNARE component coimmunoprecipitation. The results show that the Sec1p tail binds preferentially ternary Sso1p-Sec9p-Snc2p complexes and it enhances ternary SNARE complex formation in vitro. The bimolecular fluorescence complementation (BiFC) assay results suggest that, in the SNARE-deficient sso2–1 Δsso1 cells, Mso1p, a Sec1p binding protein, helps to target Sec1p(1–657) lacking the C-terminal tail to the sites of secretion. The results suggest that the Mso1p C terminus is important for Sec1p(1–657) targeting. We show that, in addition to Sec1p, Mso1p can bind the Rab-GTPase Sec4p in vitro. The BiFC results suggest that Mso1p acts in close association with Sec4p on intracellular membranes in the bud. This association depends on the Sec4p guanine nucleotide exchange factor Sec2p. Our results reveal a novel binding mode between the Sec1p C-terminal tail and the SNARE complex, and suggest a role for Mso1p as an effector of Sec4p. PMID:21119007

  1. Sec1p and Mso1p C-terminal tails cooperate with the SNAREs and Sec4p in polarized exocytosis.

    PubMed

    Weber-Boyvat, Marion; Aro, Nina; Chernov, Konstantin G; Nyman, Tuula; Jäntti, Jussi

    2011-01-15

    The Sec1/Munc18 protein family members perform an essential, albeit poorly understood, function in association with soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes in membrane fusion. The Saccharomyces cerevisiae Sec1p has a C-terminal tail that is missing in its mammalian homologues. Here we show that deletion of the Sec1p tail (amino acids 658-724) renders cells temperature sensitive for growth, reduces sporulation efficiency, causes a secretion defect, and abolishes Sec1p-SNARE component coimmunoprecipitation. The results show that the Sec1p tail binds preferentially ternary Sso1p-Sec9p-Snc2p complexes and it enhances ternary SNARE complex formation in vitro. The bimolecular fluorescence complementation (BiFC) assay results suggest that, in the SNARE-deficient sso2-1 Δsso1 cells, Mso1p, a Sec1p binding protein, helps to target Sec1p(1-657) lacking the C-terminal tail to the sites of secretion. The results suggest that the Mso1p C terminus is important for Sec1p(1-657) targeting. We show that, in addition to Sec1p, Mso1p can bind the Rab-GTPase Sec4p in vitro. The BiFC results suggest that Mso1p acts in close association with Sec4p on intracellular membranes in the bud. This association depends on the Sec4p guanine nucleotide exchange factor Sec2p. Our results reveal a novel binding mode between the Sec1p C-terminal tail and the SNARE complex, and suggest a role for Mso1p as an effector of Sec4p. PMID:21119007

  2. SEC31A-ALK Fusion Gene in Lung Adenocarcinoma.

    PubMed

    Kim, Ryong Nam; Choi, Yoon-La; Lee, Mi-Sook; Lira, Maruja E; Mao, Mao; Mann, Derrick; Stahl, Joshua; Licon, Abel; Choi, So Jung; Van Vrancken, Michael; Han, Joungho; Wlodarska, Iwona; Kim, Jhingook

    2016-01-01

    Anaplastic lymphoma kinase (ALK) fusion is a common mechanism underlying pathogenesis of non-small cell lung carcinoma (NSCLC) where these rearrangements represent important diagnostic and therapeutic targets. In this study, we found a new ALK fusion gene, SEC31A-ALK, in lung carcinoma from a 53-year-old Korean man. The conjoined region in the fusion transcript was generated by the fusion of SEC31A exon 21 and ALK exon 20 by genomic rearrangement, which contributed to generation of an intact, in-frame open reading frame. SEC31A-ALK encodes a predicted fusion protein of 1,438 amino acids comprising the WD40 domain of SEC31A at the N-terminus and ALK kinase domain at the C-terminus. Fluorescence in situ hybridization studies suggested that SEC31A-ALK was generated by an unbalanced genomic rearrangement associated with loss of the 3'-end of SEC31A. This is the first report of SEC31A-ALK fusion transcript in clinical NSCLC, which could be a novel diagnostic and therapeutic target for patients with NSCLC. PMID:25715771

  3. 46 CFR Sec. 10 - Bonds.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 10 Bonds. (a... awarded work and the furnishing of the performance and payment bonds required by Article 14 of the NSA... of the NSA-LUMPSUMREP Contract, the standard form of individual performance bond (Standard Form...

  4. 46 CFR Sec. 10 - Bonds.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 10 Bonds. (a... awarded work and the furnishing of the performance and payment bonds required by Article 14 of the NSA... of the NSA-LUMPSUMREP Contract, the standard form of individual performance bond (Standard Form...

  5. 46 CFR Sec. 10 - Bonds.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 10 Bonds. (a... awarded work and the furnishing of the performance and payment bonds required by Article 14 of the NSA... of the NSA-LUMPSUMREP Contract, the standard form of individual performance bond (Standard Form...

  6. 46 CFR Sec. 10 - Bonds.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 10 Bonds. (a... awarded work and the furnishing of the performance and payment bonds required by Article 14 of the NSA... of the NSA-LUMPSUMREP Contract, the standard form of individual performance bond (Standard Form...

  7. 46 CFR Sec. 10 - Bonds.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 10 Bonds. (a... awarded work and the furnishing of the performance and payment bonds required by Article 14 of the NSA... of the NSA-LUMPSUMREP Contract, the standard form of individual performance bond (Standard Form...

  8. 46 CFR Sec. 3 - Premiums.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 8 2012-10-01 2012-10-01 false Premiums. Sec. 3 Section 3 Shipping MARITIME... Premiums. The bonds provided for shall be furnished without cost to the National Shipping Authority, but the cost of the premiums of such bonds shall be included in the overhead expense of the General Agent....

  9. 46 CFR Sec. 3 - Premiums.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Premiums. Sec. 3 Section 3 Shipping MARITIME... Premiums. The bonds provided for shall be furnished without cost to the National Shipping Authority, but the cost of the premiums of such bonds shall be included in the overhead expense of the General Agent....

  10. 46 CFR Sec. 3 - Premiums.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 8 2013-10-01 2013-10-01 false Premiums. Sec. 3 Section 3 Shipping MARITIME... Premiums. The bonds provided for shall be furnished without cost to the National Shipping Authority, but the cost of the premiums of such bonds shall be included in the overhead expense of the General Agent....

  11. 46 CFR Sec. 3 - Premiums.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Premiums. Sec. 3 Section 3 Shipping MARITIME... Premiums. The bonds provided for shall be furnished without cost to the National Shipping Authority, but the cost of the premiums of such bonds shall be included in the overhead expense of the General Agent....

  12. 46 CFR Sec. 3 - Premiums.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 8 2014-10-01 2014-10-01 false Premiums. Sec. 3 Section 3 Shipping MARITIME... Premiums. The bonds provided for shall be furnished without cost to the National Shipping Authority, but the cost of the premiums of such bonds shall be included in the overhead expense of the General Agent....

  13. Public Files of the SEC.

    ERIC Educational Resources Information Center

    Woodward, Steve

    Some 180 different forms are used by the Securities and Exchange Commission (SEC) to cover the broad range of business activities regulated by the agency. This report examines the forms that are of greatest use to those seeking information about business. These forms are grouped in the following categories: general business information,…

  14. 46 CFR Sec. 1 - Purpose.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION B-CONTROL AND UTILIZATION OF PORTS OPERATING CONTRACT Sec. 1 Purpose. This part prescribes the standard form of marine terminal contract to be entered into by the United States of America, acting by and through the Director, National Shipping Authority (NSA) of the...

  15. 46 CFR Sec. 1 - Purpose.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION B-CONTROL AND UTILIZATION OF PORTS OPERATING CONTRACT Sec. 1 Purpose. This part prescribes the standard form of marine terminal contract to be entered into by the United States of America, acting by and through the Director, National Shipping Authority (NSA) of the...

  16. Selenocysteine Positional Variants Reveal Contributions to Copper Binding From Cysteine Residues in Domains 2 And 3 of Human Copper Chaperone for Superoxide Dismutase

    SciTech Connect

    Barry, A.N.; Clark, K.M.; Otoikhian, A.; Donk, W.A.van der; Blackburn, N.J.

    2009-05-11

    The human copper chaperone for superoxide dismutase binds copper both in an Atx1-like MTCQSC motif in domain 1 and via a multinuclear cluster formed by two CXC motifs at the D3 dimer interface. The composition of the Cu(I) cluster has been investigated previously by mutagenesis of the CXC motif, and by construction of a CXU selenocysteine derivative, which has permitted XAS studies at both Cu and Se absorption edges. Here, we report the semisynthesis and spectroscopic characterization of a series of derivatives with the sequences 243-CACA, 243-CAUA, 243-UACA, and 243-UAUA in the D1 double mutant (C22AC25A) background, prepared by expressed protein ligation of Sec-containing tetrapeptides to an hCCS-243 truncation. By varying the position of the Se atom in the CXC motif, we have been able to show that Se is always bridging (2 Se-Cu) rather than terminal (1 Se-Cu). Substitution of both D3 Cys residues by Sec in the UAUA variant does not eliminate the Cu-S contribution, confirming our previous description of the cluster as most likely a Cu{sub 4}S{sub 6} species, and suggesting that D2 Cys residues contribute to the cluster. As predicted by this model, when Cys residues C141, C144, and C227 are mutated to alanine either individually or together as a triple mutant, the cluster nuclearity is dramatically attenuated. These data suggest that Cys residues in D2 of hCCS are involved in the formation, stability, and redox potential of the D3 cluster. The significance of these finding to the SOD1 thiol/disulfide oxidase activity are discussed in terms of a model in which a similar multinuclear cluster may form in the CCS-SOD heterodimer.

  17. Production of Se-methylselenocysteine in transgenic plants expressing selenocysteine methyltransferase

    PubMed Central

    Ellis, Danielle R; Sors, Thomas G; Brunk, Dennis G; Albrecht, Carrie; Orser, Cindy; Lahner, Brett; Wood, Karl V; Harris, Hugh H; Pickering, Ingrid J; Salt, David E

    2004-01-01

    Background It has become increasingly evident that dietary Se plays a significant role in reducing the incidence of lung, colorectal and prostate cancer in humans. Different forms of Se vary in their chemopreventative efficacy, with Se-methylselenocysteine being one of the most potent. Interestingly, the Se accumulating plant Astragalus bisulcatus (Two-grooved poison vetch) contains up to 0.6% of its shoot dry weight as Se-methylselenocysteine. The ability of this Se accumulator to biosynthesize Se-methylselenocysteine provides a critical metabolic shunt that prevents selenocysteine and selenomethionine from entering the protein biosynthetic machinery. Such a metabolic shunt has been proposed to be vital for Se tolerance in A. bisulcatus. Utilization of this mechanism in other plants may provide a possible avenue for the genetic engineering of Se tolerance in plants ideally suited for the phytoremediation of Se contaminated land. Here, we describe the overexpression of a selenocysteine methyltransferase from A. bisulcatus to engineer Se-methylselenocysteine metabolism in the Se non-accumulator Arabidopsis thaliana (Thale cress). Results By over producing the A. bisulcatus enzyme selenocysteine methyltransferase in A. thaliana, we have introduced a novel biosynthetic ability that allows the non-accumulator to accumulate Se-methylselenocysteine and γ-glutamylmethylselenocysteine in shoots. The biosynthesis of Se-methylselenocysteine in A. thaliana also confers significantly increased selenite tolerance and foliar Se accumulation. Conclusion These results demonstrate the feasibility of developing transgenic plant-based production of Se-methylselenocysteine, as well as bioengineering selenite resistance in plants. Selenite resistance is the first step in engineering plants that are resistant to selenate, the predominant form of Se in the environment. PMID:15005814

  18. Protection of rat liver against hepatic ischemia-reperfusion injury by a novel selenocysteine-containing 7-mer peptide.

    PubMed

    Jiang, Qianqian; Pan, Yu; Cheng, Yupeng; Li, Huiling; Li, Hui

    2016-09-01

    Hepatic ischemia-reperfusion (I-R) injury causes acute organ damage or dysfunction, and remains a problem for liver transplantation. In the I-R phase, the generation of reactive oxygen species aggravates the injury. In the current study, a novel selenocysteine-containing 7‑mer peptide (H-Arg-Sec-Gly-Arg-Asn-Ala-Gln-OH) was constructed to imitate the active site of an antioxidant enzyme, glutathione peroxidase (GPX). The 7‑mer peptide which has a lower molecular weight, and improved water‑solubility, higher stability and improved cell membrane permeability compared with other GPX mimics. Its GPX activity reached 13 U/µmol, which was 13 times that of ebselen (a representative GPX mimic). The effect of this GPX mimic on I‑R injury of the liver was assessed in rats. The 7‑mer peptide significantly inhibited the increase in serum hepatic amino‑transferases, tissue malondialdehyde, nitric oxide contents, myeloperoxidase activity and decrease of GPX activity compared with I‑R tissue. Following treatment with the 7‑mer peptide, the expression of B‑cell CLL/lymphoma‑2 (Bcl‑2) was significantly upregulated at the mRNA and protein level compared with the I‑R group, as determined by reverse transcription‑polymerase chain reaction and immunohistochemistry, respectively. By contrast, Bcl‑2 associated X protein (Bax) was downregulated by the 7‑mer peptide compared the I‑R group. Histological and ultrastructural changes of the rat liver tissue were also compared among the experimental groups. The results of the current study suggest that the 7‑mer peptide protected the liver against hepatic I‑R injury via suppression of oxygen‑derived free radicals and regulation of Bcl‑2 and Bax expression, which are involved in the apoptosis of liver cells. The findings of the present study will further the investigation of the 7-mer peptide as an effective therapeutic agent in hepatic I-R injury. PMID:27431272

  19. The Basis of Asymmetry in the SecA-SecB Complex

    PubMed Central

    Suo, Yuying; Hardy, Simon J. S.; Randall, Linda L.

    2015-01-01

    During export in Escherichia coli, SecB, a homotetramer, structurally organized as a dimer of dimers, forms a complex with two protomers of SecA, which is the ATPase that provides energy to transfer a precursor polypeptide through the membrane via the SecYEG translocon. There are two areas of contact on SecB that stabilize the SecA:SecB complex: one, the flat sides of the SecB tetramer and the second, the C-terminal thirteen residues of SecB. These contacts within the complex are distributed asymmetrically. Breaking contact between SecA and the sides of SecB results in release of only one protomer of SecA yielding a complex of stoichiometry SecA1:SecB4. This complex mediates export; however, the coupling of ATP hydrolysis to movements of the precursor through the translocon is much less efficient than the coupling by the SecA2:SecB4 complex. Here we used heterotetrameric species of SecB to understand the source of the asymmetry in the contacts and its role in the functioning of the complex. The model of interactions presented suggests a way that binding between SecA and SecB might decrease the affinity of precursor polypeptides for SecB and facilitate the transfer to SecA. PMID:25534082

  20. Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1.

    PubMed

    Morgera, Francesca; Sallah, Margaret R; Dubuke, Michelle L; Gandhi, Pallavi; Brewer, Daniel N; Carr, Chavela M; Munson, Mary

    2012-01-01

    Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function-it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6-Sec1 interaction is exclusive of Sec6-Sec9 but compatible with Sec6-exocyst assembly. In contrast, the Sec6-exocyst interaction is incompatible with Sec6-Sec9. Therefore, upon vesicle arrival, Sec6 is proposed to release Sec9 in favor of Sec6-exocyst assembly and to simultaneously recruit Sec1 to sites of secretion for coordinated SNARE complex formation and membrane fusion. PMID:22114349

  1. A tRNA-dependent cysteine biosynthesis enzyme recognizes the selenocysteine-specific tRNA in Escherichia coli

    PubMed Central

    Yuan, Jing; Hohn, Michael J.; Sherrer, R. Lynn; Palioura, Sotiria; Su, Dan; Söll, Dieter

    2010-01-01

    The essential methanogen enzyme Sep-tRNA:Cys-tRNA synthase (SepCysS) converts O-phosphoseryl-tRNACys (Sep-tRNACys) into Cys-tRNACys in the presence of a sulfur donor. Likewise, Sep-tRNA:Sec-tRNA synthase (SepSecS) converts O-phosphoseryl-tRNASec (Sep-tRNASec) to selenocysteinyl-tRNASec (Sec-tRNASec) using a selenium donor. While the Sep moiety of the aminoacyl-tRNA substrates is the same in both reactions, tRNACys and tRNASec differ greatly in sequence and structure. In an Escherichia coli genetic approach that tests for formate dehydrogenase activity in the absence of selenium donor we show that SeptRNASec is a substrate for SepCysS. Since Sec and Cys are the only active site amino acids known to sustain FDH activity, we conclude that SepCysS converts Sep-tRNASec to Cys-tRNASec, and that Sep is crucial for SepCysS recognition. PMID:20493852

  2. Determination of seleno-amino acids bound to proteins in extra virgin olive oils.

    PubMed

    Torres, Sabier; Gil, Raul; Silva, María Fernanda; Pacheco, Pablo

    2016-04-15

    An analytical method has been developed to determine seleno-amino acids in proteins extracted from extra virgin olive oils (EVOOs). Different aqueous/organic solvents were tested to isolate proteins, an acetone:n-hexane combination being the best protein precipitant. In a first dimension chromatography, extracted proteins were analysed by size exclusion chromatography (SEC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) to identify S and Se associations as proteins marker. Two fractions of 66 kDa (A) and 443 kDa (B) were identified. These fractions were submitted to microwave-assisted acid hydrolysis (MAAH) to release seleno-amino acids. In a second dimension chromatography seleno-amino acids were determined by reversed-phase chromatography (RPC) coupled to ICP-MS. Seleno-methylselenocysteine was determined with values ranging from 1.03-2.03±0.2 μg kg(-1) and selenocysteine at a concentration of 1.47±0.1 μg kg(-1). Variations of protein and seleno-amino acid concentrations were observed between EVOO varieties, contributing to EVOO cultivar differentiation. PMID:26616967

  3. Unlocking the Bacterial SecY Translocon

    PubMed Central

    Corey, Robin A.; Allen, William J.; Komar, Joanna; Masiulis, Simonas; Menzies, Sam; Robson, Alice; Collinson, Ian

    2016-01-01

    Summary The Sec translocon performs protein secretion and membrane protein insertion at the plasma membrane of bacteria and archaea (SecYEG/β), and the endoplasmic reticular membrane of eukaryotes (Sec61). Despite numerous structures of the complex, the mechanism underlying translocation of pre-proteins, driven by the ATPase SecA in bacteria, remains unresolved. Here we present a series of biochemical and computational analyses exploring the consequences of signal sequence binding to SecYEG. The data demonstrate that a signal sequence-induced movement of transmembrane helix 7 unlocks the translocon and that this conformational change is communicated to the cytoplasmic faces of SecY and SecE, involved in SecA binding. Our findings progress the current understanding of the dynamic action of the translocon during the translocation initiation process. The results suggest that the converging effects of the signal sequence and SecA at the cytoplasmic face of SecYEG are decisive for the intercalation and translocation of pre-protein through the SecY channel. PMID:26973090

  4. Multitasking SecB chaperones in bacteria

    PubMed Central

    Sala, Ambre; Bordes, Patricia; Genevaux, Pierre

    2014-01-01

    Protein export in bacteria is facilitated by the canonical SecB chaperone, which binds to unfolded precursor proteins, maintains them in a translocation competent state and specifically cooperates with the translocase motor SecA to ensure their proper targeting to the Sec translocon at the cytoplasmic membrane. Besides its key contribution to the Sec pathway, SecB chaperone tasking is critical for the secretion of the Sec-independent heme-binding protein HasA and actively contributes to the cellular network of chaperones that control general proteostasis in Escherichia coli, as judged by the significant interplay found between SecB and the trigger factor, DnaK and GroEL chaperones. Although SecB is mainly a proteobacterial chaperone associated with the presence of an outer membrane and outer membrane proteins, secB-like genes are also found in Gram-positive bacteria as well as in certain phages and plasmids, thus suggesting alternative functions. In addition, a SecB-like protein is also present in the major human pathogen Mycobacterium tuberculosis where it specifically controls a stress-responsive toxin–antitoxin system. This review focuses on such very diverse chaperone functions of SecB, both in E. coli and in other unrelated bacteria. PMID:25538690

  5. Functional characterization of the eukaryotic SECIS elements which direct selenocysteine insertion at UGA codons.

    PubMed Central

    Berry, M J; Banu, L; Harney, J W; Larsen, P R

    1993-01-01

    We investigated the requirements for selenocysteine insertion at single or multiple UGA codons in eukaryotic selenoproteins. Two functional SECIS elements were identified in the 3' untranslated region of the rat selenoprotein P mRNA, with predicted stem-loops and critical nucleotides similar to those in the SECIS elements in the type I iodothyronine 5' deiodinase (5'DI) and glutathione peroxidase selenoprotein mRNAs. Site-directed mutational analyses of three SECIS elements confirmed that conserved nucleotides in the loop and in unpaired regions of the stem are critical for activity. This indicates that multiple contact sites are required for SECIS function. Stop codon function at any of five out-of-context UGA codons in the 5'DI mRNA was suppressed by SECIS elements from the 5'DI or selenoprotein P genes linked downstream. Thus, the presence of SECIS elements in eukaryotic selenoprotein mRNAs permits complete flexibility in UGA codon position. Images PMID:8344267

  6. The American cranberry mitochondrial genome reveals the presence of selenocysteine (tRNA-Sec and SECIS) insertion machinery in land plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The American cranberry (Vaccinium macrocarpon Ait.) mitochondrial genome was assembled and reconstructed from whole genome 454 Roche GS-FLX and Illumina shotgun sequences. Compared with other Asterids, the reconstruction of the genome revealed an average size mitochondrion (459,678 nt) with comparat...

  7. SecA: a potential antimicrobial target.

    PubMed

    Chaudhary, Arpana S; Chen, Weixuan; Jin, Jinshan; Tai, Phang C; Wang, Binghe

    2015-01-01

    There is a consensus in the medical profession of the pressing need for novel antimicrobial agents due to issues related to drug resistance. In practice, solutions to this problem to a large degree lie with the identification of new and vital targets in bacteria and subsequently designing their inhibitors. We consider SecA a very promising antimicrobial target. In this review, we compile and analyze information available on SecA to show that inhibition of SecA has a multitude of consequences. Furthermore, we discuss issues critical to the design and evaluation of SecA inhibitors. PMID:26062397

  8. SecA: a potential antimicrobial target

    PubMed Central

    Chaudhary, Arpana S; Chen, Weixuan; Jin, Jinshan; Tai, Phang C; Wang, Binghe

    2015-01-01

    There is a consensus in the medical profession of the pressing need for novel antimicrobial agents due to issues related to drug resistance. In practice, solutions to this problem to a large degree lie with the identification of new and vital targets in bacteria and subsequently designing their inhibitors. We consider SecA a very promising antimicrobial target. In this review, we compile and analyze information available on SecA to show that inhibition of SecA has a multitude of consequences. Furthermore, we discuss issues critical to the design and evaluation of SecA inhibitors. PMID:26062397

  9. Anomalous behavior of water inside the SecY translocon.

    PubMed

    Capponi, Sara; Heyden, Matthias; Bondar, Ana-Nicoleta; Tobias, Douglas J; White, Stephen H

    2015-07-21

    The heterotrimeric SecY translocon complex is required for the cotranslational assembly of membrane proteins in bacteria and archaea. The insertion of transmembrane (TM) segments during nascent-chain passage through the translocon is generally viewed as a simple partitioning process between the water-filled translocon and membrane lipid bilayer, suggesting that partitioning is driven by the hydrophobic effect. Indeed, the apparent free energy of partitioning of unnatural aliphatic amino acids on TM segments is proportional to accessible surface area, which is a hallmark of the hydrophobic effect [Öjemalm K, et al. (2011) Proc Natl Acad Sci USA 108(31):E359-E364]. However, the apparent partitioning solvation parameter is less than one-half the value expected for simple bulk partitioning, suggesting that the water in the translocon departs from bulk behavior. To examine the state of water in a SecY translocon complex embedded in a lipid bilayer, we carried out all-atom molecular-dynamics simulations of the Pyrococcus furiosus SecYE, which was determined to be in a "primed" open state [Egea PF, Stroud RM (2010) Proc Natl Acad Sci USA 107(40):17182-17187]. Remarkably, SecYE remained in this state throughout our 450-ns simulation. Water molecules within SecY exhibited anomalous diffusion, had highly retarded rotational dynamics, and aligned their dipoles along the SecY transmembrane axis. The translocon is therefore not a simple water-filled pore, which raises the question of how anomalous water behavior affects the mechanism of translocon function and, more generally, the partitioning of hydrophobic molecules. Because large water-filled cavities are found in many membrane proteins, our findings may have broader implications. PMID:26139523

  10. Anomalous behavior of water inside the SecY translocon

    PubMed Central

    Capponi, Sara; Heyden, Matthias; Bondar, Ana-Nicoleta; Tobias, Douglas J.; White, Stephen H.

    2015-01-01

    The heterotrimeric SecY translocon complex is required for the cotranslational assembly of membrane proteins in bacteria and archaea. The insertion of transmembrane (TM) segments during nascent-chain passage through the translocon is generally viewed as a simple partitioning process between the water-filled translocon and membrane lipid bilayer, suggesting that partitioning is driven by the hydrophobic effect. Indeed, the apparent free energy of partitioning of unnatural aliphatic amino acids on TM segments is proportional to accessible surface area, which is a hallmark of the hydrophobic effect [Öjemalm K, et al. (2011) Proc Natl Acad Sci USA 108(31):E359–E364]. However, the apparent partitioning solvation parameter is less than one-half the value expected for simple bulk partitioning, suggesting that the water in the translocon departs from bulk behavior. To examine the state of water in a SecY translocon complex embedded in a lipid bilayer, we carried out all-atom molecular-dynamics simulations of the Pyrococcus furiosus SecYE, which was determined to be in a “primed” open state [Egea PF, Stroud RM (2010) Proc Natl Acad Sci USA 107(40):17182–17187]. Remarkably, SecYE remained in this state throughout our 450-ns simulation. Water molecules within SecY exhibited anomalous diffusion, had highly retarded rotational dynamics, and aligned their dipoles along the SecY transmembrane axis. The translocon is therefore not a simple water-filled pore, which raises the question of how anomalous water behavior affects the mechanism of translocon function and, more generally, the partitioning of hydrophobic molecules. Because large water-filled cavities are found in many membrane proteins, our findings may have broader implications. PMID:26139523

  11. Sec-containing TrxR1 is essential for self-sufficiency of cells by control of glucose-derived H2O2

    PubMed Central

    Peng, X; Mandal, P K; Kaminskyy, V O; Lindqvist, A; Conrad, M; Arnér, E S J

    2014-01-01

    It is commonly recognized that diabetic complications involve increased oxidative stress directly triggered by hyperglycemia. The most important cellular protective systems against such oxidative stress have yet remained unclear. Here we show that the selenoprotein thioredoxin reductase 1 (TrxR1), encoded by the Txnrd1 gene, is an essential enzyme for such protection. Individually grown Txnrd1 knockout (Txnrd1−/−) mouse embryonic fibroblasts (MEFs) underwent massive cell death directly linked to glucose-induced H2O2 production. This death and excessive H2O2 levels could be reverted by reconstituted expression of selenocysteine (Sec)-containing TrxR1, but not by expression of Sec-devoid variants of the enzyme. Our results show that Sec-containing TrxR1 is absolutely required for self-sufficient growth of MEFs under high-glucose conditions, owing to an essential importance of this enzyme for elimination of glucose-derived H2O2. To our knowledge, this is the first time a strict Sec-dependent function of TrxR1 has been identified as being essential for mammalian cells. PMID:24853413

  12. 46 CFR Sec. 19 - Ship Repair Summaries.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

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  13. 46 CFR Sec. 19 - Ship Repair Summaries.

    Code of Federal Regulations, 2013 CFR

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  14. 46 CFR Sec. 19 - Ship Repair Summaries.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

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  15. 46 CFR Sec. 19 - Ship Repair Summaries.

    Code of Federal Regulations, 2011 CFR

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  16. 46 CFR Sec. 19 - Ship Repair Summaries.

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  17. 46 CFR Sec. 4 - Method of payment.

    Code of Federal Regulations, 2011 CFR

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  18. 46 CFR Sec. 7 - Job order numbering.

    Code of Federal Regulations, 2012 CFR

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  19. 46 CFR Sec. 7 - Job order numbering.

    Code of Federal Regulations, 2013 CFR

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  20. 46 CFR Sec. 7 - Job order numbering.

    Code of Federal Regulations, 2014 CFR

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  1. 46 CFR Sec. 7 - Job order numbering.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

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  2. 46 CFR Sec. 7 - Job order numbering.

    Code of Federal Regulations, 2011 CFR

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  3. 46 CFR Sec. 16 - Liquidated damages.

    Code of Federal Regulations, 2013 CFR

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  4. 46 CFR Sec. 16 - Liquidated damages.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

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  5. 46 CFR Sec. 16 - Liquidated damages.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

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  6. 46 CFR Sec. 16 - Liquidated damages.

    Code of Federal Regulations, 2011 CFR

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  7. 46 CFR Sec. 3 - Standby agreements.

    Code of Federal Regulations, 2013 CFR

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  8. 46 CFR Sec. 16 - Liquidated damages.

    Code of Federal Regulations, 2014 CFR

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  9. 46 CFR Sec. 3 - Standby agreements.

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  10. 46 CFR Sec. 4 - Funding of operations.

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  11. 46 CFR Sec. 4 - Funding of operations.

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  12. 46 CFR Sec. 4 - Funding of operations.

    Code of Federal Regulations, 2014 CFR

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  13. 46 CFR Sec. 4 - General provisions.

    Code of Federal Regulations, 2010 CFR

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  14. 46 CFR Sec. 3 - General Agents' responsibilities.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

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  15. 46 CFR Sec. 2 - Bank account.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

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  16. 46 CFR Sec. 2 - Bank account.

    Code of Federal Regulations, 2012 CFR

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  17. 46 CFR Sec. 2 - Bank account.

    Code of Federal Regulations, 2013 CFR

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  18. 46 CFR Sec. 2 - Bank account.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

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  19. 46 CFR Sec. 2 - Bank account.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

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  20. 46 CFR Sec. 2 - Amount of bond.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

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  1. 46 CFR Sec. 2 - Amount of bond.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Amount of bond. Sec. 2 Section 2 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY BONDING OF SHIP'S PERSONNEL Sec. 2 Amount of bond. The amount of the bond must be governed by the amount of monies advanced or value of...

  2. 46 CFR Sec. 4 - Posting of bond.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Posting of bond. Sec. 4 Section 4 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY BONDING OF SHIP'S PERSONNEL Sec. 4 Posting of bond. The General Agent shall retain an executed copy of each such bond in its principal...

  3. 46 CFR Sec. 4 - Posting of bond.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

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  4. 46 CFR Sec. 3 - Terminal operating contract.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Terminal operating contract. Sec. 3 Section 3 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION B-CONTROL AND UTILIZATION OF PORTS OPERATING CONTRACT Sec. 3 Terminal operating contract. Contract MA Terminal Operating Contract This agreement, made as of _________, 19__, between the...

  5. Protein Translocation: SecA-SecY Conformational Crosstalk Opens Channel.

    PubMed

    Kuhn, Andreas; Dalbey, Ross E

    2016-09-12

    A new study of the bacterial Sec translocase complex reports that ADP/ATP binding to SecA triggers multiple conformational changes in the SecYEG channel that may allow the passive directional movement of the polypeptide chain. PMID:27623265

  6. Sequence Variability in Staphylococcal Enterotoxin Genes seb, sec, and sed

    PubMed Central

    Johler, Sophia; Sihto, Henna-Maria; Macori, Guerrino; Stephan, Roger

    2016-01-01

    Ingestion of staphylococcal enterotoxins preformed by Staphylococcus aureus in food leads to staphylococcal food poisoning, the most prevalent foodborne intoxication worldwide. There are five major staphylococcal enterotoxins: SEA, SEB, SEC, SED, and SEE. While variants of these toxins have been described and were linked to specific hosts or levels or enterotoxin production, data on sequence variation is still limited. In this study, we aim to extend the knowledge on promoter and gene variants of the major enterotoxins SEB, SEC, and SED. To this end, we determined seb, sec, and sed promoter and gene sequences of a well-characterized set of enterotoxigenic Staphylococcus aureus strains originating from foodborne outbreaks, human infections, human nasal colonization, rabbits, and cattle. New nucleotide sequence variants were detected for all three enterotoxins and a novel amino acid sequence variant of SED was detected in a strain associated with human nasal colonization. While the seb promoter and gene sequences exhibited a high degree of variability, the sec and sed promoter and gene were more conserved. Interestingly, a truncated variant of sed was detected in all tested sed harboring rabbit strains. The generated data represents a further step towards improved understanding of strain-specific differences in enterotoxin expression and host-specific variation in enterotoxin sequences. PMID:27258311

  7. Large-Scale Evolutionary Analyses on SecB Subunits of Bacterial Sec System

    PubMed Central

    Yan, Shaomin; Wu, Guang

    2015-01-01

    Protein secretion systems are extremely important in bacteria because they are involved in many fundamental cellular processes. Of the various secretion systems, the Sec system is composed of seven different subunits in bacteria, and subunit SecB brings secreted preproteins to subunit SecA, which with SecYEG and SecDF forms a complex for the translocation of secreted preproteins through the inner membrane. Because of the wide existence of Sec system across bacteria, eukaryota, and archaea, each subunit of the Sec system has a complicated evolutionary relationship. Until very recently, 5,162 SecB sequences have been documented in UniProtKB, however no phylogenetic study has been conducted on a large sampling of SecBs from bacterial Sec secretion system, and no statistical study has been conducted on such size of SecBs in order to exhaustively investigate their variances of pairwise p-distance along taxonomic lineage from kingdom to phylum, to class, to order, to family, to genus and to organism. To fill in these knowledge gaps, 3,813 bacterial SecB sequences with full taxonomic lineage from kingdom to organism covering 4 phyla, 11 classes, 41 orders, 82 families, 269 genera, and 3,744 organisms were studied. Phylogenetic analysis revealed how the SecBs evolved without compromising their function with examples of 3-D structure comparison of two SecBs from Proteobacteria, and possible factors that affected the SecB evolution were considered. The average pairwise p-distances showed that the variance varied greatly in each taxonomic group. Finally, the variance was further partitioned into inter- and intra-clan variances, which could correspond to vertical and horizontal gene transfers, with relevance for Achromobacter, Brevundimonas, Ochrobactrum, and Pseudoxanthomonas. PMID:25775430

  8. Sec6 mutations and the Drosophila exocyst complex.

    PubMed

    Murthy, Mala; Ranjan, Ravi; Denef, Natalie; Higashi, Misao E L; Schupbach, Trudi; Schwarz, Thomas L

    2005-03-15

    To allow a detailed analysis of exocyst function in multicellular organisms, we have generated sec6 mutants in Drosophila. We have used these mutations to compare the phenotypes of sec6 and sec5 in the ovary and nervous system, and we find them to be similar. We also find that Sec5 is mislocalized in sec6 mutants. Additionally, we have generated an epitope-tagged Sec8 that localized with Sec5 on oocyte membranes and was mislocalized in sec5 and sec6 germ-line clones. This construct further revealed a genetic interaction of sec8 and sec5. These data, taken together, provide new information about the organization of the exocyst complex and suggest that Sec5, Sec6 and Sec8 act as a complex, each member dependent on the others for proper localization and function. PMID:15728258

  9. ALG-2 Attenuates COPII Budding In Vitro and Stabilizes the Sec23/Sec31A Complex

    PubMed Central

    la Cour, Jonas M.; Schindler, Adam J.; Berchtold, Martin W.; Schekman, Randy

    2013-01-01

    Coated vesicles mediate the traffic of secretory and membrane cargo proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. The coat protein complex (COPII) involved in vesicle budding is constituted by a GTPase, Sar1, the inner coat components of Sec23/Sec24 and the components of the outer coat Sec13/Sec31A. The Ca2+-binding protein ALG-2 was recently identified as a Sec31A binding partner and a possible link to Ca2+ regulation of COPII vesicle budding. Here we show that ALG-2/Ca2+ is capable of attenuating vesicle budding in vitro through interaction with an ALG-2 binding domain in the proline rich region of Sec31A. Binding of ALG-2 to Sec31A and inhibition of COPII vesicle budding is furthermore dependent on an intact Ca2+-binding site at EF-hand 1 of ALG-2. ALG-2 increased recruitment of COPII proteins Sec23/24 and Sec13/31A to artificial liposomes and was capable of mediating binding of Sec13/31A to Sec23. These results introduce a regulatory role for ALG-2/Ca2+ in COPII tethering and vesicle budding. PMID:24069399

  10. Transcriptional Response of Selenopolypeptide Genes and Selenocysteine Biosynthesis Machinery Genes in Escherichia coli during Selenite Reduction

    PubMed Central

    Tetteh, Antonia Y.; Sun, Katherine H.; Kittur, Farooqahmed S.; Ibeanu, Gordon C.

    2014-01-01

    Bacteria can reduce toxic selenite into less toxic, elemental selenium (Se0), but the mechanism on how bacterial cells reduce selenite at molecular level is still not clear. We used Escherichia coli strain K12, a common bacterial strain, as a model to study its growth response to sodium selenite (Na2SeO3) treatment and then used quantitative real-time PCR (qRT-PCR) to quantify transcript levels of three E. coli selenopolypeptide genes and a set of machinery genes for selenocysteine (SeCys) biosynthesis and incorporation into polypeptides, whose involvements in the selenite reduction are largely unknown. We determined that 5 mM Na2SeO3 treatment inhibited growth by ∼50% while 0.001 to 0.01 mM treatments stimulated cell growth by ∼30%. Under 50% inhibitory or 30% stimulatory Na2SeO3 concentration, selenopolypeptide genes (fdnG, fdoG, and fdhF) whose products require SeCys but not SeCys biosynthesis machinery genes were found to be induced ≥2-fold. In addition, one sulfur (S) metabolic gene iscS and two previously reported selenite-responsive genes sodA and gutS were also induced ≥2-fold under 50% inhibitory concentration. Our findings provide insight about the detoxification of selenite in E. coli via induction of these genes involved in the selenite reduction process. PMID:24839442

  11. Bioaccessibility of selenium, selenomethionine and selenocysteine from foods and influence of heat processing on the same.

    PubMed

    Khanam, Anjum; Platel, Kalpana

    2016-03-01

    Selenium (Se) is an essential nutrient with diverse physiological functions. The selenium content of commonly consumed cereals, pulses and green leafy vegetables (GLV) was determined. Bioaccessibility of Se, and its organic forms selenomethionine (SeMet), and selenocysteine (SeCys2) was also examined, and the effect of heat processing on the same was studied. The bioaccessibility of Se in cereals ranged from 10% to 24%, that of pulses was between 12% and 29%, and of GLV, 10-31%. The concentration of SeMet in the dialysates of the cereals, pulses and GLV ranged from 5.15 to 28.7, 2.7 to 36.2, and 0.03 to 5ngg(-1), respectively. The concentration of SeCys2 in the dialysates of the foods examined was negligible. Heat processing significantly decreased the bioaccessibility of Se, SeMet and SeCys2. This is the first report on the bioaccessibility of Se and its major organic forms from commonly consumed staples, and the effect of heat processing on the same. PMID:26471684

  12. Enhanced selenium tolerance and accumulation in transgenic Arabidopsis expressing a mouse selenocysteine lyase.

    PubMed

    Pilon, Marinus; Owen, Jennifer D; Garifullina, Gulnara F; Kurihara, Tatsuo; Mihara, Hisaaki; Esaki, Nobuyoshi; Pilon-Smits, Elizabeth A H

    2003-03-01

    Selenium (Se) toxicity is thought to be due to nonspecific incorporation of selenocysteine (Se-Cys) into proteins, replacing Cys. In an attempt to direct Se flow away from incorporation into proteins, a mouse (Mus musculus) Se-Cys lyase (SL) was expressed in the cytosol or chloroplasts of Arabidopsis. This enzyme specifically catalyzes the decomposition of Se-Cys into elemental Se and alanine. The resulting SL transgenics were shown to express the mouse enzyme in the expected intracellular location, and to have SL activities up to 2-fold (cytosolic lines) or 6-fold (chloroplastic lines) higher than wild-type plants. Se incorporation into proteins was reduced 2-fold in both types of SL transgenics, indicating that the approach successfully redirected Se flow in the plant. Both the cytosolic and chloroplastic SL plants showed enhanced shoot Se concentrations, up to 1.5-fold compared with wild type. The cytosolic SL plants showed enhanced tolerance to Se, presumably because of their reduced protein Se levels. Surprisingly, the chloroplastic SL transgenics were less tolerant to Se, indicating that (over) production of elemental Se in the chloroplast is toxic. Expression of SL in the cytosol may be a useful approach for the creation of plants with enhanced Se phytoremediation capacity. PMID:12644675

  13. Kinetic consequences of introducing a proximal selenocysteine ligand into cytochrome P450cam.

    PubMed

    Vandemeulebroucke, An; Aldag, Caroline; Stiebritz, Martin T; Reiher, Markus; Hilvert, Donald

    2015-11-10

    The structural, electronic, and catalytic properties of cytochrome P450cam are subtly altered when the cysteine that coordinates to the heme iron is replaced with a selenocysteine. To map the effects of the sulfur-to-selenium substitution on the individual steps of the catalytic cycle, we conducted a comparative kinetic analysis of the selenoenzyme and its cysteine counterpart. Our results show that the more electron-donating selenolate ligand has only negligible effects on substrate, product, and oxygen binding, electron transfer, catalytic turnover, and coupling efficiency. Off-pathway reduction of oxygen to give superoxide is the only step significantly affected by the mutation. Incorporation of selenium accelerates this uncoupling reaction approximately 50-fold compared to sulfur, but because the second electron transfer step is much faster, the impact on overall catalytic turnover is minimal. Density functional theory calculations with pure and hybrid functionals suggest that superoxide formation is governed by a delicate interplay of spin distribution, spin state, and structural effects. In light of the remarkably similar electronic structures and energies calculated for the sulfur- and selenium-containing enzymes, the ability of the heavier atom to enhance the rate of spin crossover may account for the experimental observations. Because the selenoenzyme closely mimics wild-type P450cam, even at the level of individual steps in the reaction cycle, selenium represents a unique mechanistic probe for analyzing the role of the proximal ligand and spin crossovers in P450 chemistry. PMID:26460790

  14. 46 CFR Sec. 20 - Reports of awards.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 20 Reports... submitted by the General Agents pursuant to section 3(d) of NSA Order 34 (SRM-3, Revised)....

  15. 46 CFR Sec. 4 - Method of payment.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... AGENTS IN PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 4 Method of payment. The General Agent shall prepare check drawn on the NSA Special bank...

  16. 46 CFR Sec. 11 - Guarantee obligations.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 11 Guarantee obligations. (a) Under the provisions of Article 10 of the NSA-LUMPSUMREP...

  17. 46 CFR Sec. 20 - Reports of awards.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 20 Reports... submitted by the General Agents pursuant to section 3(d) of NSA Order 34 (SRM-3, Revised)....

  18. 46 CFR Sec. 11 - Guarantee obligations.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 11 Guarantee obligations. (a) Under the provisions of Article 10 of the NSA-LUMPSUMREP...

  19. 46 CFR Sec. 4 - Method of payment.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... AGENTS IN PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 4 Method of payment. The General Agent shall prepare check drawn on the NSA Special bank...

  20. 46 CFR Sec. 11 - Guarantee obligations.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 11 Guarantee obligations. (a) Under the provisions of Article 10 of the NSA-LUMPSUMREP...

  1. 46 CFR Sec. 20 - Reports of awards.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 20 Reports... submitted by the General Agents pursuant to section 3(d) of NSA Order 34 (SRM-3, Revised)....

  2. 46 CFR Sec. 11 - Guarantee obligations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 11 Guarantee obligations. (a) Under the provisions of Article 10 of the NSA-LUMPSUMREP...

  3. 46 CFR Sec. 20 - Reports of awards.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 20 Reports... submitted by the General Agents pursuant to section 3(d) of NSA Order 34 (SRM-3, Revised)....

  4. 46 CFR Sec. 20 - Reports of awards.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 20 Reports... submitted by the General Agents pursuant to section 3(d) of NSA Order 34 (SRM-3, Revised)....

  5. 46 CFR Sec. 4 - Method of payment.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... AGENTS IN PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 4 Method of payment. The General Agent shall prepare check drawn on the NSA Special bank...

  6. 46 CFR Sec. 11 - Guarantee obligations.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 11 Guarantee obligations. (a) Under the provisions of Article 10 of the NSA-LUMPSUMREP...

  7. 46 CFR Sec. 4 - Method of payment.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... AGENTS IN PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 4 Method of payment. The General Agent shall prepare check drawn on the NSA Special bank...

  8. 46 CFR Sec. 2 - General Agents' authority.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... RESPONSIBILITY OF GENERAL AGENTS TO UNDERTAKE EMERGENCY REPAIRS IN FOREIGN PORTS Sec. 2 General Agents' authority. The General Agents are hereby delegated authority to undertake for the account of the...

  9. SEC sensor parametric test and evaluation system

    NASA Technical Reports Server (NTRS)

    1978-01-01

    This system provides the necessary automated hardware required to carry out, in conjunction with the existing 70 mm SEC television camera, the sensor evaluation tests which are described in detail. The Parametric Test Set (PTS) was completed and is used in a semiautomatic data acquisition and control mode to test the development of the 70 mm SEC sensor, WX 32193. Data analysis of raw data is performed on the Princeton IBM 360-91 computer.

  10. Alkbh8 Regulates Selenocysteine-Protein Expression to Protect against Reactive Oxygen Species Damage

    PubMed Central

    Endres, Lauren; Dziergowska, Agnieszka; Małkiewicz, Andrzej; Melendez, J. Andres; Dedon, Peter C.; Begley, Thomas J.

    2015-01-01

    Environmental and metabolic sources of reactive oxygen species (ROS) can damage DNA, proteins and lipids to promote disease. Regulation of gene expression can prevent this damage and can include increased transcription, translation and post translational modification. Cellular responses to ROS play important roles in disease prevention, with deficiencies linked to cancer, neurodegeneration and ageing. Here we detail basal and damage-induced translational regulation of a group of oxidative-stress response enzymes by the tRNA methyltransferase Alkbh8. Using a new gene targeted knockout mouse cell system, we show that Alkbh8-/- embryonic fibroblasts (MEFs) display elevated ROS levels, increased DNA and lipid damage and hallmarks of cellular stress. We demonstrate that Alkbh8 is induced in response to ROS and is required for the efficient expression of selenocysteine-containing ROS detoxification enzymes belonging to the glutathione peroxidase (Gpx1, Gpx3, Gpx6 and likely Gpx4) and thioredoxin reductase (TrxR1) families. We also show that, in response to oxidative stress, the tRNA modification 5-methoxycarbonylmethyl-2′-O-methyluridine (mcm5Um) increases in normal MEFs to drive the expression of ROS detoxification enzymes, with this damage-induced reprogramming of tRNA and stop-codon recoding corrupted in Alkbh8-/- MEFS. These studies define Alkbh8 and tRNA modifications as central regulators of cellular oxidative stress responses in mammalian systems. In addition they highlight a new animal model for use in environmental and cancer studies and link translational regulation to the prevention of DNA and lipid damage. PMID:26147969

  11. Diverse effects of mutation on the activity of the Escherichia coli export chaperone SecB.

    PubMed

    Kimsey, H H; Dagarag, M D; Kumamoto, C A

    1995-09-29

    The Escherichia coli SecB protein binds newly synthesized precursor maltose-binding protein (preMBP) and promotes its rapid export from the cytoplasm. Site-directed mutagenesis of two regions of SecB was carried out to better understand factors governing the SecB.preMBP interaction. 30 aminoacyl substitution mutants were analyzed, revealing two distinct classes of secB mutants. Substitutions at the alternating positions Phe-74, Cys-76, Val-78, or Gln-80 reduced the ability of SecB to form stable complexes with preMBP, but caused only mild defects in the rate of MBP export from living cells. The pattern revealed by this class of mutants suggests that a primary binding site for preMBP is hydrophobic and contains beta-sheet secondary structure. In contrast, substitutions at Asp-20, Glu-24, Leu-75, or Glu-77 caused a severe slowing in the rate of MBP export but did not disrupt SecB.preMBP complex formation. These largely acidic residues may function to regulate the opening of a preprotein binding site, allowing both high affinity preprotein binding and rapid dissociation of SecB.preprotein complexes at the membrane translocation site. PMID:7559415

  12. Structure of the Sec13-Sec16 edge element, a template for assembly of the COPII vesicle coat

    SciTech Connect

    Whittle, James R.R.; Schwartz, Thomas U

    2010-09-03

    Ancestral coatomer element 1 (ACE1) proteins assemble latticework coats for COPII vesicles and the nuclear pore complex. The ACE1 protein Sec31 and Sec13 make a 2:2 tetramer that forms the edge element of the COPII outer coat. In this study, we report that the COPII accessory protein Sec16 also contains an ACE1. The 165-kD crystal structure of the central domain of Sec16 in complex with Sec13 was solved at 2.7-Å resolution. Sec16 and Sec13 also make a 2:2 tetramer, another edge element for the COPII system. Domain swapping at the ACE1-ACE1 interface is observed both in the prior structure of Sec13-Sec31 and in Sec13-Sec16. A Sec31 mutant in which domain swapping is prevented adopts an unprecedented laminated structure, solved at 2.8-Å resolution. Our in vivo data suggest that the ACE1 element of Sec31 can functionally replace the ACE1 element of Sec16. Our data support Sec16 as a scaffold for the COPII system and a template for the Sec13-Sec31 coat.

  13. Covalently dimerized SecA is functional in protein translocation.

    PubMed

    de Keyzer, Jeanine; van der Sluis, Eli O; Spelbrink, Robin E J; Nijstad, Niels; de Kruijff, Ben; Nouwen, Nico; van der Does, Chris; Driessen, Arnold J M

    2005-10-21

    The ATPase SecA provides the driving force for the transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. SecA exists as a dimer in solution, but the exact oligomeric state of SecA during membrane binding and preprotein translocation is a topic of debate. To study the requirements of oligomeric changes in SecA during protein translocation, a non-dissociable SecA dimer was formed by oxidation of the carboxyl-terminal cysteines. The cross-linked SecA dimer interacts with the SecYEG complex with a similar stoichiometry as non-cross-linked SecA. Cross-linking reversibly disrupts the SecB binding site on SecA. However, in the absence of SecB, the activity of the disulfide-bonded SecA dimer is indistinguishable from wild-type SecA. Moreover, SecYEG binding stabilizes a cold sodium dodecylsulfate-resistant dimeric state of SecA. The results demonstrate that dissociation of the SecA dimer is not an essential feature of the protein translocation reaction. PMID:16115882

  14. 46 CFR Sec. 3 - Master's requirements.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY SLOP CHESTS Sec. 3 Master's requirements. The Master shall: (a) Receive and receipt for the quantities of slop chest items delivered on board. (b) Upon the termination of each voyage complete the Slop Chest Statement referred to...

  15. 46 CFR Sec. 2 - General Agent's requirements.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY SLOP CHESTS Sec. 2... chest items required for the intended voyage. Purchase for the account of the NSA, from recognized bona fide slop chest suppliers, at prices not in excess of the fair and reasonable level prevailing at...

  16. 46 CFR Sec. 2 - General Agent's requirements.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY SLOP CHESTS Sec. 2... chest items required for the intended voyage. Purchase for the account of the NSA, from recognized bona fide slop chest suppliers, at prices not in excess of the fair and reasonable level prevailing at...

  17. 46 CFR Sec. 3 - Master's requirements.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY SLOP CHESTS Sec. 3 Master's requirements. The Master shall: (a) Receive and receipt for the quantities of slop chest items delivered on board. (b) Upon the termination of each voyage complete the Slop Chest Statement referred to...

  18. 46 CFR Sec. 4 - General provisions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY SLOP CHESTS Sec. 4 General provisions. (a) All slop chest items, damaged or otherwise, shall be removed or transferred only in compliance... General Agent to another General Agent the physical transfer of the complete slop chest shall also...

  19. 46 CFR Sec. 2 - General Agent's requirements.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY SLOP CHESTS Sec. 2... chest items required for the intended voyage. Purchase for the account of the NSA, from recognized bona fide slop chest suppliers, at prices not in excess of the fair and reasonable level prevailing at...

  20. 46 CFR Sec. 3 - Master's requirements.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY SLOP CHESTS Sec. 3 Master's requirements. The Master shall: (a) Receive and receipt for the quantities of slop chest items delivered on board. (b) Upon the termination of each voyage complete the Slop Chest Statement referred to...

  1. 46 CFR Sec. 2 - General Agent's requirements.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY SLOP CHESTS Sec. 2... chest items required for the intended voyage. Purchase for the account of the NSA, from recognized bona fide slop chest suppliers, at prices not in excess of the fair and reasonable level prevailing at...

  2. 46 CFR Sec. 3 - Master's requirements.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY SLOP CHESTS Sec. 3 Master's requirements. The Master shall: (a) Receive and receipt for the quantities of slop chest items delivered on board. (b) Upon the termination of each voyage complete the Slop Chest Statement referred to...

  3. 46 CFR Sec. 2 - General Agent's requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY SLOP CHESTS Sec. 2... chest items required for the intended voyage. Purchase for the account of the NSA, from recognized bona fide slop chest suppliers, at prices not in excess of the fair and reasonable level prevailing at...

  4. SEC Vidicon spectra of Geminid meteors, 1972

    NASA Technical Reports Server (NTRS)

    Millman, P. M.; Clifton, K. S.

    1975-01-01

    The SEC Vidicon, a low light level closed circuit television system, was used to obtain 137 spectrographic records of meteors at Mt. Hopkins, Arizona, during the Geminid meteor shower in December 1972. Seven of the best Geminid meteor spectra are studied here in detail. The near infrared, out to wavelengths near 9000 A, is recorded for the first time for Geminids. The spectra, in general, exhibit the elements previously found in photographic records of this shower but show a surprising frequency of occurrence of the forbidden green line of O I at 5577 A. This line is normally absent from meteors moving as slowly as the Geminids (36 km/sec) and its presence in these records may be due to the added sensitivity available with the SEC Vidicon. The average green line duration in Geminid meteors with a luminosity near zero absolute visual magnitude is 0.73 sec at a mean height of 95 km, 11 km lower than the green line peak in Perseid meteors of the same luminosity.

  5. 46 CFR Sec. 3 - Preparation of invoices.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... NSA ORDER NO. 47 Sec. 3 Preparation of invoices. (a) Pursuant to Article 4 of the Service Agreement... under the applicable provisions of NSA Order No. 47. (1) Invoices shall be prepared so as to show... provisions of NSA Order No. 47 due the undersigned General Agent for the month of _____ under...

  6. 46 CFR Sec. 3 - Preparation of invoices.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... NSA ORDER NO. 47 Sec. 3 Preparation of invoices. (a) Pursuant to Article 4 of the Service Agreement... under the applicable provisions of NSA Order No. 47. (1) Invoices shall be prepared so as to show... provisions of NSA Order No. 47 due the undersigned General Agent for the month of _____ under...

  7. 46 CFR Sec. 18 - Group classification.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 8 2014-10-01 2014-10-01 false Group classification. Sec. 18 Section 18 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURE FOR ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR...

  8. 46 CFR Sec. 18 - Group classification.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Group classification. Sec. 18 Section 18 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURE FOR ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR...

  9. 46 CFR Sec. 3 - Preparation of invoices.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... NSA ORDER NO. 47 Sec. 3 Preparation of invoices. (a) Pursuant to Article 4 of the Service Agreement... under the applicable provisions of NSA Order No. 47. (1) Invoices shall be prepared so as to show... provisions of NSA Order No. 47 due the undersigned General Agent for the month of _____ under...

  10. 46 CFR Sec. 18 - Group classification.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 8 2012-10-01 2012-10-01 false Group classification. Sec. 18 Section 18 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURE FOR ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR...

  11. 46 CFR Sec. 3 - Preparation of invoices.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... NSA ORDER NO. 47 Sec. 3 Preparation of invoices. (a) Pursuant to Article 4 of the Service Agreement... under the applicable provisions of NSA Order No. 47. (1) Invoices shall be prepared so as to show... provisions of NSA Order No. 47 due the undersigned General Agent for the month of _____ under...

  12. 46 CFR Sec. 18 - Group classification.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 8 2013-10-01 2013-10-01 false Group classification. Sec. 18 Section 18 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURE FOR ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR...

  13. 46 CFR Sec. 3 - Preparation of invoices.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... NSA ORDER NO. 47 Sec. 3 Preparation of invoices. (a) Pursuant to Article 4 of the Service Agreement... under the applicable provisions of NSA Order No. 47. (1) Invoices shall be prepared so as to show... provisions of NSA Order No. 47 due the undersigned General Agent for the month of _____ under...

  14. 46 CFR Sec. 2 - General Agents' authority.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false General Agents' authority. Sec. 2 Section 2 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY AUTHORITY AND RESPONSIBILITY OF GENERAL AGENTS TO UNDERTAKE IN CONTINENTAL UNITED STATES PORTS VOYAGE REPAIRS AND...

  15. 46 CFR Sec. 3 - Master's requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY SLOP CHESTS Sec. 3.... (g) Retain on board, all damaged slop chest items, for survey, removal and disposition by the General... section 2(c) of this order, as to quantities and total value sold, quantities and total cost value on...

  16. 46 CFR Sec. 4 - Service agreements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... as an independent contractor, to exercise delegated authority of the Director, NSA, in the control of... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION B-CONTROL AND UTILIZATION OF PORTS FEDERAL PORT CONTROLLERS Sec. 4... the Director, National Shipping Authority of the Maritime Administration, Department of...

  17. 46 CFR Sec. 6 - Awarding of work.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Awarding of work. Sec. 6 Section 6 Shipping MARITIME... of work. (a) Those portions of all bids reflecting the total aggregate cost of the work involved shall be opened publicly. The work shall be awarded to the contractor submitting the lowest...

  18. 46 CFR Sec. 6 - Awarding of work.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Awarding of work. Sec. 6 Section 6 Shipping MARITIME... of work. (a) Those portions of all bids reflecting the total aggregate cost of the work involved shall be opened publicly. The work shall be awarded to the contractor submitting the lowest...

  19. 46 CFR Sec. 3 - General provisions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... VESSELS OPERATED FOR THE ACCOUNT OF THE NATIONAL SHIPPING AUTHORITY UNDER GENERAL AGENCY AGREEMENT Sec. 3... Agents within the limitation specified under the Master Repair Contract if the contractor is a holder thereof or if the contractor does not hold a Master Repair Contract under NSA-WORKSMALREP if the...

  20. 46 CFR Sec. 3 - General provisions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... VESSELS OPERATED FOR THE ACCOUNT OF THE NATIONAL SHIPPING AUTHORITY UNDER GENERAL AGENCY AGREEMENT Sec. 3... Agents within the limitation specified under the Master Repair Contract if the contractor is a holder thereof or if the contractor does not hold a Master Repair Contract under NSA-WORKSMALREP if the...

  1. 46 CFR Sec. 10 - Lost documents.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... TRANSACTIONS UNDER AGENCY AGREEMENTS Documents Sec. 10 Lost documents. In the event of the loss of documents, photostat, carbon, or other suitable copies may be substituted therefor, in which event the following certification shall be placed on such copies: I certify that, to the best of my knowledge and belief, this is...

  2. 46 CFR Sec. 10 - Lost documents.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... TRANSACTIONS UNDER AGENCY AGREEMENTS Documents Sec. 10 Lost documents. In the event of the loss of documents, photostat, carbon, or other suitable copies may be substituted therefor, in which event the following certification shall be placed on such copies: I certify that, to the best of my knowledge and belief, this is...

  3. 46 CFR Sec. 10 - Lost documents.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... TRANSACTIONS UNDER AGENCY AGREEMENTS Documents Sec. 10 Lost documents. In the event of the loss of documents, photostat, carbon, or other suitable copies may be substituted therefor, in which event the following certification shall be placed on such copies: I certify that, to the best of my knowledge and belief, this is...

  4. 46 CFR Sec. 10 - Lost documents.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... TRANSACTIONS UNDER AGENCY AGREEMENTS Documents Sec. 10 Lost documents. In the event of the loss of documents, photostat, carbon, or other suitable copies may be substituted therefor, in which event the following certification shall be placed on such copies: I certify that, to the best of my knowledge and belief, this is...

  5. 46 CFR Sec. 5 - Repatriation charges.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Repatriation charges. Sec. 5 Section 5 Shipping MARITIME... Repatriation charges. (a) If it is deemed necessary to repatriate a seaman as a passenger aboard a privately... flat transportation charge of $5.00 per day shall be made for every day spent aboard the...

  6. 46 CFR Sec. 5 - Repatriation charges.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Repatriation charges. Sec. 5 Section 5 Shipping MARITIME... Repatriation charges. (a) If it is deemed necessary to repatriate a seaman as a passenger aboard a privately... flat transportation charge of $5.00 per day shall be made for every day spent aboard the...

  7. 14 CFR Sec. 19-1 - Applicability.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Applicability. Sec. 19-1 Section 19-1 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION (AVIATION PROCEEDINGS) ECONOMIC REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS Operating...

  8. 14 CFR Sec. 19-1 - Applicability.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Applicability. Sec. 19-1 Section 19-1 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION (AVIATION PROCEEDINGS) ECONOMIC REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS Operating...

  9. 14 CFR Sec. 19-1 - Applicability.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 4 2013-01-01 2013-01-01 false Applicability. Sec. 19-1 Section 19-1 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION (AVIATION PROCEEDINGS) ECONOMIC REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS Operating...

  10. 14 CFR Sec. 19-1 - Applicability.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 4 2014-01-01 2014-01-01 false Applicability. Sec. 19-1 Section 19-1 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION (AVIATION PROCEEDINGS) ECONOMIC REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS Operating...

  11. Decatransin, a new natural product inhibiting protein translocation at the Sec61/SecYEG translocon

    PubMed Central

    Junne, Tina; Wong, Joanne; Studer, Christian; Aust, Thomas; Bauer, Benedikt W.; Beibel, Martin; Bhullar, Bhupinder; Bruccoleri, Robert; Eichenberger, Jürg; Estoppey, David; Hartmann, Nicole; Knapp, Britta; Krastel, Philipp; Melin, Nicolas; Oakeley, Edward J.; Oberer, Lukas; Riedl, Ralph; Roma, Guglielmo; Schuierer, Sven; Petersen, Frank; Tallarico, John A.; Rapoport, Tom A.; Spiess, Martin; Hoepfner, Dominic

    2015-01-01

    ABSTRACT A new cyclic decadepsipeptide was isolated from Chaetosphaeria tulasneorum with potent bioactivity on mammalian and yeast cells. Chemogenomic profiling in S. cerevisiae indicated that the Sec61 translocon complex, the machinery for protein translocation and membrane insertion at the endoplasmic reticulum, is the target. The profiles were similar to those of cyclic heptadepsipeptides of a distinct chemotype (including HUN-7293 and cotransin) that had previously been shown to inhibit cotranslational translocation at the mammalian Sec61 translocon. Unbiased, genome-wide mutagenesis followed by full-genome sequencing in both fungal and mammalian cells identified dominant mutations in Sec61p (yeast) or Sec61α1 (mammals) that conferred resistance. Most, but not all, of these mutations affected inhibition by both chemotypes, despite an absence of structural similarity. Biochemical analysis confirmed inhibition of protein translocation into the endoplasmic reticulum of both co- and post-translationally translocated substrates by both chemotypes, demonstrating a mechanism independent of a translating ribosome. Most interestingly, both chemotypes were found to also inhibit SecYEG, the bacterial Sec61 translocon homolog. We suggest ‘decatransin’ as the name for this new decadepsipeptide translocation inhibitor. PMID:25616894

  12. What's driving Gigabit/sec channels

    SciTech Connect

    Rupert, P.R.

    1991-09-01

    This paper summarizes the rationale for the drive towards Gigabit/sec communications for individual users. A description is given of a resulting prototype LAN which will deliver this capability to each user. The prototype is being built for the Lawrence Livermore National Laboratory. It will be delivered this winter and should be available for users by the end of the first quarter of next year. 19 refs., 8 figs.

  13. SEC sets guidelines for climate risk

    SciTech Connect

    2010-04-15

    In a 3--2 vote in late January 2010, the U.S. Securities & Exchange Commission (SEC), the agency in charge of making sure investors are aware of risks associated with financial investments, approved new 'interpretive guidance' for 'disclosure of climate-related business risks.' The new guidelines call for disclosure of anticipated impact of climate change on assets and financial risks associated with compliance costs for existing and pending climate regulations.

  14. Sec14 related proteins in yeast.

    PubMed

    Griac, Peter

    2007-06-01

    Lipid transport between membranes of eukaryotic organisms represents an essential aspect of organelle biogenesis. This transport must be strictly selective and directional to assure specific lipid composition of individual membranes. Despite the intensive research effort in the last few years, our understanding of how lipids are sorted and moved within cells is still rather limited. Evidence indicates that at least some of the mechanisms generating and maintaining non-random distribution of lipids in cells are linked to the action of phosphatidylinositol transfer proteins (PITPs). The major PITP in yeast Saccharomyces cerevisiae, Sec14p, is essential in promoting Golgi secretory function by modulating of its membrane lipid composition. This review focuses on a group of five yeast proteins that share significant sequence homology with Sec14p. Based on this sequence identity, they were termed Sfh (Sec fourteen homologue) proteins. It is a diverse group of proteins with distinct subcellular localizations and varied physiological functions related to lipid metabolism, phosphoinositide mediated signaling and membrane trafficking. PMID:17395532

  15. SEC14 Phospholipid Transfer Protein Is Involved in Lipid Signaling-Mediated Plant Immune Responses in Nicotiana benthamiana

    PubMed Central

    Kiba, Akinori; Galis, Ivan; Hojo, Yuko; Ohnishi, Kouhei; Yoshioka, Hirofumi; Hikichi, Yasufumi

    2014-01-01

    We previously identified a gene related to the SEC14-gene phospholipid transfer protein superfamily that is induced in Nicotiana benthamiana (NbSEC14) in response to infection with Ralstonia solanacearum. We here report that NbSEC14 plays a role in plant immune responses via phospholipid-turnover. NbSEC14-silencing compromised expression of defense–related PR-4 and accumulation of jasmonic acid (JA) and its derivative JA-Ile. Transient expression of NbSEC14 induced PR-4 gene expression. Activities of diacylglycerol kinase, phospholipase C and D, and the synthesis of diacylglycerol and phosphatidic acid elicited by avirulent R. solanacearum were reduced in NbSEC14-silenced plants. Accumulation of signaling lipids and activation of diacylglycerol kinase and phospholipases were enhanced by transient expression of NbSEC14. These results suggest that the NbSEC14 protein plays a role at the interface between lipid signaling-metabolism and plant innate immune responses. PMID:24845602

  16. Nonlethal sec71-1 and sec72-1 mutations eliminate proteins associated with the Sec63p-BiP complex from S. cerevisiae.

    PubMed Central

    Fang, H; Green, N

    1994-01-01

    The sec71-1 and sec72-1 mutations were identified by a genetic assay that monitored membrane protein integration into the endoplasmic reticulum (ER) membrane of the yeast Saccharomyces cerevisiae. The mutations inhibited integration of various chimeric membrane proteins and translocation of a subset of water soluble proteins. In this paper we show that SEC71 encodes the 31.5-kDa transmembrane glycoprotein (p31.5) and SEC72 encodes the 23-kDa protein (p23) of the Sec63p-BiP complex. SEC71 is therefore identical to SEC66 (HSS1), which was previously shown to encode p31.5. DNA sequence analyses reveal that sec71-1 cells contain a nonsense mutation that removes approximately two-thirds of the cytoplasmic C-terminal domain of p31.5. The sec72-1 mutation shifts the reading frame of the gene encoding p23. Unexpectedly, the sec71-1 mutant lacks p31.5 and p23. Neither mutation is lethal, although sec71-1 cells exhibit a growth defect at 37 degrees C. These results show that p31.5 and p23 are important for the trafficking of a subset of proteins to the ER membrane. Images PMID:7841522

  17. Dynamic Organization of SecA and SecY Secretion Complexes in the B. subtilis Membrane.

    PubMed

    Dajkovic, Alex; Hinde, Elizabeth; MacKichan, Calum; Carballido-Lopez, Rut

    2016-01-01

    In prokaryotes, about one third of cellular proteins are translocated across the plasma membrane or inserted into it by concerted action of the cytoplasmic ATPase SecA and the universally conserved SecYEG heterotrimeric polypeptide-translocating pore. Secretion complexes have been reported to localize in specific subcellular sites in Bacillus subtilis. In this work, we used a combination of total internal reflection microscopy, scanning fluorescence correlation spectroscopy, and pair correlation function to study the localization and dynamics of SecA and SecY in growing Bacillus subtilis cells. Both SecA and SecY localized in transient and dynamic foci in the cytoplasmic membrane, which displayed no higher-level organization in helices. Foci of SecA and SecY were in constant flux with freely diffusing SecA and SecY molecules. Scanning FCS confirmed the existence of populations of cellular SecA and SecY molecules with a wide range of diffusion coefficients. Diffusion of SecY as an uncomplexed molecular species was short-lived and only local while SecY complexed with its protein partners traversed distances of over half a micrometer in the cell. PMID:27336478

  18. Dynamic Organization of SecA and SecY Secretion Complexes in the B. subtilis Membrane

    PubMed Central

    Dajkovic, Alex; Hinde, Elizabeth; MacKichan, Calum; Carballido-Lopez, Rut

    2016-01-01

    In prokaryotes, about one third of cellular proteins are translocated across the plasma membrane or inserted into it by concerted action of the cytoplasmic ATPase SecA and the universally conserved SecYEG heterotrimeric polypeptide-translocating pore. Secretion complexes have been reported to localize in specific subcellular sites in Bacillus subtilis. In this work, we used a combination of total internal reflection microscopy, scanning fluorescence correlation spectroscopy, and pair correlation function to study the localization and dynamics of SecA and SecY in growing Bacillus subtilis cells. Both SecA and SecY localized in transient and dynamic foci in the cytoplasmic membrane, which displayed no higher-level organization in helices. Foci of SecA and SecY were in constant flux with freely diffusing SecA and SecY molecules. Scanning FCS confirmed the existence of populations of cellular SecA and SecY molecules with a wide range of diffusion coefficients. Diffusion of SecY as an uncomplexed molecular species was short-lived and only local while SecY complexed with its protein partners traversed distances of over half a micrometer in the cell. PMID:27336478

  19. Nascent SecM Chain Outside the Ribosome Reinforces Translation Arrest

    PubMed Central

    Funatsu, Takashi

    2015-01-01

    SecM, a bacterial secretion monitor protein, contains a specific amino acid sequence at its C-terminus, called arrest sequence, which interacts with the ribosomal tunnel and arrests its own translation. The arrest sequence is sufficient and necessary for stable translation arrest. However, some previous studies have suggested that the nascent chain outside the ribosome affects the stability of translation arrest. To clarify this issue, we performed in vitro translation assays with HaloTag proteins fused to the C-terminal fragment of E. coli SecM containing the arrest sequence or the full-length SecM. We showed that the translation of HaloTag proteins, which are fused to the fragment, is not effectively arrested, whereas the translation of HaloTag protein fused to full-length SecM is arrested efficiently. In addition, we observed that the nascent SecM chain outside the ribosome markedly stabilizes the translation arrest. These results indicate that changes in the nascent polypeptide chain outside the ribosome can affect the stability of translation arrest; the nascent SecM chain outside the ribosome stabilizes the translation arrest. PMID:25806953

  20. Epithelial organization and cyst lumen expansion require efficient Sec13-Sec31-driven secretion.

    PubMed

    Townley, Anna K; Schmidt, Katy; Hodgson, Lorna; Stephens, David J

    2012-02-01

    Epithelial morphogenesis is directed by interactions with the underlying extracellular matrix. Secretion of collagen and other matrix components requires efficient coat complex II (COPII) vesicle formation at the endoplasmic reticulum. Here, we show that suppression of the outer layer COPII component, Sec13, in zebrafish embryos results in a disorganized gut epithelium. In human intestinal epithelial cells (Caco-2), Sec13 depletion causes defective epithelial polarity and organization on permeable supports. Defects are seen in the ability of cells to adhere to the substrate, form a monolayer and form intercellular junctions. When embedded in a three-dimensional matrix, Sec13-depleted Caco-2 cells form cysts but, unlike controls, are defective in lumen expansion. Incorporation of primary fibroblasts within the three-dimensional culture substantially restores normal morphogenesis. We conclude that efficient COPII-dependent secretion, notably assembly of Sec13-Sec31, is required to drive epithelial morphogenesis in both two- and three-dimensional cultures in vitro, as well as in vivo. Our results provide insight into the role of COPII in epithelial morphogenesis and have implications for the interpretation of epithelial polarity and organization assays in cell culture. PMID:22331354

  1. Involvement of Exoc3l, a protein structurally related to the exocyst subunit Sec6, in insulin secretion.

    PubMed

    Saito, Tetsuya; Shibasaki, Tadao; Seino, Susumu

    2008-04-01

    The exocyst is an octameric complex involved in docking or tethering of secretory vesicles to fusion sites of the plasma membrane. Sec6 is the core subunit of the exocyst complex. Here we identify an isoform of Sec6, deposited as Exocyst complex component 3-like (Exoc3l) in the database, by in silico screening using rat Sec6 as a probe. The amino acid sequence of Exoc3l has 31% identity and 53% similarity with that of Sec6. RT-PCR analysis reveals that Exoc3l is expressed in insulin-secreting MIN6 cells as well as in various tissues including pancreatic islets and brain. In co-immunoprecipitation experiments, Exoc3l was found to interact with Sec5, Sec8, and Sec10, all of which are binding partners of Sec6 in the exocyst complex. Furthermore, overexpression of a deletion mutant of Exoc3l in MIN6 cells suppressed glucose-stimulated secretion. These results suggest that Exoc3l is involved in regulated exocytosis of insulin granules. PMID:18480549

  2. In Candida albicans hyphae, Sec2p is physically associated with SEC2 mRNA on secretory vesicles

    PubMed Central

    Caballero-Lima, David; Hautbergue, Guillaume M; Wilson, Stuart A; Sudbery, Peter E

    2014-01-01

    Candida albicans hyphae grow in a highly polarized fashion from their tips. This polarized growth requires the continuous delivery of secretory vesicles to the tip region. Vesicle delivery depends on Sec2p, the Guanine Exchange Factor (GEF) for the Rab GTPase Sec4p. GTP bound Sec4p is required for the transit of secretory vesicles from the trans-Golgi to sites of polarized growth. We previously showed that phosphorylation of Sec2p at residue S584 was necessary for Sec2p to support hyphal, but not yeast growth. Here we show that on secretory vesicles SEC2 mRNA is physically associated with Sec2p. Moreover, we show that the phosphorylation of S584 allows SEC2 mRNA to dissociate from Sec2p and we speculate that this is necessary for Sec2p function and/or translation. During hyphal extension, the growing tip may be separated from the nucleus by up to 15 μm. Transport of SEC2 mRNA on secretory vesicles to the tip localizes SEC2 translation to tip allowing a sufficient accumulation of this key protein at the site of polarized growth. PMID:25231350

  3. In Vitro Interaction of the Housekeeping SecA1 with the Accessory SecA2 Protein of Mycobacterium tuberculosis

    PubMed Central

    Prabudiansyah, Irfan; Kusters, Ilja; Driessen, Arnold J. M.

    2015-01-01

    The majority of proteins that are secreted across the bacterial cytoplasmic membrane leave the cell via the Sec pathway, which in its minimal form consists of the dimeric ATP-driven motor protein SecA that associates with the protein-conducting membrane pore SecYEG. Some Gram-positive bacteria contain two homologues of SecA, termed SecA1 and SecA2. SecA1 is the essential housekeeping protein, whereas SecA2 is not essential but is involved in the translocation of a subset of proteins, including various virulence factors. Some SecA2 containing bacteria also harbor a homologous SecY2 protein that may form a separate translocase. Interestingly, mycobacteria contain only one SecY protein and thus both SecA1 and SecA2 are required to interact with SecYEG, either individually or together as a heterodimer. In order to address whether SecA1 and SecA2 cooperate during secretion of SecA2 dependent proteins, we examined the oligomeric state of SecA1 and SecA2 of Mycobacterium tuberculosis and their interactions with SecA2 and the cognate SecA1, respectively. We conclude that both SecA1 and SecA2 individually form homodimers in solution but when both proteins are present simultaneously, they form dissociable heterodimers. PMID:26047312

  4. A prl mutation in SecY suppresses secretion and virulence defects of Listeria monocytogenes secA2 mutants.

    PubMed

    Durack, Juliana; Burke, Thomas P; Portnoy, Daniel A

    2015-03-01

    The bulk of bacterial protein secretion occurs through the conserved SecY translocation channel that is powered by SecA-dependent ATP hydrolysis. Many Gram-positive bacteria, including the human pathogen Listeria monocytogenes, possess an additional nonessential specialized ATPase, SecA2. SecA2-dependent secretion is required for normal cell morphology and virulence in L. monocytogenes; however, the mechanism of export via this pathway is poorly understood. L. monocytogenes secA2 mutants form rough colonies, have septation defects, are impaired for swarming motility, and form small plaques in tissue culture cells. In this study, 70 spontaneous mutants were isolated that restored swarming motility to L. monocytogenes secA2 mutants. Most of the mutants had smooth colony morphology and septated normally, but all were lysozyme sensitive. Five representative mutants were subjected to whole-genome sequencing. Four of the five had mutations in proteins encoded by the lmo2769 operon that conferred lysozyme sensitivity and increased swarming but did not rescue virulence defects. A point mutation in secY was identified that conferred smooth colony morphology to secA2 mutants, restored wild-type plaque formation, and increased virulence in mice. This secY mutation resembled a prl suppressor known to expand the repertoire of proteins secreted through the SecY translocation complex. Accordingly, the ΔsecA2prlA1 mutant showed wild-type secretion levels of P60, an established SecA2-dependent secreted autolysin. Although the prl mutation largely suppressed almost all of the measurable SecA2-dependent traits, the ΔsecA2prlA1 mutant was still less virulent in vivo than the wild-type strain, suggesting that SecA2 function was still required for pathogenesis. PMID:25535272

  5. Human ARF4 expression rescues sec7 mutant yeast cells.

    PubMed Central

    Deitz, S B; Wu, C; Silve, S; Howell, K E; Melançon, P; Kahn, R A; Franzusoff, A

    1996-01-01

    Vesicle-mediated traffic between compartments of the yeast secretory pathway involves recruitment of multiple cytosolic proteins for budding, targeting, and membrane fusion events. The SEC7 gene product (Sec7p) is a constituent of coat structures on transport vesicles en route to the Golgi complex in the yeast Saccharomyces cerevisiae. To identify mammalian homologs of Sec7p and its interacting proteins, we used a genetic selection strategy in which a human HepG2 cDNA library was transformed into conditional-lethal yeast sec7 mutants. We isolated several clones capable of rescuing sec7 mutant growth at the restrictive temperature. The cDNA encoding the most effective suppressor was identified as human ADP ribosylation factor 4 (hARF4), a member of the GTPase family proposed to regulate recruitment of vesicle coat proteins in mammalian cells. Having identified a Sec7p-interacting protein rather than the mammalian Sec7p homolog, we provide evidence that hARF4 suppressed the sec7 mutation by restoring secretory pathway function. Shifting sec7 strains to the restrictive temperature results in the disappearance of the mutant Sec7p cytosolic pool without apparent changes in the membrane-associated fraction. The introduction of hARF4 to the cells maintained the balance between cytosolic and membrane-associated Sec7p pools. These results suggest a requirement for Sec7p cycling on and off of the membranes for cell growth and vesicular traffic. In addition, overexpression of the yeast GTPase-encoding genes ARF1 and ARF2, but not that of YPT1, suppressed the sec7 mutant growth phenotype in an allele-specific manner. This allele specificity indicates that individual ARFs are recruited to perform two different Sec7p-related functions in vesicle coat dynamics. PMID:8668142

  6. Drosophila exocyst components Sec5, Sec6, and Sec15 regulate DE-Cadherin trafficking from recycling endosomes to the plasma membrane.

    PubMed

    Langevin, Johanna; Morgan, Matthew J; Sibarita, Jean-Baptiste; Aresta, Sandra; Murthy, Mala; Schwarz, Thomas; Camonis, Jacques; Bellaïche, Yohanns

    2005-09-01

    The E-Cadherin-catenin complex plays a critical role in epithelial cell-cell adhesion, polarization, and morphogenesis. Here, we have analyzed the mechanism of Drosophila E-Cadherin (DE-Cad) localization. Loss of function of the Drosophila exocyst components sec5, sec6, and sec15 in epithelial cells results in DE-Cad accumulation in an enlarged Rab11 recycling endosomal compartment and inhibits DE-Cad delivery to the membrane. Furthermore, Rab11 and Armadillo interact with the exocyst components Sec15 and Sec10, respectively. Our results support a model whereby the exocyst regulates DE-Cadherin trafficking, from recycling endosomes to sites on the epithelial cell membrane where Armadillo is located. PMID:16224820

  7. The Variable Subdomain of Escherichia coli SecA Functions To Regulate SecA ATPase Activity and ADP Release

    PubMed Central

    Das, Sanchaita; Grady, Lorry M.; Michtavy, Jennifer; Zhou, Yayan; Cohan, Frederick M.; Hingorani, Manju M.

    2012-01-01

    Bacterial SecA proteins can be categorized by the presence or absence of a variable subdomain (VAR) located within nucleotide-binding domain II of the SecA DEAD motor. Here we show that VAR is dispensable for SecA function, since the VAR deletion mutant secAΔ519–547 displayed a wild-type rate of cellular growth and protein export. Loss or gain of VAR is extremely rare in the history of bacterial evolution, indicating that it appears to contribute to secA function within the relevant species in their natural environments. VAR removal also results in additional secA phenotypes: azide resistance (Azir) and suppression of signal sequence defects (PrlD). The SecAΔ(519–547) protein was found to be modestly hyperactive for SecA ATPase activities and displayed an accelerated rate of ADP release, consistent with the biochemical basis of azide resistance. Based on our findings, we discuss models whereby VAR allosterically regulates SecA DEAD motor function at SecYEG. PMID:22389482

  8. The Sec1/Munc18 Protein Groove Plays a Conserved Role in Interaction with Sec9p/SNAP-25.

    PubMed

    Weber-Boyvat, Marion; Chernov, Konstantin G; Aro, Nina; Wohlfahrt, Gerd; Olkkonen, Vesa M; Jäntti, Jussi

    2016-02-01

    The Sec1/Munc18 (SM) proteins constitute a conserved family with essential functions in SNARE-mediated membrane fusion. Recently, a new protein-protein interaction site in Sec1p, designated the groove, was proposed. Here, we show that a sec1 groove mutant yeast strain, sec1(w24), displays temperature-sensitive growth and secretion defects. The yeast Sec1p and mammalian Munc18-1 grooves were shown to play an important role in the interaction with the SNAREs Sec9p and SNAP-25b, respectively. Incubation of SNAP-25b with the Munc18-1 groove mutant resulted in a lag in the kinetics of SNARE complex assembly in vitro when compared with wild-type Munc18-1. The SNARE regulator SRO7 was identified as a multicopy suppressor of sec1(w24) groove mutant and an intact Sec1p groove was required for the plasma membrane targeting of Sro7p-SNARE complexes. Simultaneous inactivation of Sec1p groove and SRO7 resulted in reduced levels of exocytic SNARE complexes. Our results identify the groove as a conserved interaction surface in SM proteins. The results indicate that this structural element is important for interactions with Sec9p/SNAP-25 and participates, in concert with Sro7p, in the initial steps of SNARE complex assembly. PMID:26572066

  9. The bacterial translocon SecYEG opens upon ribosome binding.

    PubMed

    Knyazev, Denis G; Lents, Alexander; Krause, Eberhard; Ollinger, Nicole; Siligan, Christine; Papinski, Daniel; Winter, Lukas; Horner, Andreas; Pohl, Peter

    2013-06-21

    In co-translational translocation, the ribosome funnel and the channel of the protein translocation complex SecYEG are aligned. For the nascent chain to enter the channel immediately after synthesis, a yet unidentified signal triggers displacement of the SecYEG sealing plug from the pore. Here, we show that ribosome binding to the resting SecYEG channel triggers this conformational transition. The purified and reconstituted SecYEG channel opens to form a large ion-conducting channel, which has the conductivity of the plug deletion mutant. The number of ion-conducting channels inserted into the planar bilayer per fusion event roughly equals the number of SecYEG channels counted by fluorescence correlation spectroscopy in a single proteoliposome. Thus, the open probability of the channel must be close to unity. To prevent the otherwise lethal proton leak, a closed post-translational conformation of the SecYEG complex bound to a ribosome must exist. PMID:23645666

  10. The Bacterial Translocon SecYEG Opens upon Ribosome Binding*

    PubMed Central

    Knyazev, Denis G.; Lents, Alexander; Krause, Eberhard; Ollinger, Nicole; Siligan, Christine; Papinski, Daniel; Winter, Lukas; Horner, Andreas; Pohl, Peter

    2013-01-01

    In co-translational translocation, the ribosome funnel and the channel of the protein translocation complex SecYEG are aligned. For the nascent chain to enter the channel immediately after synthesis, a yet unidentified signal triggers displacement of the SecYEG sealing plug from the pore. Here, we show that ribosome binding to the resting SecYEG channel triggers this conformational transition. The purified and reconstituted SecYEG channel opens to form a large ion-conducting channel, which has the conductivity of the plug deletion mutant. The number of ion-conducting channels inserted into the planar bilayer per fusion event roughly equals the number of SecYEG channels counted by fluorescence correlation spectroscopy in a single proteoliposome. Thus, the open probability of the channel must be close to unity. To prevent the otherwise lethal proton leak, a closed post-translational conformation of the SecYEG complex bound to a ribosome must exist. PMID:23645666

  11. Complete Genome Sequence of Amino Acid-Utilizing Eubacterium acidaminophilum al-2 (DSM 3953)

    PubMed Central

    Poehlein, Anja; Andreesen, Jan R.

    2014-01-01

    Eubacterium acidaminophilum is a strictly anaerobic, Gram-positive, rod-shaped bacterium which belongs to cluster XI of the Clostridia. It ferments amino acids by a Stickland reaction. The genome harbors a chromosome (2.25 Mb) and a megaplasmid (0.8 Mb). It contains several gene clusters coding for selenocysteine-containing, glycine-derived, and amino acid-degrading reductases. PMID:24926057

  12. 46 CFR Sec. 5 - Measures to protect ship's payrolls.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Measures to protect ship's payrolls. Sec. 5 Section 5... SHIP'S PERSONNEL Sec. 5 Measures to protect ship's payrolls. (a) General Agents are not required to... paying off the crew should be either the Master, or purser, or some other member of the ship's...

  13. 46 CFR Sec. 5 - Measures to protect ship's payrolls.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 8 2013-10-01 2013-10-01 false Measures to protect ship's payrolls. Sec. 5 Section 5... SHIP'S PERSONNEL Sec. 5 Measures to protect ship's payrolls. (a) General Agents are not required to... paying off the crew should be either the Master, or purser, or some other member of the ship's...

  14. 46 CFR Sec. 5 - Measures to protect ship's payrolls.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 8 2014-10-01 2014-10-01 false Measures to protect ship's payrolls. Sec. 5 Section 5... SHIP'S PERSONNEL Sec. 5 Measures to protect ship's payrolls. (a) General Agents are not required to... paying off the crew should be either the Master, or purser, or some other member of the ship's...

  15. 46 CFR Sec. 2 - Description of NSA-WORKSMALREP Contract.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Description of NSA-WORKSMALREP Contract. Sec. 2 Section... REPAIRS-NSA-WORKSMALREP Sec. 2 Description of NSA-WORKSMALREP Contract. This is an individual fixed price contract which may be awarded to any firm not holding an NSA-LUMPSUMREP Contract, as a result of...

  16. 46 CFR Sec. 2 - Description of NSA-WORKSMALREP Contract.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 8 2014-10-01 2014-10-01 false Description of NSA-WORKSMALREP Contract. Sec. 2 Section... REPAIRS-NSA-WORKSMALREP Sec. 2 Description of NSA-WORKSMALREP Contract. This is an individual fixed price contract which may be awarded to any firm not holding an NSA-LUMPSUMREP Contract, as a result of...

  17. 46 CFR Sec. 2 - Description of NSA-WORKSMALREP Contract.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Description of NSA-WORKSMALREP Contract. Sec. 2 Section... REPAIRS-NSA-WORKSMALREP Sec. 2 Description of NSA-WORKSMALREP Contract. This is an individual fixed price contract which may be awarded to any firm not holding an NSA-LUMPSUMREP Contract, as a result of...

  18. 46 CFR Sec. 2 - Description of NSA-WORKSMALREP Contract.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 8 2012-10-01 2012-10-01 false Description of NSA-WORKSMALREP Contract. Sec. 2 Section... REPAIRS-NSA-WORKSMALREP Sec. 2 Description of NSA-WORKSMALREP Contract. This is an individual fixed price contract which may be awarded to any firm not holding an NSA-LUMPSUMREP Contract, as a result of...

  19. 46 CFR Sec. 2 - Description of NSA-WORKSMALREP Contract.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 8 2013-10-01 2013-10-01 false Description of NSA-WORKSMALREP Contract. Sec. 2 Section... REPAIRS-NSA-WORKSMALREP Sec. 2 Description of NSA-WORKSMALREP Contract. This is an individual fixed price contract which may be awarded to any firm not holding an NSA-LUMPSUMREP Contract, as a result of...

  20. 14 CFR Sec. 19-4 - Service classes.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Service classes. Sec. 19-4 Section 19-4... Classifications Sec. 19-4 Service classes. The statistical classifications are designed to reflect the operating elements attributable to each distinctive class of service offered. The operating elements shall be...

  1. 14 CFR Sec. 2-5 - Revenue and accounting practices.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Revenue and accounting practices. Sec. 2-5... General Accounting Provisions Sec. 2-5 Revenue and accounting practices. (a) Revenue accounting practices... physically verify the reliability of its passenger revenue accounting practice at least once each...

  2. 46 CFR Sec. 3 - Federal control of port facilities.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Federal control of port facilities. Sec. 3 Section 3 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION B-CONTROL AND UTILIZATION OF PORTS RESTRICTIONS UPON THE TRANSFER OR CHANGE IN USE OR IN TERMS GOVERNING UTILIZATION OF PORT FACILITIES Sec....

  3. 46 CFR Sec. 8 - Extra work and changes.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Extra work and changes. Sec. 8 Section 8 Shipping... Sec. 8 Extra work and changes. (a) At any time after the award of an original job order and during the time the work thereunder is being performed, additional or extra work or changes in the work covered...

  4. 46 CFR Sec. 8 - Extra work and changes.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Extra work and changes. Sec. 8 Section 8 Shipping... Sec. 8 Extra work and changes. (a) At any time after the award of an original job order and during the time the work thereunder is being performed, additional or extra work or changes in the work covered...

  5. 46 CFR Sec. 6 - Surety and form of bond.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Surety and form of bond. Sec. 6 Section 6 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY BONDING OF SHIP'S PERSONNEL Sec. 6 Surety and form of bond. Each bond provided for by this order shall be duly executed by an authorized surety appearing on the...

  6. 14 CFR Sec. 2-5 - Revenue and accounting practices.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Revenue and accounting practices. Sec. 2-5... General Accounting Provisions Sec. 2-5 Revenue and accounting practices. (a) Revenue accounting practices... physically verify the reliability of its passenger revenue accounting practice at least once each...

  7. 14 CFR Sec. 2-4 - Accounting period.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Accounting period. Sec. 2-4 Section 2-4... REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS General Accounting Provisions Sec. 2-4 Accounting period. (a) The accounting year of each air carrier subject to this...

  8. 14 CFR Sec. 2-4 - Accounting period.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 4 2013-01-01 2013-01-01 false Accounting period. Sec. 2-4 Section 2-4... REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS General Accounting Provisions Sec. 2-4 Accounting period. (a) The accounting year of each air carrier subject to this...

  9. 14 CFR Sec. 1-6 - Accounting entities.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 4 2013-01-01 2013-01-01 false Accounting entities. Sec. 1-6 Section 1-6... REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS General Accounting Provisions Sec. 1-6 Accounting entities. (a) Separate accounting records shall be maintained for each...

  10. 14 CFR Sec. 1-6 - Accounting entities.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 4 2014-01-01 2014-01-01 false Accounting entities. Sec. 1-6 Section 1-6... REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS General Accounting Provisions Sec. 1-6 Accounting entities. (a) Separate accounting records shall be maintained for each...

  11. 14 CFR Sec. 1-6 - Accounting entities.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Accounting entities. Sec. 1-6 Section 1-6... REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS General Accounting Provisions Sec. 1-6 Accounting entities. (a) Separate accounting records shall be maintained for each...

  12. 14 CFR Sec. 2-4 - Accounting period.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 4 2014-01-01 2014-01-01 false Accounting period. Sec. 2-4 Section 2-4... REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS General Accounting Provisions Sec. 2-4 Accounting period. (a) The accounting year of each air carrier subject to this...

  13. 14 CFR Sec. 1-6 - Accounting entities.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Accounting entities. Sec. 1-6 Section 1-6... REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS General Accounting Provisions Sec. 1-6 Accounting entities. (a) Separate accounting records shall be maintained for each...

  14. 14 CFR Sec. 2-4 - Accounting period.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Accounting period. Sec. 2-4 Section 2-4... REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS General Accounting Provisions Sec. 2-4 Accounting period. (a) The accounting year of each air carrier subject to this...

  15. 14 CFR Sec. 19-2 - Maintenance of data.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Maintenance of data. Sec. 19-2 Section 19-2 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION (AVIATION PROCEEDINGS) ECONOMIC REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS Operating Statistics Classifications Sec....

  16. 14 CFR Sec. 19-2 - Maintenance of data.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 4 2013-01-01 2013-01-01 false Maintenance of data. Sec. 19-2 Section 19-2 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION (AVIATION PROCEEDINGS) ECONOMIC REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS Operating Statistics Classifications Sec....

  17. 14 CFR Sec. 19-2 - Maintenance of data.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 4 2014-01-01 2014-01-01 false Maintenance of data. Sec. 19-2 Section 19-2 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION (AVIATION PROCEEDINGS) ECONOMIC REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS Operating Statistics Classifications Sec....

  18. 46 CFR Sec. 6 - Surety and form of bond.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... PERSONNEL Sec. 6 Surety and form of bond. Each bond provided for by this order shall be duly executed by an... 46 Shipping 8 2012-10-01 2012-10-01 false Surety and form of bond. Sec. 6 Section 6 Shipping... published by the U.S. Treasury Department. The form of bond required by the National Shipping Authority...

  19. 46 CFR Sec. 6 - Surety and form of bond.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... PERSONNEL Sec. 6 Surety and form of bond. Each bond provided for by this order shall be duly executed by an... 46 Shipping 8 2011-10-01 2011-10-01 false Surety and form of bond. Sec. 6 Section 6 Shipping... published by the U.S. Treasury Department. The form of bond required by the National Shipping Authority...

  20. 46 CFR Sec. 6 - Surety and form of bond.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 8 2013-10-01 2013-10-01 false Surety and form of bond. Sec. 6 Section 6 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY BONDING OF SHIP'S PERSONNEL Sec. 6 Surety and form of bond. Each bond provided for by this order shall be duly executed by an authorized surety appearing on the...

  1. 46 CFR Sec. 6 - Surety and form of bond.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... PERSONNEL Sec. 6 Surety and form of bond. Each bond provided for by this order shall be duly executed by an... 46 Shipping 8 2014-10-01 2014-10-01 false Surety and form of bond. Sec. 6 Section 6 Shipping... published by the U.S. Treasury Department. The form of bond required by the National Shipping Authority...

  2. 46 CFR Sec. 5 - Measures to protect ship's payrolls.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Measures to protect ship's payrolls. Sec. 5 Section 5... SHIP'S PERSONNEL Sec. 5 Measures to protect ship's payrolls. (a) General Agents are not required to consider the amount of the payroll delivered to the Master at the conclusion of a voyage in determining...

  3. 14 CFR Sec. 19-2 - Maintenance of data.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Maintenance of data. Sec. 19-2 Section 19-2... REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS Operating Statistics Classifications Sec. 19-2 Maintenance of data. (a) Each air carrier required to file Form 41 Schedule T-100...

  4. Structure of external aldimine of Escherichia coli CsdB, an IscS/NifS homolog: implications for its specificity toward selenocysteine.

    PubMed

    Mihara, Hisaaki; Fujii, Tomomi; Kato, Shin-Ichiro; Kurihara, Tatsuo; Hata, Yasuo; Esaki, Nobuyoshi

    2002-05-01

    Escherichia coli CsdB is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes both cysteine desulfuration and selenocysteine deselenation. The enzyme has a high specific activity for L-selenocysteine relative to L-cysteine. On the other hand, its paralog, IscS, exhibits higher activity for L-cysteine, which acts as a sulfur donor during the biosynthesis of the iron-sulfur cluster and 4-thiouridine. The structure of CsdB complexed with L-propargylglycine was determined by X-ray crystallography at 2.8 A resolution. The overall polypeptide fold of the complex is similar to that of the uncomplexed enzyme, indicating that no significant structural change occurs upon formation of the complex. In the complex, propargylglycine forms a Schiff base with PLP, providing the features of the external aldimine formed in the active site. The Cys364 residue, which is essential for the activity of CsdB toward L-cysteine but not toward L-selenocysteine, is clearly visible on a loop of the extended lobe (Thr362-Arg375) in all enzyme forms studied, in contrast to the corresponding disordered loop (Ser321-Arg332) of the Thermotoga maritima NifS-like protein, which is closely related to IscS. The extended lobe of CsdB has an 11-residue deletion compared with that of the NifS-like protein. These facts suggest that the restricted flexibility of the Cys364-anchoring extended lobe in CsdB may be responsible for the ability of the enzyme to discriminate between selenium and sulfur. PMID:11983074

  5. A role for Sec8 in oligodendrocyte morphological differentiation.

    PubMed

    Anitei, Mihaela; Ifrim, Marius; Ewart, Marie-Ann; Cowan, Ann E; Carson, John H; Bansal, Rashmi; Pfeiffer, Steven E

    2006-03-01

    In the central nervous system, oligodendrocytes synthesize vast amounts of myelin, a multilamellar membrane wrapped around axons that dramatically enhances nerve transmission. A complex apparatus appears to coordinate trafficking of proteins and lipids during myelin synthesis, but the molecular interactions involved are not well understood. We demonstrate that oligodendrocytes express several key molecules necessary for the targeting of transport vesicles to areas of rapid membrane growth, including the exocyst components Sec8 and Sec6 and the multidomain scaffolding proteins CASK and Mint1. Sec8 overexpression significantly promotes oligodendrocyte morphological differentiation and myelin-like membrane formation in vitro; conversely, siRNA-mediated interference with Sec8 expression inhibits this process, and anti-Sec8 antibody induces a reduction in oligodendrocyte areas. In addition, Sec8 colocalizes, coimmunoprecipitates and cofractionates with the major myelin protein OSP/Claudin11 and with CASK in oligodendrocytes. These results suggest that Sec8 plays a central role in oligodendrocyte membrane formation by regulating the recruitment of vesicles that transport myelin proteins such as OSP/Claudin11 to sites of membrane growth. PMID:16478790

  6. Differing views of the role of selenium in thioredoxin reductase

    PubMed Central

    Ruggles, Erik L.

    2010-01-01

    This review covers three different chemical explanations that could account for the requirement of selenium in the form of selenocysteine in the active site of mammalian thioredoxin reductase. These views are the following: (1) the traditional view of selenocysteine as a superior nucleophile relative to cysteine, (2) the superior leaving group ability of a selenol relative to a thiol due to its significantly lower pKa and, (3) the superior ability of selenium to accept electrons (electrophilicity) relative to sulfur. We term these chemical explanations as the “chemico-enzymatic” function of selenium in an enzyme. We formally define the chemico-enzymatic function of selenium as its specific chemical property that allows a selenoenzyme to catalyze its individual reaction. However we, and others, question whether selenocysteine is chemically necessary to catalyze an enzymatic reaction since cysteine-homologs of selenocysteine-containing enzymes catalyze their specific enzymatic reactions with high catalytic efficiency. There must be a unique chemical reason for the presence of selenocysteine in enzymes that explains the biological pressure on the genome to maintain the complex selenocysteine-insertion machinery. We term this biological pressure the “chemico-biological” function of selenocysteine. We discuss evidence that this chemico-biological function is the ability of selenoenzymes to resist inactivation by irreversible oxidation. The way in which selenocysteine confers resistance to oxidation could be due to the superior ability of the oxidized form of selenocysteine (Sec-SeO2−, seleninic acid) to be recycled back to its parent form (Sec-SeH, selenocysteine) in comparison to the same cycling of cysteine-sulfinic acid to cysteine (Cys-SO2− to Cys-SH). PMID:20397034

  7. HOPS prevents the disassembly of trans-SNARE complexes by Sec17p/Sec18p during membrane fusion

    PubMed Central

    Xu, Hao; Jun, Youngsoo; Thompson, James; Yates, John; Wickner, William

    2010-01-01

    SNARE-dependent membrane fusion requires the disassembly of cis-SNARE complexes (formed by SNAREs anchored to one membrane) followed by the assembly of trans-SNARE complexes (SNAREs anchored to two apposed membranes). Although SNARE complex disassembly and assembly might be thought to be opposing reactions, the proteins promoting disassembly (Sec17p/Sec18p) and assembly (the HOPS complex) work synergistically to support fusion. We now report that trans-SNARE complexes formed during vacuole fusion are largely associated with Sec17p. Using a reconstituted proteoliposome fusion system, we show that trans-SNARE complex, like cis-SNARE complex, is sensitive to Sec17p/Sec18p mediated disassembly. Strikingly, HOPS inhibits the disassembly of SNARE complexes in the trans-, but not in the cis-, configuration. This selective HOPS preservation of trans-SNARE complexes requires HOPS:SNARE recognition and is lost when the apposed bilayers are dissolved in Triton X-100; it is also observed during fusion of isolated vacuoles. HOPS thus directs the Sec17p/Sec18p chaperone system to maximize functional trans-SNARE complex for membrane fusion, a new role of tethering factors during membrane traffic. PMID:20473271

  8. 30. Pennsylvania Railroad: Newark Station. Newark, Essex Co., NJ. Sec. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    30. Pennsylvania Railroad: Newark Station. Newark, Essex Co., NJ. Sec. 1401, MP 8.60. - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ

  9. Susquehanna River Bridge. Havre de Grace, Hareford Co., MD. Sec. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Susquehanna River Bridge. Havre de Grace, Hareford Co., MD. Sec. 1201, MP 60.07. - Northeast Railroad Corridor, Amtrak route between District of Columbia/Maryland state line & Maryland/Delaware state line, Baltimore, Independent City, MD

  10. 52. Housatonic River Bridge. Stratford, Fairfield Co., CT. Sec. 9108, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    52. Housatonic River Bridge. Stratford, Fairfield Co., CT. Sec. 9108, MP 60.44. - Northeast Railroad Corridor, Amtrak Route between New York/Connecticut & Connecticut/Rhode Island State Lines, New Haven, New Haven County, CT

  11. 49. Housatonic River Bridge. Stratford, Fairfield Co., CT. Sec. 9108, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    49. Housatonic River Bridge. Stratford, Fairfield Co., CT. Sec. 9108, MP 60.44. - Northeast Railroad Corridor, Amtrak Route between New York/Connecticut & Connecticut/Rhode Island State Lines, New Haven, New Haven County, CT

  12. Juniata Street Culvert. Havre de Grace, Hareford Co., MD. Sec. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Juniata Street Culvert. Havre de Grace, Hareford Co., MD. Sec. 1201, MP 60.77. - Northeast Railroad Corridor, Amtrak route between District of Columbia/Maryland state line & Maryland/Delaware state line, Baltimore, Independent City, MD

  13. 53. Housatonic River Bridge. Stratford, Fairfield Co., CT. Sec. 9108, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    53. Housatonic River Bridge. Stratford, Fairfield Co., CT. Sec. 9108, MP 60.44. - Northeast Railroad Corridor, Amtrak Route between New York/Connecticut & Connecticut/Rhode Island State Lines, New Haven, New Haven County, CT

  14. 51. Housatonic River Bridge. Stratford, Fairfield Co., CT. Sec. 9108, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    51. Housatonic River Bridge. Stratford, Fairfield Co., CT. Sec. 9108, MP 60.44. - Northeast Railroad Corridor, Amtrak Route between New York/Connecticut & Connecticut/Rhode Island State Lines, New Haven, New Haven County, CT

  15. 50. Housatonic River Bridge. Stratford, Fairfield Co., CT. Sec. 9108, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    50. Housatonic River Bridge. Stratford, Fairfield Co., CT. Sec. 9108, MP 60.44. - Northeast Railroad Corridor, Amtrak Route between New York/Connecticut & Connecticut/Rhode Island State Lines, New Haven, New Haven County, CT

  16. 57. Housatonic River Bridge. Stratford, Fairfield Co., CT. Sec. 9108, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    57. Housatonic River Bridge. Stratford, Fairfield Co., CT. Sec. 9108, MP 60.44. - Northeast Railroad Corridor, Amtrak Route between New York/Connecticut & Connecticut/Rhode Island State Lines, New Haven, New Haven County, CT

  17. 31. Pennsylvania Railroad: Newark Station. Newark, Essex Co., NJ. Sec. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    31. Pennsylvania Railroad: Newark Station. Newark, Essex Co., NJ. Sec. 1401, MP 8.60. - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ

  18. 55. Housatonic River Bridge. Stratford, Fairfield Co., CT. Sec. 9108, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    55. Housatonic River Bridge. Stratford, Fairfield Co., CT. Sec. 9108, MP 60.44. - Northeast Railroad Corridor, Amtrak Route between New York/Connecticut & Connecticut/Rhode Island State Lines, New Haven, New Haven County, CT

  19. 56. Housatonic River Bridge. Stratford, Fairfield Co., CT. Sec. 9108, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    56. Housatonic River Bridge. Stratford, Fairfield Co., CT. Sec. 9108, MP 60.44. - Northeast Railroad Corridor, Amtrak Route between New York/Connecticut & Connecticut/Rhode Island State Lines, New Haven, New Haven County, CT

  20. 54. Housatonic River Bridge. Stratford, Fairfield Co., CT. Sec. 9108, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    54. Housatonic River Bridge. Stratford, Fairfield Co., CT. Sec. 9108, MP 60.44. - Northeast Railroad Corridor, Amtrak Route between New York/Connecticut & Connecticut/Rhode Island State Lines, New Haven, New Haven County, CT

  1. 20. Typical circuit breaker gantry. Norwalk, Fairfield Co., CT. Sec. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    20. Typical circuit breaker gantry. Norwalk, Fairfield Co., CT. Sec. 9108, MP 41.20. - Northeast Railroad Corridor, Amtrak Route between New York/Connecticut & Connecticut/Rhode Island State Lines, New Haven, New Haven County, CT

  2. 5. ISLAND ROAD BRIDGE. COLWYN, DELAWARE CO., PA. Sec. 1101, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. ISLAND ROAD BRIDGE. COLWYN, DELAWARE CO., PA. Sec. 1101, MP 5.58. - Northeast Railroad Corridor, Amtrak route between Delaware-Pennsylvania & Pennsylvania-New Jersey state lines, Philadelphia, Philadelphia County, PA

  3. 4. COBBS CREEK BRIDGE. COLWYN, DELAWARE CO., PA. Sec. 1101, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    4. COBBS CREEK BRIDGE. COLWYN, DELAWARE CO., PA. Sec. 1101, MP 5.73 - Northeast Railroad Corridor, Amtrak route between Delaware-Pennsylvania & Pennsylvania-New Jersey state lines, Philadelphia, Philadelphia County, PA

  4. 38. Saga Interlocking Tower. Greens Farms, Fairfield Co., CT. Sec. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    38. Saga Interlocking Tower. Greens Farms, Fairfield Co., CT. Sec. 9108, MP 47.00. - Northeast Railroad Corridor, Amtrak Route between New York/Connecticut & Connecticut/Rhode Island State Lines, New Haven, New Haven County, CT

  5. 37. Saga Interlocking Tower. Greens Farms, Fairfield Co., CT. Sec. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    37. Saga Interlocking Tower. Greens Farms, Fairfield Co., CT. Sec. 9108, MP 47.00. - Northeast Railroad Corridor, Amtrak Route between New York/Connecticut & Connecticut/Rhode Island State Lines, New Haven, New Haven County, CT

  6. CoSEC: Connecting Living With a Star Research

    NASA Astrophysics Data System (ADS)

    Hurlburt, N.; Freeland, S.; Bose, P.; Zimdars, A.; Slater, G.

    2006-12-01

    The Collaborative Sun-Earth Connector (CoSEC) provide the means for heliophysics researchers to compose the data sources and processing services published by their peers into processing workflows that reliably generate publication-worthy data. It includes: composition of computational and data services into easy-to- read workflows with data quality and version traceability; straightforward translation of existing services into workflow components, and advertisement of those components to other members of the CoSEC community; annotation of published services with functional attributes to enable discovery of capabilities required by particular workflows and identify peer subgroups in the CoSEC community; and annotation of published services with nonfunctional attributes to enable selection on the basis of quality of service (QoS). We present an overview and demonstration of the CoSEC system, discuss applications, the lessons learned and future developments.

  7. 124. Mystic River Bridge. Mystic, New London Co., CT. Sec. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    124. Mystic River Bridge. Mystic, New London Co., CT. Sec. 4215, MP 132.16. - Northeast Railroad Corridor, Amtrak Route between New York/Connecticut & Connecticut/Rhode Island State Lines, New Haven, New Haven County, CT

  8. 20. Zoo Substation. Philadelphia, Philadelphia Co., PA. Sec. 1101, MP ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    20. Zoo Substation. Philadelphia, Philadelphia Co., PA. Sec. 1101, MP 87.25. - Northeast Railroad Corridor, Amtrak route between Delaware-Pennsylvania & Pennsylvania-New Jersey state lines, Philadelphia, Philadelphia County, PA

  9. 123. Mystic River Bridge. Mystic, New London Co., CT. Sec. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    123. Mystic River Bridge. Mystic, New London Co., CT. Sec. 4215, MP 132.16. - Northeast Railroad Corridor, Amtrak Route between New York/Connecticut & Connecticut/Rhode Island State Lines, New Haven, New Haven County, CT

  10. Preliminary design package for Sunair SEC-601 solar collector

    NASA Technical Reports Server (NTRS)

    1978-01-01

    The preliminary design of the Owens-Illinois model Sunair SEC-601 tubular air solar collector is presented. Information in this package includes the subsystem design and development approaches, hazard analysis, and detailed drawings available as the preliminary design review.

  11. Insights into COPll Coat Nucleation from the Structure of Sec23 Sar1 Complexed with the Active Fragment of Sec31

    SciTech Connect

    Bi,X.; Mancias, J.; Goldberg, J.

    2007-01-01

    The COPII vesicular coat forms on the endoplasmic reticulum from Sar1-GTP, Sec23/24 and Sec13/31 protein subunits. Here, we define the interaction between Sec23/24{center_dot}Sar1 and Sec13/31, involving a 40 residue Sec31 fragment. In the crystal structure of the ternary complex, Sec31 binds as an extended polypeptide across a composite surface of the Sec23 and Sar1-GTP molecules, explaining the stepwise character of Sec23/24{center_dot}Sar1 and Sec13/31 recruitment to the membrane. The Sec31 fragment stimulates GAP activity of Sec23/24, and a convergence of Sec31 and Sec23 residues at the Sar1 GTPase active site explains how GTP hydrolysis is triggered leading to COPII coat disassembly. The Sec31 active fragment is accommodated in a binding groove supported in part by Sec23 residue Phe380. Substitution of the corresponding residue F382L in human Sec23A causes cranio-lenticulo-sutural dysplasia, and we suggest that this mutation disrupts the nucleation of COPII coat proteins at endoplasmic reticulum exit sites.

  12. Mutations of the molecular chaperone protein SecB which alter the interaction between SecB and maltose-binding protein.

    PubMed

    Gannon, P M; Kumamoto, C A

    1993-01-25

    SecB is a 16-kDa cytosolic chaperone protein that is required for efficient export of particular proteins in Escherichia coli. To identify regions of SecB that contribute to efficient protein export, we isolated secB point mutants that are defective for protein export in vivo. We obtained missense mutations at residues Leu75 (SecBL75Q), Cys76 (SecBC76Y), and Glu77 (SecBE77K) in the center of the secB gene. In vivo, mutant SecBL75Q and SecBE77K proteins are capable of binding to precursor maltose-binding protein (MBP) and preventing the formation of export-incompetent precursor MBP; however, export of MBP is still defective. In vitro, purified SecBL75Q and SecBE77K proteins bound to unfolded MBP and blocked its refolding. SecBL75Q and SecBE77K were more effective than wild-type SecB at blocking the refolding of unfolded MBP, suggesting that SecBL75Q and SecBE77K have a higher affinity for unfolded MBP. PMID:8420934

  13. Direct determination of seleno-amino acids in biological tissues by anion-exchange separation and electrochemical detection.

    PubMed

    Cavalli, S; Cardellicchio, N

    1995-07-01

    Several studies have described the determination of selenium in protein extracts from tissues of marine or terrestrial animals, but have not identified the different chemical forms of selenium that are present. Selenium may be present as seleno-amino acids. Selenocysteine, for example, is a normal component of glutathione peroxidase, an antioxidant enzyme which may behave like other antioxidants, such as vitamin E, protecting tissues against methylmercury toxicity. The present study illustrates a method for the characterization of seleno-amino acids, such as selenocysteine and selenomethionine, in proteins extracted from the liver of marine mammals. The mechanism of detoxification of methylmercury, which involves seleno-compounds, is identified. The analytical determination was carried out using high-performance anion-exchange chromatography coupled with integrated pulsed amperometric detection (HPAEC-IPAD). This method allows the direct determination of underivatized amino acids, eliminating the procedure of pre- or postcolumn derivatization. The chromatographic separation was carried out on an anion-exchange column using a quaternary gradient elution. In order to optimize this method, interferences of amino acids and the influence of pH and ionic strength on the separation and electrochemical detection were studied. The IPAD response for the direct detection of amino acids is optimum at pH > 11. The detection limit (S/N = 3) for selenocysteine was found to be 450 micrograms/l. The application of this method for the identification of seleno-amino acids in protein hydrolysates is also shown. PMID:7640774

  14. Eukaryotic selenoprotein synthesis: mechanistic insight incorporating new factors and new functions for old factors.

    PubMed

    Squires, Jeffrey E; Berry, Marla J

    2008-04-01

    Selenium is an essential micronutrient that has been linked to various aspects of human health. Selenium exerts its biological activity through the incorporation of the amino acid, selenocysteine (Sec), into a unique class of proteins termed selenoproteins. Sec incorporation occurs cotranslationally at UGA codons in archaea, prokaryotes, and eukaryotes. UGA codons specify Sec coding rather than termination by the presence of specific secondary structures in mRNAs termed selenocysteine insertion (SECIS) elements, and trans-acting factors that associate with SECIS elements. Herein, we discuss the various proteins known to function in eukaryotic selenoprotein biosynthesis, including several players whose roles have only been elucidated very recently. PMID:18344183

  15. Dimerization of the exocyst protein Sec6p and its interaction with the t-SNARE Sec9p.

    PubMed

    Sivaram, Mylavarapu V S; Saporita, Jennifer A; Furgason, Melonnie L M; Boettcher, Angela J; Munson, Mary

    2005-04-26

    Vesicles in eukaryotic cells transport cargo between functionally distinct membrane-bound organelles and the plasma membrane for growth and secretion. Trafficking and fusion of vesicles to specific target sites are highly regulated processes that are not well understood at the molecular level. At the plasma membrane, tethering and fusion of secretory vesicles require the exocyst complex. As a step toward elucidation of the molecular architecture and biochemical function(s) of the exocyst complex, we expressed and purified the exocyst subunit Sec6p and demonstrated that it is a predominantly helical protein. Biophysical characterization of purified Sec6p by gel filtration and analytical ultracentrifugation experiments revealed that Sec6p is a dimer. Limited proteolysis defined an independently folded C-terminal domain (residues 300-805) that equilibrated between a dimer and monomer in solution. Removal of residues 300-410 from this construct yielded a well-folded, monomeric domain. These results demonstrate that residues 300-410 are necessary for dimerization, and the presence of the N-terminal region (1-299) increases dimer stability. Moreover, we found that the dimer of Sec6p binds to the plasma membrane t-SNARE Sec9p and inhibits the interaction between Sec9p and its partner t-SNARE Sso1p. This direct interaction between the exocyst complex and the t-SNARE implicates the exocyst in SNARE complex regulation. PMID:15835919

  16. Exo84 and Sec5 are competitive regulatory Sec6/8 effectors to the RalA GTPase.

    PubMed

    Jin, Rongsheng; Junutula, Jagath R; Matern, Hugo T; Ervin, Karen E; Scheller, Richard H; Brunger, Axel T

    2005-06-15

    The Sec6/8 complex, also known as the exocyst complex, is an octameric protein complex that has been implicated in tethering of secretory vesicles to specific regions on the plasma membrane. Two subunits of the Sec6/8 complex, Exo84 and Sec5, have recently been shown to be effector targets for active Ral GTPases. However, the mechanism by which Ral proteins regulate the Sec6/8 activities remains unclear. Here, we present the crystal structure of the Ral-binding domain of Exo84 in complex with active RalA. The structure reveals that the Exo84 Ral-binding domain adopts a pleckstrin homology domain fold, and that RalA interacts with Exo84 via an extended interface that includes both switch regions. Key residues of Exo84 and RalA were found that determine the specificity of the complex interactions; these interactions were confirmed by mutagenesis binding studies. Structural and biochemical data show that Exo84 and Sec5 competitively bind to active RalA. Taken together, these results further strengthen the proposed role of RalA-regulated assembly of the Sec6/8 complex. PMID:15920473

  17. Homotypic vacuole fusion requires Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF).

    PubMed Central

    Haas, A; Wickner, W

    1996-01-01

    In Saccharomyces cerevisiae, vacuoles are inherited by the formation of tubular and vesicular structures from the mother vacuole, the directed projection of these structures into the bud and the homotypic fusion of these vesicles. We have previously exploited a cell-free inheritance assay to show that the fusion step of vacuole inheritance requires cytosol, ATP and the GTPase Ypt7p. Here we demonstrate, using affinity-purified antibodies and purified recombinant proteins, a requirement for Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF) in homotypic vacuole fusion in vitro. Thus, Sec17p and Sec18p, which are typically involved in heterotypic transport steps, can also be involved in homotypic organelle fusion. We further show that vacuole-to-vacuole fusion is stimulated by certain fatty acyl-coenzyme A compounds in a Sec18p-dependent fashion. Finally, our data suggest the presence of a cytosolic factor which activates vacuole membrane-bound Sec18p. Images PMID:8670830

  18. Function and Evolution of Two Forms of SecDF Homologs in Streptomyces coelicolor

    PubMed Central

    Zhou, Zhan; Li, Yudong; Sun, Ning; Sun, Zhihao; Lv, Longxian; Wang, Yufeng; Shen, Libing; Li, Yong-Quan

    2014-01-01

    The general secretion (Sec) pathway plays a prominent role in bacterial protein export, and the accessory component SecDF has been shown to improve transportation efficiency. Inspection of Streptomyces coelicolor genome reveals the unexpected presence of two different forms of secDF homologous genes: one in fused form (secDF) and the other in separated form (secD and secF). However, the functional role of two SecDF homologs in S. coelicolor has not yet been determined. Transcriptional analysis of secDF homologs reveals that these genes are constitutively expressed. However, the transcript levels of secD and secF are much higher than that of secDF in S. coelicolor. Deletion of secDF or/and secD/secF in S. coelicolor did result in reduced secretion efficiency of Xylanase A and Amylase C, suggesting that they may have redundant functions for Sec-dependent translocation pathway. Moreover, our results also indicate that SecD/SecF plays a more prominent role than SecDF in protein translocation. Evolutionary analysis suggests that the fused and separated SecDF homologs in Streptomyces may have disparate evolutionary ancestries. SecD/SecF may be originated from vertical transmission of existing components from ancestor of Streptomyces species. However, SecDF may be derived from bacterial ancestors through horizontal gene transfer. Alternately, it is also plausible that SecDF may have arisen through additional gene duplication and fusion events. The acquisition of a second copy may confer a selective benefit to Streptomyces by enhancing protein transport capacity. Taken together, our results provide new insights into the potential biological function and evolutionary aspects of the prokaryotic SecDF complex. PMID:25140821

  19. ATPase active-site electrostatic interactions control the global conformation of the 100 kDa SecA translocase

    PubMed Central

    Kim, Dorothy M.; Zheng, Haiyan; Huang, Yuanpeng J.; Montelione, Gaetano T.; Hunt, John F.

    2013-01-01

    SecA is an intensively studied mechanoenzyme that uses ATP hydrolysis to drive processive extrusion of secreted proteins through a protein-conducting channel in the cytoplasmic membrane of eubacteria. The ATPase motor of SecA is strongly homologous to that in DEAD-box RNA helicases. It remains unclear how local chemical events in its ATPase active site control the overall conformation of an ~100 kDa multidomain enzyme and drive protein transport. In this paper, we use biophysical methods to establish that a single electrostatic charge in the ATPase active site controls the global conformation of SecA. The enzyme undergoes an ATP-modulated endothermic conformational transition (ECT) believed to involve similar structural mechanics to the protein transport reaction. We have characterized the effects of an isosteric glutamate-to-glutamine mutation in the catalytic base, which mimics the immediate electrostatic consequences of ATP hydrolysis in the active site. Calorimetric studies demonstrate that this mutation facilitates the ECT in E. coli SecA and triggers it completely in B. subtilis SecA. Consistent with the substantial increase in entropy observed in the course of the ECT, hydrogen-deuterium exchange mass spectrometry demonstrates that it increases protein backbone dynamics in domain-domain interfaces at remote locations from the ATPase active site. The catalytic glutamate is one of ~250 charged amino acids in SecA, and yet neutralization of its sidechain charge is sufficient to trigger a global order-disorder transition in this 100 kDa enzyme. The intricate network of structural interactions mediating this effect couples local electrostatic changes during ATP hydrolysis to global conformational and dynamic changes in SecA. This network forms the foundation of the allosteric mechanochemistry that efficiently harnesses the chemical energy stored in ATP to drive complex mechanical processes. PMID:23167435

  20. Structure of the native Sec61 protein-conducting channel

    PubMed Central

    Pfeffer, Stefan; Burbaum, Laura; Unverdorben, Pia; Pech, Markus; Chen, Yuxiang; Zimmermann, Richard; Beckmann, Roland; Förster, Friedrich

    2015-01-01

    In mammalian cells, secretory and membrane proteins are translocated across or inserted into the endoplasmic reticulum (ER) membrane by the universally conserved protein-conducting channel Sec61, which has been structurally studied in isolated, detergent-solubilized states. Here we structurally and functionally characterize native, non-solubilized ribosome-Sec61 complexes on rough ER vesicles using cryo-electron tomography and ribosome profiling. Surprisingly, the 9-Å resolution subtomogram average reveals Sec61 in a laterally open conformation, even though the channel is not in the process of inserting membrane proteins into the lipid bilayer. In contrast to recent mechanistic models for polypeptide translocation and insertion, our results indicate that the laterally open conformation of Sec61 is the only conformation present in the ribosome-bound translocon complex, independent of its functional state. Consistent with earlier functional studies, our structure suggests that the ribosome alone, even without a nascent chain, is sufficient for lateral opening of Sec61 in a lipid environment. PMID:26411746

  1. Modeling and gene knockdown to assess the contribution of nonsense-mediated decay, premature termination, and selenocysteine insertion to the selenoprotein hierarchy

    PubMed Central

    Meplan, Catherine; Huguenin, Grazielle V.B.; Hesketh, John E.; Shanley, Daryl P.

    2016-01-01

    The expression of selenoproteins, a specific group of proteins that incorporates selenocysteine, is hierarchically regulated by the availability of Se, with some, but not all selenoprotein mRNA transcripts decreasing in abundance with decreasing Se. Selenocysteine insertion into the peptide chain occurs during translation following recoding of an internal UGA stop codon. There is increasing evidence that this UGA recoding competes with premature translation termination, which is followed by nonsense-mediated decay (NMD) of the transcript. In this study, we tested the hypothesis that the susceptibility of different selenoprotein mRNAs to premature termination during translation and differential sensitivity of selenoprotein transcripts to NMD are major factors in the selenoprotein hierarchy. Selenoprotein transcript abundance was measured in Caco-2 cells using real-time PCR under different Se conditions and the data obtained fitted to mathematical models of selenoprotein translation. A calibrated model that included a combination of differential sensitivity of selenoprotein transcripts to NMD and different frequency of non-NMD related premature translation termination was able to fit all the measurements. The model predictions were tested using SiRNA to knock down expression of the crucial NMD factor UPF1 (up-frameshift protein 1) and selenoprotein mRNA expression. The calibrated model was able to predict the effect of UPF1 knockdown on gene expression for all tested selenoproteins, except SPS2 (selenophosphate synthetase), which itself is essential for selenoprotein synthesis. These results indicate an important role for NMD in the hierarchical regulation of selenoprotein mRNAs, with the exception of SPS2 whose expression is likely regulated by a different mechanism. PMID:27208313

  2. Modeling and gene knockdown to assess the contribution of nonsense-mediated decay, premature termination, and selenocysteine insertion to the selenoprotein hierarchy.

    PubMed

    Zupanic, Anze; Meplan, Catherine; Huguenin, Grazielle V B; Hesketh, John E; Shanley, Daryl P

    2016-07-01

    The expression of selenoproteins, a specific group of proteins that incorporates selenocysteine, is hierarchically regulated by the availability of Se, with some, but not all selenoprotein mRNA transcripts decreasing in abundance with decreasing Se. Selenocysteine insertion into the peptide chain occurs during translation following recoding of an internal UGA stop codon. There is increasing evidence that this UGA recoding competes with premature translation termination, which is followed by nonsense-mediated decay (NMD) of the transcript. In this study, we tested the hypothesis that the susceptibility of different selenoprotein mRNAs to premature termination during translation and differential sensitivity of selenoprotein transcripts to NMD are major factors in the selenoprotein hierarchy. Selenoprotein transcript abundance was measured in Caco-2 cells using real-time PCR under different Se conditions and the data obtained fitted to mathematical models of selenoprotein translation. A calibrated model that included a combination of differential sensitivity of selenoprotein transcripts to NMD and different frequency of non-NMD related premature translation termination was able to fit all the measurements. The model predictions were tested using SiRNA to knock down expression of the crucial NMD factor UPF1 (up-frameshift protein 1) and selenoprotein mRNA expression. The calibrated model was able to predict the effect of UPF1 knockdown on gene expression for all tested selenoproteins, except SPS2 (selenophosphate synthetase), which itself is essential for selenoprotein synthesis. These results indicate an important role for NMD in the hierarchical regulation of selenoprotein mRNAs, with the exception of SPS2 whose expression is likely regulated by a different mechanism. PMID:27208313

  3. The 12 Item Social and Economic Conservatism Scale (SECS)

    PubMed Central

    Everett, Jim A. C.

    2013-01-01

    Recent years have seen a surge in psychological research on the relationship between political ideology (particularly conservatism) and cognition, affect, behaviour, and even biology. Despite this flurry of investigation, however, there is as yet no accepted, validated, and widely used multi-item scale of conservatism that is concise, that is modern in its conceptualisation, and that includes both social and economic conservatism subscales. In this paper the 12-Item Social and Economic Conservatism Scale (SECS) is proposed and validated to help fill this gap. The SECS is suggested to be an important and useful tool for researchers working in political psychology. PMID:24349200

  4. Glutathione peroxidase's reaction intermediate selenenic acid is stabilized by the protein microenvironment.

    PubMed

    Li, Fei; Liu, Jun; Rozovsky, Sharon

    2014-11-01

    Selenenic acids are highly reactive intermediates of selenoproteins' enzymatic reactions. Knowledge of how the protein environment protects and stabilizes them is fundamental not only to descriptions of selenoproteins' reactivity but also potentially for proteomics and therapeutics. However, selenenic acids are considered particularly short-lived and are not yet identified in wild-type selenoproteins. Here, we report trapping the selenenic acid in glutathione peroxidase, an antioxidant enzyme that efficiently eliminates hydroperoxides. It has long been thought that selenium-containing glutathione peroxidases form a selenenic acid intermediate. However, this putative species has eluded detection. Here, we report its identification. The selenenic acid in bovine glutathione peroxidase 1 was chemically trapped using dimedone, an alkylating agent specific to sulfenic and selenenic acids. The alkylation of the catalytic selenocysteine was verified by electrospray ionization mass spectrometry. In the presence of glutathione, the selenocysteine was not alkylated because the selenenic acid condenses faster with glutathione than the alkylation reaction. In the absence of thiols, the selenenic acid was surprisingly long-lived with 95% of the protein still able to react with dimedone 10 min after hydrogen peroxide was removed, indicating that the protein environment stabilizes the selenenic acid by shielding it from reactive groups in the protein. After 30 min, the selenocysteine was no longer modified but became accessible once the protein was exposed to reducing agents. This suggests that the selenenic acid reacted with a protein's amide or amine to form a selenylamide bond. Such a modification may play a role in protecting glutathione peroxidase׳' reactivity. PMID:25124921

  5. Glutathione Peroxidase’s Reaction Intermediate Selenenic Acid is Stabilized by the Protein Microenvironment

    PubMed Central

    Li, Fei; Liu, Jun; Rozovsky, Sharon

    2014-01-01

    Selenenic acids are highly reactive intermediates of selenoproteins’ enzymatic reactions. Knowledge of how the protein environment protects and stabilizes them is fundamental not only to descriptions of selenoproteins’ reactivity but also potentially for proteomics and therapeutics. However, selenenic acids are considered particularly short-lived and were not yet identified in wild-type selenoproteins. Here, we report trapping the selenenic acid in glutathione peroxidase, an anti-oxidant enzyme that efficiently eliminates hydroperoxides. It has long been thought that selenium-containing glutathione peroxidases form a selenenic acid intermediate. However, this putative species has eluded detection. Here, we report its identification. The selenenic acid in bovine glutathione peroxidase 1 was chemically trapped using dimedone, an alkylating agent specific to sulfenic and selenenic acids. The alkylation of the catalytic selenocysteine was verified by electrospray ionization mass spectrometry. In the presence of glutathione, the selenocysteine was not alkylated because the selenenic acid condenses faster with glutathione than the alkylation reaction. In the absence of thiols, the selenenic acid was surprisingly long-lived with 95% of the protein still able to react with dimedone 10 min after hydrogen peroxide was removed, indicating that the protein environment stabilizes the selenenic acid by shielding it from reactive groups in the protein. After 30 min, the selenocysteine was no longer modified but became accessible once the protein was exposed to reducing agents. This suggests that the selenenic acid reacted with a protein’s amide or amine to form a selenylamide bond. Such a modification may play a role in protecting glutathione peroxidase’s reactivity. PMID:25124921

  6. 46 CFR Sec. 4 - Procedure for securing competitive bids.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... CONTRACT-NSA-LUMPSUMREP Sec. 4 Procedure for securing competitive bids. (a) The geographical area within... Bids the NSA form entitled “Invitation for Bids, Instruction for Bidders, and Specifications for... appointed representative of the time and place of opening the “Spot Bids,” and if practicable, the...

  7. 46 CFR Sec. 4 - Procedure for securing competitive bids.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... CONTRACT-NSA-LUMPSUMREP Sec. 4 Procedure for securing competitive bids. (a) The geographical area within... Bids the NSA form entitled “Invitation for Bids, Instruction for Bidders, and Specifications for... appointed representative of the time and place of opening the “Spot Bids,” and if practicable, the...

  8. 46 CFR Sec. 4 - Procedure for securing competitive bids.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... CONTRACT-NSA-LUMPSUMREP Sec. 4 Procedure for securing competitive bids. (a) The geographical area within... Bids the NSA form entitled “Invitation for Bids, Instruction for Bidders, and Specifications for... appointed representative of the time and place of opening the “Spot Bids,” and if practicable, the...

  9. 46 CFR Sec. 4 - Procedure for securing competitive bids.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... CONTRACT-NSA-LUMPSUMREP Sec. 4 Procedure for securing competitive bids. (a) The geographical area within... Bids the NSA form entitled “Invitation for Bids, Instruction for Bidders, and Specifications for... appointed representative of the time and place of opening the “Spot Bids,” and if practicable, the...

  10. 46 CFR Sec. 4 - Procedure for securing competitive bids.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CONTRACT-NSA-LUMPSUMREP Sec. 4 Procedure for securing competitive bids. (a) The geographical area within... Bids the NSA form entitled “Invitation for Bids, Instruction for Bidders, and Specifications for... appointed representative of the time and place of opening the “Spot Bids,” and if practicable, the...

  11. 14 CFR Sec. 2-1 - Generally accepted accounting principles.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Generally accepted accounting principles... AIR CARRIERS General Accounting Provisions Sec. 2-1 Generally accepted accounting principles. (a) The accounting provisions contained in this part are based on generally accepted accounting principles...

  12. 46 CFR Sec. 6 - Disbursements at other domestic ports.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Disbursements at other domestic ports. Sec. 6 Section 6 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURAL... domestic ports. Disbursements at domestic ports other than the principal office of the agent for...

  13. 46 CFR Sec. 7 - Disbursements at foreign ports.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Disbursements at foreign ports. Sec. 7 Section 7... ports. Disbursement procedures at foreign ports may differ in the case of individual agents and in view of existing conditions. Disbursements at foreign ports shall be made by one of the following...

  14. Size-exclusion chromatography (SEC) of branched polymers and polysaccharides.

    PubMed

    Gaborieau, Marianne; Castignolles, Patrice

    2011-02-01

    Branched polymers are among the most important polymers, ranging from polyolefins to polysaccharides. Branching plays a key role in the chain dynamics. It is thus very important for application properties such as mechanical and adhesive properties and digestibility. It also plays a key role in viscous properties, and thus in the mechanism of the separation of these polymers in size-exclusion chromatography (SEC). Critically reviewing the literature, particularly on SEC of polyolefins, polyacrylates and starch, we discuss common pitfalls but also highlight some unexplored possibilities to characterize branched polymers. The presence of a few long-chain branches has been shown to lead to a poor separation in SEC, as evidenced by multiple-detection SEC or multidimensional liquid chromatography. The local dispersity can be large in that case, and the accuracy of molecular weight determination achieved by current methods is poor, although hydrodynamic volume distributions offer alternatives. In contrast, highly branched polymers do not suffer from this extensive incomplete separation in terms of molecular weight. PMID:20967430

  15. 14 CFR Sec. 2-1 - Generally accepted accounting principles.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Generally accepted accounting principles... AIR CARRIERS General Accounting Provisions Sec. 2-1 Generally accepted accounting principles. (a) The accounting provisions contained in this part are based on generally accepted accounting principles...

  16. 14 CFR Sec. 1-7 - Interpretation of accounts.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 4 2013-01-01 2013-01-01 false Interpretation of accounts. Sec. 1-7 Section 1-7 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION (AVIATION PROCEEDINGS) ECONOMIC REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR...

  17. 14 CFR Sec. 1-7 - Interpretation of accounts.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Interpretation of accounts. Sec. 1-7 Section 1-7 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION (AVIATION PROCEEDINGS) ECONOMIC REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR...

  18. 14 CFR Sec. 1-7 - Interpretation of accounts.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Interpretation of accounts. Sec. 1-7 Section 1-7 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION (AVIATION PROCEEDINGS) ECONOMIC REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR...

  19. 14 CFR Sec. 1-7 - Interpretation of accounts.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 4 2014-01-01 2014-01-01 false Interpretation of accounts. Sec. 1-7 Section 1-7 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION (AVIATION PROCEEDINGS) ECONOMIC REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR...

  20. 10 CFR Appendix A to Subpart A of... - Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141 (42 U.S.C. 2161), Sec...

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Amended, Sec. 141 (42 U.S.C. 2161), Sec. 145 (42 U.S.C. 2165), Sec. 161 (42 U.S.C. 2201) A Appendix A to Subpart A of Part 710 Energy DEPARTMENT OF ENERGY CRITERIA AND PROCEDURES FOR DETERMINING ELIGIBILITY FOR... Eligibility for Access to Classified Matter or Special Nuclear Material Pt. 710, Subpt. A, App. A Appendix...

  1. 10 CFR Appendix A to Subpart A of... - Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141 (42 U.S.C. 2161), Sec...

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Amended, Sec. 141 (42 U.S.C. 2161), Sec. 145 (42 U.S.C. 2165), Sec. 161 (42 U.S.C. 2201) A Appendix A to Subpart A of Part 710 Energy DEPARTMENT OF ENERGY CRITERIA AND PROCEDURES FOR DETERMINING ELIGIBILITY FOR... Eligibility for Access to Classified Matter or Special Nuclear Material Pt. 710, Subpt. A, App. A Appendix...

  2. 10 CFR Appendix A to Subpart A of... - Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141 (42 U.S.C. 2161), Sec...

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Amended, Sec. 141 (42 U.S.C. 2161), Sec. 145 (42 U.S.C. 2165), Sec. 161 (42 U.S.C. 2201) A Appendix A to Subpart A of Part 710 Energy DEPARTMENT OF ENERGY CRITERIA AND PROCEDURES FOR DETERMINING ELIGIBILITY FOR... Eligibility for Access to Classified Matter or Special Nuclear Material Pt. 710, Subpt. A, App. A Appendix...

  3. 10 CFR Appendix A to Subpart A of... - Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141 (42 U.S.C. 2161), Sec...

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Amended, Sec. 141 (42 U.S.C. 2161), Sec. 145 (42 U.S.C. 2165), Sec. 161 (42 U.S.C. 2201) A Appendix A to Subpart A of Part 710 Energy DEPARTMENT OF ENERGY CRITERIA AND PROCEDURES FOR DETERMINING ELIGIBILITY FOR... Eligibility for Access to Classified Matter or Special Nuclear Material Pt. 710, Subpt. A, App. A Appendix...

  4. 10 CFR Appendix A to Subpart A of... - Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141 (42 U.S.C. 2161), Sec...

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Amended, Sec. 141 (42 U.S.C. 2161), Sec. 145 (42 U.S.C. 2165), Sec. 161 (42 U.S.C. 2201) A Appendix A to Subpart A of Part 710 Energy DEPARTMENT OF ENERGY CRITERIA AND PROCEDURES FOR DETERMINING ELIGIBILITY FOR... Eligibility for Access to Classified Matter or Special Nuclear Material Pt. 710, Subpt. A, App. A Appendix...

  5. Functional characterization of the trans-membrane domain interactions of the Sec61 protein translocation complex beta-subunit

    PubMed Central

    Zhao, Xueqiang; Jäntti, Jussi

    2009-01-01

    Background In eukaryotic cells co- and post-translational protein translocation is mediated by the trimeric Sec61 complex. Currently, the role of the Sec61 complex β-subunit in protein translocation is poorly understood. We have shown previously that in Saccharomyces cerevisiae the trans-membrane domain alone is sufficient for the function of the β-subunit Sbh1p in co-translational protein translocation. In addition, Sbh1p co-purifies not only with the protein translocation channel subunits Sec61p and Sss1p, but also with the reticulon family protein Rtn1p. Results We used random mutagenesis to generate novel Sbh1p mutants in order to functionally map the Sbh1p trans-membrane domain. These mutants were analyzed for their interactions with Sec61p and how they support co-translational protein translocation. The distribution of mutations identifies one side of the Sbh1p trans-membrane domain α-helix that is involved in interactions with Sec61p and that is important for Sbh1p function in protein translocation. At the same time, these mutations do not affect Sbh1p interaction with Rtn1p. Furthermore we show that Sbh1p is found in protein complexes containing not only Rtn1p, but also the two other reticulon-like proteins Rtn2p and Yop1p. Conclusion Our results identify functionally important amino acids in the Sbh1p trans-membrane domain. In addition, our results provide additional support for the involvement of Sec61β in processes unlinked to protein translocation. PMID:19857245

  6. Dynamic Interaction of the Sec Translocon with the Chaperone PpiD*

    PubMed Central

    Sachelaru, Ilie; Petriman, Narcis-Adrian; Kudva, Renuka; Koch, Hans-Georg

    2014-01-01

    The Sec translocon constitutes a ubiquitous protein transport channel that consists in bacteria of the three core components: SecY, SecE, and SecG. Additional proteins interact with SecYEG during different stages of protein transport. During targeting, SecYEG interacts with SecA, the SRP receptor, or the ribosome. Protein transport into or across the membrane is then facilitated by the interaction of SecYEG with YidC and the SecDFYajC complex. During protein transport, SecYEG is likely to interact also with the protein quality control machinery, but details about this interaction are missing. By in vivo and in vitro site-directed cross-linking, we show here that the periplasmic chaperone PpiD is located in front of the lateral gate of SecY, through which transmembrane domains exit the SecY channel. The strongest contacts were found to helix 2b of SecY. Blue native PAGE analyses verify the presence of a SecYEG-PpiD complex in native Escherichia coli membranes. The PpiD-SecY interaction was not influenced by the addition of SecA and only weakly influenced by binding of nontranslating ribosomes to SecYEG. In contrast, PpiD lost contact to the lateral gate of SecY during membrane protein insertion. These data identify PpiD as an additional and transient subunit of the bacterial SecYEG translocon. The data furthermore demonstrate the highly modular and versatile composition of the Sec translocon, which is probably essential for its ability to transport a wide range of substrates across membranes in bacteria and eukaryotes. PMID:24951590

  7. A New Class of Endoplasmic Reticulum Export Signal ΦXΦXΦ for Transmembrane Proteins and Its Selective Interaction with Sec24C*

    PubMed Central

    Otsu, Wataru; Kurooka, Takao; Otsuka, Yayoi; Sato, Kota; Inaba, Mutsumi

    2013-01-01

    Protein export from the endoplasmic reticulum (ER) depends on the interaction between a signal motif on the cargo and a cargo recognition site on the coatomer protein complex II. A hydrophobic sequence in the N terminus of the bovine anion exchanger 1 (AE1) anion exchanger facilitated the ER export of human AE1Δ11, an ER-retained AE1 mutant, through interaction with a specific Sec24 isoform. The cell surface expression and N-glycan processing of various substitution mutants or chimeras of human and bovine AE1 proteins and their Δ11 mutants in HEK293 cells were examined. The N-terminal sequence (V/L/F)X(I/L)X(M/L), 26VSIPM30 in bovine AE1, which is comparable with ΦXΦXΦ, acted as the ER export signal for AE1 and AE1Δ11 (Φ is a hydrophobic amino acid, and X is any amino acid). The AE1-Ly49E chimeric protein possessing the ΦXΦXΦ motif exhibited effective cell surface expression and N-glycan maturation via the coatomer protein complex II pathway, whereas a chimera lacking this motif was retained in the ER. A synthetic polypeptide containing the N terminus of bovine AE1 bound the Sec23A-Sec24C complex through a selective interaction with Sec24C. Co-transfection of Sec24C-AAA, in which the residues 895LIL897 (the binding site for another ER export signal motif IXM on Sec24C and Sec24D) were mutated to 895AAA897, specifically increased ER retention of the AE1-Ly49E chimera. These findings demonstrate that the ΦXΦXΦ sequence functions as a novel signal motif for the ER export of cargo proteins through an exclusive interaction with Sec24C. PMID:23658022

  8. SecA Alone Can Promote Protein Translocation and Ion Channel Activity

    PubMed Central

    Hsieh, Ying-hsin; Zhang, Hao; Lin, Bor-ruei; Cui, Ningren; Na, Bing; Yang, Hsiuchin; Jiang, Chun; Sui, Sen-fang; Tai, Phang C.

    2011-01-01

    SecA is an essential component of the Sec-dependent protein translocation pathway across cytoplasmic membranes in bacteria. Escherichia coli SecA binds to cytoplasmic membranes at SecYEG high affinity sites and at phospholipid low affinity sites. It has been widely viewed that SecYEG functions as the essential protein-conducting channel through which precursors cross the membranes in bacterial Sec-dependent pathways, and that SecA functions as a motor to hydrolyze ATP in translocating precursors through SecYEG channels. We have now found that SecA alone can promote precursor translocation into phospholiposomes. Moreover, SecA-liposomes elicit ionic currents in Xenopus oocytes. Patch-clamp recordings further show that SecA alone promotes signal peptide- or precursor-dependent single channel activity. These activities were observed with the functional SecA at about 1–2 μm. The results show that SecA alone is sufficient to promote protein translocation into liposomes and to elicit ionic channel activity at the phospholipids low affinity binding sites, thus indicating that SecA is able to form the protein-conducting channels. Even so, such SecA-liposomes are less efficient than those with a full complement of Sec proteins, and lose the signal-peptide proofreading function, resembling the effects of PrlA mutations. Addition of purified SecYEG restores the signal peptide specificity and increases protein translocation and ion channel activities. These data show that SecA can promote protein translocation and ion channel activities both when it is bound to lipids at low affinity sites and when it is bound to SecYEG with high affinity. The latter of the two interactions confers high efficiency and specificity. PMID:22033925

  9. Disulfide bridge formation between SecY and a translocating polypeptide localizes the translocation pore to the center of SecY.

    PubMed

    Cannon, Kurt S; Or, Eran; Clemons, William M; Shibata, Yoko; Rapoport, Tom A

    2005-04-25

    During their biosynthesis, many proteins pass through the membrane via a hydrophilic channel formed by the heterotrimeric Sec61/SecY complex. Whether this channel forms at the interface of multiple copies of Sec61/SecY or is intrinsic to a monomeric complex, as suggested by the recently solved X-ray structure of the Methanococcus jannaschii SecY complex, is a matter of contention. By introducing a single cysteine at various positions in Escherichia coli SecY and testing its ability to form a disulfide bond with a single cysteine in a translocating chain, we provide evidence that translocating polypeptides pass through the center of the SecY complex. The strongest cross-links were observed with residues that would form a constriction in an hourglass-shaped pore. This suggests that the channel makes only limited contact with a translocating polypeptide, thus minimizing the energy required for translocation. PMID:15851514

  10. Mammalian COPII coat component SEC24C is required for embryonic development in mice.

    PubMed

    Adams, Elizabeth J; Chen, Xiao-Wei; O'Shea, K Sue; Ginsburg, David

    2014-07-25

    COPII-coated vesicles mediate the transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi. SEC24 is the COPII component primarily responsible for recruitment of protein cargoes into nascent vesicles. There are four Sec24 paralogs in mammals, with mice deficient in SEC24A, -B, and -D exhibiting a wide range of phenotypes. We now report the characterization of mice with deficiency in the fourth Sec24 paralog, SEC24C. Although mice haploinsufficient for Sec24c exhibit no apparent abnormalities, homozygous deficiency results in embryonic lethality at approximately embryonic day 7. Tissue-specific deletion of Sec24c in hepatocytes, pancreatic cells, smooth muscle cells, and intestinal epithelial cells results in phenotypically normal mice. Thus, SEC24C is required in early mammalian development but is dispensable in a number of tissues, likely as a result of compensation by other Sec24 paralogs. The embryonic lethality resulting from loss of SEC24C occurs considerably later than the lethality previously observed in SEC24D deficiency; it is clearly distinct from the restricted neural tube phenotype of Sec24b null embryos and the mild hypocholesterolemic phenotype of adult Sec24a null mice. Taken together, these results demonstrate that the four Sec24 paralogs have developed unique functions over the course of vertebrate evolution. PMID:24876386

  11. Sec16 alternative splicing dynamically controls COPII transport efficiency.

    PubMed

    Wilhelmi, Ilka; Kanski, Regina; Neumann, Alexander; Herdt, Olga; Hoff, Florian; Jacob, Ralf; Preußner, Marco; Heyd, Florian

    2016-01-01

    The transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi depends on COPII-coated vesicles. While the basic principles of the COPII machinery have been identified, it remains largely unknown how COPII transport is regulated to accommodate tissue- or activation-specific differences in cargo load and identity. Here we show that activation-induced alternative splicing of Sec16 controls adaptation of COPII transport to increased secretory cargo upon T-cell activation. Using splice-site blocking morpholinos and CRISPR/Cas9-mediated genome engineering, we show that the number of ER exit sites, COPII dynamics and transport efficiency depend on Sec16 alternative splicing. As the mechanistic basis, we suggest the C-terminal Sec16 domain to be a splicing-controlled protein interaction platform, with individual isoforms showing differential abilities to recruit COPII components. Our work connects the COPII pathway with alternative splicing, adding a new regulatory layer to protein secretion and its adaptation to changing cellular environments. PMID:27492621

  12. Sec16 alternative splicing dynamically controls COPII transport efficiency

    PubMed Central

    Wilhelmi, Ilka; Kanski, Regina; Neumann, Alexander; Herdt, Olga; Hoff, Florian; Jacob, Ralf; Preußner, Marco; Heyd, Florian

    2016-01-01

    The transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi depends on COPII-coated vesicles. While the basic principles of the COPII machinery have been identified, it remains largely unknown how COPII transport is regulated to accommodate tissue- or activation-specific differences in cargo load and identity. Here we show that activation-induced alternative splicing of Sec16 controls adaptation of COPII transport to increased secretory cargo upon T-cell activation. Using splice-site blocking morpholinos and CRISPR/Cas9-mediated genome engineering, we show that the number of ER exit sites, COPII dynamics and transport efficiency depend on Sec16 alternative splicing. As the mechanistic basis, we suggest the C-terminal Sec16 domain to be a splicing-controlled protein interaction platform, with individual isoforms showing differential abilities to recruit COPII components. Our work connects the COPII pathway with alternative splicing, adding a new regulatory layer to protein secretion and its adaptation to changing cellular environments. PMID:27492621

  13. FunSecKB: the Fungal Secretome KnowledgeBase

    PubMed Central

    Lum, Gengkon; Min, Xiang Jia

    2011-01-01

    The Fungal Secretome KnowledgeBase (FunSecKB) provides a resource of secreted fungal proteins, i.e. secretomes, identified from all available fungal protein data in the NCBI RefSeq database. The secreted proteins were identified using a well evaluated computational protocol which includes SignalP, WolfPsort and Phobius for signal peptide or subcellular location prediction, TMHMM for identifying membrane proteins, and PS-Scan for identifying endoplasmic reticulum (ER) target proteins. The entries were mapped to the UniProt database and any annotations of subcellular locations that were either manually curated or computationally predicted were included in FunSecKB. Using a web-based user interface, the database is searchable, browsable and downloadable by using NCBI’s RefSeq accession or gi number, UniProt accession number, keyword or by species. A BLAST utility was integrated to allow users to query the database by sequence similarity. A user submission tool was implemented to support community annotation of subcellular locations of fungal proteins. With the complete fungal data from RefSeq and associated web-based tools, FunSecKB will be a valuable resource for exploring the potential applications of fungal secreted proteins. Database URL: http://proteomics.ysu.edu/secretomes/fungi.php PMID:21300622

  14. Recombinant forms of M13 procoat with an OmpA leader sequence or a large carboxy-terminal extension retain their independence of secY function.

    PubMed Central

    Kuhn, A; Kreil, G; Wickner, W

    1987-01-01

    The assembly of phage M13 procoat protein into the plasma membrane of Escherichia coli is independent of the secY protein. To test whether this is caused by the unusually small size of procoat, we fused DNA encoding 103 amino acids to the carboxy-terminal end of the procoat gene. The resulting fusion protein, which attains the same membrane-spanning conformation as mature coat protein, still does not require the secY function for membrane assembly. To determine whether the leader sequence governs interaction with the secY protein, we genetically exchanged the leader peptides between procoat and pro-OmpA, a protein which does require secY for its membrane assembly. Each of the resulting hybrid proteins assembles across the plasma membrane, though at a reduced rate. Membrane assembly of the fusion of procoat leader and OmpA required secY function, whereas assembly of the pro-OmpA leader/coat protein fusion was independent of secY. Properties of the entire procoat molecule, rather than its small size or a specific property of its leader peptide determines its mode of membrane assembly. Images Fig. 1. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:3034592

  15. Sec63 and Xbp1 regulate IRE1α activity and polycystic disease severity

    PubMed Central

    Fedeles, Sorin V.; So, Jae-Seon; Shrikhande, Amol; Lee, Seung Hun; Gallagher, Anna-Rachel; Barkauskas, Christina E.; Somlo, Stefan; Lee, Ann-Hwee

    2015-01-01

    The HSP40 cochaperone SEC63 is associated with the SEC61 translocon complex in the ER. Mutations in the gene encoding SEC63 cause polycystic liver disease in humans; however, it is not clear how altered SEC63 influences disease manifestations. In mice, loss of SEC63 induces cyst formation both in liver and kidney as the result of reduced polycystin-1 (PC1). Here we report that inactivation of SEC63 induces an unfolded protein response (UPR) pathway that is protective against cyst formation. Specifically, using murine genetic models, we determined that SEC63 deficiency selectively activates the IRE1α-XBP1 branch of UPR and that SEC63 exists in a complex with PC1. Concomitant inactivation of both SEC63 and XBP1 exacerbated the polycystic kidney phenotype in mice by markedly suppressing cleavage at the G protein–coupled receptor proteolysis site (GPS) in PC1. Enforced expression of spliced XBP1 (XBP1s) enhanced GPS cleavage of PC1 in SEC63-deficient cells, and XBP1 overexpression in vivo ameliorated cystic disease in a murine model with reduced PC1 function that is unrelated to SEC63 inactivation. Collectively, the findings show that SEC63 function regulates IRE1α/XBP1 activation, SEC63 and XBP1 are required for GPS cleavage and maturation of PC1, and activation of XBP1 can protect against polycystic disease in the setting of impaired biogenesis of PC1. PMID:25844898

  16. An improved definition of the RNA-binding specificity of SECIS-binding protein 2, an essential component of the selenocysteine incorporation machinery

    PubMed Central

    Cléry, A.; Bourguignon-Igel, V.; Allmang, C.; Krol, A.; Branlant, C.

    2007-01-01

    By binding to SECIS elements located in the 3′-UTR of selenoprotein mRNAs, the protein SBP2 plays a key role in the assembly of the selenocysteine incorporation machinery. SBP2 contains an L7Ae/L30 RNA-binding domain similar to that of protein 15.5K/Snu13p, which binds K-turn motifs with a 3-nt bulge loop closed by a tandem of G.A and A.G pairs. Here, by SELEX experiments, we demonstrate the capacity of SBP2 to bind such K-turn motifs with a protruding U residue. However, we show that conversion of the bulge loop into an internal loop reinforces SBP2 affinity and to a greater extent RNP stability. Opposite variations were found for Snu13p. Accordingly, footprinting assays revealed strong contacts of SBP2 with helices I and II and the 5′-strand of the internal loop, as opposed to the loose interaction of Snu13p. Our data also identifies new determinants for SBP2 binding which are located in helix II. Among the L7Ae/L30 family members, these determinants are unique to SBP2. Finally, in accordance with functional data on SECIS elements, the identity of residues at positions 2 and 3 in the loop influences SBP2 affinity. Altogether, the data provide a very precise definition of the SBP2 RNA specificity. PMID:17332014

  17. The Sec6/8 complex in mammalian cells: characterization of mammalian Sec3, subunit interactions, and expression of subunits in polarized cells.

    PubMed

    Matern, H T; Yeaman, C; Nelson, W J; Scheller, R H

    2001-08-14

    The yeast exocyst complex (also called Sec6/8 complex in higher eukaryotes) is a multiprotein complex essential for targeting exocytic vesicles to specific docking sites on the plasma membrane. It is composed of eight proteins (Sec3, -5, -6, -8, -10, and -15, and Exo70 and -84), with molecular weights ranging from 70 to 144 kDa. Mammalian orthologues for seven of these proteins have been described and here we report the cloning and initial characterization of the remaining subunit, Sec3. Human Sec3 (hSec3) shares 17% sequence identity with yeast Sec3p, interacts in the two-hybrid system with other subunits of the complex (Sec5 and Sec8), and is expressed in almost all tissues tested. In yeast, Sec3p has been proposed to be a spatial landmark for polarized secretion (1), and its localization depends on its interaction with Rho1p (2). We demonstrate here that hSec3 lacks the potential Rho1-binding site and GFP-fusions of hSec3 are cytosolic. Green fluorescent protein (GFP)-fusions of nearly every subunit of the mammalian Sec6/8 complex were expressed in Madin-Darby canine kidney (MDCK) cells, but they failed to assemble into a complex with endogenous proteins and localized in the cytosol. Of the subunits tested, only GFP-Exo70 localized to lateral membrane sites of cell-cell contact when expressed in MDCK cells. Cells overexpressing GFP-Exo70 fail to form a tight monolayer, suggesting the Exo70 targeting interaction is critical for normal development of polarized epithelial cells. PMID:11493706

  18. Characterization of the Saccharomyces cerevisiae sec6-4 mutation and tools to create S. cerevisiae strains containing the sec6-4 allele.

    PubMed

    Lamping, Erwin; Tanabe, Koichi; Niimi, Masakazu; Uehara, Yoshimasa; Monk, Brian C; Cannon, Richard D

    2005-11-21

    The highly conserved exocyst complex of eukaryotic cells allows the polarized transport and fusion of late secretory vesicles with the plasma membrane. In Saccharomyces cerevisiae the Sec6p component of the exocyst complex is essential for cell growth. The sec6-4 temperature-sensitive mutation of the S. cerevisiae SEC6 gene leads to the accumulation of large amounts of mature late post-Golgi secretory vesicles in the cytosol of mutant cells at the restrictive temperature of 37 degrees C. These readily isolated, inside-out and tightly sealed vesicles contain mature post-translationally modified plasma membrane and secretory proteins and provide a valuable tool for the study of plasma membrane protein function. This study shows that the single point mutation L633P in the SEC6 coding region defines the sec6-4 phenotype. We followed the localization of the wild type Sec6p and the mutant Sec6-4p proteins (C-terminally tagged with the green fluorescent protein yEGfp3p) in the presence or absence of heterologously over-expressed Candida albicans plasma membrane ATP-binding cassette (ABC) transporter CaCdr1p (C-terminally tagged with the red fluorescent protein mRfp1p). The Sec6-4p protein localized to buds and septa, like wild type Sec6p, at the permissive temperature of 23 degrees C and the sec6-4 mutant cells grew at the same rate as the wild type control cells. Sec6-4p was mislocalized at the restrictive temperature of 37 degrees C and heterogenous vesicles accumulated in cells but sec6-4 cells also accumulated homogenous secretory vesicles at the permissive temperature. PMID:16185821

  19. Functional Analysis of the Exocyst Subunit Sec15 in Candida albicans

    PubMed Central

    Chavez-Dozal, Alba A.; Bernardo, Stella M.; Rane, Hallie S.

    2015-01-01

    In prior studies of exocyst-mediated late secretion in Candida albicans, we have determined that Sec6 contributes to cell wall integrity, secretion, and filamentation. A conditional mutant lacking SEC6 expression exhibits markedly reduced lateral hyphal branching. In addition, lack of the related t-SNAREs Sso2 and Sec9 also leads to defects in secretion and filamentation. To further understand the role of the exocyst in the fundamental processes of polarized secretion and filamentation in C. albicans, we studied the exocyst subunit Sec15. Since Saccharomyces cerevisiae SEC15 is essential for viability, we generated a C. albicans conditional mutant strain in which SEC15 was placed under the control of a tetracycline-regulated promoter. In the repressed state, cell death occurred after 5 h in the tetR-SEC15 strain. Prior to this time point, the tetR-SEC15 mutant was markedly defective in Sap and lipase secretion and demonstrated increased sensitivity to Zymolyase and chitinase. Notably, tetR-SEC15 mutant hyphae were characterized by a hyperbranching phenotype, in direct contrast to strain tetR-SEC6, which had minimal lateral branching. We further studied the localization of the Spitzenkörper, polarisomes, and exocysts in the tetR-SEC15 and tetR-SEC6 mutants during filamentation. Mlc1-GFP (marking the Spitzenkörper), Spa2-GFP (the polarisome), and Exo70-GFP (exocyst) localizations were normal in the tetR-SEC6 mutant, whereas these structures were mislocalized in the tetR-SEC15 mutant. Following alleviation of gene repression by removing doxycycline, first Spitzenkörper, then polarisome, and finally exocyst localizations were recovered sequentially. These results indicate that the exocyst subunits Sec15 and Sec6 have distinct roles in mediating polarized secretion and filamentation in C. albicans. PMID:26453654

  20. A complete Sec61 complex in Nosema bombycis and its comparative genomics analyses.

    PubMed

    Wu, Zhengli; Li, Yanhong; Pan, Guoqing; Li, Chunfeng; Hu, Junhua; Liu, Handeng; Zhou, Zeyang; Xiang, Zhonghuai

    2007-01-01

    We characterized a complete Sec61 complex in Nosema bombycis, which has been shown to consist of Sec61alpha, Sec61beta, and Sec61gamma genes. Comparing the genomic regions that harbor the respective subunit genes between N. bombycis, Encephalitozoon cuniculi, and Antonospora locustae, we found that microsporidian genomes have high degree of synteny in short genomic fragment, and that this gene synteny in general might exist throughout microsporidian genomes. PMID:17669164

  1. Functional Analysis of the Exocyst Subunit Sec15 in Candida albicans.

    PubMed

    Chavez-Dozal, Alba A; Bernardo, Stella M; Rane, Hallie S; Lee, Samuel A

    2015-12-01

    In prior studies of exocyst-mediated late secretion in Candida albicans, we have determined that Sec6 contributes to cell wall integrity, secretion, and filamentation. A conditional mutant lacking SEC6 expression exhibits markedly reduced lateral hyphal branching. In addition, lack of the related t-SNAREs Sso2 and Sec9 also leads to defects in secretion and filamentation. To further understand the role of the exocyst in the fundamental processes of polarized secretion and filamentation in C. albicans, we studied the exocyst subunit Sec15. Since Saccharomyces cerevisiae SEC15 is essential for viability, we generated a C. albicans conditional mutant strain in which SEC15 was placed under the control of a tetracycline-regulated promoter. In the repressed state, cell death occurred after 5 h in the tetR-SEC15 strain. Prior to this time point, the tetR-SEC15 mutant was markedly defective in Sap and lipase secretion and demonstrated increased sensitivity to Zymolyase and chitinase. Notably, tetR-SEC15 mutant hyphae were characterized by a hyperbranching phenotype, in direct contrast to strain tetR-SEC6, which had minimal lateral branching. We further studied the localization of the Spitzenkörper, polarisomes, and exocysts in the tetR-SEC15 and tetR-SEC6 mutants during filamentation. Mlc1-GFP (marking the Spitzenkörper), Spa2-GFP (the polarisome), and Exo70-GFP (exocyst) localizations were normal in the tetR-SEC6 mutant, whereas these structures were mislocalized in the tetR-SEC15 mutant. Following alleviation of gene repression by removing doxycycline, first Spitzenkörper, then polarisome, and finally exocyst localizations were recovered sequentially. These results indicate that the exocyst subunits Sec15 and Sec6 have distinct roles in mediating polarized secretion and filamentation in C. albicans. PMID:26453654

  2. Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria

    PubMed Central

    Ishii, Eiji; Chiba, Shinobu; Hashimoto, Narimasa; Kojima, Seiji; Homma, Michio; Ito, Koreaki; Akiyama, Yoshinori; Mori, Hiroyuki

    2015-01-01

    SecDF interacts with the SecYEG translocon in bacteria and enhances protein export in a proton-motive-force-dependent manner. Vibrio alginolyticus, a marine-estuarine bacterium, contains two SecDF paralogs, V.SecDF1 and V.SecDF2. Here, we show that the export-enhancing function of V.SecDF1 requires Na+ instead of H+, whereas V.SecDF2 is Na+-independent, presumably requiring H+. In accord with the cation-preference difference, V.SecDF2 was only expressed under limited Na+ concentrations whereas V.SecDF1 was constitutive. However, it is not the decreased concentration of Na+ per se that the bacterium senses to up-regulate the V.SecDF2 expression, because marked up-regulation of the V.SecDF2 synthesis was observed irrespective of Na+ concentrations under certain genetic/physiological conditions: (i) when the secDF1VA gene was deleted and (ii) whenever the Sec export machinery was inhibited. VemP (Vibrio export monitoring polypeptide), a secretory polypeptide encoded by the upstream ORF of secDF2VA, plays the primary role in this regulation by undergoing regulated translational elongation arrest, which leads to unfolding of the Shine–Dalgarno sequence for translation of secDF2VA. Genetic analysis of V. alginolyticus established that the VemP-mediated regulation of SecDF2 is essential for the survival of this marine bacterium in low-salinity environments. These results reveal that a class of marine bacteria exploits nascent-chain ribosome interactions to optimize their protein export pathways to propagate efficiently under different ionic environments that they face in their life cycles. PMID:26392525

  3. Phylogenetic analysis and delineation of phytoplasmas based on the secY gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The secY gene, located in the operator-distal part of the spc ribosomal protein operon, codes for a protein translocase subunit secY. The secY gene sequence is more variable than that of the 16S rRNA gene. Comparative phylogenetic analyses with 16S rRNA and secY gene sequences from 80 and 83 phytop...

  4. Structural basis of the interaction between RalA and Sec5, a subunit of the sec6/8 complex.

    PubMed

    Fukai, Shuya; Matern, Hugo T; Jagath, Junutula R; Scheller, Richard H; Brunger, Axel T

    2003-07-01

    The sec6/8 complex or exocyst is an octameric protein complex that functions during cell polarization by regulating the site of exocytic vesicle docking to the plasma membrane, in concert with small GTP-binding proteins. The Sec5 subunit of the mammalian sec6/8 complex binds Ral in a GTP-dependent manner. Here we report the crystal structure of the complex between the Ral-binding domain of Sec5 and RalA bound to a non-hydrolyzable GTP analog (GppNHp) at 2.1 A resolution, providing the first structural insights into the mechanism and specificity of sec6/8 regulation. The Sec5 Ral-binding domain folds into an immunoglobulin-like beta-sandwich structure, which represents a novel fold for an effector of a GTP-binding protein. The interface between the two proteins involves a continuous antiparallel beta-sheet, similar to that found in other effector/G-protein complexes, such as Ras and Rap1A. Specific interactions unique to the RalA.Sec5 complex include Sec5 Thr11 and Arg27, and RalA Glu38, which we show are required for complex formation by isothermal titration calorimetry. Comparison of the structures of GppNHp- and GDP-bound RalA suggests a nucleotide-dependent switch mechanism for Sec5 binding. PMID:12839989

  5. Cryo-electron microscopic structure of SecA protein bound to the 70S ribosome.

    PubMed

    Singh, Rajkumar; Kraft, Christian; Jaiswal, Rahul; Sejwal, Kushal; Kasaragod, Vikram Babu; Kuper, Jochen; Bürger, Jörg; Mielke, Thorsten; Luirink, Joen; Bhushan, Shashi

    2014-03-01

    SecA is an ATP-dependent molecular motor pumping secretory and outer membrane proteins across the cytoplasmic membrane in bacteria. SecA associates with the protein-conducting channel, the heterotrimeric SecYEG complex, in a so-called posttranslational manner. A recent study further showed binding of a monomeric state of SecA to the ribosome. However, the true oligomeric state of SecA remains controversial because SecA can also form functional dimers, and high-resolution crystal structures exist for both the monomer and the dimer. Here we present the cryo-electron microscopy structures of Escherichia coli SecA bound to the ribosome. We show that not only a monomeric SecA binds to the ribosome but also that two copies of SecA can be observed that form an elongated dimer. Two copies of SecA completely surround the tunnel exit, providing a unique environment to the nascent polypeptides emerging from the ribosome. We identified the N-terminal helix of SecA required for a stable association with the ribosome. The structures indicate a possible function of the dimeric form of SecA at the ribosome. PMID:24443566

  6. SecB-mediated protein export need not occur via kinetic partitioning.

    PubMed

    Krishnan, Beena; Kulothungan, S Rajendra; Patra, Ashish K; Udgaonkar, Jayant B; Varadarajan, Raghavan

    2009-01-30

    In Escherichia coli, the cytosolic chaperone SecB is responsible for the selective entry of a subset of precursor proteins into the Sec pathway. In vitro, SecB binds to a variety of unfolded substrates without apparent sequence specificity, but not native proteins. Selectivity has therefore been suggested to occur by kinetic partitioning of substrates between protein folding and SecB association. Evidence for kinetic partitioning is based on earlier observations that SecB blocks the refolding of the precursor form of maltose-binding protein (preMBP)(5) and slow-folding maltose-binding protein (MBP) mutants, but not faster-folding mature wild-type MBP. In order to quantitatively validate the kinetic partitioning model, we have independently measured each of the rate constants involved in the interaction of SecB with refolding preMBP (a physiological substrate of SecB) and mature MBP. The measured rate constants correctly predict substrate folding kinetics over a wide range of SecB, MBP, and preMBP concentrations. Analysis of the data reveals that, for many substrates, kinetic partitioning is unlikely to be responsible for SecB-mediated protein export. Instead, the ability of SecB-bound substrates to continue folding while bound to SecB and their ability to interact with other components of the secretory machinery such as SecA may be key opposing determinants that inhibit and promote protein export, respectively. PMID:19028503

  7. Cryo-electron Microscopic Structure of SecA Protein Bound to the 70S Ribosome*

    PubMed Central

    Singh, Rajkumar; Kraft, Christian; Jaiswal, Rahul; Sejwal, Kushal; Kasaragod, Vikram Babu; Kuper, Jochen; Bürger, Jörg; Mielke, Thorsten; Luirink, Joen; Bhushan, Shashi

    2014-01-01

    SecA is an ATP-dependent molecular motor pumping secretory and outer membrane proteins across the cytoplasmic membrane in bacteria. SecA associates with the protein-conducting channel, the heterotrimeric SecYEG complex, in a so-called posttranslational manner. A recent study further showed binding of a monomeric state of SecA to the ribosome. However, the true oligomeric state of SecA remains controversial because SecA can also form functional dimers, and high-resolution crystal structures exist for both the monomer and the dimer. Here we present the cryo-electron microscopy structures of Escherichia coli SecA bound to the ribosome. We show that not only a monomeric SecA binds to the ribosome but also that two copies of SecA can be observed that form an elongated dimer. Two copies of SecA completely surround the tunnel exit, providing a unique environment to the nascent polypeptides emerging from the ribosome. We identified the N-terminal helix of SecA required for a stable association with the ribosome. The structures indicate a possible function of the dimeric form of SecA at the ribosome. PMID:24443566

  8. Essential function of Drosophila Sec6 in apical exocytosis of epithelial photoreceptor cells.

    PubMed

    Beronja, Slobodan; Laprise, Patrick; Papoulas, Ophelia; Pellikka, Milena; Sisson, John; Tepass, Ulrich

    2005-05-23

    Polarized exocytosis plays a major role in development and cell differentiation but the mechanisms that target exocytosis to specific membrane domains in animal cells are still poorly understood. We characterized Drosophila Sec6, a component of the exocyst complex that is believed to tether secretory vesicles to specific plasma membrane sites. sec6 mutations cause cell lethality and disrupt plasma membrane growth. In developing photoreceptor cells (PRCs), Sec6 but not Sec5 or Sec8 shows accumulation at adherens junctions. In late PRCs, Sec6, Sec5, and Sec8 colocalize at the rhabdomere, the light sensing subdomain of the apical membrane. PRCs with reduced Sec6 function accumulate secretory vesicles and fail to transport proteins to the rhabdomere, but show normal localization of proteins to the apical stalk membrane and the basolateral membrane. Furthermore, we show that Rab11 forms a complex with Sec5 and that Sec5 interacts with Sec6 suggesting that the exocyst is a Rab11 effector that facilitates protein transport to the apical rhabdomere in Drosophila PRCs. PMID:15897260

  9. Drosophila sec10 is required for hormone secretion but not general exocytosis or neurotransmission.

    PubMed

    Andrews, Hillary K; Zhang, Yong Q; Trotta, Nick; Broadie, Kendal

    2002-12-01

    The sec6/8, or exocyst, complex is implicated in trafficking of secretory vesicles to fusion sites in the plasma membrane. Genetic analyses have been done primarily in yeast, where mutation of the eight protein subunits similarly disrupts polarized vesicle fusion. The goal of this study was to assay the sec6/8 complex in Drosophila, and specifically to test its widely hypothesized functions in synaptogenesis and neurotransmission. We used a transgenic RNAi approach to remove the most highly conserved complex component, Drosophila sec10 (dSec10). Ubiquitous dSec10 RNAi resulted in early postembryonic lethality, demonstrating that dSec10 is essential. Surprisingly, tissue-specific dSec10 RNAi revealed no essential requirement in nervous system, musculature, gut or epidermis. Assays of polarized secretion in all these tissues failed to reveal any role for dSec10. In particular, the neuromuscular synapse showed no defects in morphogenesis or vesicle trafficking/fusion underlying neurotransmission. The essential requirement for dSec10 was restricted to the ring gland, the Drosophila organ specialized for endocrine function. The developmental arrest of dSec10 RNAi animals was partially rescued by feeding ecdysone, suggesting dSec10 mediates steroid hormone secretion. We conclude that dSec10 has no detectable role in most forms of polarized trafficking/exocytosis, including neurotransmission, but rather is essential for endocrine secretion. PMID:12453153

  10. 75 FR 44781 - Joint CFTC-SEC Advisory Committee on Emerging Regulatory Issues

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-29

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  16. [Investigation of Protein Translocation Sec-System with Heterologous Gene Expression in Shewanella oneidensis MR-1 Bacterium Cells].

    PubMed

    Mordkovich, N N; Okorokova, N A; Veiko, V P

    2015-01-01

    A comparison of the primary structures of the protein translocation Sec-system proteins in the Shewanella oneidensis MR-1 and Escherichia coli bacteria was carried out. The process of translocation of recombinant pro-enteroxins (SEB and SEH) from Staphylococcus aureus and pro-streptavidin (SAV) from Streptomyces avidinii in the S. oneidensis MR-1 and E. coli cell periplasm was studied. It was demonstrated that these marker proteins are transferred into the periplasmic space of the S. oneidensis MR-1 transformant strain cells. The identity of N-terminal amino acid sequences of mature recombinant SEB, SEH, and SAV proteins (generated during post-translation proteolysis of leader peptide by the Sec-system both in E. coli and S. oneidensis MR-1) was established. PMID:26204774

  17. Test experience, 490 N high performance (321 sec Isp) engine

    NASA Technical Reports Server (NTRS)

    Schoenman, L.; Rosenberg, S. D.; Jassowski, D. M.

    1992-01-01

    Engines with area ratios of 44:1 and 286:1 are tested by means of hot fire tests using the NTO/MMH bipropellant to maximize the performance of the combined technologies. The low-thrust engine systems are designed with oxidation resistant materials that can operate at temperatures of more than 2204 C for tens of hours. The chamber is attached to the injector in a configuration that prevents overheating of the injector, valve, and the spacecraft interface. Three injectors with 44:1 area ratios are capable of nominal specific impulse values of 309 sec, and a performance of 321 lbf-sec/lbm is noted for an all-welded engine assembly with area ratio of 286:1. The all-welded engine is shown to have an acceptable design margin for thermal characteristics. High-performance liquid apogee engines are shown to perform optimally when based on iridium/rhenium chamber technology, use of a special platelet injector, and the minimization of losses due to fuel-film cooling.

  18. Sec24D-Dependent Transport of Extracellular Matrix Proteins Is Required for Zebrafish Skeletal Morphogenesis

    PubMed Central

    Sarmah, Swapnalee; Barrallo-Gimeno, Alejandro; Melville, David B.; Topczewski, Jacek; Solnica-Krezel, Lilianna; Knapik, Ela W.

    2010-01-01

    Protein transport from endoplasmic reticulum (ER) to Golgi is primarily conducted by coated vesicular carriers such as COPII. Here, we describe zebrafish bulldog mutations that disrupt the function of the cargo adaptor Sec24D, an integral component of the COPII complex. We show that Sec24D is essential for secretion of cartilage matrix proteins, whereas the preceding development of craniofacial primordia and pre-chondrogenic condensations does not depend on this isoform. Bulldog chondrocytes fail to secrete type II collagen and matrilin to extracellular matrix (ECM), but membrane bound receptor β1-Integrin and Cadherins appear to leave ER in Sec24D-independent fashion. Consequently, Sec24D-deficient cells accumulate proteins in the distended ER, although a subset of ER compartments and Golgi complexes as visualized by electron microscopy and NBD C6-ceramide staining appear functional. Consistent with the backlog of proteins in the ER, chondrocytes activate the ER stress response machinery and significantly upregulate BiP transcription. Failure of ECM secretion hinders chondroblast intercalations thus resulting in small and malformed cartilages and severe craniofacial dysmorphology. This defect is specific to Sec24D mutants since knockdown of Sec24C, a close paralog of Sec24D, does not result in craniofacial cartilage dysmorphology. However, craniofacial development in double Sec24C/Sec24D-deficient animals is arrested earlier than in bulldog/sec24d, suggesting that Sec24C can compensate for loss of Sec24D at initial stages of chondrogenesis, but Sec24D is indispensable for chondrocyte maturation. Our study presents the first developmental perspective on Sec24D function and establishes Sec24D as a strong candidate for cartilage maintenance diseases and craniofacial birth defects. PMID:20442775

  19. SUT1 suppresses sec14-1 through upregulation of CSR1 in Saccharomyces cerevisiae.

    PubMed

    Régnacq, Matthieu; Ferreira, Thierry; Puard, Julien; Bergès, Thierry

    2002-11-01

    SUT1 constitutive expression in aerobiosis suppressed the ts phenotype of the sec14-1 mutation, restored growth of the sec14-null mutant and corrected the translocation defect of the vacuolar carboxypeptidase Y. Therefore SUT1 was shown to be a novel potent sec14-1 suppressor. Further, the hypoxic gene CSR1 (YLR380W), a Sec14 homolog, was upregulated upon SUT1 constitutive expression. In addition, SUT1 effects on both sec14-1 suppression and on free sterol composition were abolished in a csr1-null background, showing that this gene acts downstream of SUT1. PMID:12435498

  20. Neural tube opening and abnormal extraembryonic membrane development in SEC23A deficient mice

    PubMed Central

    Zhu, Min; Tao, Jiayi; Vasievich, Matthew P.; Wei, Wei; Zhu, Guojing; Khoriaty, Rami N.; Zhang, Bin

    2015-01-01

    COPII (coat protein complex-II) vesicles transport proteins from the endoplasmic reticulum (ER) to the Golgi. Higher eukaryotes have two or more paralogs of most COPII components. Here we characterize mice deficient for SEC23A and studied interactions of Sec23a null allele with the previously reported Sec23b null allele. SEC23A deficiency leads to mid-embryonic lethality associated with defective development of extraembryonic membranes and neural tube opening in midbrain. Secretion defects of multiple collagen types are observed in different connective tissues, suggesting that collagens are primarily transported in SEC23A-containing vesicles in these cells. Other extracellular matrix proteins, such as fibronectin, are not affected by SEC23A deficiency. Intracellular accumulation of unsecreted proteins leads to strong induction of the unfolded protein response in collagen-producing cells. No collagen secretion defects are observed in SEC23B deficient embryos. We report that E-cadherin is a cargo that accumulates in acini of SEC23B deficient pancreas and salivary glands. Compensatory increase of one paralog is observed in the absence of the second paralog. Haploinsufficiency of the remaining Sec23 paralog on top of homozygous inactivation of the first paralog leads to earlier lethality of embryos. Our results suggest that mammalian SEC23A and SEC23B transport overlapping yet distinct spectra of cargo in vivo. PMID:26494538

  1. SecDF as Part of the Sec-Translocase Facilitates Efficient Secretion of Bacillus cereus Toxins and Cell Wall-Associated Proteins

    PubMed Central

    Vörös, Aniko; Simm, Roger; Slamti, Leyla; McKay, Matthew J.; Hegna, Ida K.; Nielsen-LeRoux, Christina; Hassan, Karl A.; Paulsen, Ian T.; Lereclus, Didier; Økstad, Ole Andreas; Molloy, Mark P.; Kolstø, Anne-Brit

    2014-01-01

    The aim of this study was to explore the role of SecDF in protein secretion in Bacillus cereus ATCC 14579 by in-depth characterization of a markerless secDF knock out mutant. Deletion of secDF resulted in pleiotropic effects characterized by a moderately slower growth rate, aberrant cell morphology, enhanced susceptibility to xenobiotics, reduced virulence and motility. Most toxins, including food poisoning-associated enterotoxins Nhe, Hbl, and cytotoxin K, as well as phospholipase C were less abundant in the secretome of the ΔsecDF mutant as determined by label-free mass spectrometry. Global transcriptome studies revealed profound transcriptional changes upon deletion of secDF indicating cell envelope stress. Interestingly, the addition of glucose enhanced the described phenotypes. This study shows that SecDF is an important part of the Sec-translocase mediating efficient secretion of virulence factors in the Gram-positive opportunistic pathogen B. cereus, and further supports the notion that B. cereus enterotoxins are secreted by the Sec-system. PMID:25083861

  2. Characterization of three areas of interactions stabilizing complexes between SecA and SecB, two proteins involved in protein export

    PubMed Central

    Patel, Chetan N.; Smith, Virginia F.; Randall, Linda L.

    2006-01-01

    The general secretory, Sec, system translocates precursor polypeptides from the cytosol across the cytoplasmic membrane in Escherichia coli. SecB, a small cytosolic chaperone, captures the precursor polypeptides before they fold and delivers them to the membrane translocon through interactions with SecA. Both SecB and SecA display twofold symmetry and yet the complex between the two is stabilized by contacts that are distributed asymmetrically. Two distinct regions of interaction have been defined previously and here we identify a third. Calorimetric studies of complexes stabilized by different subsets of these interactions were carried out to determine the binding affinities and the thermodynamic parameters that underlie them. We show here that there is no change in affinity when either one of two contact areas out of the three is lacking. This fact and the asymmetry of the binding contacts may be important to the function of the complex in protein export. PMID:16731972

  3. Molecular genetic manipulation of Pichia pastoris SEC4 governs cell growth and glucoamylase secretion

    SciTech Connect

    Liu, S.-H.; Chou, W.-I; Lin, S.-C.; Sheu, C.-C.; Chang, Margaret Dah-Tsyr . E-mail: dtchang@life.nthu.edu.tw

    2005-11-04

    We have previously engineered a recombinant Pichia pastoris GS115 transformant, MSPGA-7, harboring seven copies of glucoamylase (GA) fused with modified signal peptide. High yield secretion of GA was achieved as an extra copy of SEC4 was integrated to the transformant. To elucidate the physiological role of SEC4, a dominant-negative mutant of SEC4, SEC4 {sub S28N}, was overexpressed under the control of alchohol oxidase 1 (AOX1) promoter in P. pastoris strain MSPGA-7 as well as a set of host cells harboring multi-copy of wild type SEC4. We found that SEC4 {sub S28N} mutation in the key guanine nucleotide binding domain reduced guanine nucleotide binding affinity, hence it blocked the transport of vesicles required for targeting and fusion to the plasma membrane. The inhibitory levels of cell growth and GA secretion were correlated with the dosage of SEC4 {sub S28N} gene. In addition, overexpression of SEC4 driven by AOX1 promoter in MSPGA-7 improved the secretory production of GA, but demonstrated the delay of cell growth by increased gene dosage of SEC4. Interestingly, a limited level of Sec4p did not disturb the cell growth. It was because expression of only one copy of SEC4 resulted in delay of cell growth at an early stage while still maintaining high level Sec4p at long-term incubation. Accordingly, as glyceraldehyde-3-phosphate dehydrogenase promoter was used to substitute AOX1 promoter to drive the SEC4 expression, enhanced GA secretion but not inhibition of cell growth was achieved. Taken together, our results demonstrate that SEC4 is essential for P. pastoris in regulating cell growth and heterologous protein secretion in a dosage-dependent manner.

  4. Molecular genetic manipulation of Pichia pastoris SEC4 governs cell growth and glucoamylase secretion.

    PubMed

    Liu, Shi-Hwei; Chou, Wei-I; Lin, Shu-Chuan; Sheu, Chia-Chin; Chang, Margaret Dah-Tsyr

    2005-11-01

    We have previously engineered a recombinant Pichia pastoris GS115 transformant, MSPGA-7, harboring seven copies of glucoamylase (GA) fused with modified signal peptide. High yield secretion of GA was achieved as an extra copy of SEC4 was integrated to the transformant. To elucidate the physiological role of SEC4, a dominant-negative mutant of SEC4, SEC4(S28N), was overexpressed under the control of alchohol oxidase 1 (AOX1) promoter in P. pastoris strain MSPGA-7 as well as a set of host cells harboring multi-copy of wild type SEC4. We found that SEC4(S28N) mutation in the key guanine nucleotide binding domain reduced guanine nucleotide binding affinity, hence it blocked the transport of vesicles required for targeting and fusion to the plasma membrane. The inhibitory levels of cell growth and GA secretion were correlated with the dosage of SEC4(S28N) gene. In addition, overexpression of SEC4 driven by AOX1 promoter in MSPGA-7 improved the secretory production of GA, but demonstrated the delay of cell growth by increased gene dosage of SEC4. Interestingly, a limited level of Sec4p did not disturb the cell growth. It was because expression of only one copy of SEC4 resulted in delay of cell growth at an early stage while still maintaining high level Sec4p at long-term incubation. Accordingly, as glyceraldehyde-3-phosphate dehydrogenase promoter was used to substitute AOX1 promoter to drive the SEC4 expression, enhanced GA secretion but not inhibition of cell growth was achieved. Taken together, our results demonstrate that SEC4 is essential for P. pastoris in regulating cell growth and heterologous protein secretion in a dosage-dependent manner. PMID:16176807

  5. Membrane protein insertion of variant MscL proteins occurs at YidC and SecYEG of Escherichia coli.

    PubMed

    Neugebauer, Stella A; Baulig, Alexandra; Kuhn, Andreas; Facey, Sandra J

    2012-04-01

    The mechanosensitive channel MscL in the inner membrane of Escherichia coli is a homopentameric complex involved in homeostasis when cells are exposed to hypoosmotic conditions. The E. coli MscL protein is synthesized as a polypeptide of 136 amino acid residues and uses the bacterial signal recognition particle for membrane targeting. The protein is inserted into the membrane independently of the Sec translocon but requires YidC. Depletion of YidC inhibits translocation of the protein across the membrane. Insertion of MscL occurs primarily in a proton motive force-independent manner. The hydrophilic loop region of MscL has 29 residues that include 5 charged residues. Altering the charges in the periplasmic loop of MscL affects the requirements for membrane insertion. The introduction of one, two or three negatively charged amino acids makes the insertion dependent on the electrochemical membrane potential and gradually dependent on the Sec translocon, whereas the addition of five negatively charged residues as well as the addition of three positively charged residues inhibits membrane insertion of MscL. However, we find that the mutant with three uncharged residues requires both the SecYEG complex and YidC but not SecA for membrane insertion. In vivo cross-linking data showed that the newly synthesized MscL interacts with YidC and with SecY. Therefore, the MscL mutants use a membrane insertion mechanism that involves SecYEG and YidC simultaneously. PMID:22310048

  6. Mutations in exocyst complex subunit SEC6 gene impaired polar auxin transport and PIN protein recycling in Arabidopsis primary root.

    PubMed

    Tan, Xiaoyun; Feng, Yihong; Liu, Yulong; Bao, Yiqun

    2016-09-01

    Polar auxin transport, which is critical for land plant pattern formation and directional growth, is largely depended on asymmetric distribution of PIN proteins at the plasma membrane (PM). Endocytosis and recycling processes play important roles in regulating PIN protein distribution and abundance at the PM. Two subunits (SEC8, EXO70A1) of exocyst, an octameric vesicle-tethering complex, have been reported to be involved in PIN protein recycling in Arabidopsis. However, the function of exocyst complex in PIN protein recycling and polar auxin transport remains incompletely understood. In this study, we utilized two SEC6 down-regulation mutants (PRsec6-1 and PRsec6-2) to investigate the role of exocyst subunit SEC6 in the primary root development, polar auxin transport and PIN proteins recycling. We found that in PRsec6 mutants: 1. Primary root growth was retarded, and lateral root initiation were compromised. 2. Primary roots were sensitive to exogenous auxin 1-napthalene acetic acid (NAA) but not 2,4-dichlorophenoxy (2.4-D). 3. Recycling of PIN1 and PIN2 proteins from the Brefeldin A (BFA) compartment to the PM was delayed. 4. Vesicles accumulated in the primary root tip cells, especially accumulated in the cytosol closed to the PM. These results further demonstrated that the exocyst complex plays an important role in PIN protein recycling and polar auxin transport in Arabidopsis primary root. PMID:27457987

  7. The Exocyst Subunit Sec6 Interacts with Assembled Exocytic SNARE Complexes.

    PubMed

    Dubuke, Michelle L; Maniatis, Stephanie; Shaffer, Scott A; Munson, Mary

    2015-11-20

    In eukaryotic cells, membrane-bound vesicles carry cargo between intracellular compartments, to and from the cell surface, and into the extracellular environment. Many conserved families of proteins are required for properly localized vesicle fusion, including the multisubunit tethering complexes and the SNARE complexes. These protein complexes work together to promote proper vesicle fusion in intracellular trafficking pathways. However, the mechanism by which the exocyst, the exocytosis-specific multisubunit tethering complex, interacts with the exocytic SNAREs to mediate vesicle targeting and fusion is currently unknown. We have demonstrated previously that the Saccharomyces cerevisiae exocyst subunit Sec6 directly bound the plasma membrane SNARE protein Sec9 in vitro and that Sec6 inhibited the assembly of the binary Sso1-Sec9 SNARE complex. Therefore, we hypothesized that the interaction between Sec6 and Sec9 prevented the assembly of premature SNARE complexes at sites of exocytosis. To map the determinants of this interaction, we used cross-linking and mass spectrometry analyses to identify residues required for binding. Mutation of residues identified by this approach resulted in a growth defect when introduced into yeast. Contrary to our previous hypothesis, we discovered that Sec6 does not change the rate of SNARE assembly but, rather, binds both the binary Sec9-Sso1 and ternary Sec9-Sso1-Snc2 SNARE complexes. Together, these results suggest a new model in which Sec6 promotes SNARE complex assembly, similar to the role proposed for other tether subunit-SNARE interactions. PMID:26446795

  8. Mechanisms of Rose Bengal inhibition on SecA ATPase and ion channel activities.

    PubMed

    Hsieh, Ying-Hsin; Huang, Ying-Ju; Jin, Jin-Shan; Yu, Liyan; Yang, Hsiuchin; Jiang, Chun; Wang, Binghe; Tai, Phang C

    2014-11-14

    SecA is an essential protein possessing ATPase activity in bacterial protein translocation for which Rose Bengal (RB) is the first reported sub-micromolar inhibitor in ATPase activity and protein translocation. Here, we examined the mechanisms of inhibition on various forms of SecA ATPase by conventional enzymatic assays, and by monitoring the SecA-dependent channel activity in the semi-physiological system in cells. We build on the previous observation that SecA with liposomes form active protein-conducting channels in the oocytes. Such ion channel activity is enhanced by purified Escherichia coli SecYEG-SecDF·YajC liposome complexes. Inhibition by RB could be monitored, providing correlation of in vitro activity and intact cell functionality. In this work, we found the intrinsic SecA ATPase is inhibited by RB competitively at low ATP concentration, and non-competitively at high ATP concentrations while the translocation ATPase with precursors and SecYEG is inhibited non-competitively by RB. The Inhibition by RB on SecA channel activity in the oocytes with exogenous ATP-Mg(2+), mimicking translocation ATPase activity, is also non-competitive. The non-competitive inhibition on channel activity has also been observed with SecA from other bacteria which otherwise would be difficult to examine without the cognate precursors and membranes. PMID:25450394

  9. Conformational Flexibility and Peptide Interaction of the Translocation ATPase SecA

    SciTech Connect

    Zimmer, Jochen; Rapoport, Tom A.; Harvard-Med

    2010-09-21

    The SecA ATPase forms a functional complex with the protein-conducting SecY channel to translocate polypeptides across the bacterial cell membrane. SecA recognizes the translocation substrate and catalyzes its unidirectional movement through the SecY channel. The recent crystal structure of the Thermotoga maritima SecA-SecYEG complex shows the ATPase in a conformation where the nucleotide-binding domains (NBDs) have closed around a bound ADP-BeFx complex and SecA's polypeptide-binding clamp is shut. Here, we present the crystal structure of T. maritima SecA in isolation, determined in its ADP-bound form at 3.1 {angstrom} resolution. SecA alone has a drastically different conformation in which the nucleotide-binding pocket between NBD1 and NBD2 is open and the preprotein cross-linking domain has rotated away from both NBDs, thereby opening the polypeptide-binding clamp. To investigate how this clamp binds polypeptide substrates, we also determined a structure of Bacillus subtilis SecA in complex with a peptide at 2.5 {angstrom} resolution. This structure shows that the peptide augments the highly conserved {beta}-sheet at the back of the clamp. Taken together, these structures suggest a mechanism by which ATP hydrolysis can lead to polypeptide translocation.

  10. Characterization of a component of the yeast secretion machinery: identification of the SEC18 gene product.

    PubMed

    Eakle, K A; Bernstein, M; Emr, S D

    1988-10-01

    SEC18 gene function is required for secretory protein transport between the endoplasmic reticulum (ER) and the Golgi complex. We cloned the SEC18 gene by complementation of the sec18-1 mutation. Gene disruption has shown that SEC18 is essential for yeast cell growth. Sequence analysis of the gene revealed a 2,271-base-pair open reading frame which could code for a protein of 83.9 kilodaltons. The predicted protein sequence showed no significant similarity to other known protein sequences. In vitro transcription and translation of SEC18 led to the synthesis of two proteins of approximately 84 and 82 kilodaltons. Antisera raised against a Sec18-beta-galactosidase fusion protein also detected two proteins (collectively referred to as Sec18p) in extracts of 35S-labeled yeast cells identical in size to those seen by in vitro translation. Mapping of the 5' end of the SEC18 mRNA revealed only one major start site for transcription, which indicates that the multiple forms of Sec18p do not arise from mRNAs with different 5' ends. Results of pulse-chase experiments indicated that the two forms of Sec18p are not the result of posttranslational processing. We suggest that translation initiating at different in-frame AUG start codons is likely to account for the presence of two forms of Sec18p. Hydrophobicity analysis indicated that the proteins were hydrophilic in nature and lacked any region that would be predicted to serve as a signal sequence or transmembrane anchor. Although potential sites for N-linked glycosylation were present in the Sec18p sequence, the sizes of the in vivo SEC18 gene products were unaffected by the drug tunicamycin, indicating that Sec18p does not enter the secretory pathway. These results suggest that Sec18p resides in the cell cytoplasm. While preliminary cell fractionation studies showed that Sec18p is not associated with the ER or Golgi complex, association with a 100,000 x g pellet fraction was observed. This suggests that Sec18p may bind

  11. Structural analysis of the complex between penta-EF-hand ALG-2 protein and Sec31A peptide reveals a novel target recognition mechanism of ALG-2.

    PubMed

    Takahashi, Takeshi; Kojima, Kyosuke; Zhang, Wei; Sasaki, Kanae; Ito, Masaru; Suzuki, Hironori; Kawasaki, Masato; Wakatsuki, Soichi; Takahara, Terunao; Shibata, Hideki; Maki, Masatoshi

    2015-01-01

    ALG-2, a 22-kDa penta-EF-hand protein, is involved in cell death, signal transduction, membrane trafficking, etc., by interacting with various proteins in mammalian cells in a Ca2+-dependent manner. Most known ALG-2-interacting proteins contain proline-rich regions in which either PPYPXnYP (type 1 motif) or PXPGF (type 2 motif) is commonly found. Previous X-ray crystal structural analysis of the complex between ALG-2 and an ALIX peptide revealed that the peptide binds to the two hydrophobic pockets. In the present study, we resolved the crystal structure of the complex between ALG-2 and a peptide of Sec31A (outer shell component of coat complex II, COPII; containing the type 2 motif) and found that the peptide binds to the third hydrophobic pocket (Pocket 3). While amino acid substitution of Phe85, a Pocket 3 residue, with Ala abrogated the interaction with Sec31A, it did not affect the interaction with ALIX. On the other hand, amino acid substitution of Tyr180, a Pocket 1 residue, with Ala caused loss of binding to ALIX, but maintained binding to Sec31A. We conclude that ALG-2 recognizes two types of motifs at different hydrophobic surfaces. Furthermore, based on the results of serial mutational analysis of the ALG-2-binding sites in Sec31A, the type 2 motif was newly defined. PMID:25667979

  12. Construction of novel chimeric proteins through the truncation of SEC2 and Sak from Staphylococcus aureus.

    PubMed

    Hui, Jing; Yu, Xiao-jie; Cui, Xiao-jin; Mu, Teng; Lin, Jia-shuai; Ni, Pei; Li, Hui; You, Song; Hu, Feng-qing

    2014-01-01

    It is an usual clinical phenomenon that cancer patients are prone to thrombosis. Until now, there have been no efficient methods or appropriate drugs to prevent and cure tumor thrombus. Therefore, the construction of a bifunctional chimeric protein for the treatment of cancer, complicated with thrombosis, is of great significance. Utilizing the superantigenic activity of staphylococcal enterotoxin C2 (SEC2) and the thrombolytic activity of staphylokinase (Sak), Sak-linker-SEC2 and SEC2-linker-Sak were constructed which had good anti-tumor and thrombolytic activities at the same time. Due to the intrinsic emetic activity of SEC2 and high molecular weight (MW) of chimeric proteins (44 kDa), their clinical applications will be restricted. In this study, novel chimeric proteins including ΔSEC2-ΔSak and ΔSak-ΔSEC2 were constructed through the truncation of SEC2 and Sak without 9-Ala linker and His-tag. Compared with the former, both the truncated proteins preserved nearly the same anti-tumor and thrombolytic activities. In addition, their MWs were only 29 kDa and their immunoreactivities were slightly lower than that of Sak-linker-SEC2 and SEC2-linker-Sak, respectively. Therefore, the novel chimeric proteins possessed merits and characteristics, such as low MS, low immunogenicity, and difunctionality which the former had not. It will be of great interest if the above-mentioned proteins can be used to cure Trousseau syndrome in clinic. PMID:25209498

  13. Cell cycle-dependent phosphorylation of Sec4p controls membrane deposition during cytokinesis.

    PubMed

    Lepore, Dante; Spassibojko, Olya; Pinto, Gabrielle; Collins, Ruth N

    2016-09-12

    Intracellular trafficking is an essential and conserved eukaryotic process. Rab GTPases are a family of proteins that regulate and provide specificity for discrete membrane trafficking steps by harnessing a nucleotide-bound cycle. Global proteomic screens have revealed many Rab GTPases as phosphoproteins, but the effects of this modification are not well understood. Using the Saccharomyces cerevisiae Rab GTPase Sec4p as a model, we have found that phosphorylation negatively regulates Sec4p function by disrupting the interaction with the exocyst complex via Sec15p. We demonstrate that phosphorylation of Sec4p is a cell cycle-dependent process associated with cytokinesis. Through a genomic kinase screen, we have also identified the polo-like kinase Cdc5p as a positive regulator of Sec4p phosphorylation. Sec4p spatially and temporally localizes with Cdc5p exclusively when Sec4p phosphorylation levels peak during the cell cycle, indicating Sec4p is a direct Cdc5p substrate. Our data suggest the physiological relevance of Sec4p phosphorylation is to facilitate the coordination of membrane-trafficking events during cytokinesis. PMID:27621363

  14. N-acetylation and phosphorylation of Sec complex subunits in the ER membrane

    PubMed Central

    2012-01-01

    Background Covalent modifications of proteins provide a mechanism to control protein function. Here, we have investigated modifications of the heptameric Sec complex which is responsible for post-translational protein import into the endoplasmic reticulum (ER). It consists of the Sec61 complex (Sec61p, Sbh1p, Sss1p) which on its own mediates cotranslational protein import into the ER and the Sec63 complex (Sec63p, Sec62p, Sec71p, Sec72p). Little is known about the biogenesis and regulation of individual Sec complex subunits. Results We show that Sbh1p when it is part of the Sec61 complex is phosphorylated on T5 which is flanked by proline residues. The phosphorylation site is conserved in mammalian Sec61ß, but only partially in birds, and not in other vertebrates or unicellular eukaryotes, suggesting convergent evolution. Mutation of T5 to A did not affect the ability of mutant Sbh1p to complement the growth defect in a Δsbh1Δsbh2 strain, and did not result in a hypophosphorylated protein which shows that alternate sites can be used by the T5 kinase. A survey of yeast phosphoproteome data shows that Sbh1p can be phosphorylated on multiple sites which are organized in two patches, one at the N-terminus of its cytosolic domain, the other proximal to the transmembrane domain. Surprisingly, although N-acetylation has been shown to interfere with ER targeting, we found that both Sbh1p and Sec62p are cotranslationally N-acetylated by NatA, and N-acetyl-proteome data indicate that Sec61p is modified by the same enzyme. Mutation of the N-acetylation site, however, did not affect Sec62p function in posttranslational protein import into the ER. Disabling NatA resulted in growth retardation, but not in co- or posttranslational translocation defects or instability of Sec62p or Sbh1p. Conclusions We conclude that N-acetylation of transmembrane and tail-anchored proteins does not interfere with their ER-targeting, and that Sbh1p phosphorylation on T5, which is not present in

  15. Sec16 influences transitional ER sites by regulating rather than organizing COPII

    PubMed Central

    Bharucha, Nike; Liu, Yang; Papanikou, Effrosyni; McMahon, Conor; Esaki, Masatoshi; Jeffrey, Philip D.; Hughson, Frederick M.; Glick, Benjamin S.

    2013-01-01

    During the budding of coat protein complex II (COPII) vesicles from transitional endoplasmic reticulum (tER) sites, Sec16 has been proposed to play two distinct roles: negatively regulating COPII turnover and organizing COPII assembly at tER sites. We tested these ideas using the yeast Pichia pastoris. Redistribution of Sec16 to the cytosol accelerates tER dynamics, supporting a negative regulatory role for Sec16. To evaluate a possible COPII organization role, we dissected the functional regions of Sec16. The central conserved domain, which had been implicated in coordinating COPII assembly, is actually dispensable for normal tER structure. An upstream conserved region (UCR) localizes Sec16 to tER sites. The UCR binds COPII components, and removal of COPII from tER sites also removes Sec16, indicating that COPII recruits Sec16 rather than the other way around. We propose that Sec16 does not in fact organize COPII. Instead, regulation of COPII turnover can account for the influence of Sec16 on tER sites. PMID:24006484

  16. GTP/GDP exchange by Sec12p enables COPII vesicle bud formation on synthetic liposomes

    PubMed Central

    Futai, Eugene; Hamamoto, Susan; Orci, Lelio; Schekman, Randy

    2004-01-01

    The generation of COPII vesicles from synthetic liposome membranes requires the minimum coat components Sar1p, Sec23/24p, Sec13/31p, and a nonhydrolyzable GTP analog such as GMP-PNP. However, in the presence of GTP and the full complement of coat subunits, nucleotide hydrolysis by Sar1p renders the coat insufficiently stable to sustain vesicle budding. In order to recapitulate a more authentic, GTP-dependent budding event, we introduced the Sar1p nucleotide exchange catalyst, Sec12p, and evaluated the dynamics of coat assembly and disassembly by light scattering and tryptophan fluorescence measurements. The catalytic, cytoplasmic domain of Sec12p (Sec12ΔCp) activated Sar1p with a turnover 10-fold higher than the GAP activity of Sec23p stimulated by the full coat. COPII assembly was stabilized on liposomes incubated with Sec12ΔCp and GTP. Numerous COPII budding profiles were visualized on membranes, whereas a parallel reaction conducted in the absence of Sec12ΔCp produced no such profiles. We suggest that Sec12p participates actively in the growth of COPII vesicles by charging new Sar1p-GTP molecules that insert at the boundary between a bud and the surrounding endoplasmic reticulum membrane. PMID:15457212

  17. ERAD and protein import defects in a sec61 mutant lacking ER-lumenal loop 7

    PubMed Central

    2013-01-01

    Background The Sec61 channel mediates protein translocation across the endoplasmic reticulum (ER) membrane during secretory protein biogenesis, and likely also during export of misfolded proteins for ER-associated degradation (ERAD). The mechanisms of channel opening for the different modes of translocation are not understood so far, but the position of the large ER-lumenal loop 7 of Sec61p suggests a decisive role. Results We show here that the Y345H mutation in L7 which causes diabetes in the mouse displays no ER import defects in yeast, but a delay in misfolded protein export. A complete deletion of L7 in Sec61p resulted in viable, cold- and tunicamycin-hypersensitive yeast cells with strong defects in posttranslational protein import of soluble proteins into the ER, and in ERAD of soluble substrates. Membrane protein ERAD was only moderately slower in sec61∆L7 than in wildtype cells. Although Sec61∆L7 channels were unstable in detergent, co-translational protein integration into the ER membrane, proteasome binding to Sec61∆L7 channels, and formation of hetero-heptameric Sec complexes were not affected. Conclusions We conclude that L7 of Sec61p is required for initiation of posttranslational soluble protein import into and misfolded soluble protein export from the ER, suggesting a key role for L7 in transverse gating of the Sec61 channel. PMID:24314051

  18. The exocyst gene Sec10 regulates renal epithelial monolayer homeostasis and apoptotic sensitivity.

    PubMed

    Polgar, Noemi; Lee, Amanda J; Lui, Vanessa H; Napoli, Josephine A; Fogelgren, Ben

    2015-08-01

    The highly conserved exocyst protein complex regulates polarized exocytosis of subsets of secretory vesicles. A previous study reported that shRNA knockdown of an exocyst central subunit, Sec10 (Sec10-KD) in Madin-Darby canine kidney (MDCK) cells disrupted primary cilia assembly and 3D cyst formation. We used three-dimensional collagen cultures of MDCK cells to further investigate the mechanisms by which Sec10 and the exocyst regulate epithelial polarity, morphogenesis, and homeostasis. Sec10-KD cysts initially demonstrated undisturbed lumen formation although later displayed significantly fewer and shorter primary cilia than controls. Later in cystogenesis, control cells maintained normal homeostasis, while Sec10-KD cysts displayed numerous apoptotic cells extruded basally into the collagen matrix. Sec10-KD MDCK cells were also more sensitive to apoptotic triggers than controls. These phenotypes were reversed by restoring Sec10 expression with shRNA-resistant human Sec10. Apico-basal polarity appeared normal in Sec10-KD cysts, whereas mitotic spindle angles differed significantly from controls, suggesting a planar cell polarity defect. In addition, analysis of renal tubules in a newly generated kidney-specific Sec10-knockout mouse model revealed significant defects in primary cilia assembly and in the targeted renal tubules; abnormal epithelial cell extrusion was also observed, supporting our in vitro results. We hypothesize that, in Sec10-KD cells, the disrupted exocyst activity results in increased apoptotic sensitivity through defective primary cilia signaling and that, in combination with an increased basal cell extrusion rate, it affects epithelial barrier integrity and homeostasis. PMID:26040895

  19. Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis.

    PubMed

    Yang, Wan Seok; Kim, Katherine J; Gaschler, Michael M; Patel, Milesh; Shchepinov, Mikhail S; Stockwell, Brent R

    2016-08-23

    Ferroptosis is form of regulated nonapoptotic cell death that is involved in diverse disease contexts. Small molecules that inhibit glutathione peroxidase 4 (GPX4), a phospholipid peroxidase, cause lethal accumulation of lipid peroxides and induce ferroptotic cell death. Although ferroptosis has been suggested to involve accumulation of reactive oxygen species (ROS) in lipid environments, the mediators and substrates of ROS generation and the pharmacological mechanism of GPX4 inhibition that generates ROS in lipid environments are unknown. We report here the mechanism of lipid peroxidation during ferroptosis, which involves phosphorylase kinase G2 (PHKG2) regulation of iron availability to lipoxygenase enzymes, which in turn drive ferroptosis through peroxidation of polyunsaturated fatty acids (PUFAs) at the bis-allylic position; indeed, pretreating cells with PUFAs containing the heavy hydrogen isotope deuterium at the site of peroxidation (D-PUFA) prevented PUFA oxidation and blocked ferroptosis. We further found that ferroptosis inducers inhibit GPX4 by covalently targeting the active site selenocysteine, leading to accumulation of PUFA hydroperoxides. In summary, we found that PUFA oxidation by lipoxygenases via a PHKG2-dependent iron pool is necessary for ferroptosis and that the covalent inhibition of the catalytic selenocysteine in Gpx4 prevents elimination of PUFA hydroperoxides; these findings suggest new strategies for controlling ferroptosis in diverse contexts. PMID:27506793

  20. Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis

    PubMed Central

    Yang, Wan Seok; Kim, Katherine J.; Gaschler, Michael M.; Patel, Milesh; Shchepinov, Mikhail S.

    2016-01-01

    Ferroptosis is form of regulated nonapoptotic cell death that is involved in diverse disease contexts. Small molecules that inhibit glutathione peroxidase 4 (GPX4), a phospholipid peroxidase, cause lethal accumulation of lipid peroxides and induce ferroptotic cell death. Although ferroptosis has been suggested to involve accumulation of reactive oxygen species (ROS) in lipid environments, the mediators and substrates of ROS generation and the pharmacological mechanism of GPX4 inhibition that generates ROS in lipid environments are unknown. We report here the mechanism of lipid peroxidation during ferroptosis, which involves phosphorylase kinase G2 (PHKG2) regulation of iron availability to lipoxygenase enzymes, which in turn drive ferroptosis through peroxidation of polyunsaturated fatty acids (PUFAs) at the bis-allylic position; indeed, pretreating cells with PUFAs containing the heavy hydrogen isotope deuterium at the site of peroxidation (D-PUFA) prevented PUFA oxidation and blocked ferroptosis. We further found that ferroptosis inducers inhibit GPX4 by covalently targeting the active site selenocysteine, leading to accumulation of PUFA hydroperoxides. In summary, we found that PUFA oxidation by lipoxygenases via a PHKG2-dependent iron pool is necessary for ferroptosis and that the covalent inhibition of the catalytic selenocysteine in Gpx4 prevents elimination of PUFA hydroperoxides; these findings suggest new strategies for controlling ferroptosis in diverse contexts. PMID:27506793

  1. Examination of Sec22 Homodimer Formation and Role in SNARE-dependent Membrane Fusion*

    PubMed Central

    Flanagan, John J.; Mukherjee, Indrani; Barlowe, Charles

    2015-01-01

    Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein complexes play essential roles in catalyzing intracellular membrane fusion events although the assembly pathway and molecular arrangement of SNARE complexes in membrane fusion reactions are not well understood. Here we monitored interactions of the R-SNARE protein Sec22 through a cysteine scanning approach and detected efficient formation of cross-linked Sec22 homodimers in cellular membranes when cysteine residues were positioned in the SNARE motif or C terminus of the transmembrane domain. When specific Sec22 cysteine derivatives are present on both donor COPII vesicles and acceptor Golgi membranes, the formation of disulfide cross-links provide clear readouts on trans- and cis-SNARE arrangements during this fusion event. The Sec22 transmembrane domain was required for efficient homodimer formation and for membrane fusion suggesting a functional role for Sec22 homodimers. We propose that Sec22 homodimers promote assembly of higher-order SNARE complexes to catalyze membrane fusion. Sec22 is also reported to function in macroautophagy and in formation of endoplasmic reticulum-plasma membrane contact sites therefore homodimer assembly may regulate Sec22 activity across a range of cellular processes. PMID:25750128

  2. 75 FR 13555 - Compliance Policy Guide Sec. 540.375 Canned Salmon - Adulteration Involving Decomposition (CPG...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-22

    ... Register on March 28, 2006 (71 FR 15422 at 15453), FDA included the Compliance Policy Guides Manual, which.... 540.375 Canned Salmon -- Adulteration Involving Decomposition (CPG 7108.10); Withdrawal of Guidance... -- Adulteration Involving Decomposition (CPG 7108.10) (CPG Sec. 540.375). CPG Sec. 540.375 is included in...

  3. 46 CFR Sec. 4 - Persons authorized to make awards under the NSA-WORKSMALREP Contract.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Persons authorized to make awards under the NSA-WORKSMALREP Contract. Sec. 4 Section 4 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A... INDIVIDUAL CONTRACT FOR MINOR REPAIRS-NSA-WORKSMALREP Sec. 4 Persons authorized to make awards under the...

  4. 46 CFR Sec. 5 - Responsibility for duplicating copies of NSA-WORKSMALREP Contract.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Responsibility for duplicating copies of NSA-WORKSMALREP Contract. Sec. 5 Section 5 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL... INDIVIDUAL CONTRACT FOR MINOR REPAIRS-NSA-WORKSMALREP Sec. 5 Responsibility for duplicating copies of...

  5. 46 CFR Sec. 5 - Responsibility for duplicating copies of NSA-WORKSMALREP Contract.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 8 2012-10-01 2012-10-01 false Responsibility for duplicating copies of NSA-WORKSMALREP Contract. Sec. 5 Section 5 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL... INDIVIDUAL CONTRACT FOR MINOR REPAIRS-NSA-WORKSMALREP Sec. 5 Responsibility for duplicating copies of...

  6. 46 CFR Sec. 3 - When the NSA-WORKSMALREP Contract may be used.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 8 2013-10-01 2013-10-01 false When the NSA-WORKSMALREP Contract may be used. Sec. 3 Section 3 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY... REPAIRS-NSA-WORKSMALREP Sec. 3 When the NSA-WORKSMALREP Contract may be used. This contract may be...

  7. 46 CFR Sec. 5 - Responsibility for duplicating copies of NSA-WORKSMALREP Contract.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 8 2014-10-01 2014-10-01 false Responsibility for duplicating copies of NSA-WORKSMALREP Contract. Sec. 5 Section 5 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL... INDIVIDUAL CONTRACT FOR MINOR REPAIRS-NSA-WORKSMALREP Sec. 5 Responsibility for duplicating copies of...

  8. 46 CFR Sec. 4 - Persons authorized to make awards under the NSA-WORKSMALREP Contract.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Persons authorized to make awards under the NSA-WORKSMALREP Contract. Sec. 4 Section 4 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A... INDIVIDUAL CONTRACT FOR MINOR REPAIRS-NSA-WORKSMALREP Sec. 4 Persons authorized to make awards under the...

  9. 46 CFR Sec. 5 - Responsibility for duplicating copies of NSA-WORKSMALREP Contract.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Responsibility for duplicating copies of NSA-WORKSMALREP Contract. Sec. 5 Section 5 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL... INDIVIDUAL CONTRACT FOR MINOR REPAIRS-NSA-WORKSMALREP Sec. 5 Responsibility for duplicating copies of...

  10. 46 CFR Sec. 3 - When the NSA-WORKSMALREP Contract may be used.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false When the NSA-WORKSMALREP Contract may be used. Sec. 3 Section 3 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY... REPAIRS-NSA-WORKSMALREP Sec. 3 When the NSA-WORKSMALREP Contract may be used. This contract may be...

  11. 46 CFR Sec. 3 - When the NSA-WORKSMALREP Contract may be used.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 8 2012-10-01 2012-10-01 false When the NSA-WORKSMALREP Contract may be used. Sec. 3 Section 3 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY... REPAIRS-NSA-WORKSMALREP Sec. 3 When the NSA-WORKSMALREP Contract may be used. This contract may be...

  12. 46 CFR Sec. 4 - Persons authorized to make awards under the NSA-WORKSMALREP Contract.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 8 2012-10-01 2012-10-01 false Persons authorized to make awards under the NSA-WORKSMALREP Contract. Sec. 4 Section 4 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A... INDIVIDUAL CONTRACT FOR MINOR REPAIRS-NSA-WORKSMALREP Sec. 4 Persons authorized to make awards under the...

  13. 46 CFR Sec. 4 - Persons authorized to make awards under the NSA-WORKSMALREP Contract.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 8 2013-10-01 2013-10-01 false Persons authorized to make awards under the NSA-WORKSMALREP Contract. Sec. 4 Section 4 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A... INDIVIDUAL CONTRACT FOR MINOR REPAIRS-NSA-WORKSMALREP Sec. 4 Persons authorized to make awards under the...

  14. 46 CFR Sec. 4 - Persons authorized to make awards under the NSA-WORKSMALREP Contract.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 8 2014-10-01 2014-10-01 false Persons authorized to make awards under the NSA-WORKSMALREP Contract. Sec. 4 Section 4 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A... INDIVIDUAL CONTRACT FOR MINOR REPAIRS-NSA-WORKSMALREP Sec. 4 Persons authorized to make awards under the...

  15. 46 CFR Sec. 3 - When the NSA-WORKSMALREP Contract may be used.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 8 2014-10-01 2014-10-01 false When the NSA-WORKSMALREP Contract may be used. Sec. 3 Section 3 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY... REPAIRS-NSA-WORKSMALREP Sec. 3 When the NSA-WORKSMALREP Contract may be used. This contract may be...

  16. 46 CFR Sec. 3 - When the NSA-WORKSMALREP Contract may be used.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false When the NSA-WORKSMALREP Contract may be used. Sec. 3 Section 3 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY... REPAIRS-NSA-WORKSMALREP Sec. 3 When the NSA-WORKSMALREP Contract may be used. This contract may be...

  17. 46 CFR Sec. 5 - Responsibility for duplicating copies of NSA-WORKSMALREP Contract.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 8 2013-10-01 2013-10-01 false Responsibility for duplicating copies of NSA-WORKSMALREP Contract. Sec. 5 Section 5 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL... INDIVIDUAL CONTRACT FOR MINOR REPAIRS-NSA-WORKSMALREP Sec. 5 Responsibility for duplicating copies of...

  18. 7 CFR 22.203 - Major responsibilities under title VI, Sec. 603.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 1 2012-01-01 2012-01-01 false Major responsibilities under title VI, Sec. 603. 22... Roles and Responsibilities of Federal Government § 22.203 Major responsibilities under title VI, Sec. 603. (a) Title VI, section 603(b). (1) Section 603(b) of the Rural Development Act charges...

  19. 7 CFR 22.203 - Major responsibilities under title VI, Sec. 603.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 1 2014-01-01 2014-01-01 false Major responsibilities under title VI, Sec. 603. 22... Roles and Responsibilities of Federal Government § 22.203 Major responsibilities under title VI, Sec. 603. (a) Title VI, section 603(b). (1) Section 603(b) of the Rural Development Act charges...

  20. 7 CFR 22.203 - Major responsibilities under title VI, Sec. 603.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 1 2011-01-01 2011-01-01 false Major responsibilities under title VI, Sec. 603. 22... Roles and Responsibilities of Federal Government § 22.203 Major responsibilities under title VI, Sec. 603. (a) Title VI, section 603(b). (1) Section 603(b) of the Rural Development Act charges...

  1. 14 CFR Sec. 19-6 - Public disclosure of traffic data.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 4 2014-01-01 2014-01-01 false Public disclosure of traffic data. Sec. 19-6 Section 19-6 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION (AVIATION... Operating Statistics Classifications Sec. 19-6 Public disclosure of traffic data. (a) Detailed domestic...

  2. Biochemical methods for studying kinetic regulation of Arf1 activation by Sec7

    PubMed Central

    Richardson, Brian C.; Fromme, J. Christopher

    2015-01-01

    The Arf family of small GTPases regulates vesicular transport at several locations within the cell, and is in turn regulated by guanine nucleotide exchange factors (GEFs) via a conserved catalytic domain, termed the Sec7 domain. The catalytic activity of the Sec7 domain is well characterized in the context of a few GEFs acting at the periphery of the cell. This chapter describes techniques used to extend biochemical analysis of activity to the much larger GEFs acting on the Arf family in the core secretory pathway, using the activity of S. cerevisiae Sec7 on Arf1, regulating export from the trans-Golgi network (TGN), as a model. Complete methods for purification to near-homogeneity of all proteins required, including several Sec7 constructs and multiple relevant small GTPases, are detailed. These are followed by methods for quantification of the nucleotide exchange activity of Sec7 in a physiologically relevant context, including modifications required to dissect the signal integration functions of Sec7 as an effector of several other small GTPases, and methods for identifying stable Sec7-small GTPase interactions in the presence of membranes. These techniques may be extended to analysis of similar members of the Sec7 GEF subfamily in other species and acting elsewhere in the secretory pathway. PMID:26360031

  3. 17 CFR 200.80b - Appendix B-SEC releases.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 17 Commodity and Securities Exchanges 2 2010-04-01 2010-04-01 false Appendix B-SEC releases. 200.80b Section 200.80b Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION ORGANIZATION; CONDUCT AND ETHICS; AND INFORMATION AND REQUESTS Information and Requests § 200.80b Appendix B—SEC releases. Free mailing list distribution...

  4. Design, Synthesis and Evaluation of Triazole-Pyrimidine Analogues as SecA Inhibitors.

    PubMed

    Cui, Jianmei; Jin, Jinshan; Chaudhary, Arpana Sagwal; Hsieh, Ying-hsin; Zhang, Hao; Dai, Chaofeng; Damera, Krishna; Chen, Weixuan; Tai, Phang C; Wang, Binghe

    2016-01-01

    SecA, a key component of the bacterial Sec-dependent secretion pathway, is an attractive target for the development of new antimicrobial agents. Through a combination of virtual screening and experimental exploration of the surrounding chemical space, we identified a hit bistriazole SecA inhibitor, SCA-21, and studied a series of analogues by systematic dissections of the core scaffold. Evaluation of these analogues allowed us to establish an initial structure-activity relationship in SecA inhibition. The best compounds in this group are potent inhibitors of SecA-dependent protein-conducting channel activity and protein translocation activity at low- to sub-micromolar concentrations. They also have minimal inhibitory concentration (MIC) values against various strains of bacteria that correlate well with the SecA and protein translocation inhibition data. These compounds are effective against methicillin-resistant Staphylococcus aureus strains with various levels of efflux pump activity, indicating the capacity of SecA inhibitors to null the effect of multidrug resistance. Results from studies of drug-affinity-responsive target stability and protein pull-down assays are consistent with SecA as a target for these compounds. PMID:26607404

  5. Polarized Exocytosis Induces Compensatory Endocytosis by Sec4p-Regulated Cortical Actin Polymerization.

    PubMed

    Johansen, Jesper; Alfaro, Gabriel; Beh, Christopher T

    2016-08-01

    Polarized growth is maintained by both polarized exocytosis, which transports membrane components to specific locations on the cell cortex, and endocytosis, which retrieves these components before they can diffuse away. Despite functional links between these two transport pathways, they are generally considered to be separate events. Using live cell imaging, in vivo and in vitro protein binding assays, and in vitro pyrene-actin polymerization assays, we show that the yeast Rab GTPase Sec4p couples polarized exocytosis with cortical actin polymerization, which induces endocytosis. After polarized exocytosis to the plasma membrane, Sec4p binds Las17/Bee1p (yeast Wiskott-Aldrich Syndrome protein [WASp]) in a complex with Sla1p and Sla2p during actin patch assembly. Mutations that inactivate Sec4p, or its guanine nucleotide exchange factor (GEF) Sec2p, inhibit actin patch formation, whereas the activating sec4-Q79L mutation accelerates patch assembly. In vitro assays of Arp2/3-dependent actin polymerization established that GTPγS-Sec4p overrides Sla1p inhibition of Las17p-dependent actin nucleation. These results support a model in which Sec4p relocates along the plasma membrane from polarized sites of exocytic vesicle fusion to nascent sites of endocytosis. Activated Sec4p then promotes actin polymerization and triggers compensatory endocytosis, which controls surface expansion and kinetically refines cell polarization. PMID:27526190

  6. 46 CFR Sec. 14 - Anti-Kickback and Davis-Bacon Acts.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Anti-Kickback and Davis-Bacon Acts. Sec. 14 Section 14... Sec. 14 Anti-Kickback and Davis-Bacon Acts. (a) All work awarded under the NSA-LUMPSUMREP Contract is subject to the provisions of the Anti-Kickback Act, and is also subject to the provisions of the...

  7. Regulation of cytokinesis by exocyst subunit SEC6 and KEULE in Arabidopsis thaliana.

    PubMed

    Wu, Jiandong; Tan, Xiaoyun; Wu, Chengyun; Cao, Kun; Li, Yan; Bao, Yiqun

    2013-11-01

    Proper vesicle tethering and membrane fusion at the cell plate are essential for cytokinesis. Both the vesicle tethering complex exocyst and membrane fusion regulator KEULE were shown to function in cell plate formation, but the exact mechanisms still remain to be explored. In this study, using yeast two-hybrid (Y-2-H) assay, we found that SEC6 interacted with KEULE, and that a small portion of C-terminal region of KEULE was required for the interaction. The direct SEC6-KEULE interaction was supported by further studies using in vitro pull-down assay, immunoprecipitation, and in vivo bimolecular fluorescence complementation (BIFC) microscopy. sec6 mutants were male gametophytic lethal as reported; however, pollen-rescued sec6 mutants (PRsec6) displayed cytokinesis defects in the embryonic cells and later in the leaf pavement cells and the guard cells. SEC6 and KEULE proteins were co-localized to the cell plate during cytokinesis in transgenic Arabidopsis. Furthermore, only SEC6 but not other exocyst subunits located in the cell plate interacted with KEULE in vitro. These results demonstrated that, like KEULE, SEC6 plays a physiological role in cytokinesis, and the SEC6-KEULE interaction may serve as a novel molecular linkage between arriving vesicles and membrane fusion machinery or directly regulate membrane fusion during cell plate formation in plants. PMID:23702595

  8. 78 FR 21432 - SEC Advisory Committee on Small and Emerging Companies

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-10

    ... on the Commission's Web site at www.sec.gov . Persons needing special accommodations to take part... only one method. The Commission will post all statements on the Advisory Committee's Web site ( http://www.sec.gov ./info/smallbus/acsec.shtml). Statements also will be available for Web site viewing...

  9. Polarized Exocytosis Induces Compensatory Endocytosis by Sec4p-Regulated Cortical Actin Polymerization

    PubMed Central

    Johansen, Jesper; Alfaro, Gabriel; Beh, Christopher T.

    2016-01-01

    Polarized growth is maintained by both polarized exocytosis, which transports membrane components to specific locations on the cell cortex, and endocytosis, which retrieves these components before they can diffuse away. Despite functional links between these two transport pathways, they are generally considered to be separate events. Using live cell imaging, in vivo and in vitro protein binding assays, and in vitro pyrene-actin polymerization assays, we show that the yeast Rab GTPase Sec4p couples polarized exocytosis with cortical actin polymerization, which induces endocytosis. After polarized exocytosis to the plasma membrane, Sec4p binds Las17/Bee1p (yeast Wiskott—Aldrich Syndrome protein [WASp]) in a complex with Sla1p and Sla2p during actin patch assembly. Mutations that inactivate Sec4p, or its guanine nucleotide exchange factor (GEF) Sec2p, inhibit actin patch formation, whereas the activating sec4-Q79L mutation accelerates patch assembly. In vitro assays of Arp2/3-dependent actin polymerization established that GTPγS-Sec4p overrides Sla1p inhibition of Las17p-dependent actin nucleation. These results support a model in which Sec4p relocates along the plasma membrane from polarized sites of exocytic vesicle fusion to nascent sites of endocytosis. Activated Sec4p then promotes actin polymerization and triggers compensatory endocytosis, which controls surface expansion and kinetically refines cell polarization. PMID:27526190

  10. Cell-Free Synthesis of SecYEG Translocon as the Fundamental Protein Transport Machinery

    NASA Astrophysics Data System (ADS)

    Matsubayashi, Hideaki; Kuruma, Yutetsu; Ueda, Takuya

    2014-12-01

    The cell membrane has many indispensable functions for sustaining cell alive besides a role as merely outer envelope. The most of such functions are implemented by membrane embedded proteins that are emerged through the membrane integration machinery, SecYEG translocon. Here, we synthesized SecYEG by expressing the corresponding gene in vitro to study the process of functionalization of the cell membrane.

  11. 78 FR 53489 - SEC Advisory Committee on Small and Emerging Companies

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-29

    ... on the Commission's Web site at www.sec.gov . Persons needing special accommodations to take part... only one method. The Commission will post all statements on the Advisory Committee's Web site ( http://www.sec.gov ./info/smallbus/acsec.shtml ). Statements also will be available for Web site viewing...

  12. 14 CFR Sec. 1-1 - Applicability of system of accounts and reports.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Applicability of system of accounts and reports. Sec. 1-1 Section 1-1 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION... AIR CARRIERS General Accounting Provisions Sec. 1-1 Applicability of system of accounts and...

  13. 14 CFR Sec. 1-1 - Applicability of system of accounts and reports.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Applicability of system of accounts and reports. Sec. 1-1 Section 1-1 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION... AIR CARRIERS General Accounting Provisions Sec. 1-1 Applicability of system of accounts and...

  14. 14 CFR Sec. 1-1 - Applicability of system of accounts and reports.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 4 2014-01-01 2014-01-01 false Applicability of system of accounts and reports. Sec. 1-1 Section 1-1 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION... AIR CARRIERS General Accounting Provisions Sec. 1-1 Applicability of system of accounts and...

  15. 14 CFR Sec. 1-1 - Applicability of system of accounts and reports.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 4 2013-01-01 2013-01-01 false Applicability of system of accounts and reports. Sec. 1-1 Section 1-1 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION... AIR CARRIERS General Accounting Provisions Sec. 1-1 Applicability of system of accounts and...

  16. 14 CFR Sec. 2-2 - Basis of allocation between entities.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Basis of allocation between entities. Sec. 2-2 Section 2-2 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION... AIR CARRIERS General Accounting Provisions Sec. 2-2 Basis of allocation between entities. (a)...

  17. 14 CFR Sec. 1-2 - Waivers from this system of accounts and reports.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Waivers from this system of accounts and reports. Sec. 1-2 Section 1-2 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION... AIR CARRIERS General Accounting Provisions Sec. 1-2 Waivers from this system of accounts and...

  18. Apratoxin Kills Cells by Direct Blockade of the Sec61 Protein Translocation Channel.

    PubMed

    Paatero, Anja O; Kellosalo, Juho; Dunyak, Bryan M; Almaliti, Jehad; Gestwicki, Jason E; Gerwick, William H; Taunton, Jack; Paavilainen, Ville O

    2016-05-19

    Apratoxin A is a cytotoxic natural product that prevents the biogenesis of secretory and membrane proteins. Biochemically, apratoxin A inhibits cotranslational translocation into the ER, but its cellular target and mechanism of action have remained controversial. Here, we demonstrate that apratoxin A prevents protein translocation by directly targeting Sec61α, the central subunit of the protein translocation channel. Mutagenesis and competitive photo-crosslinking studies indicate that apratoxin A binds to the Sec61 lateral gate in a manner that differs from cotransin, a substrate-selective Sec61 inhibitor. In contrast to cotransin, apratoxin A does not exhibit a substrate-selective inhibitory mechanism, but blocks ER translocation of all tested Sec61 clients with similar potency. Our results suggest that multiple structurally unrelated natural products have evolved to target overlapping but non-identical binding sites on Sec61, thereby producing distinct biological outcomes. PMID:27203376

  19. Interaction of SecB with intermediates along the folding pathway of maltose-binding protein.

    PubMed Central

    Diamond, D. L.; Strobel, S.; Chun, S. Y.; Randall, L. L.

    1995-01-01

    SecB, a molecular chaperone involved in protein export in Escherichia coli, displays the remarkable ability to selectively bind many different polypeptide ligands whose only common feature is that of being nonnative. The selectivity is explained in part by a kinetic partitioning between the folding of a polypeptide and its association with SecB. SecB has no affinity for native, stably folded polypeptides but interacts tightly with polypeptides that are nonnative. In order to better understand the nature of the binding, we have examined the interaction of SecB with intermediates along the folding pathway of maltose-binding protein. Taking advantage of forms of maltose-binding protein that are altered in their folding properties, we show that the first intermediate in folding, represented by the collapsed state, binds to SecB, and that the polypeptide remains active as a ligand until it crosses the final energy barrier to attain the native state. PMID:7549876

  20. Subcellular localization of yeast Sec14 homologues and their involvement in regulation of phospholipid turnover.

    PubMed

    Schnabl, Martina; Oskolkova, Olga V; Holic, Roman; Brezná, Barbara; Pichler, Harald; Zágorsek, Milos; Kohlwein, Sepp D; Paltauf, Fritz; Daum, Günther; Griac, Peter

    2003-08-01

    Sec14p of the yeast Saccharomyces cerevisiae is involved in protein secretion and regulation of lipid synthesis and turnover in vivo, but acts as a phosphatidylinositol-phosphatidylcholine transfer protein in vitro. In this work, the five homologues of Sec14p, Sfh1p-Sfh5p, were subjected to biochemical and cell biological analysis to get a better view of their physiological role. We show that overexpression of SFH2 and SFH4 suppressed the sec14 growth defect in a more and SFH1 in a less efficient way, whereas overexpression of SFH3 and SFH5 did not complement sec14. Using C-terminal yEGFP fusions, Sfh2p, Sfh4p and Sfh5p are mainly localized to the cytosol and microsomes similar to Sec14p. Sfh1p was detected in the nucleus and Sfh3p in lipid particles and in microsomes. In contrast to Sec14p, which inhibits phospholipase D1 (Pld1p), overproduction of Sfh2p and Sfh4p resulted in the activation of Pld1p-mediated phosphatidylcholine turnover. Interestingly, Sec14p and the two homologues Sfh2p and Sfh4p downregulate phospholipase B1 (Plb1p)-mediated turnover of phosphatidylcholine in vivo. In summary, Sfh2p and Sfh4p are the Sec14p homologues with the most pronounced functional similarity to Sec14p, whereas the other Sfh proteins appear to be functionally less related to Sec14p. PMID:12869188

  1. Development of a Microemulsion Formulation for Antimicrobial SecA Inhibitors

    PubMed Central

    Wang, Nian

    2016-01-01

    In our previous study, we have identified five antimicrobial small molecules via structure based design, which inhibit SecA of Candidatus Liberibacter asiaticus (Las). SecA is a critical protein translocase ATPase subunit and is involved in pre-protein translocation across and integration into the cellular membrane in bacteria. In this study, eleven compounds were identified using similarity search method based on the five lead SecA inhibitors identified previously. The identified SecA inhibitors have poor aqueous solubility. Thus a microemulsion master mix (MMX) was developed to address the solubility issue and for application of the antimicrobials. MMX consists of N-methyl-2-pyrrolidone and dimethyl sulfoxide as solvent and co-solvent, as well as polyoxyethylated castor oil, polyalkylene glycol, and polyoxyethylene tridecyl ether phosphate as surfactants. MMX has significantly improved the solubility of SecA inhibitors and has no or little phytotoxic effects at concentrations less than 5.0% (v/v). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the SecA inhibitors and streptomycin against eight bacteria including Agrobacterium tumefaciens, Liberibacter crescens, Rhizobium etli, Bradyrhizobium japonicum, Mesorhizobium loti, and Sinorhizobium meliloti phylogenetically related to Las were determined using the broth microdilution method. MIC and MBC results showed that the 16 SecA inhibitors have antibacterial activities comparable to that of streptomycin. Overall, we have identified 11 potent SecA inhibitors using similarity search method. We have developed a microemulsion formulation for SecA inhibitors which improved the antimicrobial activities of SecA inhibitors. PMID:26963811

  2. Development of a Microemulsion Formulation for Antimicrobial SecA Inhibitors.

    PubMed

    Hu, Jiahuai; Akula, Nagaraju; Wang, Nian

    2016-01-01

    In our previous study, we have identified five antimicrobial small molecules via structure based design, which inhibit SecA of Candidatus Liberibacter asiaticus (Las). SecA is a critical protein translocase ATPase subunit and is involved in pre-protein translocation across and integration into the cellular membrane in bacteria. In this study, eleven compounds were identified using similarity search method based on the five lead SecA inhibitors identified previously. The identified SecA inhibitors have poor aqueous solubility. Thus a microemulsion master mix (MMX) was developed to address the solubility issue and for application of the antimicrobials. MMX consists of N-methyl-2-pyrrolidone and dimethyl sulfoxide as solvent and co-solvent, as well as polyoxyethylated castor oil, polyalkylene glycol, and polyoxyethylene tridecyl ether phosphate as surfactants. MMX has significantly improved the solubility of SecA inhibitors and has no or little phytotoxic effects at concentrations less than 5.0% (v/v). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the SecA inhibitors and streptomycin against eight bacteria including Agrobacterium tumefaciens, Liberibacter crescens, Rhizobium etli, Bradyrhizobium japonicum, Mesorhizobium loti, and Sinorhizobium meliloti phylogenetically related to Las were determined using the broth microdilution method. MIC and MBC results showed that the 16 SecA inhibitors have antibacterial activities comparable to that of streptomycin. Overall, we have identified 11 potent SecA inhibitors using similarity search method. We have developed a microemulsion formulation for SecA inhibitors which improved the antimicrobial activities of SecA inhibitors. PMID:26963811

  3. Sec3-containing exocyst complex is required for desmosome assembly in mammalian epithelial cells.

    PubMed

    Andersen, Nicholas J; Yeaman, Charles

    2010-01-01

    The Exocyst is a conserved multisubunit complex involved in the docking of post-Golgi transport vesicles to sites of membrane remodeling during cellular processes such as polarization, migration, and division. In mammalian epithelial cells, Exocyst complexes are recruited to nascent sites of cell-cell contact in response to E-cadherin-mediated adhesive interactions, and this event is an important early step in the assembly of intercellular junctions. Sec3 has been hypothesized to function as a spatial landmark for the development of polarity in budding yeast, but its role in epithelial cells has not been investigated. Here, we provide evidence in support of a function for a Sec3-containing Exocyst complex in the assembly or maintenance of desmosomes, adhesive junctions that link intermediate filament networks to sites of strong intercellular adhesion. We show that Sec3 associates with a subset of Exocyst complexes that are enriched at desmosomes. Moreover, we found that membrane recruitment of Sec3 is dependent on cadherin-mediated adhesion but occurs later than that of the known Exocyst components Sec6 and Sec8 that are recruited to adherens junctions. RNA interference-mediated suppression of Sec3 expression led to specific impairment of both the morphology and function of desmosomes, without noticeable effect on adherens junctions. These results suggest that two different exocyst complexes may function in basal-lateral membrane trafficking and will enable us to better understand how exocytosis is spatially organized during development of epithelial plasma membrane domains. PMID:19889837

  4. Mutations in the exocyst component Sec5 disrupt neuronal membrane traffic, but neurotransmitter release persists.

    PubMed

    Murthy, Mala; Garza, Dan; Scheller, Richard H; Schwarz, Thomas L

    2003-02-01

    The exocyst (Sec6/8) complex is necessary for secretion in yeast and has been postulated to establish polarity by directing vesicle fusion to specific sites along the plasma membrane. The complex may also function in the nervous system, but its precise role is unknown. We have investigated exocyst function in Drosophila with mutations in one member of the complex, sec5. Null alleles die as growth-arrested larvae, whose neuromuscular junctions fail to expand. In culture, neurite outgrowth fails in sec5 mutants once maternal Sec5 is exhausted. Using a trafficking assay, we found impairments in the membrane addition of newly synthesized proteins. In contrast, synaptic vesicle fusion was not impaired. Thus, Sec5 differentiates between two forms of vesicle trafficking: trafficking for cell growth and membrane protein insertion depend on sec5, whereas transmitter secretion does not. In this regard, sec5 differs from the homologs of other yeast exocytosis genes that are required for both neuronal trafficking pathways. PMID:12575951

  5. Identification of Sec14-like 3 as a novel lipid-packing sensor in the lung.

    PubMed

    Hishikawa, Daisuke; Shindou, Hideo; Harayama, Takeshi; Ogasawara, Rie; Suwabe, Akira; Shimizu, Takao

    2013-12-01

    Pulmonary surfactant, a complex composed primarily of lipids and associated proteins, is synthesized in alveolar type II (ATII) cells and secreted into alveoli to prevent collapse during respiration. Although numerous studies have clarified the fundamental roles of pulmonary surfactant, the molecular mechanisms of transport and secretion of pulmonary surfactant remain totally unknown. Thus, we screened candidate genes by comparing genes with the expressed sequence tag (EST) libraries of embryonic and adult lungs by using the digital differential display method in the National Center for Biotechnology Information (NCBI) database. We identified Sec14-like 3 (Sec14L3) as a new class of lipid-associated proteins highly expressed in ATII cells. We found that Sec14L3 expression is >100-fold increased during the perinatal period in the lung. Furthermore, Sec14L3 bound to small-sized liposomes (30 nm in diameter), but not to large-sized liposomes (100 nm diameter), through its Sec14 domain. Because of the increased curvature, lipid-packing defects are more likely to occur in small-sized liposomes than in large-sized liposomes. Based on these results, we conclude that Sec14L3 is a new class of lipid-packing sensor. Sec14L3 may play important roles in the lung, such as intracellular lipid transport, surfactant maturation, and endo/exocytosis. PMID:24018064

  6. Mutation of sec63 in zebrafish causes defects in myelinated axons and liver pathology

    PubMed Central

    Monk, Kelly R.; Voas, Matthew G.; Franzini-Armstrong, Clara; Hakkinen, Ian S.; Talbot, William S.

    2013-01-01

    SUMMARY Mutations in SEC63 cause polycystic liver disease in humans. Sec63 is a member of the endoplasmic reticulum (ER) translocon machinery, although it is unclear how mutations in SEC63 lead to liver cyst formation in humans. Here, we report the identification and characterization of a zebrafish sec63 mutant, which was discovered in a screen for mutations that affect the development of myelinated axons. Accordingly, we show that disruption of sec63 in zebrafish leads to abnormalities in myelinating glia in both the central and peripheral nervous systems. In the vertebrate nervous system, segments of myelin are separated by the nodes of Ranvier, which are unmyelinated regions of axonal membrane containing a high density of voltage-gated sodium channels. We show that sec63 mutants have morphologically abnormal and reduced numbers of clusters of voltage-gated sodium channels in the spinal cord and along peripheral nerves. Additionally, we observed reduced myelination in both the central and peripheral nervous systems, as well as swollen ER in myelinating glia. Markers of ER stress are upregulated in sec63 mutants. Finally, we show that sec63 mutants develop liver pathology. As in glia, the primary defect, detectable at 5 dpf, is fragmentation and swelling of the ER, indicative of accumulation of proteins in the lumen. At 8 dpf, ER swelling is severe; other pathological features include disrupted bile canaliculi, altered cytoplasmic matrix and accumulation of large lysosomes. Together, our analyses of sec63 mutant zebrafish highlight the possible role of ER stress in polycystic liver disease and suggest that these mutants will serve as a model for understanding the pathophysiology of this disease and other abnormalities involving ER stress. PMID:22864019

  7. Sec35p, a Novel Peripheral Membrane Protein, Is Required for ER to Golgi Vesicle Docking

    PubMed Central

    VanRheenen, Susan M.; Cao, Xiaochun; Lupashin, Vladimir V.; Barlowe, Charles; Gerard Waters, M.

    1998-01-01

    SEC35 was identified in a novel screen for temperature-sensitive mutants in the secretory pathway of the yeast Saccharomyces cerevisiae (Wuestehube et al., 1996. Genetics. 142:393–406). At the restrictive temperature, the sec35-1 strain exhibits a transport block between the ER and the Golgi apparatus and accumulates numerous vesicles. SEC35 encodes a novel cytosolic protein of 32 kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec35-1 is efficiently suppressed by YPT1, which encodes the rab-like GTPase required early in the secretory pathway, or by SLY1-20, which encodes a dominant form of the ER to Golgi target -SNARE–associated protein Sly1p. Weaker suppression is evident upon overexpression of genes encoding the vesicle-SNAREs SEC22, BET1, or YKT6. The cold-sensitive lethality that results from deleting SEC35 is suppressed by YPT1 or SLY1-20. These genetic relationships suggest that Sec35p acts upstream of, or in conjunction with, Ypt1p and Sly1p as was previously found for Uso1p. Using a cell-free assay that measures distinct steps in vesicle transport from the ER to the Golgi, we find Sec35p is required for a vesicle docking stage catalyzed by Uso1p. These genetic and biochemical results suggest Sec35p acts with Uso1p to dock ER-derived vesicles to the Golgi complex. PMID:9606204

  8. The Candida albicans Exocyst Subunit Sec6 Contributes to Cell Wall Integrity and Is a Determinant of Hyphal Branching

    PubMed Central

    Chavez-Dozal, Alba A.; Bernardo, Stella M.; Rane, Hallie S.; Herrera, Gloria; Kulkarny, Vibhati; Wagener, Jeanette; Cunningham, Iain; Brand, Alexandra C.; Gow, Neil A. R.

    2015-01-01

    The yeast exocyst is a multiprotein complex comprised of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) which orchestrates trafficking of exocytic vesicles to specific docking sites on the plasma membrane during polarized secretion. To study SEC6 function in Candida albicans, we generated a conditional mutant strain in which SEC6 was placed under the control of a tetracycline-regulated promoter. In the repressed state, the tetR-SEC6 mutant strain (denoted tSEC6) was viable for up to 27 h; thus, all phenotypic analyses were performed at 24 h or earlier. Strain tSEC6 under repressing conditions had readily apparent defects in cytokinesis and endocytosis and accumulated both post-Golgi apparatus secretory vesicles and structures suggestive of late endosomes. Strain tSEC6 was markedly defective in secretion of aspartyl proteases and lipases as well as filamentation under repressing conditions. Lack of SEC6 expression resulted in markedly reduced lateral hyphal branching, which requires the establishment of a new axis of polarized secretion. Aberrant localization of chitin at the septum and increased resistance to zymolyase activity were observed, suggesting that C. albicans Sec6 plays an important role in mediating trafficking and delivery of cell wall components. The tSEC6 mutant was also markedly defective in macrophage killing, indicating a role of SEC6 in C. albicans virulence. Taken together, these studies indicate that the late secretory protein Sec6 is required for polarized secretion, hyphal morphogenesis, and the pathogenesis of C. albicans. PMID:26002719

  9. Role of Human Sec63 in Modulating the Steady-State Levels of Multi-Spanning Membrane Proteins

    PubMed Central

    Awe, Karin; Stieler, Jens; Döring, Tatjana; Füser, Sabine; Prange, Reinhild

    2012-01-01

    The Sec61 translocon of the endoplasmic reticulum (ER) membrane forms an aqueous pore, allowing polypeptides to be transferred across or integrated into membranes. Protein translocation into the ER can occur co- and posttranslationally. In yeast, posttranslational translocation involves the heptameric translocase complex including its Sec62p and Sec63p subunits. The mammalian ER membrane contains orthologs of yeast Sec62p and Sec63p, but their function is poorly understood. Here, we analyzed the effects of excess and deficit Sec63 on various ER cargoes using human cell culture systems. The overexpression of Sec63 reduces the steady-state levels of viral and cellular multi-spanning membrane proteins in a cotranslational mode, while soluble and single-spanning ER reporters are not affected. Consistent with this, the knock-down of Sec63 increases the steady-state pools of polytopic ER proteins, suggesting a substrate-specific and regulatory function of Sec63 in ER import. Overexpressed Sec63 exerts its down-regulating activity on polytopic protein levels independent of its Sec62-interacting motif, indicating that it may not act in conjunction with Sec62 in human cells. The specific action of Sec63 is further sustained by our observations that the up-regulation of either Sec62 or two other ER proteins with lumenal J domains, like ERdj1 and ERdj4, does not compromise the steady-state level of a multi-spanning membrane reporter. A J domain-specific mutation of Sec63, proposed to weaken its interaction with the ER resident BiP chaperone, reduces the down-regulating capacity of excess Sec63, suggesting an involvement of BiP in this process. Together, these results suggest that Sec63 may perform a substrate-selective quantity control function during cotranslational ER import. PMID:23166619

  10. The Candida albicans Exocyst Subunit Sec6 Contributes to Cell Wall Integrity and Is a Determinant of Hyphal Branching.

    PubMed

    Chavez-Dozal, Alba A; Bernardo, Stella M; Rane, Hallie S; Herrera, Gloria; Kulkarny, Vibhati; Wagener, Jeanette; Cunningham, Iain; Brand, Alexandra C; Gow, Neil A R; Lee, Samuel A

    2015-07-01

    The yeast exocyst is a multiprotein complex comprised of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) which orchestrates trafficking of exocytic vesicles to specific docking sites on the plasma membrane during polarized secretion. To study SEC6 function in Candida albicans, we generated a conditional mutant strain in which SEC6 was placed under the control of a tetracycline-regulated promoter. In the repressed state, the tetR-SEC6 mutant strain (denoted tSEC6) was viable for up to 27 h; thus, all phenotypic analyses were performed at 24 h or earlier. Strain tSEC6 under repressing conditions had readily apparent defects in cytokinesis and endocytosis and accumulated both post-Golgi apparatus secretory vesicles and structures suggestive of late endosomes. Strain tSEC6 was markedly defective in secretion of aspartyl proteases and lipases as well as filamentation under repressing conditions. Lack of SEC6 expression resulted in markedly reduced lateral hyphal branching, which requires the establishment of a new axis of polarized secretion. Aberrant localization of chitin at the septum and increased resistance to zymolyase activity were observed, suggesting that C. albicans Sec6 plays an important role in mediating trafficking and delivery of cell wall components. The tSEC6 mutant was also markedly defective in macrophage killing, indicating a role of SEC6 in C. albicans virulence. Taken together, these studies indicate that the late secretory protein Sec6 is required for polarized secretion, hyphal morphogenesis, and the pathogenesis of C. albicans. PMID:26002719

  11. Microprocessor-controlled tester for evaluation of the Self-Energized Credential System (SECS)

    SciTech Connect

    Corlis, N.E.

    1980-03-01

    The Self-Energized Credential System (SECS) was developed for use in the Plutonium Protection System (PPS) installed at Hanford, Washington. Evaluation and development of the SECS system was enhanced by the use of a microprocessor-controlled portal tester. This tester used infrared (ir) beam sensors to provide information on the direction of travel of the credential wearer and to detect inoperative credentials. A printed record of the portal number, actual code read, time, and direction of the credential passage provided information essential to an assessment of the operability of the SECS.

  12. 46 CFR Sec. 17 - Performance of work resulting from damage sustained while undergoing repairs.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 17 Performance of work resulting... performance of repairs under the NSA Master Contract, negotiations for accomplishment of work necessary...

  13. 46 CFR Sec. 17 - Performance of work resulting from damage sustained while undergoing repairs.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 17 Performance of work resulting... performance of repairs under the NSA Master Contract, negotiations for accomplishment of work necessary...

  14. 46 CFR Sec. 17 - Performance of work resulting from damage sustained while undergoing repairs.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 17 Performance of work resulting... performance of repairs under the NSA Master Contract, negotiations for accomplishment of work necessary...

  15. 46 CFR Sec. 17 - Performance of work resulting from damage sustained while undergoing repairs.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 17 Performance of work resulting... performance of repairs under the NSA Master Contract, negotiations for accomplishment of work necessary...

  16. 46 CFR Sec. 17 - Performance of work resulting from damage sustained while undergoing repairs.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 17 Performance of work resulting... performance of repairs under the NSA Master Contract, negotiations for accomplishment of work necessary...

  17. Structure of the Sec61 channel opened by a signal sequence.

    PubMed

    Voorhees, Rebecca M; Hegde, Ramanujan S

    2016-01-01

    Secreted and integral membrane proteins compose up to one-third of the biological proteome. These proteins contain hydrophobic signals that direct their translocation across or insertion into the lipid bilayer by the Sec61 protein-conducting channel. The molecular basis of how hydrophobic signals within a nascent polypeptide trigger channel opening is not understood. Here, we used cryo-electron microscopy to determine the structure of an active Sec61 channel that has been opened by a signal sequence. The signal supplants helix 2 of Sec61α, which triggers a rotation that opens the central pore both axially across the membrane and laterally toward the lipid bilayer. Comparisons with structures of Sec61 in other states suggest a pathway for how hydrophobic signals engage the channel to gain access to the lipid bilayer. PMID:26721998

  18. Size Interplay between Polymer and Nanopores for the Band Broadening of SEC

    NASA Astrophysics Data System (ADS)

    Weiss, Ian; Ryu, Chang Yeol; Chang, Taihyun

    2012-02-01

    The size interplay between polymer chains and nanopores plays a key role in governing the retention time of polymer chains in liquid chromatography. These nanopores also contribute to the band broadening of the resulting peaks seen in most liquid chromatography systems including size exclusion chromatography (SEC). We have studied how the relationship between the size of the nanopores and the hydrodynamic radius of the polymers affects the band broadening during SEC. This related to Brown random motion of polymer chains in solution, whose motions are restricted by the presence of nanoporous stationary phase for the SEC. We have prepared model polystyrene samples with extremely narrow polydispersity (PDI < 1.0001) using temperature gradient interaction chromatography. Those model samples allow us to directly measure the band broadening of SEC using different size pore columns at various solvent conditions.

  19. The structure of the exocyst subunit Sec6p defines a conserved architecture with diverse roles.

    PubMed

    Sivaram, Mylavarapu V S; Furgason, Melonnie L M; Brewer, Daniel N; Munson, Mary

    2006-06-01

    The exocyst is a conserved protein complex essential for trafficking secretory vesicles to the plasma membrane. The structure of the C-terminal domain of the exocyst subunit Sec6p reveals multiple helical bundles, which are structurally and topologically similar to Exo70p and the C-terminal domains of Exo84p and Sec15, despite <10% sequence identity. The helical bundles appear to be evolutionarily related molecular scaffolds that have diverged to create functionally distinct exocyst proteins. PMID:16699513

  20. OsSEC24, a functional SEC24-like protein in rice, improves tolerance to iron deficiency and high pH by enhancing H(+) secretion mediated by PM-H(+)-ATPase.

    PubMed

    Li, Shuang; Pan, Xiao-Xi; Berry, James O; Wang, Yi; Naren; Ma, Shuang; Tan, Song; Xiao, Wei; Zhao, Wei-Zhong; Sheng, Xian-Yong; Yin, Li-Ping

    2015-04-01

    Iron is abundant in the soil, but its low solubility in neutral or alkaline soils limits its uptake. Plants can rely on rhizosphere acidification to increase iron solubility. OsSEC27p was previously found to be a highly up-regulated gene in iron-deficient rice roots. Here, pH-dependent complementation assays using yeast mutants sec24Δ/SEC24 and sec27Δ/SEC27 showed that OsSEC27 could functionally complement SEC24 but not SEC27 in yeast; thus, it was renamed as OsSEC24. We found that OsSEC24-transgenic tobacco plants increased the length and number of roots under iron deficiency at pH 8.0. To explore how OsSEC24 confers tolerance to iron deficiency, we utilized transgenic tobacco, rice and rice protoplasts. H(+) flux measurements using Non-invasive Micro-test Technology (NMT) indicated that the transgenic OsSEC24 tobacco and rice enhanced H(+) efflux under iron deficiency. Conversely, the application of plasma membrane PM-H(+)-ATPase inhibitor vanadate elucidated that H(+) secretion increased by OsSEC24 was mediated by PM-H(+)-ATPase. OsPMA2 was used as a representative of iron deficiency-responsive PM-H(+)-ATPases in rice root via RT-PCR analysis. In transgenic rice protoplasts OsPMA2 was packaged into OsSEC24 vesicles after export from the ER through confocal-microscopy observation. Together, OsSEC24 vesicles, along with PM-H(+)-ATPases stimulate roots formation under iron deficiency by enhancing rhizosphere acidification. PMID:25711814

  1. The ribosome quality control pathway can access nascent polypeptides stalled at the Sec61 translocon.

    PubMed

    von der Malsburg, Karina; Shao, Sichen; Hegde, Ramanujan S

    2015-06-15

    Cytosolic ribosomes that stall during translation are split into subunits, and nascent polypeptides trapped in the 60S subunit are ubiquitinated by the ribosome quality control (RQC) pathway. Whether the RQC pathway can also target stalls during cotranslational translocation into the ER is not known. Here we report that listerin and NEMF, core RQC components, are bound to translocon-engaged 60S subunits on native ER membranes. RQC recruitment to the ER in cultured cells is stimulated by translation stalling. Biochemical analyses demonstrated that translocon-targeted nascent polypeptides that subsequently stall are polyubiquitinated in 60S complexes. Ubiquitination at the translocon requires cytosolic exposure of the polypeptide at the ribosome-Sec61 junction. This exposure can result from either failed insertion into the Sec61 channel or partial backsliding of translocating nascent chains. Only Sec61-engaged nascent chains early in their biogenesis were relatively refractory to ubiquitination. Modeling based on recent 60S-RQC and 80S-Sec61 structures suggests that the E3 ligase listerin accesses nascent polypeptides via a gap in the ribosome-translocon junction near the Sec61 lateral gate. Thus the RQC pathway can target stalled translocation intermediates for degradation from the Sec61 channel. PMID:25877867

  2. The Structure of Sec12 Implicates Potassium Ion Coordination in Sar1 Activation*

    PubMed Central

    McMahon, Conor; Studer, Sean M.; Clendinen, Chaevia; Dann, Geoffrey P.; Jeffrey, Philip D.; Hughson, Frederick M.

    2012-01-01

    Coat protein II (COPII)-coated vesicles transport proteins and lipids from the endoplasmic reticulum to the Golgi. Crucial for the initiation of COPII coat assembly is Sec12, a guanine nucleotide exchange factor responsible for activating the small G protein Sar1. Once activated, Sar1/GTP binds to endoplasmic reticulum membranes and recruits COPII coat components (Sec23/24 and Sec13/31). Here, we report the 1.36 Å resolution crystal structure of the catalytically active, 38-kDa cytoplasmic portion of Saccharomyces cerevisiae Sec12. Sec12 adopts a β propeller fold. Conserved residues cluster around a loop we term the “K loop,” which extends from the N-terminal propeller blade. Structure-guided site-directed mutagenesis, in conjunction with in vitro and in vivo functional studies, reveals that this region of Sec12 is catalytically essential, presumably because it makes direct contact with Sar1. Strikingly, the crystal structure also reveals that a single potassium ion stabilizes the K loop; bound potassium is, moreover, essential for optimum guanine nucleotide exchange activity in vitro. Thus, our results reveal a novel role for a potassium-stabilized loop in catalyzing guanine nucleotide exchange. PMID:23109340

  3. Structure of the mammalian ribosome-Sec61 complex to 3.4 Å resolution.

    PubMed

    Voorhees, Rebecca M; Fernández, Israel S; Scheres, Sjors H W; Hegde, Ramanujan S

    2014-06-19

    Cotranslational protein translocation is a universally conserved process for secretory and membrane protein biosynthesis. Nascent polypeptides emerging from a translating ribosome are either transported across or inserted into the membrane via the ribosome-bound Sec61 channel. Here, we report structures of a mammalian ribosome-Sec61 complex in both idle and translating states, determined to 3.4 and 3.9 Å resolution. The data sets permit building of a near-complete atomic model of the mammalian ribosome, visualization of A/P and P/E hybrid-state tRNAs, and analysis of a nascent polypeptide in the exit tunnel. Unprecedented chemical detail is observed for both the ribosome-Sec61 interaction and the conformational state of Sec61 upon ribosome binding. Comparison of the maps from idle and translating complexes suggests how conformational changes to the Sec61 channel could facilitate translocation of a secreted polypeptide. The high-resolution structure of the mammalian ribosome-Sec61 complex provides a valuable reference for future functional and structural studies. PMID:24930395

  4. The mammalian and yeast translocon complexes comprise a characteristic Sec61 channel.

    PubMed

    Erdmann, Frank; Jung, Martin; Maurer, Patrick; Harsman, Anke; Zimmermann, Richard; Wagner, Richard

    2010-06-01

    In eukaryotes, protein translocation across and insertion into the membrane of the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex or translocon. In our previous electrophysiological studies, we characterized the mammalian Sec61 channel from Canis familiaris. Here we extended these initial results to the Sec61 channel from the yeast Saccharomyces cerevisiae and compared the basic electrophysiological properties of both channel preparations with respect to the gating behaviour, distribution of channel open states, ionic conductance, approximated pore dimensions, reversal potential and selectivity as well as voltage-dependent open probability. We found that the Sec61 complexes from both species displayed conformable characteristics of the highly dynamic channel in an intrinsically open state. In contrast, the bacterial Sec61-homologue, the SecYEG complex from Escherichia coli, displayed under the same experimental conditions significantly different properties residing in an intrinsically closed state. We therefore propose that considerable differences between the respective eukaryote and prokaryote protein-conducting channel units and their regulation exist. PMID:20450886

  5. The glycine transporter GLYT1 interacts with Sec3, a component of the exocyst complex.

    PubMed

    Cubelos, Beatriz; Giménez, Cecilio; Zafra, Francisco

    2005-11-01

    Evidence is accumulating that the glycine transporter GLYT1 regulates NMDA receptor function by modulating the glycine concentration in glutamatergic synapses. In this article, we describe a physical and functional interaction between GLYT1 and the exocyst complex. Through a yeast two-hybrid screen to search for proteins capable of interacting with the intracellular C-terminal tail of GLYT1, we identified a protein that is highly homologous to the human and mouse Sec3 protein, a component of the exocyst complex. Pull-down and immunoprecipitation assays confirmed the physical interaction between the C-terminus of GLYT1 and Sec3. Subsequently, immunofluorescence experiments indicated that Sec3-GFP was partially recruited to the plasma membrane upon coexpression with GLYT1. The interaction of GLYT1 with exocyst components was also observed in the native rat brain since complexes immunoprecipitated from brain extracts with anti-GLYT1 antibodies contained both Sec6 and Sec8. Functional assays revealed that Sec3 increased the transporter capacity of GLYT1, suggesting that the exocyst favors insertion of GLYT1 into the plasma membrane. PMID:16181645

  6. The Serotonin Transporter Is an Exclusive Client of the Coat Protein Complex II (COPII) Component SEC24C*

    PubMed Central

    Sucic, Sonja; El-Kasaby, Ali; Kudlacek, Oliver; Sarker, Subhodeep; Sitte, Harald H.; Marin, Philippe; Freissmuth, Michael

    2011-01-01

    The transporters for serotonin (SERT), dopamine, and noradrenaline have a conserved hydrophobic core but divergent N and C termini. The C terminus harbors the binding site for the coat protein complex II (COPII) cargo-binding protein SEC24. Here we explored which SEC24 isoform was required for export of SERT from the endoplasmic reticulum (ER). Three lines of evidence argue that SERT can only exit the ER by recruiting SEC24C: (i) Mass spectrometry showed that a peptide corresponding to the C terminus of SERT recruited SEC24C-containing COPII complexes from mouse brain lysates. (ii) Depletion of individual SEC24 isoforms by siRNAs revealed that SERT was trapped in the ER only if SEC24C was down-regulated, in both, cells that expressed SERT endogenously or after transfection. The combination of all siRNAs was not more effective than that directed against SEC24C. A SERT mutant in which the SEC24C-binding motif (607RI608) was replaced by alanine was insensitive to down-regulation of SEC24C levels. (iii) Overexpression of a SEC24C variant with a mutation in the candidate cargo-binding motif (SEC24C-D796V/D797N) but not of the corresponding mutant SEC24D-D733V/D734N reduced SERT surface levels. In contrast, noradrenaline and dopamine transporters and the more distantly related GABA transporter 1 relied on SEC24D for ER export. These observations demonstrate that closely related transporters are exclusive client cargo proteins for different SEC24 isoforms. The short promoter polymorphism results in reduced SERT cell surface levels and renders affected individuals more susceptible to depression. By inference, variations in the Sec24C gene may also affect SERT cell surface levels and thus be linked to mood disorders. PMID:21454670

  7. Mutations in Drosophila sec15 reveal a function in neuronal targeting for a subset of exocyst components.

    PubMed

    Mehta, Sunil Q; Hiesinger, P Robin; Beronja, Slobodan; Zhai, R Grace; Schulze, Karen L; Verstreken, Patrik; Cao, Yu; Zhou, Yi; Tepass, Ulrich; Crair, Michael C; Bellen, Hugo J

    2005-04-21

    The exocyst is a complex of proteins originally identified in yeast that has been implicated in polarized secretion. Components of the exocyst have been implicated in neurite outgrowth, cell polarity, and cell viability. We have isolated an exocyst component, sec15, in a screen for genes required for synaptic specificity. Loss of sec15 causes a targeting defect of photoreceptors that coincides with mislocalization of specific cell adhesion and signaling molecules. Additionally, sec15 mutant neurons fail to localize other exocyst members like Sec5 and Sec8, but not Sec6, to neuronal terminals. However, loss of sec15 does not cause cell lethality in contrast to loss of sec5 or sec6. Our data suggest a role of Sec15 in an exocyst-like subcomplex for the targeting and subcellular distribution of specific proteins. The data also show that functions of other exocyst components persist in the absence of sec15, suggesting that different exocyst components have separable functions. PMID:15848801

  8. The product of gene secC is involved in the synthesis of exported proteins in E. coli.

    PubMed

    Ferro-Novick, S; Honma, M; Beckwith, J

    1984-08-01

    To obtain additional mutants in the secretory apparatus of E. coli we have isolated suppressors of a mutant (secAts) that is temperature-sensitive for secretion. One of these, secC, can suppress the secretion defect of secA and has a phenotype of its own. At 23 degrees C, the secC mutant is cold-sensitive for growth and blocks the synthesis of transported proteins. The synthesis of at least one secreted protein, maltose-binding protein (MBP), can be restored by mutations that alter the hydrophobic region of the signal sequence of MBP. The phenotype of the secC mutant suggests that the SecC protein may be a component of the secretory apparatus of E. coli; it also supports the notion that in procaryotes secretion and gene expression are coupled. The secC gene maps at 68.5 minutes on the E. coli chromosome. PMID:6088066

  9. The SEC6 protein is required for contractile vacuole function in Chlamydomonas reinhardtii.

    PubMed

    Komsic-Buchmann, Karin; Stephan, Lisa Marie; Becker, Burkhard

    2012-06-15

    Contractile vacuoles (CVs) are essential for osmoregulation in many protists. To investigate the mechanism of CV function in Chlamydomonas, we isolated novel osmoregulatory mutants. Four of the isolated mutant cell lines carried the same 33,641 base deletion, rendering the cell lines unable to grow under strong hypotonic conditions. One mutant cell line (Osmo75) was analyzed in detail. The CV morphology was variable in mutant cells, and most cells had multiple small CVs. In addition, one or two enlarged CVs or no visible CVs at all, were observed by light microscopy. These findings suggest that the mutant is impaired in homotypic vacuolar and exocytotic membrane fusion. Furthermore the mutants had long flagella. One of the affected genes is the only SEC6 homologue in Chlamydomonas (CreSEC6). The SEC6 protein is a component of the exocyst complex that is required for efficient exocytosis. Transformation of the Osmo75 mutant with a CreSEC6-GFP construct rescued the mutant completely (osmoregulation and flagellar length). Rescued strains overexpressed CreSEC6 (as a GFP-tagged protein) and displayed a modified CV activity. CVs were larger, whereas the CV contraction interval remained unchanged, leading to increased water efflux rates. Electron microscopy analysis of Osmo75 cells showed that the mutant is able to form the close contact zones between the plasma membrane and the CV membrane observed during late diastole and systole. These results indicate that CreSEC6 is essential for CV function and required for homotypic vesicle fusion during diastole and water expulsion during systole. In addition, CreSEC6 is not only necessary for CV function, but possibly influences the CV cycle in an indirect manner and flagellar length in Chlamydomonas. PMID:22427688

  10. Somatic overgrowth associated with homozygous mutations in both MAN1B1 and SEC23A

    PubMed Central

    Gupta, Swati; Fahiminiya, Somayyeh; Wang, Tracy; Dempsey Nunez, Laura; Rosenblatt, David S.; Gibson, William T.; Gilfix, Brian; Bergeron, John J. M.; Jerome-Majewska, Loydie A.

    2016-01-01

    Using whole-exome sequencing, we identified homozygous mutations in two unlinked genes, SEC23A c.1200G>C (p.M400I) and MAN1B1 c.1000C>T (p.R334C), associated with congenital birth defects in two patients from a consanguineous family. Patients presented with carbohydrate-deficient transferrin, tall stature, obesity, macrocephaly, and maloccluded teeth. The parents were healthy heterozygous carriers for both mutations and an unaffected sibling with tall stature carried the heterozygous mutation in SEC23A only. Mutations in SEC23A are responsible for craniolenticosultura dysplasia (CLSD). CLSD patients are short, have late-closing fontanels, and have reduced procollagen (pro-COL1A1) secretion because of abnormal pro-COL1A1 retention in the endoplasmic reticulum (ER). The mutation we identified in MAN1B1 was previously associated with reduced MAN1B1 protein and congenital disorders of glycosylation (CDG). CDG patients are also short, are obese, and have abnormal glycan remodeling. Molecular analysis of fibroblasts from the family revealed normal levels of SEC23A in all cells and reduced levels of MAN1B1 in cells with heterozygous or homozygous mutations in SEC23A and MAN1B1. Secretion of pro-COL1A1 was increased in fibroblasts from the siblings and patients, and pro-COL1A1 was retained in Golgi of heterozygous and homozygous mutant cells, although intracellular pro-COL1A1 was increased in patient fibroblasts only. We postulate that increased pro-COL1A1 secretion is responsible for tall stature in these patients and an unaffected sibling, and not previously discovered in patients with mutations in either SEC23A or MAN1B1. The patients in this study share biochemical and cellular characteristics consistent with mutations in MAN1B1 and SEC23A, indicating a digenic disease. PMID:27148587

  11. Somatic overgrowth associated with homozygous mutations in both MAN1B1 and SEC23A.

    PubMed

    Gupta, Swati; Fahiminiya, Somayyeh; Wang, Tracy; Dempsey Nunez, Laura; Rosenblatt, David S; Gibson, William T; Gilfix, Brian; Bergeron, John J M; Jerome-Majewska, Loydie A

    2016-05-01

    Using whole-exome sequencing, we identified homozygous mutations in two unlinked genes, SEC23A c.1200G>C (p.M400I) and MAN1B1 c.1000C>T (p.R334C), associated with congenital birth defects in two patients from a consanguineous family. Patients presented with carbohydrate-deficient transferrin, tall stature, obesity, macrocephaly, and maloccluded teeth. The parents were healthy heterozygous carriers for both mutations and an unaffected sibling with tall stature carried the heterozygous mutation in SEC23A only. Mutations in SEC23A are responsible for craniolenticosultura dysplasia (CLSD). CLSD patients are short, have late-closing fontanels, and have reduced procollagen (pro-COL1A1) secretion because of abnormal pro-COL1A1 retention in the endoplasmic reticulum (ER). The mutation we identified in MAN1B1 was previously associated with reduced MAN1B1 protein and congenital disorders of glycosylation (CDG). CDG patients are also short, are obese, and have abnormal glycan remodeling. Molecular analysis of fibroblasts from the family revealed normal levels of SEC23A in all cells and reduced levels of MAN1B1 in cells with heterozygous or homozygous mutations in SEC23A and MAN1B1. Secretion of pro-COL1A1 was increased in fibroblasts from the siblings and patients, and pro-COL1A1 was retained in Golgi of heterozygous and homozygous mutant cells, although intracellular pro-COL1A1 was increased in patient fibroblasts only. We postulate that increased pro-COL1A1 secretion is responsible for tall stature in these patients and an unaffected sibling, and not previously discovered in patients with mutations in either SEC23A or MAN1B1. The patients in this study share biochemical and cellular characteristics consistent with mutations in MAN1B1 and SEC23A, indicating a digenic disease. PMID:27148587

  12. Sec6-dependent sorting of fungal extracellular exosomes and laccase of Cryptococcus neoformans.

    PubMed

    Panepinto, John; Komperda, Kazimierz; Frases, Susana; Park, Yoon-Dong; Djordjevic, Julianne T; Casadevall, Arturo; Williamson, Peter R

    2009-03-01

    The cell wall of pathogenic fungi such as Cryptococcus neoformans, provides a formidable barrier to secrete virulence factors that produce host cell damage. To study secretion of virulence factors to the cell periphery, sec6 RNAi mutant strains of C. neoformans were tested for virulence factor expression. The studies reported here show that SEC6 RNAi mutant strains were defective in a number of virulence factors including laccase, urease as well as soluble polysaccharide and demonstrated attenuated virulence in mice. Further analysis by transmission electron microscopy detected the production of abundant extracellular exosomes in wild-type strains containing empty plasmid, but a complete absence in the iSEC6 strain. In addition, a green fluorescent protein-laccase fusion protein demonstrated aberrant localization within cytoplasmic vesicles in iSEC6 strains. In contrast, iSEC6 strains retained normal growth at 37 degrees C, as well as substantially normal capsule formation, phospholipase activity and total secreted protein. These results provide the first molecular evidence for the existence of fungal exosomes and associate these vesicles with the virulence of C. neoformans. PMID:19210702

  13. Crystal structure of the Sec18p N-terminal domain

    PubMed Central

    Babor, S. Mariana; Fass, Deborah

    1999-01-01

    Yeast Sec18p and its mammalian orthologue N-ethylmaleimide-sensitive fusion protein (NSF) are hexameric ATPases with a central role in vesicle trafficking. Aided by soluble adapter factors (SNAPs), Sec18p/NSF induces ATP-dependent disassembly of a complex of integral membrane proteins from the vesicle and target membranes (SNAP receptors). During the ATP hydrolysis cycle, the Sec18p/NSF homohexamer undergoes a large-scale conformational change involving repositioning of the most N terminal of the three domains of each protomer, a domain that is required for SNAP-mediated interaction with SNAP receptors. Whether an internal conformational change in the N-terminal domains accompanies their reorientation with respect to the rest of the hexamer remains to be addressed. We have determined the structure of the N-terminal domain from Sec18p by x-ray crystallography. The Sec18p N-terminal domain consists of two β-sheet-rich subdomains connected by a short linker. A conserved basic cleft opposite the linker may constitute a SNAP-binding site. Despite structural variability in the linker region and in an adjacent loop, all three independent molecules in the crystal asymmetric unit have the identical subdomain interface, supporting the notion that this interface is a preferred packing arrangement. However, the linker flexibility allows for the possibility that other subdomain orientations may be sampled. PMID:10611286

  14. Plasmodium falciparum Sec24 marks transitional ER that exports a model cargo via a diacidic motif

    PubMed Central

    Lee, Marcus C. S.; Moura, Pedro A.; Miller, Elizabeth A.; Fidock, David A.

    2009-01-01

    Summary Exit from the endoplasmic reticulum (ER) often occurs at distinct sites of vesicle formation known as transitional ER (tER) that are enriched for COPII vesicle coat proteins. We have characterized the organization of ER export in the malaria parasite, Plasmodium falciparum, by examining the localization of two components of the COPII machinery, PfSec12 and PfSec24a. PfSec12 was found throughout the ER, whereas the COPII cargo adaptor, PfSec24a, was concentrated at distinct foci that likely correspond to tER sites. These foci were closely apposed to cis-Golgi sites marked by PfGRASP-GFP, and upon treatment with brefeldin A they accumulated a model cargo protein via a process dependent on the presence of an intact diacidic export motif. Our data suggest that the cargo-binding function of PfSec24a is conserved and that accumulation of cargo in discrete tER sites depends upon positive sorting signals. Furthermore, the number and position of tER sites with respect to the cis-Golgi suggests a co-ordinated biogenesis of these domains. PMID:18410493

  15. SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes.

    PubMed

    Bruno, Joanne; Brumfield, Alexandria; Chaudhary, Natasha; Iaea, David; McGraw, Timothy E

    2016-07-01

    RAB10 is a regulator of insulin-stimulated translocation of the GLUT4 glucose transporter to the plasma membrane (PM) of adipocytes, which is essential for whole-body glucose homeostasis. We establish SEC16A as a novel RAB10 effector in this process. Colocalization of SEC16A with RAB10 is augmented by insulin stimulation, and SEC16A knockdown attenuates insulin-induced GLUT4 translocation, phenocopying RAB10 knockdown. We show that SEC16A and RAB10 promote insulin-stimulated mobilization of GLUT4 from a perinuclear recycling endosome/TGN compartment. We propose RAB10-SEC16A functions to accelerate formation of the vesicles that ferry GLUT4 to the PM during insulin stimulation. Because GLUT4 continually cycles between the PM and intracellular compartments, the maintenance of elevated cell-surface GLUT4 in the presence of insulin requires accelerated biogenesis of the specialized GLUT4 transport vesicles. The function of SEC16A in GLUT4 trafficking is independent of its previously characterized activity in ER exit site formation and therefore independent of canonical COPII-coated vesicle function. However, our data support a role for SEC23A, but not the other COPII components SEC13, SEC23B, and SEC31, in the insulin stimulation of GLUT4 trafficking, suggesting that vesicles derived from subcomplexes of COPII coat proteins have a role in the specialized trafficking of GLUT4. PMID:27354378

  16. The SecB-like chaperone Rv1957 from Mycobacterium tuberculosis: crystallization and X-ray crystallographic analysis.

    PubMed

    Lu, Zuokun; Wang, Han; Yu, TingTing

    2016-06-01

    Protein export is important in all bacteria, and bacteria have evolved specialized export machineries to fulfil this task. In Mycobacterium tuberculosis, the causative agent of tuberculosis, the general secretion pathway (Sec pathway) is conserved and is essential in performing the export of proteins. The bacterial Sec pathway post-translationally exports unfolded proteins out of the cytoplasm, and the core of the Sec pathway is composed of a heterotrimeric membrane-embedded channel, SecYEG, and two cytosolic components, SecA and SecB. SecB functions by stabilizing unfolded proteins, maintaining them in an export-competent state. Although SecB is mainly found in Proteobacteria, a SecB-like protein, Rv1957, that controls a stress-response toxin-antitoxin system, is found in M. tuberculosis. Rv1957 can also functionally replace the Escherichia coli SecB chaperone both in vivo and in vitro. In this work, the production, crystallization and X-ray crystallographic analysis of Rv1957 are reported. Notably, diffraction-quality crystals were obtained only at high concentrations of dimethyl sulfoxide, i.e. about 12%(v/v). The crystals of Rv1957 belonged to space group P212121, with unit-cell parameters a = 64.5, b = 92.0, c = 115.4 Å. PMID:27303898

  17. Continuous Cold-Atom Inertial Sensor with 1 nrad /sec Rotation Stability

    NASA Astrophysics Data System (ADS)

    Dutta, I.; Savoie, D.; Fang, B.; Venon, B.; Garrido Alzar, C. L.; Geiger, R.; Landragin, A.

    2016-05-01

    We report the operation of a cold-atom inertial sensor which continuously captures the rotation signal. Using a joint interrogation scheme, where we simultaneously prepare a cold-atom source and operate an atom interferometer (AI), enables us to eliminate the dead times. We show that such continuous operation improves the short-term sensitivity of AIs, and demonstrate a rotation sensitivity of 100 nrad /sec /√{Hz } in a cold-atom gyroscope of 11 cm2 Sagnac area. We also demonstrate a rotation stability of 1 nrad /sec at 104 sec of integration time, which represents the state of the art for atomic gyroscopes. The continuous operation of cold-atom inertial sensors will lead to large area AIs at their full sensitivity potential, determined by the quantum noise limit.

  18. Continuous Cold-Atom Inertial Sensor with 1  nrad/sec Rotation Stability.

    PubMed

    Dutta, I; Savoie, D; Fang, B; Venon, B; Garrido Alzar, C L; Geiger, R; Landragin, A

    2016-05-01

    We report the operation of a cold-atom inertial sensor which continuously captures the rotation signal. Using a joint interrogation scheme, where we simultaneously prepare a cold-atom source and operate an atom interferometer (AI), enables us to eliminate the dead times. We show that such continuous operation improves the short-term sensitivity of AIs, and demonstrate a rotation sensitivity of 100  nrad/sec/sqrt[Hz] in a cold-atom gyroscope of 11  cm^{2} Sagnac area. We also demonstrate a rotation stability of 1  nrad/sec at 10^{4}  sec of integration time, which represents the state of the art for atomic gyroscopes. The continuous operation of cold-atom inertial sensors will lead to large area AIs at their full sensitivity potential, determined by the quantum noise limit. PMID:27203320

  19. Mutations that improve export of maltose-binding protein in SecB- cells of Escherichia coli.

    PubMed

    Collier, D N; Bassford, P J

    1989-09-01

    It previously has been proposed that the Escherichia coli SecB protein promotes the export of the maltose-binding protein (MBP) from the cytoplasm by preventing the folding of the precursor MBP (preMBP) into a translocation-incompetent conformation. The export of wild-type MBP is only partially blocked in SecB- cells. In contrast, the export of MBP16-1, an MBP species with a defective signal peptide, is totally dependent on SecB; hence, SecB- cells that synthesize MBP16-1 are unable to utilize maltose as a sole carbon source. The selection of Mal+ revertants primarily yielded mutants with alterations in the MBP16-1 signal peptide that permitted SecB-independent MBP export to the periplasm to various extents. Although each of these alterations increased the overall hydrophobicity of the signal peptide, it was not possible to strictly equate changes in hydrophobicity with the degree of SecB-independent export. Somewhat unexpectedly, two mutants were obtained in which MBP export in SecB- cells was markedly superior to that of the wild-type MBP. Although wild-type MBP is not cotranslationally translocated in SecB- cells, the two mutant proteins designated MBP172 and MBP173 exhibited significant cotranslational export in the absence of SecB. Thus, the role of SecB was partially supplanted by a signal peptide that promoted more rapid movement of MBP through the export pathway. When preMBP included the MBP172 signal peptide as well as an alteration in the mature moiety that slows folding, the SecB requirement for maximal MBP export efficiency was almost totally eliminated. These results provide additional strong support for the proposed antifolding role of SecB in MBP export. PMID:2670890

  20. The Nuclear Pore Complex Function of Sec13 Protein Is Required for Cell Survival during Retinal Development*

    PubMed Central

    Niu, Xubo; Hong, Jian; Zheng, Xiaofeng; Melville, David B.; Knapik, Ela W.; Meng, Anming; Peng, Jinrong

    2014-01-01

    Sec13 is a dual function protein, being a core component of both the COPII coat, which mediates protein trafficking from the endoplasmic reticulum to the Golgi apparatus, and the nuclear pore complex (NPC), which facilitates nucleo-cytoplasmic traffic. Here, we present a genetic model to differentiate the roles of these two functions of Sec13 in vivo. We report that sec13sq198 mutant embryos develop small eyes that exhibit disrupted retinal lamination and that the mutant retina contains an excessive number of apoptotic cells. Surprisingly, we found that loss of COPII function by oligonucleotide-mediated gene knockdown of sec31a and sec31b or brefeldin A treatment did not disrupt retinal lamination, although it did result in digestive organ defects similar to those seen in sec13sq198, suggesting that the digestive organ defects observed in sec13sq198 are due to loss of COPII function, whereas the retinal lamination defects are due to loss of the NPC function. We showed that the retinal cells of sec13sq198 failed to form proper nuclear pores, leading to a nuclear accumulation of total mRNA and abnormal activation of the p53-dependent apoptosis pathway, causing the retinal defect in sec13sq198. Furthermore, we found that a mutant lacking Nup107, a key NPC-specific component, phenocopied the retinal lamination phenotype as observed in sec13sq198. Our results demonstrate a requirement for the nuclear pore function of Sec13 in development of the retina and provide the first genetic evidence to differentiate the contributions of the NPC and the COPII functions of Sec13 during organogenesis. PMID:24627485