scholarly journals SEC62 encodes a putative membrane protein required for protein translocation into the yeast endoplasmic reticulum.

1989 ◽  
Vol 109 (6) ◽  
pp. 2653-2664 ◽  
Author(s):  
R J Deshaies ◽  
R Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Alpha Helix ◽  
Wild Type ◽  
Cytosol Fraction ◽  
Arginine Residues ◽  
Mutant Cells

Yeast sec62 mutant cells are defective in the translocation of several secretory precursor proteins into the lumen of the endoplasmic reticulum (Rothblatt et al., 1989). The deficiency, which is most restrictive for alpha-factor precursor (pp alpha F) and preprocarboxypeptidase Y, has been reproduced in vitro. Membranes isolated from mutant cells display low and labile translocation activity with pp alpha F translated in a wild-type cytosol fraction. The defect is unique to the membrane fraction because cytosol from mutant cells supports translocation into membranes from wild-type yeast. Invertase assembly is only partly affected by the sec62 mutation in vivo and is nearly normal with mutant membranes in vitro. A potential membrane location for the SEC62 gene product is supported by evaluation of the molecular clone. DNA sequence analysis reveals a 32-kD protein with no obvious NH2-terminal signal sequence but with two domains of sufficient length and hydrophobicity to span a lipid bilayer. Sec62p is predicted to display significant NH2- and COOH-terminal hydrophilic domains on the cytoplasmic surface of the ER membrane. The last 30 amino acids of the COOH terminus may form an alpha-helix with 14 lysine and arginine residues arranged uniformly about the helix. This domain may allow Sec62p to interact with other proteins of the putative translocation complex.

scholarly journals Signal sequence insufficiency contributes to neurodegeneration caused by transmembrane prion protein

2010 ◽  
Vol 188 (4) ◽  
pp. 515-526 ◽  
Author(s):  
Neena S. Rane ◽  
Oishee Chakrabarti ◽  
Lionel Feigenbaum ◽  
Ramanujan S. Hegde
Keyword(s):  
Prion Protein ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Sequences ◽  
Hydrophobic Domain ◽  
Sequence Variations ◽  
Sufficient Magnitude ◽  
The Impact

Protein translocation into the endoplasmic reticulum is mediated by signal sequences that vary widely in primary structure. In vitro studies suggest that such signal sequence variations may correspond to subtly different functional properties. Whether comparable functional differences exist in vivo and are of sufficient magnitude to impact organism physiology is unknown. Here, we investigate this issue by analyzing in transgenic mice the impact of signal sequence efficiency for mammalian prion protein (PrP). We find that replacement of the average efficiency signal sequence of PrP with more efficient signals rescues mice from neurodegeneration caused by otherwise pathogenic PrP mutants in a downstream hydrophobic domain (HD). This effect is explained by the demonstration that efficient signal sequence function precludes generation of a cytosolically exposed, disease-causing transmembrane form of PrP mediated by the HD mutants. Thus, signal sequences are functionally nonequivalent in vivo, with intrinsic inefficiency of the native PrP signal being required for pathogenesis of a subset of disease-causing PrP mutations.

scholarly journals Secretion ofSaccharomyces cerevisiaeKiller Toxin: Processing of the Glycosylated Precursor

1983 ◽  
Vol 3 (8) ◽  
pp. 1362-1370 ◽  
Author(s):  
H. Bussey ◽  
D. Saville ◽  
D. Greene ◽  
D. J. Tipper ◽  
K. A. Bostian
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Killer Toxin ◽  
Membrane System ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Endoproteolytic Cleavage ◽  
Toxin Secretion

Killer toxin secretion was blocked at the restrictive temperature inSaccharomyces cerevisiae secmutants with conditional defects in theS. cerevisiaesecretory pathway leading to accumulation of endoplasmic reticulum (sec18), Golgi (sec7), or secretory vesicles (sec1). A 43,000-molecular-weight (43K) glycosylated protoxin was found by pulse-labeling in allsecmutants at the restrictive temperature. Insec18the protoxin was stable after a chase; but insec7andsec1the protoxin was unstable, and insec111K toxin was detected in cell lysates. The chymotrypsin inhibitor tosyl-l-phenylalanyl chloromethyl ketone (TPCK) blocked toxin secretion in vivo in wild-type cells by inhibiting protoxin cleavage. The unstable protoxin in wild-type and insec7andsec1cells at the restrictive temperature was stabilized by TPCK, suggesting that the protoxin cleavage was post-sec18and was mediated by a TPCK-inhibitable protease. Protoxin glycosylation was inhibited by tunicamycin, and a 36K protoxin was detected in inhibited cells. This 36K protoxin was processed, but toxin secretion was reduced 10-fold. We examined twokexmutants defective in toxin secretion; both synthesized a 43K protoxin, which was stable inkex1but unstable inkex2. Protoxin stability inkex1 kex2double mutants indicated the orderkex1→kex2in the protoxin processing pathway. TPCK did not block protoxin instability inkex2mutants. This suggested that theKEX1- andKEX2-dependent steps preceded thesec7Golgi block. We attempted to localize the protoxin inS. cerevisiaecells. Use of an in vitro rabbit reticulocyte-dog pancreas microsomal membrane system indicated that protoxin synthesized in vitro could be inserted into and glycosylated by the microsomal membranes. This membrane-associated protoxin was protected from trypsin proteolysis. Pulse-chased cells or spheroplasts, with or without TPCK, failed to secrete protoxin. The protoxin may not be secreted into the lumen of the endoplasmic reticulum, but may remain membrane associated and may require endoproteolytic cleavage for toxin secretion.

scholarly journals Loss of the F-Actin Binding and Vesicle-Associated Protein Comitin Leads to a Phagocytosis Defect

2002 ◽  
Vol 1 (6) ◽  
pp. 906-914 ◽  
Author(s):  
Thomas Schreiner ◽  
Martina R. Mohrs ◽  
Rosemarie Blau-Wasser ◽  
Alfred von Krempelhuber ◽  
Michael Steinert ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Gene Replacement ◽  
Actin Binding ◽  
Wild Type ◽  
Hyperosmotic Shock ◽  
Latex Beads ◽  
Knockout Mutants ◽  
Mutant Cells

ABSTRACT Comitin is an F-actin binding and membrane-associated protein from Dictyostelium discoideum, which is present on Golgi and vesicle membranes and changes its localization in response to agents affecting the cytoskeleton. To investigate its in vivo functions we have generated knockout mutants by gene replacement. Based on comitin's in vitro functions we examined properties related to vesicular transport and microfilament function. Whereas cell growth, pinocytosis, secretion, chemotaxis, motility, and development were unaltered, comitin-lacking cells were impaired in the early steps of phagocytosis of Saccharomyces cerevisiae particles and of Escherichia coli, whereas uptake of latex beads was unaffected. Furthermore, the lack of comitin positively affected survival of pathogenic bacteria. Mutant cells also showed an altered response to hyperosmotic shock in comparison to the wild type. The redistribution of comitin during hyperosmotic shock in wild-type cells and its presence on early phagosomes suggest a direct involvement of comitin in these processes.

scholarly journals Roles of the C-Terminal End of SecY in Protein Translocation and Viability of Escherichia coli

2002 ◽  
Vol 184 (8) ◽  
pp. 2243-2250 ◽  
Author(s):  
Kazuhiko Chiba ◽  
Hiroyuki Mori ◽  
Koreaki Ito
Keyword(s):  
Initial Phase ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Central Component ◽  
Cytosolic Domains ◽  
Functional Consequences ◽  
Sequence Region ◽  
Functional Defects

ABSTRACT SecY, a central component of the membrane-embedded sector of protein translocase, contains six cytosolic domains. Here, we examined the importance of the C-terminal cytosolic region of SecY by systematically shortening the C-terminal end and examining the functional consequences of these mutations in vivo and in vitro. It was indicated that the C-terminal five residues are dispensable without any appreciable functional defects in SecY. Mutants missing the C-terminal six to seven residues were partially compromised, especially at low temperature or in the absence of SecG. In vitro analyses indicated that the initial phase of the translocation reaction, in which the signal sequence region of the preprotein is inserted into the membrane, was affected by the lack of the C-terminal residues. SecA binding was normal, but SecA insertion in response to ATP and a preprotein was impaired. It is suggested that the C-terminal SecY residues are required for SecA-dependent translocation initiation.

scholarly journals Detection of Transient In Vivo Interactions between Substrate and Transporter during Protein Translocation into the Endoplasmic Reticulum

1999 ◽  
Vol 10 (2) ◽  
pp. 329-344 ◽  
Author(s):  
Martin Dünnwald ◽  
Alexander Varshavsky ◽  
Nils Johnsson
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Interactions ◽  
Polypeptide Chain ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Living Cells ◽  
C Terminus ◽  
Identical Test ◽  
Split Ubiquitin

The split-ubiquitin technique was used to detect transient protein interactions in living cells. Nub, the N-terminal half of ubiquitin (Ub), was fused to Sec62p, a component of the protein translocation machinery in the endoplasmic reticulum ofSaccharomyces cerevisiae. Cub, the C-terminal half of Ub, was fused to the C terminus of a signal sequence. The reconstitution of a quasi-native Ub structure from the two halves of Ub, and the resulting cleavage by Ub-specific proteases at the C terminus of Cub, serve as a gauge of proximity between the two test proteins linked to Nub and Cub. Using this assay, we show that Sec62p is spatially close to the signal sequence of the prepro-α-factor in vivo. This proximity is confined to the nascent polypeptide chain immediately following the signal sequence. In addition, the extent of proximity depends on the nature of the signal sequence. Cub fusions that bore the signal sequence of invertase resulted in a much lower Ub reconstitution with Nub-Sec62p than otherwise identical test proteins bearing the signal sequence of prepro-α-factor. An inactive derivative of Sec62p failed to interact with signal sequences in this assay. These in vivo findings are consistent with Sec62p being part of a signal sequence-binding complex.

scholarly journals Mutations in RNA Polymerase II and Elongation Factor SII Severely Reduce mRNA Levels in Saccharomyces cerevisiae

1998 ◽  
Vol 18 (10) ◽  
pp. 5771-5779 ◽  
Author(s):  
J. Cale Lennon ◽  
Megan Wind ◽  
Laura Saunders ◽  
M. Benjamin Hock ◽  
Daniel Reines
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Genetic Interaction ◽  
Elongation Factor ◽  
Mrna Levels ◽  
Wild Type ◽  
Mutant Cells ◽  
Rna Levels

ABSTRACT Elongation factor SII interacts with RNA polymerase II and enables it to transcribe through arrest sites in vitro. The set of genes dependent upon SII function in vivo and the effects on RNA levels of mutations in different components of the elongation machinery are poorly understood. Using yeast lacking SII and bearing a conditional allele of RPB2, the gene encoding the second largest subunit of RNA polymerase II, we describe a genetic interaction between SII and RPB2. An SII gene disruption or therpb2-10 mutation, which yields an arrest-prone enzyme in vitro, confers sensitivity to 6-azauracil (6AU), a drug that depresses cellular nucleoside triphosphates. Cells with both mutations had reduced levels of total poly(A)+ RNA and specific mRNAs and displayed a synergistic level of drug hypersensitivity. In cells in which the SII gene was inactivated, rpb2-10 became dominant, as if template-associated mutant RNA polymerase II hindered the ability of wild-type polymerase to transcribe. Interestingly, while 6AU depressed RNA levels in both wild-type and mutant cells, wild-type cells reestablished normal RNA levels, whereas double-mutant cells could not. This work shows the importance of an optimally functioning elongation machinery for in vivo RNA synthesis and identifies an initial set of candidate genes with which SII-dependent transcription can be studied.

scholarly journals A stealth adhesion factor contributes toVibrio vulnificuspathogenicity: Flp pili play roles in host invasion, survival in the blood stream and resistance to complement activation

2019 ◽  
Author(s):  
Tra–My Duong–Nu ◽  
Kwangjoon Jeong ◽  
Soo Young Kim ◽  
Wenzhi Tan ◽  
Sao Puth ◽  
...  
Keyword(s):  
Host Cell ◽  
Complement Activation ◽  
Vibrio Vulnificus ◽  
Alternative Pathway ◽  
Molecular Genetic ◽  
Wild Type ◽  
Triple Mutant ◽  
Mutant Cells

AbstractThe tad operons encode the machinery required for adhesive Flp (fimbrial low-molecular-weight protein) pili biogenesis.Vibrio vulnificus, an opportunistic pathogen, harbors three distincttadloci. Among them, onlytad1locus was highly upregulated inin vivogrowing bacteria compared toin vitroculture condition. To understand the pathogenic roles of the threetadloci during infection, we constructed single, double and triple tad loci deletion mutants. Interestingly, only theΔtad123triple mutant cells exhibited significantly decreased lethality in mice. Ultrastructural observations revealed short, thin filamentous projections disappeared on theΔtad123mutant cells. Since the pilin was paradoxically non-immunogenic, a V5 tag was fused to Flp to visualize the pilin protein by using immunogold EM and immunofluorescence microscopy. TheΔtad123mutant cells showed attenuated host cell adhesion, delayed RtxA1 exotoxin secretion and subsequently impaired translocation across the intestinal epithelium compared to wild type, which could be partially complemented with each wild type operon. TheΔtad123mutant was susceptible to complement-mediated bacteriolysis, predominantly via the alternative pathway, suggesting stealth hiding role of the Tad pili. Taken together, all threetadloci cooperate to confer successful invasion ofV. vulnificusinto deeper tissue and evasion from host defense mechanisms, ultimately resulting in septicemia.Author SummaryTo understand the roles of the three Tad operons in the pathogenesis ofV. vulnificusinfection, we constructed mutant strain with single, double and triple Tad loci deletions. Employing a variety of mouse infection models coupled with molecular genetic analyses, we demonstrate here that all three Tad operons are required forV. vulnificuspathogenicity as the cryptic pili contribute to host cell and tissue invasion, survival in the blood, and resistance to complement activation.

Sec62p, A Component of the Endoplasmic Reticulum Protein Translocation Machinery, Contains Multiple Binding Sites for the Sec-Complex

2000 ◽  
Vol 11 (11) ◽  
pp. 3859-3871 ◽  
Author(s):  
Sandra Wittke ◽  
Martin Dünnwald ◽  
Nils Johnsson
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Endoplasmic Reticulum Membrane ◽  
Sequence Recognition ◽  
Multiple Binding Sites ◽  
Oligomeric Protein ◽  
Multiple Binding ◽  
Endoplasmic Reticulum Protein

SEC62 encodes an essential component of the Sec-complex that is responsible for posttranslational protein translocation across the membrane of the endoplasmic reticulum in Saccharomyces cerevisiae. The specific role of Sec62p in translocation was not known and difficult to identify because it is part of an oligomeric protein complex in the endoplasmic reticulum membrane. An in vivo competition assay allowed us to characterize and dissect physical and functional interactions between Sec62p and components of the Sec-complex. We could show that Sec62p binds via its cytosolic N- and C-terminal domains to the Sec-complex. The N-terminal domain, which harbors the major interaction site, binds directly to the last 14 residues of Sec63p. The C-terminal binding site of Sec62p is less important for complex stability, but adjoins the region in Sec62p that might be involved in signal sequence recognition.

scholarly journals A signal sequence suppressor mutant that stabilizes an assembled state of the twin arginine translocase

2017 ◽  
Vol 114 (10) ◽  
pp. E1958-E1967 ◽  
Author(s):  
Qi Huang ◽  
Felicity Alcock ◽  
Holger Kneuper ◽  
Justin C. Deme ◽  
Sarah E. Rollauer ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Transmembrane Helix ◽  
Peptide Binding ◽  
Signal Peptides ◽  
Arginine Residues ◽  
Tat System ◽  
Twin Arginine Translocase

The twin-arginine protein translocation (Tat) system mediates transport of folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. The Tat system ofEscherichia coliis made up of TatA, TatB, and TatC components. TatBC comprise the substrate receptor complex, and active Tat translocases are formed by the substrate-induced association of TatA oligomers with this receptor. Proteins are targeted to TatBC by signal peptides containing an essential pair of arginine residues. We isolated substitutions, locating to the transmembrane helix of TatB that restored transport activity to Tat signal peptides with inactivating twin arginine substitutions. A subset of these variants also suppressed inactivating substitutions in the signal peptide binding site on TatC. The suppressors did not function by restoring detectable signal peptide binding to the TatBC complex. Instead, site-specific cross-linking experiments indicate that the suppressor substitutions induce conformational change in the complex and movement of the TatB subunit. The TatB F13Y substitution was associated with the strongest suppressing activity, even allowing transport of a Tat substrate lacking a signal peptide. In vivo analysis using a TatA–YFP fusion showed that the TatB F13Y substitution resulted in signal peptide-independent assembly of the Tat translocase. We conclude that Tat signal peptides play roles in substrate targeting and in triggering assembly of the active translocase.

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