scholarly journals SSI1 encodes a novel Hsp70 of the Saccharomyces cerevisiae endoplasmic reticulum.

1996 ◽  
Vol 16 (11) ◽  
pp. 6444-6456 ◽  
Author(s):  
B K Baxter ◽  
P James ◽  
T Evans ◽  
E A Craig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Synthetic Lethality ◽  
Subcellular Fractionation ◽  
Cold Sensitivity ◽  
Direct Role ◽  
Yeast Saccharomyces Cerevisiae ◽  
Hsp70 Family ◽  
C Terminus ◽  
The Unfolded Protein Response

The endoplasmic reticulum (ER) of the budding yeast Saccharomyces cerevisiae contains a well-characterized, essential member of the Hsp70 family of molecular chaperones, Kar2p. Kar2p has been shown to be involved in the translocation of proteins into the ER as well as the proper folding of proteins in that compartment. We report the characterization of a novel Hsp70 of the ER, Ssi1p. Ssi1p, which shares 24% of the amino acids of Kar2p, is not essential for growth under normal conditions. However, deletion of SSI1 results in cold sensitivity as well as enhanced resistance to manganese. The localization of Ssi1p to the ER, suggested by the presence of a conserved S. cerevisiae ER retention signal at its C terminus, was confirmed by subcellular fractionation, protease protection assays, and immunofluorescence. The SSI1 promoter contains an element with similarity to the unfolded protein response element of KAR2. Like KAR2, SSI1 is induced both in the presence of tunicamycin and in a kar2-159 mutant strain, conditions which lead to an accumulation of unfolded proteins in the ER. Unlike KAR2, however, SSI1 is not induced by heat shock. Deletion of SSI1 shows a complex pattern of genetic interactions with various conditional alleles of KAR2, ranging from synthetic lethality to synthetic rescue. Interestingly, SSI1 deletion strains show a partial block in translocation of multiple proteins into the ER, suggesting that Ssi1p plays a direct role in the translocation process.

Vam3p, a new member of syntaxin related protein, is required for vacuolar assembly in the yeast Saccharomyces cerevisiae

1997 ◽  
Vol 110 (11) ◽  
pp. 1299-1306 ◽  
Author(s):  
Y. Wada ◽  
N. Nakamura ◽  
Y. Ohsumi ◽  
A. Hirata
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Trafficking ◽  
Subcellular Fractionation ◽  
Carboxypeptidase Y ◽  
Loss Of Function ◽  
Yeast Saccharomyces Cerevisiae ◽  
C Terminus ◽  
Late Step ◽  
Spherical Structures ◽  
Vacuolar Membranes

Syntaxins are thought to participate in the specific interactions between vesicles and acceptor membranes in intracellular protein trafficking. VAM3 of Saccharomyces cerevisiae encodes a 33 kDa protein (Vam3p) with a hydrophobic transmembrane segment at its C terminus. Vam3p has structural similarities to syntaxins of yeast, animal and plant cells. delta vam3 cells accumulated spherical structures of 200–600 nm in diameter, but lacked normal large vacuolar compartments. Loss of function of Vam3p resulted in inefficient processing of vacuolar proteins proteinase A, proteinase B and carboxypeptidase Y, and defective maturation of alkaline phosphatase. Subcellular fractionation and immunofluorescence microscopy showed that Vam3p was localized to the vacuolar membranes. Vam3p was accumulated in certain regions of the vacuolar membranes. We conclude from these observations that Vam3p is a novel member of syntaxin in the vacuoles and it provides the t-SNARE function in a late step of the vacuolar assembly.

scholarly journals The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane.

1997 ◽  
Vol 8 (9) ◽  
pp. 1805-1814 ◽  
Author(s):  
J S Cox ◽  
R E Chapman ◽  
P Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Unfolded Protein Response ◽  
Secretory Pathway ◽  
Lipid Synthesis ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Yeast Saccharomyces Cerevisiae ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

The endoplasmic reticulum (ER) is a multifunctional organelle responsible for production of both lumenal and membrane components of secretory pathway compartments. Secretory proteins are folded, processed, and sorted in the ER lumen and lipid synthesis occurs on the ER membrane itself. In the yeast Saccharomyces cerevisiae, synthesis of ER components is highly regulated: the ER-resident proteins by the unfolded protein response and membrane lipid synthesis by the inositol response. We demonstrate that these two responses are intimately linked, forming different branches of the same pathway. Furthermore, we present evidence indicating that this coordinate regulation plays a role in ER biogenesis.

scholarly journals The a-factor transporter (STE6 gene product) and cell polarity in the yeast Saccharomyces cerevisiae.

1993 ◽  
Vol 120 (5) ◽  
pp. 1203-1215 ◽  
Author(s):  
K Kuchler ◽  
H G Dohlman ◽  
J Thorner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Gene Product ◽  
Cell Polarity ◽  
Polyclonal Antibodies ◽  
Apparent Molecular Weight ◽  
Subcellular Fractionation ◽  
Mating Pheromone ◽  
Yeast Saccharomyces Cerevisiae ◽  
Factor Secretion

STE6 gene product is required for secretion of the lipopeptide mating pheromone a-factor by Saccharomyces cerevisiae MATa cells. Radiolabeling and immunoprecipitation, either with specific polyclonal antibodies raised against a TrpE-Ste6 fusion protein or with mAbs that recognize c-myc epitopes in fully functional epitope-tagged Ste6 derivatives, demonstrated that Ste6 is a 145-kD phosphoprotein. Subcellular fractionation, various extraction procedures, and immunoblotting showed that Ste6 is an intrinsic plasma membrane-associated protein. The apparent molecular weight of Ste6 was unaffected by tunicamycin treatment, and the radiolabeled protein did not bind to concanavalin A, indicating that Ste6 is not glycosylated and that glycosylation is not required either for its membrane delivery or its function. The amino acid sequence of Ste6 predicts two ATP-binding folds; correspondingly, Ste6 was photoaffinity-labeled specifically with 8-azido-[alpha-32P]ATP. Indirect immunofluorescence revealed that in exponentially growing MATa cells, the majority of Ste6 showed a patchy distribution within the plasma membrane, but a significant fraction was found concentrated in a number of vesicle-like bodies subtending the plasma membrane. In contrast, in MATa cells exposed to the mating pheromone alpha-factor, which markedly induced Ste6 production, the majority of Ste6 was incorporated into the plasma membrane within the growing tip of the elongating cells. The highly localized insertion of this transporter may establish pronounced anisotropy in a-factor secretion from the MATa cell, and thereby may contribute to the establishment of the cell polarity which restricts partner selection and cell fusion during mating to one MAT alpha cell.

scholarly journals TheSaccharomyces cerevisiaePrenylcysteine Carboxyl Methyltransferase Ste14p Is in the Endoplasmic Reticulum Membrane

1998 ◽  
Vol 9 (8) ◽  
pp. 2231-2247 ◽  
Author(s):  
Julia D. Romano ◽  
Walter K. Schmidt ◽  
Susan Michaelis
Keyword(s):  
Endoplasmic Reticulum ◽  
Subcellular Fractionation ◽  
Endoplasmic Reticulum Membrane ◽  
Carboxyl Methylation ◽  
C Terminus ◽  
Eukaryotic Proteins ◽  
Internal Site ◽  
Membrane Spanning ◽  
Er Membrane

Eukaryotic proteins containing a C-terminal CAAX motif undergo a series of posttranslational CAAX-processing events that include isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. We demonstrated previously that the STE14gene product of Saccharomyces cerevisiae mediates the carboxyl methylation step of CAAX processing in yeast. In this study, we have investigated the subcellular localization of Ste14p, a predicted membrane-spanning protein, using a polyclonal antibody generated against the C terminus of Ste14p and an in vitro methyltransferase assay. We demonstrate by immunofluorescence and subcellular fractionation that Ste14p and its associated activity are localized to the endoplasmic reticulum (ER) membrane of yeast. In addition, other studies from our laboratory have shown that the CAAX proteases are also ER membrane proteins. Together these results indicate that the intracellular site of CAAX protein processing is the ER membrane, presumably on its cytosolic face. Interestingly, the insertion of a hemagglutinin epitope tag at the N terminus, at the C terminus, or at an internal site disrupts the ER localization of Ste14p and results in its mislocalization, apparently to the Golgi. We have also expressed the Ste14p homologue from Schizosaccharomyces pombe, mam4p, in S. cerevisiae and have shown that mam4p complements a Δste14 mutant. This finding, plus additional recent examples of cross-species complementation, indicates that the CAAX methyltransferase family consists of functional homologues.

scholarly journals Yeast Nuclear Envelope Subdomains with Distinct Abilities to Resist Membrane Expansion

2006 ◽  
Vol 17 (4) ◽  
pp. 1768-1778 ◽  
Author(s):  
Joseph L. Campbell ◽  
Alexander Lorenz ◽  
Keren L. Witkin ◽  
Thomas Hays ◽  
Josef Loidl ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Budding Yeast ◽  
Phospholipid Biosynthesis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nuclear Compartment ◽  
Round Shape ◽  
Nuclear Protrusion

Little is known about what dictates the round shape of the yeast Saccharomyces cerevisiae nucleus. In spo7Δ mutants, the nucleus is misshapen, exhibiting a single protrusion. The Spo7 protein is part of a phosphatase complex that represses phospholipid biosynthesis. Here, we report that the nuclear protrusion of spo7Δ mutants colocalizes with the nucleolus, whereas the nuclear compartment containing the bulk of the DNA is unaffected. Using strains in which the nucleolus is not intimately associated with the nuclear envelope, we show that the single nuclear protrusion of spo7Δ mutants is not a result of nucleolar expansion, but rather a property of the nuclear membrane. We found that in spo7Δ mutants the peripheral endoplasmic reticulum (ER) membrane was also expanded. Because the nuclear membrane and the ER are contiguous, this finding indicates that in spo7Δ mutants all ER membranes, with the exception of the membrane surrounding the bulk of the DNA, undergo expansion. Our results suggest that the nuclear envelope has distinct domains that differ in their ability to resist membrane expansion in response to increased phospholipid biosynthesis. We further propose that in budding yeast there is a mechanism, or structure, that restricts nuclear membrane expansion around the bulk of the DNA.

Uptake and trafficking of exogenous sterols in Saccharomyces cerevisiae

2006 ◽  
Vol 34 (3) ◽  
pp. 359-362 ◽  
Author(s):  
S. Raychaudhuri ◽  
W.A. Prinz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Abc Transporters ◽  
Cellular Functions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Membrane Function ◽  
Oxysterol Binding Protein ◽  
Atp Binding Cassette Transporters ◽  
Related Proteins

The proper distribution of sterols among organelles is critical for numerous cellular functions. How sterols are sorted and moved among membranes remains poorly understood, but they are transported not only in vesicles but also by non-vesicular pathways. One of these pathways moves exogenous sterols from the plasma membrane to the endoplasmic reticulum in the yeast Saccharomyces cerevisiae. We have found that two classes of proteins play critical roles in this transport, ABC transporters (ATP-binding-cassette transporters) and oxysterol-binding protein-related proteins. Transport is also regulated by phosphoinositides and the interactions of sterols with other lipids. Here, we summarize these findings and speculate on the role of non-vesicular sterol transfer in determining intracellular sterol distribution and membrane function.

scholarly journals The JNM1 gene in the yeast Saccharomyces cerevisiae is required for nuclear migration and spindle orientation during the mitotic cell cycle.

1994 ◽  
Vol 125 (1) ◽  
pp. 143-158 ◽  
Author(s):  
J N McMillan ◽  
K Tatchell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Low Temperature ◽  
Mother Cell ◽  
Significant Proportion ◽  
Nuclear Migration ◽  
Cold Sensitivity ◽  
Chain Gene ◽  
Chromosome Loss ◽  
Lacz Gene ◽  
Yeast Saccharomyces Cerevisiae

JNM1, a novel gene on chromosome XIII in the yeast Saccharomyces cerevisiae, is required for proper nuclear migration. jnm1 null mutants have a temperature-dependent defect in nuclear migration and an accompanying alteration in astral microtubules. At 30 degrees C, a significant proportion of the mitotic spindles is not properly located at the neck between the mother cell and the bud. This defect is more severe at low temperature. At 11 degrees C, 60% of the cells accumulate with large buds, most of which have two DAPI staining regions in the mother cell. Although mitosis is delayed and nuclear migration is defective in jnm1 mutant, we rarely observe more than two nuclei in a cell, nor do we frequently observe anuclear cells. No loss of viability is observed at 11 degrees C and cells continue to grow exponentially with increased doubling time. At low temperature the large budded cells of jnm1 mutants exhibit extremely long astral microtubules that often wind around the periphery of the cell. jnm1 mutants are not defective in chromosome segregation during mitosis, as assayed by the rate of chromosome loss, or nuclear migration during conjugation, as assayed by the rate of mating and cytoduction. The phenotype of a jnm1 mutant is strikingly similar to that for mutants in the dynein heavy chain gene (Eshel, D., L. A. Urrestarazu, S. Vissers, J.-C. Jauniaux, J. C. van Vliet-Reedijk, R. J. Plants, and I. R. Gibbons. 1993. Proc. Natl. Acad. Sci. USA. 90:11172-11176; Li, Y. Y., E. Yeh, T. Hays, and K. Bloom. 1993. Proc. Natl. Acad. Sci. USA. 90:10096-10100). The JNM1 gene product is predicted to encode a 44-kD protein containing three coiled coil domains. A JNM1:lacZ gene fusion is able to complement the cold sensitivity and microtubule phenotype of a jnm1 deletion strain. This hybrid protein localizes to a single spot in the cell, most often near the spindle pole body in unbudded cells and in the bud in large budded cells. Together these results point to a specific role for Jnm1p in spindle migration, possibly as a subunit or accessory protein for yeast dynein.

scholarly journals Folding of Active β-Lactamase in the Yeast Cytoplasm before Translocation into the Endoplasmic Reticulum

1998 ◽  
Vol 9 (4) ◽  
pp. 817-827 ◽  
Author(s):  
Eija Paunola ◽  
Taina Suntio ◽  
Eija Jämsä ◽  
Marja Makarow
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Disulfide Formation ◽  
Cytoplasmic Surface ◽  
C Terminus ◽  
Unfolded State ◽  
Partially Unfolded

Polypeptides targeted to the yeast endoplasmic reticulum (ER) posttranslationally are thought to be kept in the cytoplasm in an unfolded state by Hsp70 chaperones before translocation. We show here that Escherichia coli β-lactamase associated with Hsp70, but adopted a native-like conformation before translocation in living Saccharomyces cerevisiae cells. β-Lactamase is a globular trypsin-resistant molecule in authentic form. For these studies, it was linked to the C terminus of a yeast polypeptide Hsp150Δ, which conferred posttranslational translocation and provided sites for O-glycosylation. We devised conditions to retard translocation of Hsp150Δ-β-lactamase. This enabled us to show by protease protection assays that an unglycosylated precursor was associated with the cytoplasmic surface of isolated microsomes, whereas a glycosylated form resided inside the vesicles. Both proteins were trypsin resistant and had similar β-lactamase activity andK m values for nitrocefin. The enzymatically active cytoplasmic intermediate could be chased into the ER, followed by secretion of the activity to the medium. Productive folding in the cytoplasm occurred in the absence of disulfide formation, whereas in the ER lumen, proper folding required oxidation of the sulfhydryls. This suggests that the polypeptide was refolded in the ER and consequently, at least partially unfolded for translocation.

Isolation and characterization of STI1, a stress-inducible gene from Saccharomyces cerevisiae

1989 ◽  
Vol 9 (9) ◽  
pp. 3638-3646
Author(s):  
C M Nicolet ◽  
E A Craig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Saccharomyces Cerevisiae ◽  
Two Dimensional Gel Electrophoresis ◽  
Reading Frame ◽  
Hsp70 Family ◽  
Isolation And Characterization ◽  
Isoelectric Points ◽  
Amino Acid Analog ◽  
Inducible Gene

We have isolated a gene from the yeast Saccharomyces cerevisiae that encodes a 2.0-kilobase heat-inducible mRNA. This gene, which we have designated STI1, for stress inducible, was also induced by the amino acid analog canavanine and showed a slight increase in expression as cells moved into stationary phase. The STI1 gene encodes a 66-kilodalton protein, as determined from the sequence of the longest open reading frame. The putative STI1 protein, as identified by two-dimensional gel electrophoresis, migrated in the region of 73 to 75 kilodaltons as a series of four isoforms with different isoelectric points. STI1 is not homologous to the other conserved HSP70 family members in yeasts, despite similarities in size and regulation. Cells carrying a disruption mutation of the STI1 gene grew normally at 30 degrees C but showed impaired growth at higher and lower temperatures. Overexpression of the STI1 gene resulted in substantial trans-activation of SSA4 promoter-reporter gene fusions, indicating that STI1 may play a role in mediating the heat shock response of some HSP70 genes.

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