scholarly journals Genomic Screen for Vacuolar Protein Sorting Genes inSaccharomyces cerevisiae

2002 ◽  
Vol 13 (7) ◽  
pp. 2486-2501 ◽  
Author(s):  
Cecilia J. Bonangelino ◽  
Edna M. Chavez ◽  
Juan S. Bonifacino
Keyword(s):  
Alkaline Phosphatase ◽  
Actin Cytoskeleton ◽  
Protein Sorting ◽  
Carboxypeptidase Y ◽  
Gene Products ◽  
The Novel ◽  
Vacuolar Protein Sorting ◽  
Novel Gene ◽  
A Genome ◽  
Vacuolar Protein

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 ofSaccharomyces 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 α-factor. Others, such as Vps61p, Vps64p, and Vps67p, displayed defects in the actin cytoskeleton at 30°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.

scholarly journals A sorting nexin-1 homologue, Vps5p, forms a complex with Vps17p and is required for recycling the vacuolar protein-sorting receptor.

1997 ◽  
Vol 8 (8) ◽  
pp. 1529-1541 ◽  
Author(s):  
B F Horazdovsky ◽  
B A Davies ◽  
M N Seaman ◽  
S A McLaughlin ◽  
S Yoon ◽  
...  
Keyword(s):  
Gene Product ◽  
Protein Sorting ◽  
Carboxypeptidase Y ◽  
Vacuolar Protein Sorting ◽  
Sorting Nexin ◽  
Class B ◽  
Sorting Receptor ◽  
Vacuolar Protein ◽  
Mutant Cells ◽  
Sorting Nexin 1

A number of the Saccharomyces cerevisiae vacuolar protein-sorting (vps) mutants exhibit an altered vacuolar morphology. Unlike wild-type cells that contain 1-3 large vacuolar structures, the class B vps5 and vps17 mutant cells contain 10-20 smaller vacuole-like compartments. To explore the role of these VPS gene products in vacuole biogenesis, we cloned and sequenced VPS5 and characterized its protein products. The VPS5 gene is predicted to encode a very hydrophilic protein of 675 amino acids that shows significant sequence homology with mammalian sorting nexin-1. Polyclonal antiserum directed against the VPS5 gene product detects a single, cytoplasmic protein that is phosphorylated specifically on a serine residue(s). Subcellular fractionation studies indicate that Vps5p is associated peripherally with a dense membrane fraction distinct from Golgi, endosomal, and vacuolar membranes. This association was found to be dependent on the presence of another class B VPS gene product, Vps17p. Biochemical cross-linking studies demonstrated that Vps5p and Vps17p physically interact. Gene disruption experiments show that the VPS5 genes product is not essential for cell viability; however, cells carrying the null allele contain fragmented vacuoles and exhibit defects in vacuolar protein-sorting similar to vps17 null mutants. More than 95% of carboxypeptidase Y is secreted from these cells in its Golgi-modified p2 precursor form. Additionally, the Vps10p vacuolar protein-sorting receptor is mislocalized to the vacuole in vps5 mutant cells. On the basis of these and other observations, we propose that the Vps17p protein complex may participate in the intracellular trafficking of the Vps10p-sorting receptor, as well as other later-Golgi proteins.

scholarly journals Involvement of Specific COPI Subunits in Protein Sorting from the Late Endosome to the Vacuole in Yeast

2006 ◽  
Vol 27 (2) ◽  
pp. 526-540 ◽  
Author(s):  
Galina Gabriely ◽  
Rachel Kama ◽  
Jeffrey E. Gerst
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Protein Sorting ◽  
Multivesicular Body ◽  
Carboxypeptidase Y ◽  
Direct Role ◽  
Vacuolar Protein Sorting ◽  
Early Secretory Pathway ◽  
Physical Interactions ◽  
Vacuolar Protein

ABSTRACT Although COPI function on the early secretory pathway in eukaryotes is well established, earlier studies also proposed a nonconventional role for this coat complex in endocytosis in mammalian cells. Here we present results that suggest an involvement for specific COPI subunits in the late steps of endosomal protein sorting in Saccharomyces cerevisiae. First, we found that carboxypeptidase Y (CPY) was partially missorted to the cell surface in certain mutants of the COPIB subcomplex (COPIb; Sec27, Sec28, and possibly Sec33), which indicates an impairment in endosomal transport. Second, integral membrane proteins destined for the vacuolar lumen (i.e., carboxypeptidase S [CPS1]; Fur4, Ste2, and Ste3) accumulated at an aberrant late endosomal compartment in these mutants. The observed phenotypes for COPIb mutants resemble those of class E vacuolar protein sorting (vps) mutants that are impaired in multivesicular body (MVB) protein sorting and biogenesis. Third, we observed physical interactions and colocalization between COPIb subunits and an MVB-associated protein, Vps27. Together, our findings suggest that certain COPI subunits could have a direct role in vacuolar protein sorting to the MVB compartment.

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

1996 ◽  
Vol 16 (5) ◽  
pp. 2369-2377 ◽  
Author(s):  
C G Burd ◽  
P A Mustol ◽  
P V Schu ◽  
S D Emr
Keyword(s):  
Intracellular Transport ◽  
Protein Sorting ◽  
Growth Defect ◽  
Carboxypeptidase Y ◽  
Rab Gtpase ◽  
Temperature Sensitive ◽  
Electron Microscopic ◽  
Yeast Saccharomyces Cerevisiae ◽  
Vacuolar Protein Sorting ◽  
Vacuolar Protein

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.

Mutations in the yeast vacuolar ATPase result in the mislocalization of vacuolar proteins.

1992 ◽  
Vol 172 (1) ◽  
pp. 83-92 ◽  
Author(s):  
D J Klionsky ◽  
H Nelson ◽  
N Nelson ◽  
D S Yaver
Keyword(s):  
Alkaline Phosphatase ◽  
Protein Sorting ◽  
Subcellular Fractionation ◽  
Vacuolar Atpase ◽  
Vacuolar Membrane ◽  
Carboxypeptidase Y ◽  
Atpase Inhibitor ◽  
Yeast Saccharomyces Cerevisiae ◽  
Vacuolar Protein ◽  
Vacuolar System

The vacuolar ATPase of the yeast Saccharomyces cerevisiae acidifies the vacuolar lumen and generates an electrochemical gradient across the vacuole membrane. We have investigated the role of compartment acidification of the vacuolar system in the sorting of vacuolar proteins. Strains with chromosomal disruptions of genes (delta vat) encoding the A (69 x 10(3) M(r)), B (57 x 10(3) M(r)) or c (16 x 10(3) M(r)) subunits of the vacuolar ATPase accumulate and secrete precursor forms of the soluble vacuolar hydrolases carboxypeptidase Y and proteinase A. A kinetic analysis suggests that these precursor proteins accumulate in, and are secreted from, the Golgi complex or post-Golgi vesicles. In addition, subcellular fractionation shows that vacuolar hydrolase-invertase hybrid proteins are inefficiently localized to the vacuole in delta vat strains. This result suggests that the vat mutations cause a steady-state defect in vacuolar protein sorting. The vat mutations also affect the sorting of vacuolar membrane proteins. Precursor forms of alkaline phosphatase are accumulated in vat mutant cells, but to a lesser extent than is seen for the soluble vacuolar hydrolases. This finding, coupled with the insensitivity of alkaline phosphatase to the ATPase inhibitor bafilomycin A1, suggests that vacuolar membrane protein sorting is less sensitive to changes in lumenal pH when compared with the targeting of soluble vacuolar proteins. These results indicate that acidification of the vacuolar system is important for efficient sorting of soluble proteins to the vacuole.

scholarly journals Multilamellar endosome-like compartment accumulates in the yeast vps28 vacuolar protein sorting mutant.

1996 ◽  
Vol 7 (6) ◽  
pp. 985-999 ◽  
Author(s):  
S E Rieder ◽  
L M Banta ◽  
K Köhrer ◽  
J M McCaffery ◽  
S D Emr
Keyword(s):  
Protein Sorting ◽  
Retrograde Transport ◽  
Carboxypeptidase Y ◽  
Coat Proteins ◽  
Electron Microscopic ◽  
Yeast Saccharomyces Cerevisiae ◽  
Intense Staining ◽  
Vacuolar Protein Sorting ◽  
Vacuolar Protein ◽  
Class E

In the yeast Saccharomyces cerevisiae, vacuolar proteins such as carboxypeptidase Y transit from the Golgi to the lysosome-like vacuole via an endosome-like intermediate compartment. The vacuolar protein sorting (vps) mutant vps28, a member of the "class E" vps mutants, accumulates vacuolar, endocytic, and late Golgi markers in an aberrant endosome-like class E compartment. Sequence analysis of VPS28 revealed an open reading frame predicted to encode a hydrophilic protein of 242 amino acids. Consistent with this, polyclonal antiserum raised against Vps28p recognized a cytoplasmic protein of 28 kDa. Disruption of VPS28 resulted in moderate defects in both biosynthetic traffic and endocytic traffic destined for the vacuole. The transport of soluble vacuolar hydrolases to the vacuole was impaired in vps28 null mutant cells (approximately 40-50% carboxypeptidase Y missorted). Internalization of the endocytic marker FM 4-64, a vital lipophilic dye, resulted in intense staining of a small intracellular compartment adjacent to an enlarged vacuole in delta vps28 cells. Furthermore, the vacuolar H+-ATPase accumulated in the perivacuolar class E compartment in delta vps28 cells, as did a-factor receptor Ste3p that was internalized from the plasma membrane. Electron microscopic analysis revealed the presence of a novel compartment consisting of stacks of curved membrane cisternae. Immunolocalization studies demonstrated that the vacuolar H+-ATPase is associated with this cupped cisternal structure, indicating that it corresponds to the class E compartment observed by fluorescence microscopy. Our data indicate that kinetic defects in both anterograde and retrograde transport out of the prevacuolar compartment in vps28 mutants result in the accumulation of protein and membrane in an exaggerated multilamellar endosomal compartment. We propose that Vps28p, as well as other class E Vps proteins, may facilitate (possibly as coat proteins) the formation of transport intermediates required for efficient transport out of the prevacuolar endosome.

scholarly journals Isolation and Characterization of Aspergillus oryzae Vacuolar Protein Sorting Mutants

2005 ◽  
Vol 71 (8) ◽  
pp. 4856-4861 ◽  
Author(s):  
Mamoru Ohneda ◽  
Manabu Arioka ◽  
Katsuhiko Kitamoto
Keyword(s):  
Cell Polarity ◽  
Aspergillus Oryzae ◽  
Fluorescent Protein ◽  
Protein Sorting ◽  
Carboxypeptidase Y ◽  
Vacuolar Protein Sorting ◽  
Ph Response ◽  
Isolation And Characterization ◽  
Vacuolar Protein ◽  
Egfp Fluorescence

ABSTRACT The vacuolar protein sorting (vps) system in the filamentous fungus Aspergillus oryzae, which has unique cell polarity and the ability to secrete large amounts of proteins, was evaluated by using mutants that missort vacuolar proteins into the medium. Vacuolar carboxypeptidase Y (CPY) fused with enhanced green fluorescent protein (EGFP) was used as a vacuolar marker. Twenty dfc (dim EGFP fluorescence in conidia) mutants with reduced intracellular EGFP fluorescence in conidia were isolated by fluorescence-activated cell sorting from approximately 20,000 UV-treated conidia. Similarly, 22 hfm (hyper-EGFP fluorescence released into the medium) mutants with increased extracellular EGFP fluorescence were isolated by using a fluorescence microplate reader from approximately 20,000 UV-treated conidia. The dfc and hfm mutant phenotypes were pH dependent, and missorting of CPY-EGFP could vary by 10- to 40-fold depending on the ambient pH. At pH 5.5, the dfc-14 and hfm-4 mutants had an abnormal hyphal morphology that is consistent with fragmentation of vacuoles and defects in cell polarity. In contrast, the hyphal and vacuolar morphology of the dfc-14 and hfm-4 mutants was normal at pH 8.0, although CPY-EGFP accumulated in perivacuolar dot-like structures similar to the class E compartments in Saccharomyces cerevisiae vps mutants. In hfm-21, CPY-EGFP localized at the Spitzenkörper when the mutant was grown at pH 8.0 but not in vacuoles, suggesting that hfm-21 may transport CPY-EGFP via a novel pathway that involves the Spitzenkörper. Correlations between vacuolar protein sorting, pH response, and cell polarity are reported for the first time for filamentous fungi.

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