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Endosome to Golgi retrieval of the vacuolar protein sorting receptor, Vps10p, requires the function of the VPS29, VPS30, and VPS35 gene products.

Seaman MN, Marcusson EG, Cereghino JL, Emr SD - J. Cell Biol. (1997)

Bottom Line: The sequences of the VPS29, VPS30, and VPS35 genes do not yet give any clues to the functions of their products.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Cellular and Molecular Medicine, University of California, San Diego, School of Medicine, La Jolla 92093-0668, USA.

ABSTRACT
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.

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Vps10p is localized to the vacuole in vps29, vps30, and  vps35 mutants. Using an epitope-tagged version of Vps10p expressed at centromeric levels, Vps10p protein was localized in  wild-type cells, vps29, Δvps30 and Δvps35 cells, and also Δpep12/ vps6 cells. In wild-type cells the labeling pattern appeared punctate and characteristic of a Golgi and endosomal localization. In  the vps29, Δvps30, and Δvps35 cells, Vps10p was localized to the  vacuolar membrane. The redistribution of Vps10p to the vacuolar membrane is not a general phenomenon associated with disruption of the VPS pathway as in Δpep12/vps6 cells; Vps10p has a  punctate distribution indistinguishable from wild-type cells.
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Figure 7: Vps10p is localized to the vacuole in vps29, vps30, and vps35 mutants. Using an epitope-tagged version of Vps10p expressed at centromeric levels, Vps10p protein was localized in wild-type cells, vps29, Δvps30 and Δvps35 cells, and also Δpep12/ vps6 cells. In wild-type cells the labeling pattern appeared punctate and characteristic of a Golgi and endosomal localization. In the vps29, Δvps30, and Δvps35 cells, Vps10p was localized to the vacuolar membrane. The redistribution of Vps10p to the vacuolar membrane is not a general phenomenon associated with disruption of the VPS pathway as in Δpep12/vps6 cells; Vps10p has a punctate distribution indistinguishable from wild-type cells.

Mentions: The redistribution of Vps10p to the P13 pellet in vps29, vps30, and vps35 cells is indicative of Vps10p being delivered to the vacuole. However, it was possible that Vps10p could have been in one of the other organelles which fractionates with the P13 pellet, such as the plasma membrane. To determine if the vacuole was in fact the location of Vps10p in these mutant cells, indirect immunofluorescence experiments were undertaken. Previous immunofluorescence studies using a Myc-tagged version of Vps10p encoded on a centromeric plasmid showed a dispersed punctate staining pattern in diploid wild-type cells, consistent with a late-Golgi location of Vps10p (Cereghino et al., 1995). When the same procedure was undertaken with haploid wild-type cells, once again a punctate pattern was seen (Fig. 7). In contrast, in haploid Δvps29, Δvps30, and Δvps35 cells expressing the Myc-tagged version of Vps10p from a centromeric plasmid, clear vacuolar staining was observed (Fig. 7). This indicated that the shift of Vps10p from the P100 fraction to the P13 fraction in these cells is indicative of Vps10p being mislocalized to the vacuolar membrane. As a control, the staining pattern for Vps10p in pep12 (vps6) mutant haploid cells was also investigated. In these cells, as in wild-type cells, only a dispersed punctate pattern was seen (Fig. 7). This shows that vps mutants which do not cause Vps10p to shift to the P13 pellet in subcellular fractionation experiments also do not show a vacuolar staining pattern for Vps10p.


Endosome to Golgi retrieval of the vacuolar protein sorting receptor, Vps10p, requires the function of the VPS29, VPS30, and VPS35 gene products.

Seaman MN, Marcusson EG, Cereghino JL, Emr SD - J. Cell Biol. (1997)

Vps10p is localized to the vacuole in vps29, vps30, and  vps35 mutants. Using an epitope-tagged version of Vps10p expressed at centromeric levels, Vps10p protein was localized in  wild-type cells, vps29, Δvps30 and Δvps35 cells, and also Δpep12/ vps6 cells. In wild-type cells the labeling pattern appeared punctate and characteristic of a Golgi and endosomal localization. In  the vps29, Δvps30, and Δvps35 cells, Vps10p was localized to the  vacuolar membrane. The redistribution of Vps10p to the vacuolar membrane is not a general phenomenon associated with disruption of the VPS pathway as in Δpep12/vps6 cells; Vps10p has a  punctate distribution indistinguishable from wild-type cells.
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Related In: Results  -  Collection

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Figure 7: Vps10p is localized to the vacuole in vps29, vps30, and vps35 mutants. Using an epitope-tagged version of Vps10p expressed at centromeric levels, Vps10p protein was localized in wild-type cells, vps29, Δvps30 and Δvps35 cells, and also Δpep12/ vps6 cells. In wild-type cells the labeling pattern appeared punctate and characteristic of a Golgi and endosomal localization. In the vps29, Δvps30, and Δvps35 cells, Vps10p was localized to the vacuolar membrane. The redistribution of Vps10p to the vacuolar membrane is not a general phenomenon associated with disruption of the VPS pathway as in Δpep12/vps6 cells; Vps10p has a punctate distribution indistinguishable from wild-type cells.
Mentions: The redistribution of Vps10p to the P13 pellet in vps29, vps30, and vps35 cells is indicative of Vps10p being delivered to the vacuole. However, it was possible that Vps10p could have been in one of the other organelles which fractionates with the P13 pellet, such as the plasma membrane. To determine if the vacuole was in fact the location of Vps10p in these mutant cells, indirect immunofluorescence experiments were undertaken. Previous immunofluorescence studies using a Myc-tagged version of Vps10p encoded on a centromeric plasmid showed a dispersed punctate staining pattern in diploid wild-type cells, consistent with a late-Golgi location of Vps10p (Cereghino et al., 1995). When the same procedure was undertaken with haploid wild-type cells, once again a punctate pattern was seen (Fig. 7). In contrast, in haploid Δvps29, Δvps30, and Δvps35 cells expressing the Myc-tagged version of Vps10p from a centromeric plasmid, clear vacuolar staining was observed (Fig. 7). This indicated that the shift of Vps10p from the P100 fraction to the P13 fraction in these cells is indicative of Vps10p being mislocalized to the vacuolar membrane. As a control, the staining pattern for Vps10p in pep12 (vps6) mutant haploid cells was also investigated. In these cells, as in wild-type cells, only a dispersed punctate pattern was seen (Fig. 7). This shows that vps mutants which do not cause Vps10p to shift to the P13 pellet in subcellular fractionation experiments also do not show a vacuolar staining pattern for Vps10p.

Bottom Line: The sequences of the VPS29, VPS30, and VPS35 genes do not yet give any clues to the functions of their products.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Cellular and Molecular Medicine, University of California, San Diego, School of Medicine, La Jolla 92093-0668, USA.

ABSTRACT
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.

Show MeSH