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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.

Show MeSH
Vps10p is transported to the vacuole via the  prevacuolar endosome in  Δvps35 cells. The strains  MSY3501 (sec1 Δvps35) and  MSY3512 (pep12ts Δvps35)  were converted to spheroplasts before pre-shifting to  the indicated temperature  for 15 min. The cells were  then labeled for 15 min with  [35S]methionine and chased  for an additional 45 min at either 26°C or 38°C. After  dounce homogenization, the lysed cells were subjected to differential centrifugation to separate small Golgi-enriched membranes  (P100) from the larger vacuoles (P13). Vps10p was immunoprecipitated from the fractions. Transport of Vps10p to the P13 fraction  does not require Sec1p function and therefore does not occur by arrival of Vps10p at the plasma membrane and subsequent endocytosis  and delivery to the vacuole, but does require Pep12p function as Vps10p becomes trapped in a P100 membrane fraction upon inactivation of the endosomal t-SNARE Pep12p.
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Figure 8: Vps10p is transported to the vacuole via the prevacuolar endosome in Δvps35 cells. The strains MSY3501 (sec1 Δvps35) and MSY3512 (pep12ts Δvps35) were converted to spheroplasts before pre-shifting to the indicated temperature for 15 min. The cells were then labeled for 15 min with [35S]methionine and chased for an additional 45 min at either 26°C or 38°C. After dounce homogenization, the lysed cells were subjected to differential centrifugation to separate small Golgi-enriched membranes (P100) from the larger vacuoles (P13). Vps10p was immunoprecipitated from the fractions. Transport of Vps10p to the P13 fraction does not require Sec1p function and therefore does not occur by arrival of Vps10p at the plasma membrane and subsequent endocytosis and delivery to the vacuole, but does require Pep12p function as Vps10p becomes trapped in a P100 membrane fraction upon inactivation of the endosomal t-SNARE Pep12p.

Mentions: Although the immunofluorescence experiment shows that Vps10p is localized to the vacuole in Δvps29, Δvps30, and Δvps35 cells, it is still possible that Vps10p reaches the vacuole by endocytosis from the plasma membrane. This possibility was tested using the sec1 mutant strain in which VPS35 was deleted in the same fashion used to create the strain EMY18. Sec1p is required for secretory vesicles to fuse with the plasma membrane and the allele present in the sec1 strain is temperature sensitive for function (Aalto et al., 1991). Thus, it is possible to block the arrival of proteins at the plasma membrane by elevating the temperature to 38°C which is nonpermissive for the sec1 allele. Vps10p was localized in the sec1 Δvps35 strain at both the permissive and nonpermissive temperature by subcellular fractionation. At both temperatures, Vps10p was localized to the P13 fraction (Fig. 8, lanes 1–6). If Vps10p was transported to the vacuole via the plasma membrane, it would be detected in the P100 fraction which contains small vesicles including secretory vesicles. However, as the data show, the distribution of Vps10p is unaffected by inactivation of Sec1p. Thus, it seems most likely that Vps10p is transported directly to the vacuole in Δvps35 mutants presumably through a prevacuolar compartment from which Vps10p would recycle back to the Golgi in wild-type cells.


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 transported to the vacuole via the  prevacuolar endosome in  Δvps35 cells. The strains  MSY3501 (sec1 Δvps35) and  MSY3512 (pep12ts Δvps35)  were converted to spheroplasts before pre-shifting to  the indicated temperature  for 15 min. The cells were  then labeled for 15 min with  [35S]methionine and chased  for an additional 45 min at either 26°C or 38°C. After  dounce homogenization, the lysed cells were subjected to differential centrifugation to separate small Golgi-enriched membranes  (P100) from the larger vacuoles (P13). Vps10p was immunoprecipitated from the fractions. Transport of Vps10p to the P13 fraction  does not require Sec1p function and therefore does not occur by arrival of Vps10p at the plasma membrane and subsequent endocytosis  and delivery to the vacuole, but does require Pep12p function as Vps10p becomes trapped in a P100 membrane fraction upon inactivation of the endosomal t-SNARE Pep12p.
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Related In: Results  -  Collection

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Figure 8: Vps10p is transported to the vacuole via the prevacuolar endosome in Δvps35 cells. The strains MSY3501 (sec1 Δvps35) and MSY3512 (pep12ts Δvps35) were converted to spheroplasts before pre-shifting to the indicated temperature for 15 min. The cells were then labeled for 15 min with [35S]methionine and chased for an additional 45 min at either 26°C or 38°C. After dounce homogenization, the lysed cells were subjected to differential centrifugation to separate small Golgi-enriched membranes (P100) from the larger vacuoles (P13). Vps10p was immunoprecipitated from the fractions. Transport of Vps10p to the P13 fraction does not require Sec1p function and therefore does not occur by arrival of Vps10p at the plasma membrane and subsequent endocytosis and delivery to the vacuole, but does require Pep12p function as Vps10p becomes trapped in a P100 membrane fraction upon inactivation of the endosomal t-SNARE Pep12p.
Mentions: Although the immunofluorescence experiment shows that Vps10p is localized to the vacuole in Δvps29, Δvps30, and Δvps35 cells, it is still possible that Vps10p reaches the vacuole by endocytosis from the plasma membrane. This possibility was tested using the sec1 mutant strain in which VPS35 was deleted in the same fashion used to create the strain EMY18. Sec1p is required for secretory vesicles to fuse with the plasma membrane and the allele present in the sec1 strain is temperature sensitive for function (Aalto et al., 1991). Thus, it is possible to block the arrival of proteins at the plasma membrane by elevating the temperature to 38°C which is nonpermissive for the sec1 allele. Vps10p was localized in the sec1 Δvps35 strain at both the permissive and nonpermissive temperature by subcellular fractionation. At both temperatures, Vps10p was localized to the P13 fraction (Fig. 8, lanes 1–6). If Vps10p was transported to the vacuole via the plasma membrane, it would be detected in the P100 fraction which contains small vesicles including secretory vesicles. However, as the data show, the distribution of Vps10p is unaffected by inactivation of Sec1p. Thus, it seems most likely that Vps10p is transported directly to the vacuole in Δvps35 mutants presumably through a prevacuolar compartment from which Vps10p would recycle back to the Golgi in wild-type cells.

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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