scholarly journals Two New Ypt GTPases Are Required for Exit From the Yeast trans-Golgi Compartment

1997 ◽  
Vol 137 (3) ◽  
pp. 563-580 ◽  
Author(s):  
Gregory Jedd ◽  
Jon Mulholland ◽  
Nava Segev
Keyword(s):  
Protein Transport ◽  
Small Gtpases ◽  
Yeast Cells ◽  
Secretory Vesicles ◽  
Golgi Compartment ◽  
Rab Family ◽  
Vacuolar Protein ◽  
Exocytic Pathway ◽  
Mutant Cells

Small GTPases of the Ypt/rab family are involved in the regulation of vesicular transport. These GTPases apparently function during the targeting of vesicles to the acceptor compartment. Two members of the Ypt/rab family, Ypt1p and Sec4p, have been shown to regulate early and late steps of the yeast exocytic pathway, respectively. Here we tested the role of two newly identified GTPases, Ypt31p and Ypt32p. These two proteins share 81% identity and 90% similarity, and belong to the same protein subfamily as Ypt1p and Sec4p. Yeast cells can tolerate deletion of either the YPT31 or the YPT32 gene, but not both. These observations suggest that Ypt31p and Ypt32p perform identical or overlapping functions. Cells deleted for the YPT31 gene and carrying a conditional ypt32 mutation exhibit protein transport defects in the late exocytic pathway, but not in vacuolar protein sorting. The ypt31/ 32 mutant secretory defect is clearly downstream from that displayed by a ypt1 mutant and is similar to that of sec4 mutant cells. However, electron microscopy revealed that while sec4 mutant cells accumulate secretory vesicles, ypt31/32 mutant cells accumulate aberrant Golgi structures. The ypt31/32 phenotype is epistatic to that of a sec1 mutant, which accumulates secretory vesicles. Together, these results indicate that the Ypt31/32p GTPases are required for a step that occurs in the transGolgi compartment, between the reactions regulated by Ypt1p and Sec4p. This step might involve budding of vesicles from the trans-Golgi. Alternatively, Ypt31/ 32p might promote secretion indirectly, by allowing fusion of recycling vesicles with the trans-Golgi compartment.

scholarly journals Structural and Functional Analysis of a Novel Coiled-Coil Protein Involved in Ypt6 GTPase-regulated Protein Transport in Yeast

1999 ◽  
Vol 10 (1) ◽  
pp. 63-75 ◽  
Author(s):  
Miki Tsukada ◽  
Elke Will ◽  
Dieter Gallwitz
Keyword(s):  
Protein Transport ◽  
Protein Complexes ◽  
Coiled Coil ◽  
Carboxypeptidase Y ◽  
Loss Of Function ◽  
Golgi Compartment ◽  
Helical Content ◽  
Associated Proteins ◽  
Yeast Genes ◽  
Vacuolar Protein

The yeast transport GTPase Ypt6p is dispensable for cell growth and secretion, but its lack results in temperature sensitivity and missorting of vacuolar carboxypeptidase Y. We previously identified four yeast genes (SYS1, 2, 3, and 5) that on high expression suppressed these phenotypic alterations.SYS3 encodes a 105-kDa protein with a predicted high α-helical content. It is related to a variety of mammalian Golgi-associated proteins and to the yeast Uso1p, an essential protein involved in docking of endoplasmic reticulum–derived vesicles to thecis-Golgi. Like Uso1p, Sys3p is predominatly cytosolic. According to gel chromatographic, two-hybrid, and chemical cross-linking analyses, Sys3p forms dimers and larger protein complexes. Its loss of function results in partial missorting of carboxypeptidase Y. Double disruptions of SYS3and YPT6 lead to a significant growth inhibition of the mutant cells, to a massive accumulation of 40- to 50-nm vesicles, to an aggravation of vacuolar protein missorting, and to a defect in α-pheromone processing apparently attributable to a perturbation of protease Kex2p cycling between the Golgi and a post-Golgi compartment. The results of this study suggest that Sys3p, like Ypt6p, acts in vesicular transport (presumably at a vesicle-docking stage) between an endosomal compartment and the most distal Golgi compartment.

scholarly journals The Ypt1 GTPase is essential for the first two steps of the yeast secretory pathway.

1995 ◽  
Vol 131 (3) ◽  
pp. 583-590 ◽  
Author(s):  
G Jedd ◽  
C Richardson ◽  
R Litt ◽  
N Segev
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Small Gtpases ◽  
Rab Proteins ◽  
Restrictive Temperature ◽  
Rab Gtpases ◽  
Curr Opin Cell Biol ◽  
Rab Family ◽  
One Step ◽  
Mutant Cells

Small GTPases of the rab family are involved in the regulation of vesicular transport. The restricted distribution of each of these proteins in mammalian cells has led to the suggestion that different rab proteins act at different steps of transport (Pryer, N. K., L. J. Wuestehube, and R. Sheckman. 1992. Annu Rev. Biochem. 61:471-516; Zerial, M., and H. Stenmark. 1993. Curr. Opin. Cell Biol. 5:613-620). However, in this report we show that the Ypt1-GTPase, a member of the rab family, is essential for more than one step of the yeast secretory pathway. We determined the secretory defect conferred by a novel ypt1 mutation by comparing the processing of several transported glycoproteins in wild-type and mutant cells. The ypt1-A136D mutant has a change in an amino acid that is conserved among rab GTPases. This mutation leads to a rapid and tight secretory block upon a shift to the restrictive temperature, and allows for the identification of the specific steps in the secretory pathway that directly require Ypt1 protein (Ypt1p). The ypt1-A136D mutant exhibits tight blocks in two secretory steps, ER to cis-Golgi and cis- to medial-Golgi, but later steps are unaffected. Thus, it is unlikely that Ypt1p functions as the sole determinant of fusion specificity. Our results are more consistent with a role for Ypt1/rab proteins in determining the directionality or fidelity of protein sorting.

scholarly journals Inhibition of endoplasmic reticulum (ER)-to-Golgi transport induces relocalization of binding protein (BiP) within the ER to form the BiP bodies.

1994 ◽  
Vol 5 (10) ◽  
pp. 1129-1143 ◽  
Author(s):  
S Nishikawa ◽  
A Hirata ◽  
A Nakano
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Binding Protein ◽  
Yeast Cells ◽  
Restrictive Temperature ◽  
Golgi Transport ◽  
Coa Reductase ◽  
Vacuolar Protein ◽  
Er Membrane

Immunofluorescence staining of yeast cells with anti-binding protein (BiP) antibodies shows uniform staining of the endoplasmic reticulum (ER). We have found that overproduction of Sec12p, an ER membrane protein, causes a change of BiP distribution within the cell. Upon induction of Sec12p by the GAL1 promoter, the staining pattern of BiP turns into bright dots scattering in the cell, whereas the staining of Sec12p remains to be the typical ER figure. Overproduction of other ER membrane proteins, HMG-CoA reductase or Sed4 protein, does not induce such relocalization of BiP. Pulse-chase experiments and electron microscopy have revealed that the overproduction of Sec12p inhibits protein transport from the ER to the Golgi apparatus. When the transport is arrested by one of the sec mutations that block the ER-to-Golgi step at the restrictive temperature, the BiP staining also changes into the punctate pattern. In contrast, the sec mutants that block later or earlier steps of the secretory pathway do not induce such change of BiP localization. These observations indicate that relocalization of BiP is caused by the inhibition of ER-to-Golgi transport. Using immunoelectron microscopy, we have found that the punctate staining is because of the accumulation of BiP in the restricted region of the ER, which we propose to call the "BiP body." This implicates existence of ER subdomains in yeast. A vacuolar protein, proteinase A, appears to colocalize in the BiP body when the ER-to-Golgi transport is blocked, suggesting that the BiP body may have a role as the site of accumulation of cargo molecules before exit from the ER.

scholarly journals The TRAPP Complex Is a Nucleotide Exchanger for Ypt1 and Ypt31/32

2000 ◽  
Vol 11 (12) ◽  
pp. 4403-4411 ◽  
Author(s):  
Sara Jones ◽  
Christina Newman ◽  
Fengli Liu ◽  
Nava Segev
Keyword(s):  
Protein Complex ◽  
Protein Transport ◽  
Apparent Molecular Weight ◽  
Yeast Cells ◽  
Mutant Protein ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Exocytic Pathway

In yeast, the Ypt1 GTPase is required for ER-to-cis-Golgi and cis-to-medial-Golgi protein transport, while Ypt31/32 are a functional pair of GTPases essential for exit from the trans-Golgi. We have previously identified a Ypt1 guanine nucleotide exchange factor (GEF) activity and characterized it as a large membrane-associated protein complex that localizes to the Golgi and can be extracted from the membrane by salt, but not by detergent. TRAPP is a large protein complex that is required for ER-to-Golgi transport and that has properties similar to those of Ypt1 GEF. Here we show that TRAPP has Ypt1 GEF activity. GST-tagged Bet3p or Bet5p, two of the TRAPP subunits, were expressed in yeast cells and were precipitated by glutathione-agarose (GA) beads. The resulting precipitates can stimulate both GDP release and GTP uptake by Ypt1p. The majority of the Ypt1 GEF activity associated with the GST-Bet3p precipitate has an apparent molecular weight of > 670 kDa, indicating that the GEF activity resides in the TRAPP complex. Surprisingly, TRAPP can also stimulate nucleotide exchange on the Ypt31/32 GTPases, but not on Sec4p, a Ypt-family GTPase required for the last step of the exocytic pathway. Like the previously characterized Ypt1 GEF, the TRAPP Ypt1-GEF activity can be inhibited by the nucleotide-free Ypt1-D124N mutant protein. This mutant protein also inhibits the Ypt32 GEF activity of TRAPP. Coprecipitation and overexpression studies suggest that TRAPP can act as a GEF for Ypt1 and Ypt31/32 in vivo. These data suggest the exciting possibility that a GEF complex common to Ypt1 and Ypt31/32 might coordinate the function of these GTPases in entry into and exit from the Golgi.

scholarly journals Slk19 clusters kinetochores and facilitates chromosome bipolar attachment

2013 ◽  
Vol 24 (5) ◽  
pp. 566-577 ◽  
Author(s):  
Daniel Richmond ◽  
Raed Rizkallah ◽  
Fengshan Liang ◽  
Myra M. Hurt ◽  
Yanchang Wang
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Biological Significance ◽  
Yeast Cells ◽  
Eukaryotic Cells ◽  
Large Protein ◽  
Large Protein Complex ◽  
The Stability ◽  
Mutant Cells

In all eukaryotic cells, DNA is packaged into multiple chromosomes that are linked to microtubules through a large protein complex called a kinetochore. Previous data show that the kinetochores are clustered together during most of the cell cycle, but the mechanism and the biological significance of kinetochore clustering are unknown. As a kinetochore protein in budding yeast, the role of Slk19 in the stability of the anaphase spindle has been well studied, but its function in chromosome segregation has remained elusive. Here we show that Slk19 is required for kinetochore clustering when yeast cells are treated with the microtubule-depolymerizing agent nocodazole. We further find that slk19Δ mutant cells exhibit delayed kinetochore capture and chromosome bipolar attachment after the disruption of the kinetochore–microtubule interaction by nocodazole, which is likely attributed to defective kinetochore clustering. In addition, we show that Slk19 interacts with itself, suggesting that the dimerization of Slk19 may mediate the interaction between kinetochores for clustering. Therefore Slk19 likely acts as kinetochore glue that clusters kinetochores to facilitate efficient and faithful chromosome segregation.

scholarly journals Importance of catalase in the adaptive response to hydrogen peroxide: analysis of acatalasaemic Saccharomyces cerevisiae

1996 ◽  
Vol 320 (1) ◽  
pp. 61-67 ◽  
Author(s):  
Shingo IZAWA ◽  
Yoshiharu INOUE ◽  
Akira KIMURA
Keyword(s):  
Oxidative Stress ◽  
Saccharomyces Cerevisiae ◽  
Adaptive Response ◽  
Yeast Cells ◽  
Exponential Growth Phase ◽  
Wild Type ◽  
Single Mutant ◽  
Mutant Cells ◽  
Similar Growth

Controversy about the importance of catalase in the detoxification of H2O2 in human erythrocytes continues. It has been suggested that catalase has no role in the clearance of H2O2 in erythrocytes. In the present study we investigated the role of catalase in the defence mechanism against oxidative stress using Saccharomyces cerevisiae. S. cerevisiae has two catalases, catalase A and catalase T. We constructed a double mutant (acatalasaemic mutant) unable to produce either catalase A or catalase T, and compared it with wild-type and single-mutant cells. The acatalasaemic mutant cells showed a similar growth rate to wild-type cells under non-oxidative stress conditions, and showed a similar susceptibility to H2O2 stress in the exponential growth phase. The acatalasaemic mutant cells at stationary phase were, however, much more sensitive to H2O2 stress than wild-type and single-mutant cells. Moreover, the ability of acatalasaemic and single-mutant cells to show adaptation to 2 mM H2O2 was distinctly inferior to that of wild-type cells. These results suggest that catalase is not essential for yeast cells under normal conditions, but plays an important role in the acquisition of tolerance to oxidative stress in the adaptive response of these cells.

scholarly journals Caenorhabditis elegansF09E10.3 Encodes a Putative 3-Oxoacyl-Thioester Reductase of Mitochondrial Type 2 Fatty Acid Synthase FASII that Is Functional in Yeast

2009 ◽  
Vol 2009 ◽  
pp. 1-6 ◽  
Author(s):  
Aner Gurvitz
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Reductase Activity ◽  
Carbon Sources ◽  
Yeast Cells ◽  
C Elegans ◽  
Animal Physiology ◽  
Mutant Cells

Caenorhabditis elegansF09E10.3 (dhs-25) was identified as encoding a 3-oxoacyl-thioester reductase, potentially of the mitochondrial type 2 fatty acid synthase (FASII) system. Mitochondrial FASII is a relatively recent discovery in metazoans, and the relevance of this process to animal physiology has not been elucidated. A good animal model to study the role of FASII is the nematodeC. elegans. However, the components of nematode mitochondrial FASII have hitherto evaded positive identification. The nematode F09E10.3 protein was ectopically expressed without an additional mitochondrial targeting sequence inSaccharomyces cerevisiaemutant cells lacking the homologous mitochondrial FASII enzyme 3-oxoacyl-ACP reductase Oar1p. These yeastoar1Δmutants are unable to respire, grow on nonfermentable carbon sources, or synthesize sufficient levels of lipoic acid. Mutant yeast cells producing a full-length mitochondrial F09E10.3 protein containedNAD+-dependent 3-oxoacyl-thioester reductase activity and resembled the corresponding mutant overexpressing native Oar1p for the above-mentioned phenotype characteristics. This is the first identification of a metazoan 3-oxoacyl-thioester reductase (see Note Added in Proof).

scholarly journals Voa1p Functions in V-ATPase Assembly in the Yeast Endoplasmic Reticulum

2008 ◽  
Vol 19 (12) ◽  
pp. 5131-5142 ◽  
Author(s):  
Margret Ryan ◽  
Laurie A. Graham ◽  
Tom H. Stevens
Keyword(s):  
Endoplasmic Reticulum ◽  
Atp Hydrolysis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Growth Defects ◽  
The Core ◽  
Golgi Compartment ◽  
Assembly Factor ◽  
Two Sectors

The yeast Saccharomyces cerevisiae vacuolar ATPase (V-ATPase) is a multisubunit complex divided into two sectors: the V1 sector catalyzes ATP hydrolysis and the V0 sector translocates protons, resulting in acidification of its resident organelle. Four protein factors participate in V0 assembly. We have discovered a fifth V0 assembly factor, Voa1p (YGR106C); an endoplasmic reticulum (ER)-localized integral membrane glycoprotein. The role of Voa1p in V0 assembly was revealed in cells expressing an ER retrieval-deficient form of the V-ATPase assembly factor Vma21p (Vma21pQQ). Loss of Voa1p in vma21QQ yeast cells resulted in loss of V-ATPase function; cells were unable to acidify their vacuoles and exhibited growth defects typical of cells lacking V-ATPase. V0 assembly was severely compromised in voa1 vma21QQ double mutants. Isolation of V0–Vma21p complexes indicated that Voa1p associates most strongly with Vma21p and the core proteolipid ring of V0 subunits c, c′, and c″. On assembly of the remaining three V0 subunits (a, d, and e) into the V0 complex, Voa1p dissociates from the now fully assembled V0–Vma21p complex. Our results suggest Voa1p functions with Vma21p early in V0 assembly in the ER, but then it dissociates before exit of the V0–Vma21p complex from the ER for transport to the Golgi compartment.

scholarly journals Genome-Wide Identification of Saccharomyces cerevisiae Genes Required for Maximal Tolerance to Ethanol

2009 ◽  
Vol 75 (18) ◽  
pp. 5761-5772 ◽  
Author(s):  
Miguel C. Teixeira ◽  
Luís R. Raposo ◽  
Nuno P. Mira ◽  
Artur B. Lourenço ◽  
Isabel Sá-Correia
Keyword(s):  
Protein Import ◽  
Yeast Cells ◽  
Lytic Enzyme ◽  
Ethanol Stress ◽  
Minimal Growth ◽  
Data Set ◽  
Genome Wide ◽  
Vacuolar Protein ◽  
High Ethanol

ABSTRACT The understanding of the molecular basis of yeast resistance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. In this study, the yeast disruptome was screened for mutants with differential susceptibility to stress induced by high ethanol concentrations in minimal growth medium. Over 250 determinants of resistance to ethanol were identified. The most significant gene ontology terms enriched in this data set are those associated with intracellular organization, biogenesis, and transport, in particular, regarding the vacuole, the peroxisome, the endosome, and the cytoskeleton, and those associated with the transcriptional machinery. Clustering the proteins encoded by the identified determinants of ethanol resistance by their known physical and genetic interactions highlighted the importance of the vacuolar protein sorting machinery, the vacuolar H+-ATPase complex, and the peroxisome protein import machinery. Evidence showing that vacuolar acidification and increased resistance to the cell wall lytic enzyme β-glucanase occur in response to ethanol-induced stress was obtained. Based on the genome-wide results, the particular role of the FPS1 gene, encoding a plasma membrane aquaglyceroporin which mediates controlled glycerol efflux, in ethanol stress resistance was further investigated. FPS1 expression contributes to decreased [3H]ethanol accumulation in yeast cells, suggesting that Fps1p may also play a role in maintaining the intracellular ethanol level during active fermentation. The increased expression of FPS1 confirmed the important role of this gene in alcoholic fermentation, leading to increased final ethanol concentration under conditions that lead to high ethanol production.

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