scholarly journals VPS21Controls Entry of Endocytosed and Biosynthetic Proteins into the Yeast Prevacuolar Compartment

2000 ◽  
Vol 11 (2) ◽  
pp. 613-626 ◽  
Author(s):  
Sonja R. Gerrard ◽  
Nia J. Bryant ◽  
Tom H. Stevens
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Fusion ◽  
Protein Sorting ◽  
Vacuolar Protein Sorting ◽  
Endosomal Compartment ◽  
Early Endosomes ◽  
Strong Homology ◽  
Rab Family ◽  
Prevacuolar Compartment ◽  
Vacuolar Protein

Mutations in the VPS (vacuolar protein sorting) genes of Saccharomyces cerevisiae have been used to define the trafficking steps that soluble vacuolar hydrolases take en route from the late Golgi to the vacuole. The class DVPS genes include VPS21,PEP12, and VPS45, which appear to encode components of a membrane fusion complex involved in Golgi-to-endosome transport. Vps21p is a member of the Rab family of small Ras-like GTPases and shows strong homology to the mammalian Rab5 protein, which is involved in endocytosis and the homotypic fusion of early endosomes. Although Rab5 and Vps21p appear homologous at the sequence level, it has not been clear if the functions of these two Rabs are similar. We find that Vps21p is an endosomal protein that is involved in the delivery of vacuolar and endocytosed proteins to the vacuole. Vacuolar and endocytosed proteins accumulate in distinct transport intermediates in cells that lack Vps21p function. Therefore, it appears that Vps21p is involved in two trafficking steps into the prevacuolar/late endosomal compartment.

Nystatin Sensitive and Vacuolar Protein Sorting Defective (vps) Mutants of Saccharomyces cerevisiae: Their Isolation and Characterization

2001 ◽  
Vol 4 (3) ◽  
pp. 259-262
Author(s):  
M. Shah Alam Bhuiyan ◽  
Yuji Ito . ◽  
Naotaka Tanaka . ◽  
Golam Sadik . ◽  
Kiyotaka Fujita . ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Sorting ◽  
Vacuolar Protein Sorting ◽  
Isolation And Characterization ◽  
Vacuolar Protein

scholarly journals Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants.

1992 ◽  
Vol 3 (12) ◽  
pp. 1389-1402 ◽  
Author(s):  
C K Raymond ◽  
I Howald-Stevenson ◽  
C A Vater ◽  
T H Stevens
Keyword(s):  
Protein Sorting ◽  
Carboxypeptidase Y ◽  
Vacuolar Protein Sorting ◽  
Complementation Groups ◽  
Prevacuolar Compartment ◽  
Golgi Protein ◽  
Mother Cells ◽  
Vacuolar Protein ◽  
Morphological Phenotype

The collection of vacuolar protein sorting mutants (vps mutants) in Saccharomyces cerevisiae comprises of 41 complementation groups. The vacuoles in these mutant strains were examined using immunofluorescence microscopy. Most of the vps mutants were found to possess vacuolar morphologies that differed significantly from wild-type vacuoles. Furthermore, mutants representing independent vps complementation groups were found to share aberrant morphological features. Six distinct classes of vacuolar morphology were observed. Mutants from eight vps complementation groups were defective both for vacuolar segregation from mother cells into developing buds and for acidification of the vacuole. Another group of mutants, represented by 13 complementation groups, accumulated a novel organelle distinct from the vacuole that contained a late-Golgi protein, active vacuolar H(+)-ATPase complex, and soluble vacuolar hydrolases. We suggest that this organelle may represent an exaggerated endosome-like compartment. None of the vps mutants appeared to mislocalize significant amounts of the vacuolar membrane protein alkaline phosphatase. Quantitative immunoprecipitations of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) were performed to determine the extent of the sorting defect in each vps mutant. A good correlation between morphological phenotype and the extent of the CPY sorting defect was observed.

scholarly journals A putative zinc finger protein, Saccharomyces cerevisiae Vps18p, affects late Golgi functions required for vacuolar protein sorting and efficient alpha-factor prohormone maturation.

1991 ◽  
Vol 11 (12) ◽  
pp. 5813-5824 ◽  
Author(s):  
J S Robinson ◽  
T R Graham ◽  
S D Emr
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Protein Sorting ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Vacuolar Protein Sorting ◽  
Cell Biol ◽  
Alpha Factor ◽  
Vacuolar Protein

Saccharomyces cerevisiae strains carrying vps18 mutations are defective in the sorting and transport of vacuolar enzymes. The precursor forms of these proteins are missorted and secreted from the mutant cells. Most vps18 mutants are temperature sensitive for growth and are defective in vacuole biogenesis; no structure resembling a normal vacuole is seen. A plasmid complementing the temperature-sensitive growth defect of strains carrying the vps18-4 allele was isolated from a centromere-based yeast genomic library. Integrative mapping experiments indicated that the 26-kb insert in this plasmid was derived from the VPS18 locus. A 4-kb minimal complementing fragment contains a single long open reading frame predicted to encode a 918-amino-acid hydrophilic protein. Comparison of the VPS18 sequence with the PEP3 sequence reported in the accompanying paper (R. A. Preston, H. F. Manolson, K. Becherer, E. Weidenhammer, D. Kirkpatrick, R. Wright, and E. W. Jones, Mol. Cell. Biol. 11:5801-5812, 1991) shows that the two genes are identical. Disruption of the VPS18/PEP3 gene (vps18 delta 1::TRP1) is not lethal but results in the same vacuolar protein sorting and growth defects exhibited by the original temperature-sensitive vps18 alleles. In addition, vps18 delta 1::TRP1 MAT alpha strains exhibit a defect in the Kex2p-dependent processing of the secreted pheromone alpha-factor. This finding suggests that vps18 mutations alter the function of a late Golgi compartment which contains Kex2p and in which vacuolar proteins are thought to be sorted from proteins destined for the cell surface. The Vps18p sequence contains a cysteine-rich, zinc finger-like motif at the COOH terminus. A mutant in which the first cysteine of this motif was changed to serine results in a temperature-conditional carboxypeptidase Y sorting defect shortly after a shift to nonpermissive conditions. We identified a similar cysteine-rich motif near the COOH terminus of another Vps protein, the Vps11/Pep5/End1 protein. Preston et al. (Mol. Cell. Biol. 11:5801-5812, 1991) present evidence that the Vps18/Pep3 protein colocalizes with the Vps11/Pep5 protein to the cytosolic face of the vacuolar membrane. Together with the similar phenotypes exhibited by both vps11 and vps18 mutants, this finding suggests that they may function at a common step during vacuolar protein sorting and that the integrity of their zinc finger motifs may be required for this function.

scholarly journals A putative zinc finger protein, Saccharomyces cerevisiae Vps18p, affects late Golgi functions required for vacuolar protein sorting and efficient alpha-factor prohormone maturation

1991 ◽  
Vol 11 (12) ◽  
pp. 5813-5824
Author(s):  
J S Robinson ◽  
T R Graham ◽  
S D Emr
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Protein Sorting ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Vacuolar Protein Sorting ◽  
Cell Biol ◽  
Alpha Factor ◽  
Vacuolar Protein

Saccharomyces cerevisiae strains carrying vps18 mutations are defective in the sorting and transport of vacuolar enzymes. The precursor forms of these proteins are missorted and secreted from the mutant cells. Most vps18 mutants are temperature sensitive for growth and are defective in vacuole biogenesis; no structure resembling a normal vacuole is seen. A plasmid complementing the temperature-sensitive growth defect of strains carrying the vps18-4 allele was isolated from a centromere-based yeast genomic library. Integrative mapping experiments indicated that the 26-kb insert in this plasmid was derived from the VPS18 locus. A 4-kb minimal complementing fragment contains a single long open reading frame predicted to encode a 918-amino-acid hydrophilic protein. Comparison of the VPS18 sequence with the PEP3 sequence reported in the accompanying paper (R. A. Preston, H. F. Manolson, K. Becherer, E. Weidenhammer, D. Kirkpatrick, R. Wright, and E. W. Jones, Mol. Cell. Biol. 11:5801-5812, 1991) shows that the two genes are identical. Disruption of the VPS18/PEP3 gene (vps18 delta 1::TRP1) is not lethal but results in the same vacuolar protein sorting and growth defects exhibited by the original temperature-sensitive vps18 alleles. In addition, vps18 delta 1::TRP1 MAT alpha strains exhibit a defect in the Kex2p-dependent processing of the secreted pheromone alpha-factor. This finding suggests that vps18 mutations alter the function of a late Golgi compartment which contains Kex2p and in which vacuolar proteins are thought to be sorted from proteins destined for the cell surface. The Vps18p sequence contains a cysteine-rich, zinc finger-like motif at the COOH terminus. A mutant in which the first cysteine of this motif was changed to serine results in a temperature-conditional carboxypeptidase Y sorting defect shortly after a shift to nonpermissive conditions. We identified a similar cysteine-rich motif near the COOH terminus of another Vps protein, the Vps11/Pep5/End1 protein. Preston et al. (Mol. Cell. Biol. 11:5801-5812, 1991) present evidence that the Vps18/Pep3 protein colocalizes with the Vps11/Pep5 protein to the cytosolic face of the vacuolar membrane. Together with the similar phenotypes exhibited by both vps11 and vps18 mutants, this finding suggests that they may function at a common step during vacuolar protein sorting and that the integrity of their zinc finger motifs may be required for this function.

scholarly journals Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (12) ◽  
pp. 6742-6754 ◽  
Author(s):  
P K Herman ◽  
S D Emr
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Sorting ◽  
Yeast Cells ◽  
Growth Defect ◽  
Cell Fractionation ◽  
Wild Type ◽  
Vacuolar Protein Sorting ◽  
Vacuolar Compartment ◽  
Dividing Cells ◽  
Vacuolar Protein

VPS34 gene function is required for the efficient localization of a variety of vacuolar proteins. We have cloned and sequenced the wild-type VPS34 gene in order to gain a better understanding of the role of its protein product in this intracellular sorting pathway. Interestingly, disruption of the VPS34 locus resulted in a temperature-sensitive growth defect, indicating that the VPS34 gene is essential for vegetative growth only at elevated growth temperatures. As with the original vps34 alleles, vps34 null mutants exhibited severe vacuolar protein sorting defects and possessed a morphologically normal vacuolar structure. The VPS34 gene DNA sequence identifies an open reading frame that could encode a hydrophilic protein of 875 amino acids. The predicted protein sequence lacks any apparent signal sequence or membrane-spanning domains, suggesting that Vps34p does not enter the secretory pathway. Results from immunoprecipitation experiments with antiserum prepared against a TrpE-Vps34 fusion protein were consistent with this prediction: a rare, unglycosylated protein of approximately 95,000 Da was detected in extracts of wild-type Saccharomyces cerevisiae cells. Cell fractionation studies indicated that a significant portion of the Vps34p is found associated with a particulate fraction of yeast cells. This particulate Vps34p was readily solubilized by treatment with 2 M urea but not with Triton X-100, suggesting that the presence of Vps34p in this pelletable structure is mediated by protein-protein interactions. vp34 mutant cells also exhibited a defect in the normal partitioning of the vacuolar compartment between mother and daughter cells during cell division. In more than 80% of the delta vps34 dividing cells examined, no vacuolar structures were observed in the newly emerging bud, whereas in wild-type dividing cells, more than 95% of the buds had a detectable vacuolar compartment. Our results suggest that the Vps34p may act as a component of a relatively large intracellular structure that functions to facilitate specific steps of the vacuolar protein delivery and inheritance pathways.

scholarly journals Genome-Wide Screen in Saccharomyces cerevisiae Identifies Vacuolar Protein Sorting, Autophagy, Biosynthetic, and tRNA Methylation Genes Involved in Life Span Regulation

PLoS Genetics ◽  
2010 ◽  
Vol 6 (7) ◽  
pp. e1001024 ◽  
Author(s):  
Paola Fabrizio ◽  
Shawn Hoon ◽  
Mehrnaz Shamalnasab ◽  
Abdulaye Galbani ◽  
Min Wei ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Life Span ◽  
Protein Sorting ◽  
Vacuolar Protein Sorting ◽  
Genome Wide ◽  
Vacuolar Protein

scholarly journals Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (12) ◽  
pp. 6742-6754
Author(s):  
P K Herman ◽  
S D Emr
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Sorting ◽  
Yeast Cells ◽  
Growth Defect ◽  
Cell Fractionation ◽  
Wild Type ◽  
Vacuolar Protein Sorting ◽  
Vacuolar Compartment ◽  
Dividing Cells ◽  
Vacuolar Protein

VPS34 gene function is required for the efficient localization of a variety of vacuolar proteins. We have cloned and sequenced the wild-type VPS34 gene in order to gain a better understanding of the role of its protein product in this intracellular sorting pathway. Interestingly, disruption of the VPS34 locus resulted in a temperature-sensitive growth defect, indicating that the VPS34 gene is essential for vegetative growth only at elevated growth temperatures. As with the original vps34 alleles, vps34 null mutants exhibited severe vacuolar protein sorting defects and possessed a morphologically normal vacuolar structure. The VPS34 gene DNA sequence identifies an open reading frame that could encode a hydrophilic protein of 875 amino acids. The predicted protein sequence lacks any apparent signal sequence or membrane-spanning domains, suggesting that Vps34p does not enter the secretory pathway. Results from immunoprecipitation experiments with antiserum prepared against a TrpE-Vps34 fusion protein were consistent with this prediction: a rare, unglycosylated protein of approximately 95,000 Da was detected in extracts of wild-type Saccharomyces cerevisiae cells. Cell fractionation studies indicated that a significant portion of the Vps34p is found associated with a particulate fraction of yeast cells. This particulate Vps34p was readily solubilized by treatment with 2 M urea but not with Triton X-100, suggesting that the presence of Vps34p in this pelletable structure is mediated by protein-protein interactions. vp34 mutant cells also exhibited a defect in the normal partitioning of the vacuolar compartment between mother and daughter cells during cell division. In more than 80% of the delta vps34 dividing cells examined, no vacuolar structures were observed in the newly emerging bud, whereas in wild-type dividing cells, more than 95% of the buds had a detectable vacuolar compartment. Our results suggest that the Vps34p may act as a component of a relatively large intracellular structure that functions to facilitate specific steps of the vacuolar protein delivery and inheritance pathways.

scholarly journals The Saccharomyces cerevisiae MVP1 gene interacts with VPS1 and is required for vacuolar protein sorting.

1995 ◽  
Vol 15 (3) ◽  
pp. 1671-1678 ◽  
Author(s):  
K Ekena ◽  
T H Stevens
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Interaction ◽  
Protein Sorting ◽  
Dominant Negative ◽  
Yeast Cells ◽  
Vacuolar Protein Sorting ◽  
Cellular Processes ◽  
Golgi Membranes ◽  
Multicopy Suppressors ◽  
Vacuolar Protein

The VPS1 gene of Saccharomyces cerevisiae encodes an 80-kDa GTPase that associates with Golgi membranes and is required for the sorting of proteins to the yeast vacuole. Vps1p is a member of a growing family of high-molecular-weight GTPases that are found in a number of organisms and are involved in a variety of cellular processes. Vps1p is most similar to mammalian dynamin and the Drosophila Shibire protein, both of which have been shown to play a role in an early step of endocytosis. To identify proteins that interact with Vps1p, a genetic screen was designed to isolate multicopy suppressors of dominant-negative vps1 mutations. One such suppressor, MVP1, that exhibits genetic interaction with VPS1 and is itself required for vacuolar protein sorting has been isolated. Overproduction of Mvp1p will suppress several dominant alleles of VPS1, and suppression is dependent on the presence of wild-type Vps1p. MVP1 encodes a 59-kDa hydrophilic protein, Mvp1p, which appears to colocalize with Vps1p in vps1d and vps27 delta yeast cells. We therefore propose that Mvp1p and Vps1p act in concert to promote membrane traffic to the vacuole.

scholarly journals The Doa4 Deubiquitinating Enzyme Is Functionally Linked to the Vacuolar Protein-sorting and Endocytic Pathways

2000 ◽  
Vol 11 (10) ◽  
pp. 3365-3380 ◽  
Author(s):  
Alexander Y. Amerik ◽  
Jonathan Nowak ◽  
Sowmya Swaminathan ◽  
Mark Hochstrasser
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Protein Sorting ◽  
Late Endosome ◽  
Deubiquitinating Enzyme ◽  
Vacuolar Protein Sorting ◽  
Prevacuolar Compartment ◽  
Green Fluorescent ◽  
Vacuolar Protein ◽  
Class E

The Saccharomyces cerevisiae DOA4 gene encodes a deubiquitinating enzyme that is required for rapid degradation of ubiquitin–proteasome pathway substrates. Both genetic and biochemical data suggest that Doa4 acts in this pathway by facilitating ubiquitin recycling from ubiquitinated intermediates targeted to the proteasome. Here we describe the isolation of 12 spontaneous extragenic suppressors of the doa4-1 mutation; these involve seven different genes, six of which were cloned. Surprisingly, all of the clonedDID (Doa4-independent degradation) genes encode components of the vacuolar protein-sorting (Vps) pathway. In particular, all are class E Vps factors, which function in the maturation of a late endosome/prevacuolar compartment into multivesicular bodies that then fuse with the vacuole. Four of the six Did proteins are structurally related, suggesting an overlap in function. In wild-type and several vps strains, Doa4–green fluorescent protein displays a cytoplasmic/nuclear distribution. However, in cells lacking the Vps4/Did6 ATPase, a large fraction of Doa4–green fluorescent protein, like several other Vps factors, concentrates at the late endosome–like class E compartment adjacent to the vacuole. These results suggest an unanticipated connection between protein deubiquitination and endomembrane protein trafficking in which Doa4 acts at the late endosome/prevacuolar compartment to recover ubiquitin from ubiquitinated membrane proteins en route to the vacuole.

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