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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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Pep12p is epistatic to Vps35p. (A) The strain MSY 0435  (Δvps4Δvps35) harboring plasmids encoding either wild-type  VPS35 (pGPY35) or the temperature conditional allele of vps35  (pMS35-4ts) was shifted to 38°C 15 min before labeling with  [35S]methionine. After labeling for 10 min, excess unlabeled methionine was added and the cells were chased for the specified  times. Aliquots of cells (2 OD equivalents) were removed at the  specified times, the cells were lysed and Vps10p was recovered by  immunoprecipitation. Vps10p was clipped (clipped Vps10p denoted by *) with a half-time of ∼30 min which is typical for a class  E mutant. The clipping of Vps10p is unaffected by inactivation of  Vps35p. (B) The strain MSY4012 (vps4Δpep12) harboring the  plasmid encoding the temperature conditional allele of pep12  (pCBY9) was grown at 26oC, before being shifted to the specified  temperature for 10 min before labeling. The cells were labeled  and chased as described above at either 26oC or 38oC, and Vps10p  was recovered from the lysates as described above. Vps10p was  completely stabilized by inactivation of Pep12p, indicating that it  never reached the class E compartment and hence that Pep12p is  epistatic to Vps35p.
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Figure 9: Pep12p is epistatic to Vps35p. (A) The strain MSY 0435 (Δvps4Δvps35) harboring plasmids encoding either wild-type VPS35 (pGPY35) or the temperature conditional allele of vps35 (pMS35-4ts) was shifted to 38°C 15 min before labeling with [35S]methionine. After labeling for 10 min, excess unlabeled methionine was added and the cells were chased for the specified times. Aliquots of cells (2 OD equivalents) were removed at the specified times, the cells were lysed and Vps10p was recovered by immunoprecipitation. Vps10p was clipped (clipped Vps10p denoted by *) with a half-time of ∼30 min which is typical for a class E mutant. The clipping of Vps10p is unaffected by inactivation of Vps35p. (B) The strain MSY4012 (vps4Δpep12) harboring the plasmid encoding the temperature conditional allele of pep12 (pCBY9) was grown at 26oC, before being shifted to the specified temperature for 10 min before labeling. The cells were labeled and chased as described above at either 26oC or 38oC, and Vps10p was recovered from the lysates as described above. Vps10p was completely stabilized by inactivation of Pep12p, indicating that it never reached the class E compartment and hence that Pep12p is epistatic to Vps35p.

Mentions: In certain vps mutants, namely the class E mutants, the prevacuolar endosome becomes enlarged and acidic with active proteases contained within and is termed the “class E compartment” (Raymond et al., 1992; Reider et al., 1996; Piper et al., 1996). Vps10p traffics through this compartment in class E mutants and is proteolytically clipped with a half time of ∼30 min (Cereghino et al., 1995; Cooper and Stevens, 1996). The finding that Vps35p appears to be associated with membranes enriched in Kex2p as well as Vps10p (see Fig. 6) raised the possibility that Vps35p might also act at the late-Golgi, perhaps to affect the exit rate of Vps10p. Any effect that inactivation of Vps35p might have on the exit of Vps10p from the late-Golgi could manifest itself as an effect upon the kinetics of the clipping of Vps10p in a class E mutant. To address this question, a class E Δvps35 mutant was constructed using a mutant of vps4 as a representative class E mutant (Babst et al., 1997). This mutant was transformed with plasmids containing either wild-type VPS35 or the vps35ts mutant gene. The clipping of Vps10p in these strains was investigated and is shown in Fig. 9 A. Cells were incubated for 15 min at 38°C to inactivate the temperature conditional allele of vps35 before labeling with [35S]methionine for 10 min, after which an excess of unlabeled methionine was added. Aliquots of cells were removed at 0 min of chase and also after 30 min and 60 min. After cell lysis, Vps10p was recovered by immunoprecipitation. As shown in Fig. 9 A, cells harboring the wild-type VPS35 allele (lanes 1–3) have ∼50% clipped Vps10p (denoted by the *) after a 30-min chase, a phenotype which is identical to class E mutant alone (Cereghino et al., 1995). Cells which contained the temperature conditional allele of vps35 (Fig. 9 A, lanes 4–6) showed no significant difference in the kinetics of the clipping of Vps10p, suggesting that inactivation of Vps35p does not affect the exit rate of Vps10p from the late-Golgi. By contrast, however, inactivation of Pep12p in a class E mutant completely prevents the clipping of Vps10p. In Fig. 9 B, cells containing a mutant allele of vps4 and also the temperature conditional allele of pep12 (Cowles et al., 1997) were pulsed for 10 min with [35S]methionine and chased at either 26°C or 38°C as described above. Vps10p was recovered from the aliquots of cells removed after the specified chase times. At permissive temperature, Vps10p is clipped with normal kinetics for a class E mutant; however, upon raising the temperature to 38°C and inactivation of Pep12p, Vps10p is stabilized and is not proteolytically clipped even after 60 min of chase. These data strongly suggest that pep12ts is epistatic to vps35 and that the vps35 mutation does not affect the exit of Vps10p from the Golgi in any way.


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)

Pep12p is epistatic to Vps35p. (A) The strain MSY 0435  (Δvps4Δvps35) harboring plasmids encoding either wild-type  VPS35 (pGPY35) or the temperature conditional allele of vps35  (pMS35-4ts) was shifted to 38°C 15 min before labeling with  [35S]methionine. After labeling for 10 min, excess unlabeled methionine was added and the cells were chased for the specified  times. Aliquots of cells (2 OD equivalents) were removed at the  specified times, the cells were lysed and Vps10p was recovered by  immunoprecipitation. Vps10p was clipped (clipped Vps10p denoted by *) with a half-time of ∼30 min which is typical for a class  E mutant. The clipping of Vps10p is unaffected by inactivation of  Vps35p. (B) The strain MSY4012 (vps4Δpep12) harboring the  plasmid encoding the temperature conditional allele of pep12  (pCBY9) was grown at 26oC, before being shifted to the specified  temperature for 10 min before labeling. The cells were labeled  and chased as described above at either 26oC or 38oC, and Vps10p  was recovered from the lysates as described above. Vps10p was  completely stabilized by inactivation of Pep12p, indicating that it  never reached the class E compartment and hence that Pep12p is  epistatic to Vps35p.
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Figure 9: Pep12p is epistatic to Vps35p. (A) The strain MSY 0435 (Δvps4Δvps35) harboring plasmids encoding either wild-type VPS35 (pGPY35) or the temperature conditional allele of vps35 (pMS35-4ts) was shifted to 38°C 15 min before labeling with [35S]methionine. After labeling for 10 min, excess unlabeled methionine was added and the cells were chased for the specified times. Aliquots of cells (2 OD equivalents) were removed at the specified times, the cells were lysed and Vps10p was recovered by immunoprecipitation. Vps10p was clipped (clipped Vps10p denoted by *) with a half-time of ∼30 min which is typical for a class E mutant. The clipping of Vps10p is unaffected by inactivation of Vps35p. (B) The strain MSY4012 (vps4Δpep12) harboring the plasmid encoding the temperature conditional allele of pep12 (pCBY9) was grown at 26oC, before being shifted to the specified temperature for 10 min before labeling. The cells were labeled and chased as described above at either 26oC or 38oC, and Vps10p was recovered from the lysates as described above. Vps10p was completely stabilized by inactivation of Pep12p, indicating that it never reached the class E compartment and hence that Pep12p is epistatic to Vps35p.
Mentions: In certain vps mutants, namely the class E mutants, the prevacuolar endosome becomes enlarged and acidic with active proteases contained within and is termed the “class E compartment” (Raymond et al., 1992; Reider et al., 1996; Piper et al., 1996). Vps10p traffics through this compartment in class E mutants and is proteolytically clipped with a half time of ∼30 min (Cereghino et al., 1995; Cooper and Stevens, 1996). The finding that Vps35p appears to be associated with membranes enriched in Kex2p as well as Vps10p (see Fig. 6) raised the possibility that Vps35p might also act at the late-Golgi, perhaps to affect the exit rate of Vps10p. Any effect that inactivation of Vps35p might have on the exit of Vps10p from the late-Golgi could manifest itself as an effect upon the kinetics of the clipping of Vps10p in a class E mutant. To address this question, a class E Δvps35 mutant was constructed using a mutant of vps4 as a representative class E mutant (Babst et al., 1997). This mutant was transformed with plasmids containing either wild-type VPS35 or the vps35ts mutant gene. The clipping of Vps10p in these strains was investigated and is shown in Fig. 9 A. Cells were incubated for 15 min at 38°C to inactivate the temperature conditional allele of vps35 before labeling with [35S]methionine for 10 min, after which an excess of unlabeled methionine was added. Aliquots of cells were removed at 0 min of chase and also after 30 min and 60 min. After cell lysis, Vps10p was recovered by immunoprecipitation. As shown in Fig. 9 A, cells harboring the wild-type VPS35 allele (lanes 1–3) have ∼50% clipped Vps10p (denoted by the *) after a 30-min chase, a phenotype which is identical to class E mutant alone (Cereghino et al., 1995). Cells which contained the temperature conditional allele of vps35 (Fig. 9 A, lanes 4–6) showed no significant difference in the kinetics of the clipping of Vps10p, suggesting that inactivation of Vps35p does not affect the exit rate of Vps10p from the late-Golgi. By contrast, however, inactivation of Pep12p in a class E mutant completely prevents the clipping of Vps10p. In Fig. 9 B, cells containing a mutant allele of vps4 and also the temperature conditional allele of pep12 (Cowles et al., 1997) were pulsed for 10 min with [35S]methionine and chased at either 26°C or 38°C as described above. Vps10p was recovered from the aliquots of cells removed after the specified chase times. At permissive temperature, Vps10p is clipped with normal kinetics for a class E mutant; however, upon raising the temperature to 38°C and inactivation of Pep12p, Vps10p is stabilized and is not proteolytically clipped even after 60 min of chase. These data strongly suggest that pep12ts is epistatic to vps35 and that the vps35 mutation does not affect the exit of Vps10p from the Golgi in any way.

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