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

ABSTRACT: 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.

SUBMITTER: Ekena K

PROVIDER: S-EPMC230391 | biostudies-other | 1995 Mar

REPOSITORIES: biostudies-other

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

Ekena K K Stevens T H TH

Molecular and cellular biology 19950301 3

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 identi ...[more]

PMID: 7862158

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Project description: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.

Project description: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.

Dataset Information

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

Publications

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

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