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.     

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.     

Characterization of yeast Vps33p, a protein required for vacuolar protein sorting and vacuole biogenesis.

vps33 mutants missort and secrete multiple vacuolar hydrolases and exhibit extreme defects in vacuolar morphology. Toward a molecular understanding of the role of the VPS33 gene in vacuole biogenesis, we have cloned this gene from a yeast genomic library by complementation of a temperature-sensitive vps33 mutation. Gene disruption demonstrated that VPS33 was not essential but was required for growth at high temperatures. At the permissive temperature, vps33 null mutants exhibited defects in vacuolar protein localization and vacuole morphology similar to those seen in most of the original mutant alleles. Sequence analysis revealed a putative open reading frame sufficient to encode a protein of 691 amino acids. Hydropathy analysis indicated that the deduced product of the VPS33 gene is generally hydrophilic, contains no obvious signal sequence or transmembrane domains, and is therefore unlikely to enter the secretory pathway. Polyclonal antisera raised against TrpE-Vps33 fusion proteins recognized a protein in yeast cells of the expected molecular weight, approximately 75,000. In cell fractionation studies, Vps33p behaved as a cytosolic protein. The predicted VPS33 gene product possessed sequence similarity with a number of ATPases and ATP-binding proteins specifically in their ATP-binding domains. One vps33 temperature-sensitive mutant contained a missense mutation near this region of sequence similarity; the mutation resulted in a Leu-646----Pro substitution in Vps33p. This temperature-sensitive mutant strain contained normal vacuoles at the permissive temperature but lacked vacuoles specifically in the bud at the nonpermissive temperature. Our data suggest that Vps33p acts in the cytoplasm to facilitate Golgi-to-vacuole protein delivery. We propose that as a consequence of the vps33 protein-sorting defects, abnormalities in vacuolar morphology and vacuole assembly result.     

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.     

Multiple functions of the vacuolar sorting protein Ccz1p in Saccharomyces cerevisiae

The CCZ1 (YBR131w) gene encodes a protein required for fusion of various transport intermediates with the vacuole. Ccz1p, in a complex with Mon1p, is a close partner of Ypt7p in the processes of fusion of endosomes to vacuoles and homotypic vacuole fusion. In this work, we exploited the Ca(2+)-sensitivity of the ccz1Delta mutant to identify genes specifically interacting with CCZ1, basing on functional multicopy suppression of calcium toxicity. The presented results indicate that Ccz1p functions in the cell either in association with Mon1p and Ypt7p in fusion at the vacuolar membrane, or--separately--with Arl1p at early steps of vacuolar transport. We also show that suppression of calcium toxicity by the calcium pumps Pmr1p and Pmc1p is restricted only to the subset of mutants defective in vacuole morphology. The mechanisms of Ca(2+)-pump-mediated suppression also differ from each other, since the action of Pmr1p, but not Pmc1p, appears to require Arl1p function.     

Multiple protein tyrosine phosphatase-encoding genes in the yeast Saccharomyces cerevisiae.

In higher eukaryotic organisms, the regulation of tyrosine phosphorylation is known to play a major role in the control of cell division. Recently, a wide variety of protein tyrosine phosphatase (PTPase)-encoding genes (PTPs) have been identified to accompany the many tyrosine kinases previously studied. However, in the yeasts, where the cell cycle has been most extensively studied, identification of the genes involved in the direct regulation of tyrosine phosphorylation has been difficult. We have identified a pair of genes in the yeast Saccharomyces cerevisiae, which we call PTP1 and PTP2, whose products are highly homologous to PTPases identified in other systems. Both genes are poorly expressed, and contain sequence elements consistent with low-abundance proteins. We have carried out an extensive genetic analysis of PTP1 and PTP2, and found that they are not essential either singly or in combination. Neither deletion nor overexpression results in any strong phenotypes in a number of assays. Deletions also do not affect the mitotic blockage caused by deletion of the MIH1 gene (encoding a positive regulator of mitosis) and induction of the heterologous Schizosaccharomyces pombe wee1+ gene (encoding a negative regulator of mitosis). Molecular analysis has shown that PTP1 and PTP2 are quite different structurally and are not especially well conserved at the amino acid sequence level. Low-stringency Southern blots indicate that yeast may contain a family of PTPase-encoding genes. These results suggest that yeast may contain other PTPase-encoding genes that overlap functionally with PTP1 and PTP2.     

Characterization of an estrogen-binding protein in the yeast Saccharomyces cerevisiae

This paper further characterizes the estrogen-binding protein we have described in the cytosol of the yeast Saccharomyces cerevisiae. [3H]Estradiol was used as the radioprobe, and specific binding of cytosol fractions was measured by chromatography on Sephadex minicolumns. Other 3H-steroids did not exhibit specific binding. [3H]Estradiol binding was destroyed by treatment with trypsin, but not RNase, DNase, or phospholipase; N-ethylmaleimide substantially decreased the binding. The yeast did not metabolize estradiol added to the medium, and extraction and chromatography of the bound moiety showed it to be unmetabolized estradiol. Scatchard analysis of cytosol from both a and alpha mating types as well as the a/alpha diploid cell revealed similar binding properties: an apparent dissociation constant or Kd(25 degrees) for [3H]estradiol of 1.6-1.8 nM and a maximal binding capacity or Nmax of approximately 2000-2800 fmol/mg of cytosol protein. Gel exclusion chromatography on Sephacryl S-200 and high performance liquid chromatography suggested a Stokes radius of approximately 30 A. Sucrose gradient centrifugation showed a sedimentation coefficient of approximately 5 S, and the complex did not exhibit ionic dependent aggregation. The estrogen binder in S. cerevisiae differed in its steroidal specificities from classical mammalian estrogen receptors in rat uterus. 17 beta-Estradiol was the best competitor, 17 alpha-estradiol had about 5% the activity, and diethylstilbestrol exhibited negligible binding affinity as did tamoxifen, nafoxidine, and the zearalenones. In summary, a high affinity, stereospecific, steroid-selective binding protein has been demonstrated in the cytosol of the simple yeast S. cerevisiae. We speculate that this molecule may represent a primitive hormone receptor system, possibly for an estrogen-like message molecule.     

Nystatin effects on vacuolar function in Saccharomyces cerevisiae.

The effects of nystatin, a polyene antibiotic, was studied in Saccharomyces cerevisiae by isolating and characterizing nystatin-sensitive mutants. We isolated a number of nystatin-sensitive mutants by ethylmethane sulfonate mutagenesis. One of these mutants, the nss1 mutant, was characterized in detail. The mutant was sensitive to stresses such as high temperature or high concentrations of monovalent and divalent cations. The nss1 mutants showed severe vacuolar protein sorting and vacuolar morphology defects. The nss1 mutant was demonstrated to have a mutational lesion in the known VPS16 gene, which is essential for vacuolar protein sorting in S. cerevisiae. All of the vacuolar deficient mutants (vps11, vps16, vps18, and vps33) were sensitive to nystatin. Nystatin was found to cause extensive enlargement of the vacuole in wild-type S. cerevisiae cells. These results are discussed with special reference to the vacuolar function of S. cerevisiae.     

Mutants of Saccharomyces cerevisiae with defective vacuolar function.

Mutants of the yeast Saccharomyces cerevisiae that have a small vacuolar lysine pool were isolated and characterized. Mutant KL97 (lys1 slp1-1) and strain KL197-1A (slp1-1), a prototrophic derivative of KL97, did not grow well in synthetic medium supplemented with 10 mM lysine. Genetic studies indicated that the slp1-1 mutation (for small lysine pool) is recessive and is due to a single chromosomal mutation. Mutant KL97 shows the following pleiotropic defects in vacuolar functions. (i) It has small vacuolar pools for lysine, arginine, and histidine. (ii) Its growth is sensitive to lysine, histidine, Ca2+, heavy metal ions, and antibiotics. (iii) It has many small vesicles but no large central vacuole. (iv) It has a normal amount of the vacuolar membrane marker alpha-mannosidase but shows reduced activities of the vacuole sap markers proteinase A, proteinase B, and carboxypeptidase Y.     

Characterization of four B-type cyclin genes of the budding yeast Saccharomyces cerevisiae.

The previously described CLB1 and CLB2 genes encode a closely related pair of B-type cyclins. Here we present the sequences of another related pair of B-type cyclin genes, which we term CLB3 and CLB4. Although CLB1 and CLB2 mRNAs rise in abundance at the time of nuclear division, CLB3 and CLB4 are turned on earlier, rising early in S phase and declining near the end of nuclear division. When all possible single and multiple deletion mutants were constructed, some multiple mutations were lethal, whereas all single mutants were viable. All lethal combinations included the clb2 deletion, whereas the clb1 clb3 clb4 triple mutant was viable, suggesting a key role for CLB2. The inviable multiple clb mutants appeared to have a defect in mitosis. Conditional clb mutants arrested as large budded cells with a G2 DNA content but without any mitotic spindle. Electron microscopy showed that the spindle pole bodies had duplicated but not separated, and no spindle had formed. This suggests that the Clb/Cdc28 kinase may have a relatively direct role in spindle formation. The two groups of Clbs may have distinct roles in spindle formation and elongation.     

Lipid rafts in protein sorting and cell polarity in budding yeast Saccharomyces cerevisiae

Cellular membranes contain many types and species of lipids. One of the most important functional consequences of this heterogeneity is the existence of microdomains within the plane of the membrane. Sphingolipid acyl chains have the ability of forming tightly packed platforms together with sterols. These platforms or lipid rafts constitute segregation and sorting devices into which proteins specifically associate. In budding yeast, Saccharomyces cerevisiae, lipid rafts serve as sorting platforms for proteins destined to the cell surface. The segregation capacity of rafts also provides the basis for the polarization of proteins at the cell surface during mating. Here we discuss some recent findings that stress the role of lipid rafts as key players in yeast protein sorting and cell polarity.     

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.     

Biogenesis of vacuolar membrane glycoproteins of yeast Saccharomyces cerevisiae

To investigate the biogenesis of the yeast vacuole, we have sought novel marker proteins localized to the vacuolar membrane. Glycoproteins were prepared from vacuolar membrane vesicles by concanavalin A-Sepharose column chromatography and used to raise monoclonal antibodies. The antibodies obtained recognize several vacuolar proteins that have N-linked oligosaccharide chains. A set of the antibodies reacts with a vacuolar glycoprotein with a major molecular species of 72 kDa (vgp72), which appears to associate peripherally with the vacuolar membrane. The biosynthesis of vgp72 has been examined in detail by pulse-chase experiments and by analyses using various secretory mutants (sec18, sec7, and sec1) and a vacuolar protease mutant (pep4). vgp72 first appears in the endoplasmic reticulum as a 74-kDa species and is quickly modified in the Golgi apparatus to two distinct species: a 79-kDa form, and a heterogeneously glycosylated form (90-150 kDa). Subsequently, both species are proteolytically processed in the vacuole giving rise to a 72-kDa species as well as heavily glycosylated form. Thus, the biogenesis of vgp72 utilizes the early part of the secretory pathway as is the case of vacuolar soluble enzymes. A unique feature is that two species that are different in the extent of glycosylation appear to follow the same destination to the vacuolar membrane.     

Ligand recognition and domain structure of Vps10p, a vacuolar protein sorting receptor in Saccharomyces cerevisiae.

Vp10p is a receptor that sorts several different vacuolar proteins by cycling between a late Golgi compartment and the endosome. The cytoplasmic tail of Vps10p is necessary for the recycling, whereas the lumenal domain is predicted to interact with the soluble ligands. We have studied ligand binding to Vps10p by introducing deletions in the lumenal region. This region contains two domains with homology to each other. Domain 2 binds carboxypeptidase Y (CPY), proteinase A (PrA) and hybrids of these proteases with invertase. Moreover, we show that aminopeptidase Y (APY) is a ligand of Vps10p. The native proteases compete for binding to domain 2. Binding of CPY(156)-invertase or PrA(137)-invertase, on the other hand, do not interfere with binding of CPY to Vps10p. Furthermore, the Q24RPL27 sequence known to be important for vacuolar sorting of CPY, is of little importance in the Vps10p-dependent sorting of CPY-invertase. Apparently, domain 2 contains two different binding sites; one for APY, CPY and PrA, and one for CPY-invertase and PrA-invertase. The latter interaction seems not to be sequence specific, and we suggest that an unfolded structure in these ligands is recognized by Vps10p.     

The VPS1 protein, a homolog of dynamin required for vacuolar protein sorting in Saccharomyces cerevisiae, is a GTPase with two functionally separable domains

The product of the VPS1 gene, Vps1p, is required for the sorting of soluble vacuolar proteins in the yeast Saccharomyces cerevisiae. We demonstrate here that Vps1p, which contains a consensus tripartite motif for guanine nucleotide binding, is capable of binding and hydrolyzing GTP. Vps1p is a member of a subfamily of large GTP-binding proteins whose members include the vertebrate Mx proteins, the yeast MGM1 protein, the Drosophila melanogaster shibire protein, and dynamin, a bovine brain protein that bundles microtubules in vitro. Disruption of microtubules did not affect the fidelity or kinetics of vacuolar protein sorting, indicating that Vps1p function is not dependent on microtubules. Based on mutational analyses, we propose a two-domain model for Vps1p function. When VPS1 was treated with hydroxylamine, half of all mutations isolated were found to be dominant negative with respect to vacuolar protein sorting. All of the dominant-negative mutations analyzed further mapped to the amino-terminal half of Vps1p and gave rise to full-length protein products. In contrast, recessive mutations gave rise to truncated or unstable protein products. Two large deletion mutations in VPS1 were created to further investigate Vps1p function. A mutant form of Vps1p lacking the carboxy-terminal half of the protein retained the capacity to bind GTP and did not interfere with sorting in a wild-type background. A mutant form of Vps1p lacking the entire GTP-binding domain interfered with vacuolar protein sorting in wild-type cells. We suggest that the amino-terminal domain of Vps1p provides a GTP-binding and hydrolyzing activity required for vacuolar protein sorting, and the carboxy-terminal domain mediates Vps1p association with an as yet unidentified component of the sorting apparatus.     

Characterization of REC104, a gene required for early meiotic recombination in the yeast Saccharomyces cerevisiae.

The REC104 gene was initially defined by mutations that rescued the inviability of a rad52 spo 13 haploid strain in meiosis. We have observed that rec104 mutant strains undergo essentially no induction of meiotic gene conversion, and we have not been able to detect any meiotic crossing over in such strains. The REC104 gene has no apparent role in mitosis, since mutations have no observable effect on growth, mitotic recombination, or DNA repair. The DNA sequence of REC104 reveals that it is a previously unknown gene with a coding region of 549-bp, and genetic mapping has localized the gene to chromosome VIII near FUR1. Expression of the REC104 gene is induced in meiosis, and it appears that the gene is not transcribed in mitotic cells. Possible roles for the REC104 gene product in meiosis are discussed.     

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.