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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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Detection of Vps30p. (A) Yeast cells were pulselabeled with Tran35S for 20 min and chased for either 0 or 60 min.  The position of Vps30p is indicated. The strains used were  JCY3000 (Δvps30), SEY6210 (WT), and SEY6210 carrying a 2-μ  plasmid containing the VPS30 gene (WT + 2 μ VPS30). (B) Wildtype spheroplasts (SEY6210) were pulse-labeled with Tran35S for  20 min and chased for 60 min. The spheroplasts were then gently  lysed by dounce homogenization in a hypotonic buffer and then  washed in either 10 mM Tris/1 mM EDTA, pH 8.0 (Buffer), 1 M  NaCl, or 3 M urea. The homogenates were then spun in an ultracentrifuge at 100,000 g. The pellet (P) and supernatant (S) fractions were then immunoprecipitated with antiserum specific to  Vps30p and resolved by SDS-PAGE.
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Figure 4: Detection of Vps30p. (A) Yeast cells were pulselabeled with Tran35S for 20 min and chased for either 0 or 60 min. The position of Vps30p is indicated. The strains used were JCY3000 (Δvps30), SEY6210 (WT), and SEY6210 carrying a 2-μ plasmid containing the VPS30 gene (WT + 2 μ VPS30). (B) Wildtype spheroplasts (SEY6210) were pulse-labeled with Tran35S for 20 min and chased for 60 min. The spheroplasts were then gently lysed by dounce homogenization in a hypotonic buffer and then washed in either 10 mM Tris/1 mM EDTA, pH 8.0 (Buffer), 1 M NaCl, or 3 M urea. The homogenates were then spun in an ultracentrifuge at 100,000 g. The pellet (P) and supernatant (S) fractions were then immunoprecipitated with antiserum specific to Vps30p and resolved by SDS-PAGE.

Mentions: To make an antiserum specific for Vps30p, a TrpE VPS30 fusion construct was made (see Materials and Methods). Antisera raised against the resulting fusion protein was used to further analyze the VPS30 gene product. Wildtype, Δvps30, and wild-type cells carrying a multicopy plasmid containing the VPS30 gene were pulse-labeled with Tran35S-label for 20 min at 30°C. The cells were then incubated with unlabeled cysteine and methionine for 0 or 60 min. Cell lysates were immunoprecipitated with the antisera raised against the TrpE/Vps30 fusion protein. In wildtype cells, the antisera recognized a 63-kD protein which was stable over the 60-min chase period (Fig. 4 A, lanes 2 and 3). This protein was absent from Δvps30 cells (Fig. 4 A, lane 1) and was stably expressed at a higher level in the cells carrying a multicopy plasmid containing the VPS30 gene (Fig. 4 A, lanes 4 and 5). These results show that the antisera specifically recognized the VPS30 gene product.


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)

Detection of Vps30p. (A) Yeast cells were pulselabeled with Tran35S for 20 min and chased for either 0 or 60 min.  The position of Vps30p is indicated. The strains used were  JCY3000 (Δvps30), SEY6210 (WT), and SEY6210 carrying a 2-μ  plasmid containing the VPS30 gene (WT + 2 μ VPS30). (B) Wildtype spheroplasts (SEY6210) were pulse-labeled with Tran35S for  20 min and chased for 60 min. The spheroplasts were then gently  lysed by dounce homogenization in a hypotonic buffer and then  washed in either 10 mM Tris/1 mM EDTA, pH 8.0 (Buffer), 1 M  NaCl, or 3 M urea. The homogenates were then spun in an ultracentrifuge at 100,000 g. The pellet (P) and supernatant (S) fractions were then immunoprecipitated with antiserum specific to  Vps30p and resolved by SDS-PAGE.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139870&req=5

Figure 4: Detection of Vps30p. (A) Yeast cells were pulselabeled with Tran35S for 20 min and chased for either 0 or 60 min. The position of Vps30p is indicated. The strains used were JCY3000 (Δvps30), SEY6210 (WT), and SEY6210 carrying a 2-μ plasmid containing the VPS30 gene (WT + 2 μ VPS30). (B) Wildtype spheroplasts (SEY6210) were pulse-labeled with Tran35S for 20 min and chased for 60 min. The spheroplasts were then gently lysed by dounce homogenization in a hypotonic buffer and then washed in either 10 mM Tris/1 mM EDTA, pH 8.0 (Buffer), 1 M NaCl, or 3 M urea. The homogenates were then spun in an ultracentrifuge at 100,000 g. The pellet (P) and supernatant (S) fractions were then immunoprecipitated with antiserum specific to Vps30p and resolved by SDS-PAGE.
Mentions: To make an antiserum specific for Vps30p, a TrpE VPS30 fusion construct was made (see Materials and Methods). Antisera raised against the resulting fusion protein was used to further analyze the VPS30 gene product. Wildtype, Δvps30, and wild-type cells carrying a multicopy plasmid containing the VPS30 gene were pulse-labeled with Tran35S-label for 20 min at 30°C. The cells were then incubated with unlabeled cysteine and methionine for 0 or 60 min. Cell lysates were immunoprecipitated with the antisera raised against the TrpE/Vps30 fusion protein. In wildtype cells, the antisera recognized a 63-kD protein which was stable over the 60-min chase period (Fig. 4 A, lanes 2 and 3). This protein was absent from Δvps30 cells (Fig. 4 A, lane 1) and was stably expressed at a higher level in the cells carrying a multicopy plasmid containing the VPS30 gene (Fig. 4 A, lanes 4 and 5). These results show that the antisera specifically recognized the VPS30 gene product.

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
Related in: MedlinePlus