scholarly journals An Endosome-to-Plasma Membrane Pathway Involved in Trafficking of a Mutant Plasma Membrane ATPase in Yeast

2000 ◽  
Vol 11 (2) ◽  
pp. 579-592 ◽  
Author(s):  
Wen-jie Luo ◽  
Amy Chang
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Secretory Pathway ◽  
Protein Sorting ◽  
Temperature Sensitive ◽  
Plasma Membrane Atpase ◽  
Vacuolar Protein Sorting ◽  
Membrane Atpase ◽  
Vacuolar Protein ◽  
Endosomal System

The plasma membrane ATPase, encoded by PMA1, is delivered to the cell surface via the secretory pathway. Previously, we characterized a temperature-sensitive pma1 mutant in which newly synthesized Pma1-7 is not delivered to the plasma membrane but is mislocalized instead to the vacuole at 37°C. Severalvps mutants, which are defective in vacuolar protein sorting, suppress targeting-defective pma1 by allowing mutant Pma1 to move once again to the plasma membrane. In this study, we have analyzed trafficking in the endosomal system by monitoring the movement of Pma1-7 in vps36, vps1, andvps8 mutants. Upon induction of expression, mutant Pma1 accumulates in the prevacuolar compartment in vps36cells. After chase, a fraction of newly synthesized Pma1-7 is delivered to the plasma membrane. In both vps1 andvps8 cells, newly synthesized mutant Pma1 appears in small punctate structures before arrival at the cell surface. Nevertheless, biosynthetic membrane traffic appears to follow different routes in vps8 and vps1: the vacuolar protein-sorting receptor Vps10p is stable in vps8 but not in vps1. Furthermore, a defect in endocytic delivery to the vacuole was revealed in vps8 (andvps36) but not vps1 by endocytosis of the bulk membrane marker FM 4-64. Moreover, in vps8 cells, there is defective down-regulation from the cell surface of the mating receptor Ste3, consistent with persistent receptor recycling from an endosomal compartment to the plasma membrane. These data support a model in which mutant Pma1 is diverted from the Golgi to the surface invps1 cells. We hypothesize that in vps8and vps36, in contrast to vps1, mutant Pma1 moves to the surface via endosomal intermediates, implicating an endosome-to-surface traffic pathway.

scholarly journals Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases.

1988 ◽  
Vol 8 (11) ◽  
pp. 4936-4948 ◽  
Author(s):  
J S Robinson ◽  
D J Klionsky ◽  
L M Banta ◽  
S D Emr
Keyword(s):  
Cell Surface ◽  
Protein Sorting ◽  
Vacuolar Membrane ◽  
Temperature Sensitive ◽  
Vacuolar Protein Sorting ◽  
Proteinase A ◽  
Complementation Groups ◽  
The Right ◽  
Vacuolar Protein ◽  
Mutant Cells

Using a selection for spontaneous mutants that mislocalize a vacuolar carboxypeptidase Y (CPY)-invertase fusion protein to the cell surface, we identified vacuolar protein targeting (vpt) mutants in 25 new vpt complementation groups. Additional alleles in each of the eight previously identified vpt complementation groups (vpt1 through vpt8) were also obtained. Representative alleles from each of the 33 vpt complementation groups (vpt1 through vpt33) were shown to exhibit defects in the sorting and processing of several native vacuolar proteins, including the soluble hydrolases CPY, proteinase A, and proteinase B. Of the 33 complementation groups, 19 were found to contain mutant alleles that led to extreme defects. In these mutants, CPY accumulated in its Golgi complex-modified precursor form which was secreted by the mutant cells. Normal protein secretion appeared to be unaffected in the vpt mutants. The lack of significant leakage of cytosolic markers from the vpt mutant cells indicated that the vacuolar protein-sorting defects associated with these mutants do not result from cell lysis. In addition, the observation that the precursor rather than the mature forms of CPY, proteinase A, proteinase B were secreted from the vpt mutants was consistent with the fact that mislocalization occurred at a stage after Golgi complex-specific modification, but before final vacuolar sorting of these enzymes. Vacuolar membrane protein sorting appeared to be unaffected in the majority of the vpt mutants. However, a subset of the vpt mutants (vpt11, vpt16, vpt18, and vpt33) was found to exhibit defects in the sorting of a vacuolar membrane marker enzyme, alpha-mannosidase. Up to 50% of the alpha-mannosidase enzyme activity was found to be mislocalized to the cell surface in these vpt mutants. Seven of the vpt complementation groups (vpt3, vpt11, vpt15, vpt16, vpt18, vpt29, and vpt33) contained alleles that led to a conditional lethal phenotype; the mutants were temperature sensitive for vegetative cell growth. This temperature-sensitive phenotype has been shown to be recessive and to cosegregate with the vacuolar protein-sorting defect in each case. Tetrad analysis showed that vpt3 mapped to the right arm of chromosome XV and that vpt15 mapped to the right arm of chromosome II. Intercrosses with other mutants that exhibited defects in vacuolar protein sorting or function (vpl, sec, pep, and end mutants) revealed several overlaps among these different sets of genes. Together, these data indicate that more than 50 gene products are involved, directly or indirectly, in the process of vacuolar protein sorting.

scholarly journals Novel Genes Involved in Endosomal Traffic in Yeast Revealed by Suppression of a Targeting-defective Plasma Membrane ATPase Mutant

1997 ◽  
Vol 138 (4) ◽  
pp. 731-746 ◽  
Author(s):  
Wen-jie Luo ◽  
Amy Chang
Keyword(s):  
Plasma Membrane ◽  
Genetic Selection ◽  
Endocytic Pathway ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Plasma Membrane Atpase ◽  
Major Mechanism ◽  
Membrane Atpase ◽  
Bulk Membrane ◽  
Endosomal System

A novel genetic selection was used to identify genes regulating traffic in the yeast endosomal system. We took advantage of a temperature-sensitive mutant in PMA1, encoding the plasma membrane ATPase, in which newly synthesized Pma1 is mislocalized to the vacuole via the endosome. Diversion of mutant Pma1 from vacuolar delivery and rerouting to the plasma membrane is a major mechanism of suppression of pma1ts. 16 independent suppressor of pma1 (sop) mutants were isolated. Identification of the corresponding genes reveals eight that are identical with VPS genes required for delivery of newly synthesized vacuolar proteins. A second group of SOP genes participates in vacuolar delivery of mutant Pma1 but is not essential for delivery of the vacuolar protease carboxypeptidase Y. Because the biosynthetic pathway to the vacuole intersects with the endocytic pathway, internalization of a bulk membrane endocytic marker FM 4-64 was assayed in the sop mutants. By this means, defective endosome-to-vacuole trafficking was revealed in a subset of sop mutants. Another subset of sop mutants displays perturbed trafficking between endosome and Golgi: impaired pro-α factor processing in these strains was found to be due to defective recycling of the trans-Golgi protease Kex2. One of these strains defective in Kex2 trafficking carries a mutation in SOP2, encoding a homologue of mammalian synaptojanin (implicated in synaptic vesicle endocytosis and recycling). Thus, cell surface delivery of mutant Pma1 can occur as a consequence of disturbances at several different sites in the endosomal system.

scholarly journals Ubiquitin-mediated Targeting of a Mutant Plasma Membrane ATPase, Pma1-7, to the Endosomal/Vacuolar System in Yeast

2004 ◽  
Vol 15 (5) ◽  
pp. 2401-2409 ◽  
Author(s):  
Maddalena Pizzirusso ◽  
Amy Chang
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Ubiquitin Ligase ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Plasma Membrane Atpase ◽  
Membrane Atpase ◽  
Endosomal Pathway ◽  
Quality Control Mechanism ◽  
Vacuolar System

Pma1-7 is a mutant plasma membrane ATPase that is impaired in targeting to the cell surface at 37°C and is delivered instead to the endosomal/vacuolar pathway for degradation. We have proposed that Pma1-7 is a substrate for a Golgibased quality control mechanism. By contrast with wild-type Pma1, Pma1-7 is ubiquitinated. Ubiquitination and endosomal targeting of Pma1-7 is dependent on the Rsp5-Bul1-Bul2 ubiquitin ligase protein complex but not the transmembrane ubiquitin ligase Tul1. Analysis of Pma1-7 ubiquitination in mutants blocked in protein transport at various steps of the secretory pathway suggests that ubiquitination occurs after ER exit but before endosomal entry. In the absence of ubiquitination in rsp5-1 cells, Pma1-7 is delivered to the cell surface and remains stable. Nevertheless, Pma1-7 remains impaired in association with detergent-insoluble glycolipid-enriched complexes in rsp5-1 cells, suggesting that ubiquitination is not the cause of Pma1-7 exclusion from rafts. In vps1 cells in which protein transport into the endosomal pathway is blocked, Pma1-7 is routed to the cell surface. On arrival at the plasma membrane in vps1 cells, Pma1-7 remains stable and its ubiquitination disappears, suggesting deubiquitination activity at the cell surface. We suggest that Pma1-7 sorting and fate are regulated by ubiquitination.

scholarly journals Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases

1988 ◽  
Vol 8 (11) ◽  
pp. 4936-4948
Author(s):  
J S Robinson ◽  
D J Klionsky ◽  
L M Banta ◽  
S D Emr
Keyword(s):  
Cell Surface ◽  
Protein Sorting ◽  
Vacuolar Membrane ◽  
Temperature Sensitive ◽  
Vacuolar Protein Sorting ◽  
Proteinase A ◽  
Complementation Groups ◽  
The Right ◽  
Vacuolar Protein ◽  
Mutant Cells

Using a selection for spontaneous mutants that mislocalize a vacuolar carboxypeptidase Y (CPY)-invertase fusion protein to the cell surface, we identified vacuolar protein targeting (vpt) mutants in 25 new vpt complementation groups. Additional alleles in each of the eight previously identified vpt complementation groups (vpt1 through vpt8) were also obtained. Representative alleles from each of the 33 vpt complementation groups (vpt1 through vpt33) were shown to exhibit defects in the sorting and processing of several native vacuolar proteins, including the soluble hydrolases CPY, proteinase A, and proteinase B. Of the 33 complementation groups, 19 were found to contain mutant alleles that led to extreme defects. In these mutants, CPY accumulated in its Golgi complex-modified precursor form which was secreted by the mutant cells. Normal protein secretion appeared to be unaffected in the vpt mutants. The lack of significant leakage of cytosolic markers from the vpt mutant cells indicated that the vacuolar protein-sorting defects associated with these mutants do not result from cell lysis. In addition, the observation that the precursor rather than the mature forms of CPY, proteinase A, proteinase B were secreted from the vpt mutants was consistent with the fact that mislocalization occurred at a stage after Golgi complex-specific modification, but before final vacuolar sorting of these enzymes. Vacuolar membrane protein sorting appeared to be unaffected in the majority of the vpt mutants. However, a subset of the vpt mutants (vpt11, vpt16, vpt18, and vpt33) was found to exhibit defects in the sorting of a vacuolar membrane marker enzyme, alpha-mannosidase. Up to 50% of the alpha-mannosidase enzyme activity was found to be mislocalized to the cell surface in these vpt mutants. Seven of the vpt complementation groups (vpt3, vpt11, vpt15, vpt16, vpt18, vpt29, and vpt33) contained alleles that led to a conditional lethal phenotype; the mutants were temperature sensitive for vegetative cell growth. This temperature-sensitive phenotype has been shown to be recessive and to cosegregate with the vacuolar protein-sorting defect in each case. Tetrad analysis showed that vpt3 mapped to the right arm of chromosome XV and that vpt15 mapped to the right arm of chromosome II. Intercrosses with other mutants that exhibited defects in vacuolar protein sorting or function (vpl, sec, pep, and end mutants) revealed several overlaps among these different sets of genes. Together, these data indicate that more than 50 gene products are involved, directly or indirectly, in the process of vacuolar protein sorting.

scholarly journals Secretory vesicles externalize the major plasma membrane ATPase in yeast.

1988 ◽  
Vol 106 (3) ◽  
pp. 641-648 ◽  
Author(s):  
C L Holcomb ◽  
W J Hansen ◽  
T Etcheverry ◽  
R Schekman
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Biochemical Characterization ◽  
Plasma Membrane Atpase ◽  
Membrane Assembly ◽  
Yeast Cell Surface ◽  
Membrane Atpase ◽  
Constitutive Secretion ◽  
Secretory Enzyme

Yeast cell surface growth is accomplished by constitutive secretion and plasma membrane assembly, culminating in the fusion of vesicles with the bud membrane. Coordination of secretion and membrane assembly has been investigated by examining the biogenesis of plasma membrane ATPase (PM ATPase) in secretion-defective (sec) strains of Saccharomyces cerevisiae. PM ATPase is synthesized as a approximately 106-kD polypeptide that is not detectably modified by asparagine-linked glycosylation or proteolysis during transit to the plasma membrane. Export of the PM ATPase requires the secretory pathway. In sec1, a mutant defective in the last step of secretion, large amounts of Golgi-derived vesicles are accumulated. Biochemical characterization of this organelle has demonstrated that PM ATPase and the secretory enzyme, acid phosphatase, are transported in a single vesicle species.

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 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.

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