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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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Subcellular fractionation. (A) Yeast spheroplasts were pulse-labeled  with Tran35S for 20 min and  chased for 40 min. The  spheroplasts were then gently lysed by dounce homogenization in a hypotonic  buffer and the lysates were  fractionated by differential  centrifugation. The fractions  were immunoprecipitated  with an antiserum specific to  Vps10p and separated on an  8% SDS-polyacrylamide gel.  The strains used were  SEY6210 (WT), PSY129  (vps29), JCY3000 (Δvps30),  and EMY18 (Δvps35). To localize Vps35p, the same procedure was followed, except  antisera specific to Vps35p  was used to immunoprecipitate the samples. In the wildtype strain, Vps10p is localized to the P100 fraction  which contains Golgi, endosomal, and vesicular proteins; however in the vps29,  Δvps30, and Δvps35 strains,  Vps10p is localized to a P13  fraction which is rich in vacuolar markers. Vps35p appears to follow Vps10p to the P13 fraction in the vps29 strain and partially in the  Δvps30 strain. (B) The strains SEY6210 (wild-type) and MSY35-4ts (vps35ts) were shifted to the indicated temperature for 15 min before labeling and chasing at that temperature as described above. The cells were fractionated and Vps10p was immunoprecipitated as  described above. In the vps35ts strain, at the nonpermissive temperature, Vps10p is shifted to a P13 fraction (lanes 10–12). This shift of  Vps10p is not observed in wild-type cells (lanes 4–6).
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Figure 5: Subcellular fractionation. (A) Yeast spheroplasts were pulse-labeled with Tran35S for 20 min and chased for 40 min. The spheroplasts were then gently lysed by dounce homogenization in a hypotonic buffer and the lysates were fractionated by differential centrifugation. The fractions were immunoprecipitated with an antiserum specific to Vps10p and separated on an 8% SDS-polyacrylamide gel. The strains used were SEY6210 (WT), PSY129 (vps29), JCY3000 (Δvps30), and EMY18 (Δvps35). To localize Vps35p, the same procedure was followed, except antisera specific to Vps35p was used to immunoprecipitate the samples. In the wildtype strain, Vps10p is localized to the P100 fraction which contains Golgi, endosomal, and vesicular proteins; however in the vps29, Δvps30, and Δvps35 strains, Vps10p is localized to a P13 fraction which is rich in vacuolar markers. Vps35p appears to follow Vps10p to the P13 fraction in the vps29 strain and partially in the Δvps30 strain. (B) The strains SEY6210 (wild-type) and MSY35-4ts (vps35ts) were shifted to the indicated temperature for 15 min before labeling and chasing at that temperature as described above. The cells were fractionated and Vps10p was immunoprecipitated as described above. In the vps35ts strain, at the nonpermissive temperature, Vps10p is shifted to a P13 fraction (lanes 10–12). This shift of Vps10p is not observed in wild-type cells (lanes 4–6).

Mentions: Since vps29, vps30, and vps35 mutants show a vacuolar sorting phenotype similar to that of vps10 mutants, we reasoned that the function and/or location of Vps10p might be affected in these mutants. To investigate the subcellular location of Vps10p in these mutants, cells were converted to spheroplasts and then labeled for 15 min at 30°C with Tran35S-Label. The spheroplasts were then chased with unlabeled amino acids for 45 min to allow the labeled Vps10p to reach its steady state location in the cell. Gentle lysis of the spheroplasts was achieved by dounce homogenization in a hypotonic buffer. The lysates were then separated into pellet and supernatant fractions by sequential centrifugation at 500, 13,000, and 100,000 g. It has been previously shown that in wild-type cells ∼85–90% of the Vps10p is found in the same Golgi-enriched 100,000 g pellet (P100) fraction that contains the late Golgi protein Kex2p (Marcusson et al., 1994). The remainder of Vps10p was found in the 13,000-g pellet (P13) fraction, which has been shown to contain endoplasmic reticulum, mitochondria, plasma membrane, and vacuoles (Marcusson et al., 1994). When this same procedure was performed using Δvps29, Δvps30, or Δvps35 cells, the vast majority of Vps10p was found in the P13 fraction (Fig. 5 A, upper panels). This effect seems to be a general effect on proteins that reside late in the Golgi complex, as Kex2p is rapidly degraded in the vacuole in Δvps29 and Δvps35 mutant strains (Table II). The degradation of Kex2p occurred more slowly in the Δvps30 mutant, but it is still faster than in wild-type cells. This group of vps mutants does not seem to affect the distribution of all Golgi proteins as Mnn1p, a glycosyltransferase of the medial-Golgi, can still be found in the P100 fraction in a Δvps29 mutant (data not shown). Importantly, not all vps mutants cause Vps10p to be redistributed to the P13 fraction. The majority of Vps10p could be found in the P100 subcellular fraction from cells with mutations in PEP12 (VPS6), VPS8, VPS13, VPS18, VPS21, or VPS45 (data not shown).


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)

Subcellular fractionation. (A) Yeast spheroplasts were pulse-labeled  with Tran35S for 20 min and  chased for 40 min. The  spheroplasts were then gently lysed by dounce homogenization in a hypotonic  buffer and the lysates were  fractionated by differential  centrifugation. The fractions  were immunoprecipitated  with an antiserum specific to  Vps10p and separated on an  8% SDS-polyacrylamide gel.  The strains used were  SEY6210 (WT), PSY129  (vps29), JCY3000 (Δvps30),  and EMY18 (Δvps35). To localize Vps35p, the same procedure was followed, except  antisera specific to Vps35p  was used to immunoprecipitate the samples. In the wildtype strain, Vps10p is localized to the P100 fraction  which contains Golgi, endosomal, and vesicular proteins; however in the vps29,  Δvps30, and Δvps35 strains,  Vps10p is localized to a P13  fraction which is rich in vacuolar markers. Vps35p appears to follow Vps10p to the P13 fraction in the vps29 strain and partially in the  Δvps30 strain. (B) The strains SEY6210 (wild-type) and MSY35-4ts (vps35ts) were shifted to the indicated temperature for 15 min before labeling and chasing at that temperature as described above. The cells were fractionated and Vps10p was immunoprecipitated as  described above. In the vps35ts strain, at the nonpermissive temperature, Vps10p is shifted to a P13 fraction (lanes 10–12). This shift of  Vps10p is not observed in wild-type cells (lanes 4–6).
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Figure 5: Subcellular fractionation. (A) Yeast spheroplasts were pulse-labeled with Tran35S for 20 min and chased for 40 min. The spheroplasts were then gently lysed by dounce homogenization in a hypotonic buffer and the lysates were fractionated by differential centrifugation. The fractions were immunoprecipitated with an antiserum specific to Vps10p and separated on an 8% SDS-polyacrylamide gel. The strains used were SEY6210 (WT), PSY129 (vps29), JCY3000 (Δvps30), and EMY18 (Δvps35). To localize Vps35p, the same procedure was followed, except antisera specific to Vps35p was used to immunoprecipitate the samples. In the wildtype strain, Vps10p is localized to the P100 fraction which contains Golgi, endosomal, and vesicular proteins; however in the vps29, Δvps30, and Δvps35 strains, Vps10p is localized to a P13 fraction which is rich in vacuolar markers. Vps35p appears to follow Vps10p to the P13 fraction in the vps29 strain and partially in the Δvps30 strain. (B) The strains SEY6210 (wild-type) and MSY35-4ts (vps35ts) were shifted to the indicated temperature for 15 min before labeling and chasing at that temperature as described above. The cells were fractionated and Vps10p was immunoprecipitated as described above. In the vps35ts strain, at the nonpermissive temperature, Vps10p is shifted to a P13 fraction (lanes 10–12). This shift of Vps10p is not observed in wild-type cells (lanes 4–6).
Mentions: Since vps29, vps30, and vps35 mutants show a vacuolar sorting phenotype similar to that of vps10 mutants, we reasoned that the function and/or location of Vps10p might be affected in these mutants. To investigate the subcellular location of Vps10p in these mutants, cells were converted to spheroplasts and then labeled for 15 min at 30°C with Tran35S-Label. The spheroplasts were then chased with unlabeled amino acids for 45 min to allow the labeled Vps10p to reach its steady state location in the cell. Gentle lysis of the spheroplasts was achieved by dounce homogenization in a hypotonic buffer. The lysates were then separated into pellet and supernatant fractions by sequential centrifugation at 500, 13,000, and 100,000 g. It has been previously shown that in wild-type cells ∼85–90% of the Vps10p is found in the same Golgi-enriched 100,000 g pellet (P100) fraction that contains the late Golgi protein Kex2p (Marcusson et al., 1994). The remainder of Vps10p was found in the 13,000-g pellet (P13) fraction, which has been shown to contain endoplasmic reticulum, mitochondria, plasma membrane, and vacuoles (Marcusson et al., 1994). When this same procedure was performed using Δvps29, Δvps30, or Δvps35 cells, the vast majority of Vps10p was found in the P13 fraction (Fig. 5 A, upper panels). This effect seems to be a general effect on proteins that reside late in the Golgi complex, as Kex2p is rapidly degraded in the vacuole in Δvps29 and Δvps35 mutant strains (Table II). The degradation of Kex2p occurred more slowly in the Δvps30 mutant, but it is still faster than in wild-type cells. This group of vps mutants does not seem to affect the distribution of all Golgi proteins as Mnn1p, a glycosyltransferase of the medial-Golgi, can still be found in the P100 fraction in a Δvps29 mutant (data not shown). Importantly, not all vps mutants cause Vps10p to be redistributed to the P13 fraction. The majority of Vps10p could be found in the P100 subcellular fraction from cells with mutations in PEP12 (VPS6), VPS8, VPS13, VPS18, VPS21, or VPS45 (data not shown).

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