scholarly journals Sec24p and Iss1p Function Interchangeably in Transport Vesicle Formation from the Endoplasmic Reticulum inSaccharomyces cerevisiae

2000 ◽  
Vol 11 (3) ◽  
pp. 983-998 ◽  
Author(s):  
Tatsuo Kurihara ◽  
Susan Hamamoto ◽  
Ruth E. Gimeno ◽  
Chris A. Kaiser ◽  
Randy Schekman ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Temperature Sensitivity ◽  
Carboxypeptidase Y ◽  
Homologous Proteins ◽  
C Terminus ◽  
Complex Functions ◽  
Transport Vesicle ◽  
Copii Vesicles ◽  
Synthetically Lethal

The Sec23p/Sec24p complex functions as a component of the COPII coat in vesicle transport from the endoplasmic reticulum. Here we characterize Saccharomyces cerevisiae SEC24, which encodes a protein of 926 amino acids (YIL109C), and a close homologue, ISS1 (YNL049C), which is 55% identical to SEC24. SEC24 is essential for vesicular transport in vivo because depletion of Sec24p is lethal, causing exaggeration of the endoplasmic reticulum and a block in the maturation of carboxypeptidase Y. Overproduction of Sec24p suppressed the temperature sensitivity of sec23-2, and overproduction of both Sec24p and Sec23p suppressed the temperature sensitivity of sec16-2. SEC24 gene disruption could be complemented by overexpression ofISS1, indicating functional redundancy between the two homologous proteins. Deletion of ISS1 had no significant effect on growth or secretion; however, iss1Δ mutants were found to be synthetically lethal with mutations in the v-SNARE genes SEC22 and BET1. Moreover, overexpression of ISS1 could suppress mutations inSEC22. These genetic interactions suggest that Iss1p may be specialized for the packaging or the function of COPII v-SNAREs. Iss1p tagged with His6at its C terminus copurified with Sec23p. Pure Sec23p/Iss1p could replace Sec23p/Sec24p in the packaging of a soluble cargo molecule (α-factor) and v-SNAREs (Sec22p and Bet1p) into COPII vesicles. Abundant proteins in the purified vesicles produced with Sec23p/Iss1p were indistinguishable from those in the regular COPII vesicles produced with Sec23p/Sec24p.

scholarly journals The C-terminus of cytochrome b5 confers endoplasmic reticulum specificity by preventing spontaneous insertion into membranes

2007 ◽  
Vol 401 (3) ◽  
pp. 701-709 ◽  
Author(s):  
Matthew P. A. Henderson ◽  
Yeen Ting Hwang ◽  
John M. Dyer ◽  
Robert T. Mullen ◽  
David W. Andrews
Keyword(s):  
Endoplasmic Reticulum ◽  
Subcellular Localization ◽  
Lipid Bilayers ◽  
Fusion Proteins ◽  
Molecular Mechanisms ◽  
Cytochrome B5 ◽  
Terminal Sequence ◽  
C Terminus

The molecular mechanisms that determine the correct subcellular localization of proteins targeted to membranes by tail-anchor sequences are poorly defined. Previously, we showed that two isoforms of the tung oil tree [Vernicia (Aleurites) fordii] tail-anchored Cb5 (cytochrome b5) target specifically to ER (endoplasmic reticulum) membranes both in vivo and in vitro [Hwang, Pelitire, Henderson, Andrews, Dyer and Mullen (2004) Plant Cell 16, 3002–3019]. In the present study, we examine the targeting of various tung Cb5 fusion proteins and truncation mutants to purified intracellular membranes in vitro in order to assess the importance of the charged CTS (C-terminal sequence) in targeting to specific membranes. Removal of the CTS from tung Cb5 proteins resulted in efficient binding to both ER and mitochondria. Results from organelle competition, liposome-binding and membrane proteolysis experiments demonstrated that removal of the CTS results in spontaneous insertion of tung Cb5 proteins into lipid bilayers. Our results indicate that the CTSs from plant Cb5 proteins provide ER specificity by preventing spontaneous insertion into incorrect subcellular membranes.

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 Functional interaction of cytosolic hsp70 and a DnaJ-related protein, Ydj1p, in protein translocation in vivo.

1996 ◽  
Vol 16 (8) ◽  
pp. 4378-4386 ◽  
Author(s):  
J Becker ◽  
W Walter ◽  
W Yan ◽  
E A Craig
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Genetic Interaction ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Alpha Factor ◽  
Synthetically Lethal ◽  
Shock Proteins ◽  
Related Proteins

In order to analyze the in vivo role of the SSA class of cytosolic 70-kDa heat shock proteins (hsps) of Saccharomyces cerevisiae, we isolated a temperature-sensitive mutant of SSA1. The effect of a shift of mutant cells (ssa1ts ssa2 ssa3 ssa4) from the permissive temperature of 23 degrees C to the nonpermissive temperature of 37 degrees C on the processing of several precursor proteins translocated into the endoplasmic reticulum or mitochondria was assessed. Of three mitochondrial proteins tested, the processing of only one, the beta subunit of the F1F0 ATPase, was dramatically affected. Of six proteins destined for the endoplasmic reticulum, the translocation of only prepro-alpha-factor and proteinase A was inhibited. The processing of prepro-alpha-factor was inhibited within 2 min of the shift to 37 degrees C, suggesting a direct effect of the hsp70 defect on translocation. More than 50% of radiolabeled alpha-factor accumulated in the precursor form, with the remainder rapidly reaching the mature form. However, the translocation block was complete, as the precursor form could not be chased through the translocation pathway. Since DnaJ-related proteins are known to interact with hsp70s and strains containing conditional mutations in a dnaJ-related gene, YDJ1, are defective in translocation of prepro-alpha-factor, we looked for a genetic interaction between SSA genes and YDJ1 in vivo. We found that a deletion mutation of YDJ1 was synthetically lethal in a ssa1ts ssa2 ssa3 ssa4 background. In addition, a strain containing a single functional SSA gene, SSA1, and a deletion of YDJ1 accumulated the precursor form of alpha-factor. However, no genetic interaction was observed between a YDJ1 mutation and mutations in the SSB genes, which encode a second class of cytosolic hsp70 chaperones. These results are consistent with SSA proteins and Ydj1p acting together in the translocation process.

scholarly journals Recognition of a Subset of Signal Sequences by Ssh1p, a Sec61p-related Protein in the Membrane of Endoplasmic Reticulum of Yeast Saccharomyces cerevisiae

2002 ◽  
Vol 13 (7) ◽  
pp. 2223-2232 ◽  
Author(s):  
Sandra Wittke ◽  
Martin Dünnwald ◽  
Markus Albertsen ◽  
Nils Johnsson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Signal Sequence ◽  
Carboxypeptidase Y ◽  
Related Protein ◽  
Signal Sequences ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sequence Recognition ◽  
Split Ubiquitin

Ssh1p of Saccharomyces cerevisiae is related in sequence to Sec61p, a general receptor for signal sequences and the major subunit of the channel that guides proteins across the membrane of the endoplasmic reticulum. The split-ubiquitin technique was used to determine whether Ssh1p serves as an additional receptor for signal sequences in vivo. We measured the interactions between the Nub-labeled Ssh1p and Cub-translocation substrates bearing four different signal sequences. The so-determined interaction profile of Ssh1p was compared with the signal sequence interaction profile of the correspondingly modified Nub-Sec61p. The assay reveals interactions of Ssh1p with the signal sequences of Kar2p and invertase, whereas Sec61p additionally interacts with the signal sequences of Mfα1 and carboxypeptidase Y. The measured physical proximity between Ssh1p and the β-subunit of the signal sequence recognition particle receptor confirms our hypothesis that Ssh1p is directly involved in the cotranslational translocation of proteins across the membrane of the endoplasmic reticulum.

scholarly journals A Developmentally Regulated Chaperone Complex for the Endoplasmic Reticulum of Male Haploid Germ Cells

2007 ◽  
Vol 18 (8) ◽  
pp. 2795-2804 ◽  
Author(s):  
Marcel van Lith ◽  
Anna-Riikka Karala ◽  
Dave Bown ◽  
John A. Gatehouse ◽  
Lloyd W. Ruddock ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Germ Cells ◽  
Specific Protein ◽  
Developmentally Regulated ◽  
C Terminus ◽  
Pancreatic Trypsin Inhibitor ◽  
Disulfide Isomerases ◽  
Chaperone Complex

Glycoprotein folding is mediated by lectin-like chaperones and protein disulfide isomerases (PDIs) in the endoplasmic reticulum. Calnexin and the PDI homologue ERp57 work together to help fold nascent polypeptides with glycans located toward the N-terminus of a protein, whereas PDI and BiP may engage proteins that lack glycans or have sugars toward the C-terminus. In this study, we show that the PDI homologue PDILT is expressed exclusively in postmeiotic male germ cells, in contrast to the ubiquitous expression of many other PDI family members in the testis. PDILT is induced during puberty and represents the first example of a PDI family member under developmental control. We find that PDILT is not active as an oxido-reductase, but interacts with the model peptide Δ-somatostatin and nonnative bovine pancreatic trypsin inhibitor in vitro, indicative of chaperone activity. In vivo, PDILT forms a tissue-specific chaperone complex with the calnexin homologue calmegin. The identification of a redox-inactive chaperone partnership defines a new system of testis-specific protein folding with implications for male fertility.

scholarly journals Molecular Chaperones in the Yeast Endoplasmic Reticulum Maintain the Solubility of Proteins for Retrotranslocation and Degradation

2001 ◽  
Vol 153 (5) ◽  
pp. 1061-1070 ◽  
Author(s):  
Shuh-ichi Nishikawa ◽  
Sheara W. Fewell ◽  
Yoshihito Kato ◽  
Jeffrey L. Brodsky ◽  
Toshiya Endo
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Chaperone ◽  
Molecular Chaperones ◽  
Elevated Temperatures ◽  
Carboxypeptidase Y ◽  
Mutant Form ◽  
Genes Encoding

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the process by which aberrant proteins in the ER lumen are exported back to the cytosol and degraded by the proteasome. Although ER molecular chaperones are required for ERAD, their specific role(s) in this process have been ill defined. To understand how one group of interacting lumenal chaperones facilitates ERAD, the fates of pro–α-factor and a mutant form of carboxypeptidase Y were examined both in vivo and in vitro. We found that these ERAD substrates are stabilized and aggregate in the ER at elevated temperatures when BiP, the lumenal Hsp70 molecular chaperone, is mutated, or when the genes encoding the J domain–containing proteins Jem1p and Scj1p are deleted. In contrast, deletion of JEM1 and SCJ1 had little effect on the ERAD of a membrane protein. These results suggest that one role of the BiP, Jem1p, and Scj1p chaperones is to maintain lumenal ERAD substrates in a retrotranslocation-competent state.

scholarly journals Complementary and divergent roles for Ctage5 and Tango1 in zebrafish

2020 ◽  
Author(s):  
Eric M. Clark ◽  
Brian A. Link
Keyword(s):  
Endoplasmic Reticulum ◽  
Genetic Interaction ◽  
Interaction Study ◽  
Pathway Activation ◽  
Copii Vesicles ◽  
Collagen Versus ◽  
First Time ◽  
Stress Pathway ◽  
And Cartilage

AbstractCoat protein complex II (COPII) factors mediate cargo export from the endoplasmic reticulum (ER), but bulky collagens and lipoproteins are too large for traditional COPII vesicles. Mammalian CTAGE5 and TANGO1 have been well characterized individually as specialized cargo receptors at the ER that function with COPII coats to facilitate trafficking of bulky cargoes. Here, we present a genetic interaction study in zebrafish of deletions in ctage5, tango1, or both to investigate their potential distinct and complimentary functions. We found that Ctage5 and Tango1 have different roles related to organogenesis, collagen versus lipoprotein trafficking, stress-pathway activation, and survival. While deletion of both ctage5 and tango1 compounded phenotype severity, deletion of either factor alone revealed novel tissue specific defects in the building of heart, muscle, lens, and intestine, in addition to the previously described roles in the development of neural and cartilage tissues. Together, our results suggest that Ctage5 and Tango1 have overlapping, but also divergent roles in tissue development and homeostasis.SummaryIn this genetic study Ctage5 and Tango1 endoplasmic reticulum cargo receptors were investigated together in vivo for the first time. Cell differentiation, survival, trafficking, and stress pathway activation were investigated.

scholarly journals Translocation in yeast and mammalian cells: not all signal sequences are functionally equivalent.

1987 ◽  
Vol 105 (6) ◽  
pp. 2905-2914 ◽  
Author(s):  
P Bird ◽  
M J Gething ◽  
J Sambrook
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Influenza Virus ◽  
Signal Peptide ◽  
Mammalian Cells ◽  
Signal Sequence ◽  
Carboxypeptidase Y ◽  
Influenza Virus Hemagglutinin

In Saccharomyces cerevisiae, nascent carboxypeptidase Y (CPY) is directed into the endoplasmic reticulum by an NH2-terminal signal peptide that is removed before the glycosylated protein is transported to the vacuole. In this paper, we show that this signal peptide does not function in mammalian cells: CPY expressed in COS-1 cells is not glycosylated, does not associate with membranes, and retains its signal peptide. In a mammalian cell-free protein-synthesizing system, CPY is not translocated into microsomes. However, if the CPY signal is either mutated to increase its hydrophobicity or replaced with that of influenza virus hemagglutinin, the resulting precursors are efficiently translocated both in vivo and in vitro. The implications of these results for models of signal sequence function are discussed.

scholarly journals Sec31 encodes an essential component of the COPII coat required for transport vesicle budding from the endoplasmic reticulum.

1997 ◽  
Vol 8 (2) ◽  
pp. 205-217 ◽  
Author(s):  
N R Salama ◽  
J S Chuang ◽  
R W Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Protein Complex ◽  
Essential Gene ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Transport Vesicles ◽  
Transport Vesicle ◽  
Membrane Budding

The COPII vesicle coat protein promotes the formation of endoplasmic reticulum- (ER) derived transport vesicles that carry secretory proteins to the Golgi complex in Saccharomyces cerevisiae. This coat protein consists of Sar1p, the Sec23p protein complex containing Sec23p and Sec24p, and the Sec13p protein complex containing Sec13p and a novel 150-kDa protein, p150. Here, we report the cloning and characterization of the p150 gene. p150 is encoded by an essential gene. Depletion of this protein in vivo blocks the exit of secretory proteins from the ER and causes an elaboration of ER membranes, indicating that p150 is encoded by a SEC gene. Additionally, overproduction of the p150 gene product compromises the growth of two ER to Golgi sec mutants: sec16-2 and sec23-1. p150 is encoded by SEC31, a gene isolated in a genetic screen for mutations that accumulate unprocessed forms of the secretory protein alpha-factor. The sec31-1 mutation was mapped by gap repair, and sequence analysis revealed an alanine to valine change at position 1239, near the carboxyl terminus. Sec31p is a phosphoprotein and treatment of the Sec31p-containing fraction with alkaline phosphatase results in a 50-75% inhibition of transport vesicle formation activity in an ER membrane budding assay.

scholarly journals Tiered Assembly of the Yeast Far3-7-8-9-10-11 Complex at the Endoplasmic Reticulum

2013 ◽  
Vol 288 (23) ◽  
pp. 16986-16997 ◽  
Author(s):  
Tammy Pracheil ◽  
Zhengchang Liu
Keyword(s):  
Endoplasmic Reticulum ◽  
Cellular Localization ◽  
Target Of Rapamycin ◽  
Homologous Proteins ◽  
Cycle Arrest ◽  
Optimal Function ◽  
Phosphatase 2A ◽  
Cell Growth And Proliferation ◽  
Mutant Cells

Target of rapamycin signaling is a conserved, essential pathway integrating nutritional cues with cell growth and proliferation. The target of rapamycin kinase exists in two distinct complexes, TORC1 and TORC2. It has been reported that protein phosphatase 2A (PP2A) and the Far3-7-8-9-10-11 complex (Far complex) negatively regulate TORC2 signaling in yeast. The Far complex, originally identified as factors required for pheromone-induced cell cycle arrest, and PP2A form the yeast counterpart of the STRIPAK complex, which was first isolated in mammals. The cellular localization of the Far complex has yet to be fully characterized. Here, we show that the Far complex localizes to the endoplasmic reticulum (ER) by analyzing functional GFP-tagged Far proteins in vivo. We found that Far9 and Far10, two homologous proteins each with a tail-anchor domain, localize to the ER in mutant cells lacking the other Far complex components. Far3, Far7, and Far8 form a subcomplex, which is recruited to the ER by Far9/10. The Far3-7-8- complex in turn recruits Far11 to the ER. Finally, we show that the tail-anchor domain of Far9 is required for its optimal function in TORC2 signaling. Our study reveals tiered assembly of the yeast Far complex at the ER and a function for Far complex's ER localization in TORC2 signaling.

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