A yeast protein related to a mammalian Ras-binding protein, Vps9p, is required for localization of vacuolar proteins.

In the yeast Saccharomyces cerevisiae, mutations in vacuolar protein sorting (VPS) genes result in secretion of proteins normally localized to the vacuole. Characterization of the VPS pathway has provided considerable insight into mechanisms of protein sorting and vesicle-mediated intracellular transport. We have cloned VPS9 by complementation of the vacuolar protein sorting defect of vps9 cells, characterized its gene product, and investigated its role in vacuolar protein sorting. Cells with a vps9 disruption exhibit severe vacuolar protein sorting defects and a temperature-sensitive growth defect at 38 degrees C. Electron microscopic examination of delta vps9 cells revealed the appearance of novel reticular membrane structures as well as an accumulation of 40- to 50-nm-diameter vesicles, suggesting that Vps9p may be required for the consumption of transport vesicles containing vacuolar protein precursors. A temperature-conditional allele of vps9 was constructed and used to investigate the function of Vps9p. Immediately upon shifting of temperature-conditional vps9 cells to the nonpermissive temperature, newly synthesized carboxypeptidase Y was secreted, indicating that Vps9p function is directly required in the VPS pathway. Antibodies raised against Vps9p immunoprecipitate a rare 52-kDa protein that fractionates with cytosolic proteins following cell lysis and centrifugation. Analysis of the VPS9 DNA sequence predicts that Vps9p is related to human proteins that bind Ras and negatively regulate Ras-mediated signaling. We term the related regions of Vps9p and these Ras-binding proteins a GTPase binding homology domain and suggest that it defines a family of proteins that bind monomeric GTPases. Vps9p may bind and serve as an effector of a rab GTPase, like Vps2lp, required for vacuolar protein sorting.     

Vps9p is a guanine nucleotide exchange factor involved in vesicle-mediated vacuolar protein transport.

Vacuolar protein sorting (vps) mutants of Saccharomyces cerevisiae missort and secrete vacuolar hydrolases. The gene affected in one of these mutants, VPS21, encodes a member of the Sec4/Ypt/Rab family of small GTPases. Rab proteins play an essential role in vesicle-mediated protein transport. Using both yeast two-hybrid assays and chemical cross-linking, we have identified another VPS gene product, Vps9p, that preferentially interacts with a mutant form of Vps21p-S21N that binds GDP but not GTP. In vitro purified Vps9p was found to stimulate GDP release from Vps21p in a dose-dependent manner. Vps9p also stimulated GTP association as a result of facilitated GDP release. However, Vps9p did not stimulate guanine nucleotide exchange of GTP-bound Vps21p or GTP hydrolysis. We tested the ability of Vps9p to stimulate the intrinsic guanine nucleotide exchange activity of Rab5, which is a mammalian sequence homologue of Vps21p, and Ypt7p, which is another yeast Rab protein involved in vacuolar protein transport. Rab5, but not Ypt7p was responsive to Vps9p, which indicates that Vps9p recognizes sequence variation among Rab proteins. We conclude that Vps9p is a novel guanine nucleotide exchange factor that is specific for Vps21p/Rab5. Since there are no obvious Vps9p sequence homologues in yeast, Vps9p may also possess unique regulatory functions required for vacuolar protein transport.     

Mammalian cells express two VPS4 proteins both of which are involved in intracellular protein trafficking

The yeast Vps4 protein (Vps4p) is a member of the AAA protein family (ATPases associated with diverse cellular activities) and a key player in the transport of proteins out of a prevacuolar endosomal compartment. In human cells, we identified two non-allelic orthologous proteins (VPS4-A and VPS4-B) of yeast Vps4p. The human VPS4-A and VPS4-B proteins display a high degree of sequence identity to each other (80 %) and to the yeast Vps4 protein (59 and 60 %, respectively). Yeast cells lacking a functional VPS4 gene exhibit a temperature-sensitive growth defect and mislocalise a carboxypeptidase Y-invertase fusion protein to the cell surface. Heterologous expression of human VPS4 genes in vps4 mutant yeast strains led, in the case of human VPS4-A, to a partial and, in the case of human VPS4-B, to a complete suppression of the temperature-sensitive growth defect. The vacuolar protein sorting defect of vps4 mutant yeast cells was complemented completely by heterologous expressed human VPS4-B protein, and partially by the human VPS4-A protein. Expression of mutant human VPS4-A (E228Q) and VPS4-B (E235Q) proteins, harbouring single amino acid exchanges in their AAA domains, induced dominant-negative vacuolar protein sorting defects in wild-type yeast cells in both cases. Two-hybrid experiments suggest that the human VPS4-A and VPS4-B proteins can form heteromeric complexes, and subcellular localisation experiments indicate that both human VPS4 proteins associate with endosomal compartments in yeast. Based on these results, we conclude that both human VPS4 proteins are involved in intracellular protein trafficking, presumably at a late endosomal protein transport step, similar to the Vps4p in yeast.     

A putative GTP binding protein homologous to interferon-inducible Mx proteins performs an essential function in yeast protein sorting

Members of the Mx protein family promote interferon-inducible resistance to viral infection in mammals and act by unknown mechanisms. We identified an Mx-like protein in yeast and present genetic evidence for its cellular function. This protein, the VPS1 product, is essential for vacuolar protein sorting, normal organization of intracellular membranes, and growth at high temperature, implying that Mx-like proteins are engaged in fundamental cellular processes in eukaryotes. Vps1p contains a tripartite GTP binding motif, which suggests that binding to GTP is essential to its role in protein sorting. Vps1p-specific antibody labels punctate cytoplasmic structures that condense to larger structures in a Golgi-accumulating sec7 mutant; thus, Vps1p may associate with an intermediate organelle of the secretory pathway.     

A novel protein kinase homolog essential for protein sorting to the yeast lysosome-like vacuole

The VPS15 gene encodes a novel protein kinase homolog that is essential for the efficient delivery of soluble hydrolases to the yeast vacuole. Point mutations altering highly conserved residues within the Vps15p kinase domain result in the secretion of multiple vacuolar proteases. In addition, the in vivo phosphorylation of Vps15p is defective in these kinase domain mutants, suggesting that Vps15p may regulate specific protein phosphorylation reactions required for protein sorting to the yeast vacuole. Subcellular fractionation studies further demonstrate that the 1455 amino acid Vps15p is peripherally associated with the cytoplasmic face of a late Golgi or vesicle compartment. This association may be mediated by myristate as Vps15p contains a consensus signal for N-terminal myristoylation. We propose that protein phosphorylation may act as a molecular "switch" within intracellular protein sorting pathways by actively diverting proteins from a default transit pathway (e.g., secretion) to an alternative pathway (e.g., to the vacuole).     

VPS21 encodes a rab5-like GTP binding protein that is required for the sorting of yeast vacuolar proteins.

Many of the vacuolar protein sorting (vps) mutants of Saccharomyces cerevisiae exhibit severe defects in the sorting of vacuolar proteins but still retain near-normal vacuole morphology. The gene affected in one such mutant, vps21, has been cloned and found to encode a member of the ras-like GTP binding protein family. Sequence comparisons with other known GTP binding proteins indicate that Vps21p is unique but shares striking similarity with mammalian rab5 proteins (> 50% identity and > 70% similarity). Regions with highest similarity are clustered within the putative GTP binding motifs and the proposed effector domains of the Vps21/rab5 proteins. Point mutations constructed within these conserved regions inactivate Vps21p function; the mutant cells missort and secrete the soluble vacuolar hydrolase carboxypeptidase Y (CPY). Cells carrying a complete deletion of the VPS21 coding sequence (i) are viable but exhibit a growth defect at 38 degrees C, (ii) missort multiple vacuolar proteins, (iii) accumulate 40-50 nm vesicles and (iv) contain a large vacuole. VPS21 encodes a 22 kDa protein that binds GTP and fractionates with subcellular membranes. Mutant analysis indicates that the association with a membrane(s) is dependent on geranylgeranylation of the C-terminal cysteine residue(s) of Vps21p. We propose that Vps21p functions in the targeting and/or fusion of transport vesicles that mediate the delivery of proteins to the vacuole.     

Vesicle-mediated protein transport: regulatory interactions between the Vps15 protein kinase and the Vps34 PtdIns 3-kinase essential for protein sorting to the vacuole in yeast

A membrane-associated complex composed of the Vps15 protein kinase and the Vps34 phosphatidylinositol 3-kinase (PtdIns 3-kinase) is essential for the delivery of proteins to the yeast vacuole. An active Vps15p is required for the recruitment of Vps34p to the membrane and subsequent stimulation of Vps34p PtdIns 3-kinase activity. Consistent with this, mutations altering highly conserved residues in the lipid kinase domain of Vps34p lead to a dominant-negative phenotype resulting from titration of activating Vps15 proteins. In contrast, catalytically inactive Vps15p mutants do not produce a dominant mutant phenotype because they are unable to associate with Vps34p in a wild-type manner. These data indicate that an intact Vps15p protein kinase domain is necessary for the association with and activation of Vps34p, and they demonstrate that a functional Vps15p-Vps34p complex is absolutely required for the efficient delivery of proteins to the vacuole. Analysis of a temperature-conditional allele of VPS15, in which a shift to the nonpermissive temperature leads to a decrease in cellular PtdIns(3)P levels, indicates that the loss of Vps15p function leads to a defect in activation of Vps34p. In addition, characterization of a temperature-sensitive allele of VPS34 demonstrates that inactivation of Vps34p leads to the immediate missorting of soluble vacuolar proteins (e.g., carboxypeptidase Y) without an apparent defect in the sorting of the vacuolar membrane protein alkaline phosphatase. This rapid block in vacuolar protein sorting appears to be the result of loss of PtdIns 3- kinase activity since cellular PtdIns(3)P levels decrease dramatically in vps34 temperature-sensitive mutant cells that have been incubated at the nonpermissive temperature. Finally, analysis of the defects in cellular PtdIns(3)P levels in various vps15 and vsp34 mutant strains has led to additional insights into the importance of PtdIns(3)P intracellular localization, as well as the roles of Vps15p and Vps34p in vacuolar protein sorting.     

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

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.     

Characterization of Vta1p, a class E Vps protein in Saccharomyces cerevisiae

We identified VTA1 in a screen for mutations that result in altered vacuole morphology. Deletion of VTA1 resulted in delayed trafficking of the lipophilic dye FM4-64 to the vacuole and altered vacuolar morphology when cells were exposed to the dye 5-(and 6)-carboxy-2',7'-dichlorofluorescein diacetate (CDCFDA). Deletion of class E vacuolar protein sorting (VPS) genes, which encode proteins that affect multivesicular body formation, also showed altered vacuolar morphology upon exposure to high concentrations of CDCFDA. These results suggest a VPS defect for Deltavta1 cells. Deletion of VTA1 did not affect growth on raffinose and only mildly affected carboxypeptidase S sorting. Turnover of the surface protein Ste3p, the a-factor receptor, was affected in Deltavta1 cells with the protein accumulating on the vacuolar membrane. Likewise the alpha-factor receptor Ste2p accumulated on the vacuolar membrane in Deltavta1 cells. We demonstrated that many class E VPS deletion strains are hyper-resistant to the cell wall disruption agent calcofluor white. Deletion of VTA1 or VPS60, another putative class E gene, resulted in calcofluor white hypersensitivity. A Vta1p-green fluorescent protein fusion protein transiently associated with a Pep12p-positive compartment. This localization was altered by deletion of many of the class E VPS genes, indicating that Vta1p binds to endosomes in a manner dependent on the assembly of the endosomal sorting complexes required for transport. Membrane-associated Vta1p co-purified with Vps60p, suggesting that Vta1p is a class E Vps protein that interacts with Vps60p on a prevacuolar compartment.     

The Phosphatidylinositol 3-Phosphate Binding Protein Vac1p Interacts with a Rab GTPase and a Sec1p Homologue to Facilitate Vesicle-mediated Vacuolar Protein Sorting

Activated GTP-bound Rab proteins are thought to interact with effectors to elicit vesicle targeting and fusion events. Vesicle-associated v-SNARE and target membrane t-SNARE proteins are also involved in vesicular transport. Little is known about the functional relationship between Rabs and SNARE protein complexes. We have constructed an activated allele of VPS21, a yeast Rab protein involved in vacuolar protein sorting, and demonstrated an allele-specific interaction between Vps21p and Vac1p. Vac1p was found to bind the Sec1p homologue Vps45p. Although no association between Vps21p and Vps45p was seen, a genetic interaction between VPS21 and VPS45 was observed. Vac1p contains a zinc-binding FYVE finger that may bind phosphatidylinositol 3-phosphate [PtdIns(3)P]. In other FYVE domain proteins, this motif and PtdIns(3)P are necessary for membrane association. Vac1 proteins with mutant FYVE fingers still associated with membranes but showed vacuolar protein sorting defects and reduced interactions with Vps45p and activated Vps21p. Vac1p membrane association was not dependent on PtdIns(3)P, Pep12p, Vps21p, Vps45p, or the PtdIns 3-kinase, Vps34p. Vac1p FYVE finger mutant missorting phenotypes were suppressed by a defective allele of VPS34. These data indicate that PtdIns(3)P may perform a regulatory role, possibly involved in mediating Vac1p protein-protein interactions. We propose that activated-Vps21p interacts with its effector, Vac1p, which interacts with Vps45p to regulate the Golgi to endosome SNARE complex.     

Human homologues of yeast vacuolar protein sorting 29 and 35

In the yeast Saccharomyces cerevisiae, a membrane coat complex is required for endosome to Golgi retrograde transport. The vacuolar protein sorting proteins Vps29p, Vps35p, and Vps26p are required for pre-vacuolar/late endosome to Golgi retrieval of the vacuolar hydrolase receptor Vps10p. They form a cargo recognition and concentration subcomplex, termed the inner shell of the retromer coat, prior to vesicle formation by the addition of the membrane-deforming outer shell. We have cloned the human and murine homologues of yeast Vps29p and the human homologue of Vps35p. They encode 182 and 796 residue proteins, with 43 and 29% identity to their respective yeast. The 10.5 kb, 5 exon, VPS29 gene is located on chromosome 12q24 and the 29.6 kb, 17 exon, VPS35 gene is on chromosome 16. In humans, Vps29p, Vps35p, and Hbeta58, the homologue of Vps26p, may form an inner shell of the retromer coat similar to that found in yeast.     

A genetic and structural analysis of the yeast Vps15 protein kinase: evidence for a direct role of Vps15p in vacuolar protein delivery.

The yeast VPS15 gene encodes a novel protein kinase homolog that is required for the sorting of soluble hydrolases to the yeast vacuole. In this study, we extend our previous mutational analysis of the VPS15 gene and show that alterations of specific Gps15p residues, that are highly conserved among all protein kinase molecules, result in the biological inactivation of Vps15p. Furthermore, we demonstrate here that short C-terminal deletions of Vps15p result in a temperature-conditional defect in vacuolar protein sorting. Immediately following the temperature shift, soluble vacuolar hydrolases, such as carboxypeptidase Y and proteinase A, accumulate as Golgi-modified precursors within a saturable intracellular compartment distinct from the vacuole. This vacuolar protein sorting block is efficiently reversed when mutant cells are shifted back to the permissive temperature; the accumulated precursors are rapidly processed to their mature forms indicating that they have been delivered to the vacuole. This rapid and efficient reversal suggests that the accumulated vacuolar protein precursors were present within a normal transport intermediate in the vacuolar protein sorting pathway. In addition, this protein delivery block shows specificity for soluble vacuolar enzymes as the membrane protein, alkaline phosphatase, is efficiently delivered to the vacuole at the non-permissive temperature. Interestingly, the C-terminal Vps15p truncations are not phosphorylated in vivo suggesting that the phosphorylation of Vps15p may be critical for its biological activity at elevated temperatures. The rapid onset and high degree of specificity of the vacuolar protein delivery block in these mutants suggests that the primary role of Vps15p is to regulate the sorting of soluble hydrolases to the yeast vacuolar compartment.     

The human endosomal sorting complex required for transport (ESCRT-I) and its role in HIV-1 budding

Efficient human immunodeficiency virus type 1 (HIV-1) budding requires an interaction between the PTAP late domain in the viral p6(Gag) protein and the cellular protein TSG101. In yeast, Vps23p/TSG101 binds both Vps28p and Vps37p to form the soluble ESCRT-I complex, which functions in sorting ubiquitylated protein cargoes into multivesicular bodies. Human cells also contain ESCRT-I, but the VPS37 component(s) have not been identified. Bioinformatics and yeast two-hybrid screening methods were therefore used to identify four novel human proteins (VPS37A-D) that share weak but significant sequence similarity with yeast Vps37p and to demonstrate that VPS37A and VPS37B bind TSG101. Detailed studies produced four lines of evidence that human VPS37B is a Vps37p ortholog. 1) TSG101 bound to several different sites on VPS37B, including a putative coiled-coil region and a PTAP motif. 2) TSG101 and VPS28 co-immunoprecipitated with VPS37B-FLAG, and the three proteins comigrated together in soluble complexes of the correct size for human ESCRT-I ( approximately 350 kDa). 3) Like TGS101, VPS37B became trapped on aberrant endosomal compartments in the presence of VPS4A proteins lacking ATPase activity. 4) Finally, VPS37B could recruit TSG101/ESCRT-I activity and thereby rescue the budding of both mutant Gag particles and HIV-1 viruses lacking native late domains. Further studies of ESCRT-I revealed that TSG101 mutations that inhibited PTAP or VPS28 binding blocked HIV-1 budding. Taken together, these experiments define new components of the human ESCRT-I complex and characterize several TSG101 protein/protein interactions required for HIV-1 budding and infectivity.     

CUE domain-containing protein Vps901 is required for vacuolar protein transport in Schizosaccharomyces pombe

The functions of two Schizosaccharomyces pombe Vps9-like genes, SPBC4F6.10/vps901(+) and SPBC29A10.11c/vps902(+), were characterized. Genomic sequence analysis predicted that Vps901p contains a VPS9 domain, whereas cDNA analyses revealed that Vps901p contains a CUE domain (coupling of ubiquitin to ER degradation) in its C-terminal region. Deletion of vps901(+) resulted in mis-sorting and secretion of S. pombe vacuolar carboxypeptidase Cpy1p, whereas deletion of vps902(+) had no effect, suggesting that only Vps901p functions in vacuolar protein transport in S. pombe. Deletion of vps901(+) further produced pleiotropic phenotypes, including vacuolar homotypic fusion and endocytosis defects. Heterologous expression of the budding yeast VPS9 gene corrected the CPY mis-sorting defect in vps901Δ cells. These findings suggest that the VPS9 domain of Vps901p is required for vacuolar protein trafficking in S. pombe.     

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

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.     

Vps10p cycles between the late-Golgi and prevacuolar compartments in its function as the sorting receptor for multiple yeast vacuolar hydrolases

VPS10 (Vacuolar Protein Sorting) encodes a large type I transmembrane protein (Vps10p), involved in the sorting of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) to the Saccharomyces cerevisiae lysosome-like vacuole. Cells lacking Vps10p missorted greater than 90% CPY and 50% of another vacuolar hydrolase, PrA, to the cell surface. In vitro equilibrium binding studies established that the 1,380-amino acid lumenal domain of Vps10p binds CPY precursor in a 1:1 stoichiometry, further supporting the assignment of Vps10p as the CPY sorting receptor. Vps10p has been immunolocalized to the late-Golgi compartment where CPY is sorted away from the secretory pathway. Vps10p is synthesized at a rate 20-fold lower that that of its ligand CPY, which in light of the 1:1 binding stoichiometry, requires that Vps10p must recycle and perform multiple rounds of CPY sorting. The 164-amino acid Vps10p cytosolic domain is involved in receptor trafficking, as deletion of this domain resulted in delivery of the mutant Vps10p to the vacuole, the default destination for membrane proteins in yeast. A tyrosine-based signal (YSSL80) within the cytosolic domain enables Vps10p to cycle between the late-Golgi and prevacuolar/endosomal compartments. This tyrosine-based signal is homologous to the recycling signal of the mammalian mannose-6-phosphate receptor. A second yeast gene, VTH2, encodes a protein highly homologous to Vps10p which, when over-produced, is capable of suppressing the CPY and PrA missorting defects of a vps10 delta strain. These results indicate that a family of related receptors act to target soluble hydrolases to the vacuole.     

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

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.     

Endosome to Golgi Retrieval of the Vacuolar Protein Sorting Receptor, Vps10p, Requires the Function of the VPS29, VPS30, and VPS35 Gene Products

Mutations in the S. cerevisiae VPS29 and VPS30 genes lead to a selective protein sorting defect in which the vacuolar protein carboxypeptidase Y (CPY) is missorted and secreted from the cell, while other soluble vacuolar hydrolases like proteinase A (PrA) are delivered to the vacuole. This phenotype is similar to that seen in cells with mutations in the previously characterized VPS10 and VPS35 genes. Vps10p is a late Golgi transmembrane protein that acts as the sorting receptor for soluble vacuolar hydrolases like CPY and PrA, while Vps35p is a peripheral membrane protein which cofractionates with membranes enriched in Vps10p. The sequences of the VPS29, VPS30, and VPS35 genes do not yet give any clues to the functions of their products. Each is predicted to encode a hydrophilic protein with homologues in the human and C. elegans genomes. Interestingly, mutations in the VPS29, VPS30, or VPS35 genes change the subcellular distribution of the Vps10 protein, resulting in a shift of Vps10p from the Golgi to the vacuolar membrane. The route that Vps10p takes to reach the vacuole in a vps35 mutant does not depend upon Sec1p mediated arrival at the plasma membrane but does require the activity of the pre-vacuolar endosomal t-SNARE, Pep12p. A temperature conditional allele of the VPS35 gene was generated and has been found to cause missorting/secretion of CPY and also Vps10p to mislocalize to a vacuolar membrane fraction at the nonpermissive temperature. Vps35p continues to cofractionate with Vps10p in vps29 mutants, suggesting that Vps10p and Vps35p may directly interact. Together, the data indicate that the VPS29, VPS30, and VPS35 gene products are required for the normal recycling of Vps10p from the prevacuolar endosome back to the Golgi where it can initiate additional rounds of vacuolar hydrolase sorting.     

Vps52p, Vps53p, and Vps54p form a novel multisubunit complex required for protein sorting at the yeast late Golgi

The late Golgi of the yeast Saccharomyces cerevisiae receives membrane traffic from the secretory pathway as well as retrograde traffic from post-Golgi compartments, but the machinery that regulates these vesicle-docking and fusion events has not been characterized. We have identified three components of a novel protein complex that is required for protein sorting at the yeast late Golgi compartment. Mutation of VPS52, VPS53, or VPS54 results in the missorting of 70% of the vacuolar hydrolase carboxypeptidase Y as well as the mislocalization of late Golgi membrane proteins to the vacuole, whereas protein traffic through the early part of the Golgi complex is unaffected. A vps52/53/54 triple mutant strain is phenotypically indistinguishable from each of the single mutants, consistent with the model that all three are required for a common step in membrane transport. Native coimmunoprecipitation experiments indicate that Vps52p, Vps53p, and Vps54p are associated in a 1:1:1 complex that sediments as a single peak on sucrose velocity gradients. This complex, which exists both in a soluble pool and as a peripheral component of a membrane fraction, colocalizes with markers of the yeast late Golgi by immunofluorescence microscopy. Together, the phenotypic and biochemical data suggest that VPS52, VPS53, and VPS54 are required for the retrograde transport of Golgi membrane proteins from an endosomal/prevacuolar compartment. The Vps52/53/54 complex joins a growing list of distinct multisubunit complexes that regulate membrane-trafficking events.     

Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae

The biosynthetic sorting of hydrolases to the yeast vacuole involves transport along two distinct routes referred to as the carboxypeptidase Y and alkaline phosphatase pathways. To identify genes involved in sorting to the vacuole, we conducted a genome-wide screen of 4653 homozygous diploid gene deletion strains of Saccharomyces cerevisiae for missorting of carboxypeptidase Y. We identified 146 mutant strains that secreted strong-to-moderate levels of carboxypeptidase Y. Of these, only 53 of the corresponding genes had been previously implicated in vacuolar protein sorting, whereas the remaining 93 had either been identified in screens for other cellular processes or were only known as hypothetical open reading frames. Among these 93 were genes encoding: 1) the Ras-like GTP-binding proteins Arl1p and Arl3p, 2) actin-related proteins such as Arp5p and Arp6p, 3) the monensin and brefeldin A hypersensitivity proteins Mon1p and Mon2p, and 4) 15 novel proteins designated Vps61p-Vps75p. Most of the novel gene products were involved only in the carboxypeptidase Y pathway, whereas a few, including Mon1p, Mon2p, Vps61p, and Vps67p, appeared to be involved in both the carboxypeptidase Y and alkaline phosphatase pathways. Mutants lacking some of the novel gene products, including Arp5p, Arp6p, Vps64p, and Vps67p, were severely defective in secretion of mature alpha-factor. Others, such as Vps61p, Vps64p, and Vps67p, displayed defects in the actin cytoskeleton at 30 degrees C. The identification and phenotypic characterization of these novel mutants provide new insights into the mechanisms of vacuolar protein sorting, most notably the probable involvement of the actin cytoskeleton in this process.