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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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Complementation of the vps29 and vps30 mutants.  Yeast cells were pulse-labeled with Tran35S for 10 min and chased  for 30 min. The cells were then spheroplasted and separated into  intracellular (I) and extracellular (E) fractions. CPY or PrA was  immunoprecipitated from both fractions and resolved by SDSPAGE and fluorography. The strains used were PSY129 (vps29),  PSY129 carrying a centromeric plasmid containing the VPS29 gene  (PSY129 + pVPS29), JCY3000 (Δvps30), and JCY3000 carrying  a centromeric plasmid containing the VPS30 gene (JCY3000 +  pVPS30).
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Figure 2: Complementation of the vps29 and vps30 mutants. Yeast cells were pulse-labeled with Tran35S for 10 min and chased for 30 min. The cells were then spheroplasted and separated into intracellular (I) and extracellular (E) fractions. CPY or PrA was immunoprecipitated from both fractions and resolved by SDSPAGE and fluorography. The strains used were PSY129 (vps29), PSY129 carrying a centromeric plasmid containing the VPS29 gene (PSY129 + pVPS29), JCY3000 (Δvps30), and JCY3000 carrying a centromeric plasmid containing the VPS30 gene (JCY3000 + pVPS30).

Mentions: To characterize further the function of VPS29 and VPS30, the genes were cloned by complementation of the CPY sorting defect as previously described (Paravicini et al., 1992). The CPY sorting phenotypes of vps29 and vps30 mutants with and without the complementing fragments are shown in Fig. 2. The cells were pulse labeled with Tran35S for 10 min and chased for 30 min at 30°C. The cells were then converted to spheroplasts and separated into intracellular (I) and extracellular (E) fractions from which CPY was immunoprecipitated. The vps29 and vps30 mutant cells missorted and secreted virtually all of their CPY as the Golgi modified p2 form (Fig. 2, lanes 1, 2, 5, and 6). However, when the appropriate plasmid was introduced into each mutant, CPY was properly matured inside the cells, indicating that the vacuolar sorting defect had been corrected (Fig. 2, lanes 3, 4, 7, and 8). Sequencing of VPS29 showed that it encodes a hydrophilic protein of 282 amino acids and a pI of 4.3. The primary sequence of VPS29 does not contain any structural motifs or homologies with proteins of known function. VPS30 also encodes a hydrophilic protein containing 556 amino acids which has a predicted molecular mass of 65 kD and pI of 4.8. The central portion of Vps30p (residues 186 to 322) is predicted to form coiled-coil structures (Lupas et al., 1991).


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)

Complementation of the vps29 and vps30 mutants.  Yeast cells were pulse-labeled with Tran35S for 10 min and chased  for 30 min. The cells were then spheroplasted and separated into  intracellular (I) and extracellular (E) fractions. CPY or PrA was  immunoprecipitated from both fractions and resolved by SDSPAGE and fluorography. The strains used were PSY129 (vps29),  PSY129 carrying a centromeric plasmid containing the VPS29 gene  (PSY129 + pVPS29), JCY3000 (Δvps30), and JCY3000 carrying  a centromeric plasmid containing the VPS30 gene (JCY3000 +  pVPS30).
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Related In: Results  -  Collection

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Figure 2: Complementation of the vps29 and vps30 mutants. Yeast cells were pulse-labeled with Tran35S for 10 min and chased for 30 min. The cells were then spheroplasted and separated into intracellular (I) and extracellular (E) fractions. CPY or PrA was immunoprecipitated from both fractions and resolved by SDSPAGE and fluorography. The strains used were PSY129 (vps29), PSY129 carrying a centromeric plasmid containing the VPS29 gene (PSY129 + pVPS29), JCY3000 (Δvps30), and JCY3000 carrying a centromeric plasmid containing the VPS30 gene (JCY3000 + pVPS30).
Mentions: To characterize further the function of VPS29 and VPS30, the genes were cloned by complementation of the CPY sorting defect as previously described (Paravicini et al., 1992). The CPY sorting phenotypes of vps29 and vps30 mutants with and without the complementing fragments are shown in Fig. 2. The cells were pulse labeled with Tran35S for 10 min and chased for 30 min at 30°C. The cells were then converted to spheroplasts and separated into intracellular (I) and extracellular (E) fractions from which CPY was immunoprecipitated. The vps29 and vps30 mutant cells missorted and secreted virtually all of their CPY as the Golgi modified p2 form (Fig. 2, lanes 1, 2, 5, and 6). However, when the appropriate plasmid was introduced into each mutant, CPY was properly matured inside the cells, indicating that the vacuolar sorting defect had been corrected (Fig. 2, lanes 3, 4, 7, and 8). Sequencing of VPS29 showed that it encodes a hydrophilic protein of 282 amino acids and a pI of 4.3. The primary sequence of VPS29 does not contain any structural motifs or homologies with proteins of known function. VPS30 also encodes a hydrophilic protein containing 556 amino acids which has a predicted molecular mass of 65 kD and pI of 4.8. The central portion of Vps30p (residues 186 to 322) is predicted to form coiled-coil structures (Lupas et al., 1991).

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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