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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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Vps35p is associated with two discrete pools of light  membranes. Wild-type cells were spheroplasted, labeled with  [35S]methionine for 15 min, and then chased for 45 min. After lysis by dounce homogenization, large membranes were cleared by  centrifugation at 13,000 g for 10 min. The cleared lysate was then  loaded onto a 10–60% sucrose gradient and spun to equilibrium.  12 fractions were collected; Vps35p, Vps10p, and Kex2p were immunoprecipitated from the fractions and subjected to SDSPAGE and fluorography. The autoradiogram shows the distribution of Vps35p in the gradient, and the graph shows the amounts  of Vps10p and Kex2p in each fraction. Vps35p appears to associate with two discrete pools of membrane.
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Figure 6: Vps35p is associated with two discrete pools of light membranes. Wild-type cells were spheroplasted, labeled with [35S]methionine for 15 min, and then chased for 45 min. After lysis by dounce homogenization, large membranes were cleared by centrifugation at 13,000 g for 10 min. The cleared lysate was then loaded onto a 10–60% sucrose gradient and spun to equilibrium. 12 fractions were collected; Vps35p, Vps10p, and Kex2p were immunoprecipitated from the fractions and subjected to SDSPAGE and fluorography. The autoradiogram shows the distribution of Vps35p in the gradient, and the graph shows the amounts of Vps10p and Kex2p in each fraction. Vps35p appears to associate with two discrete pools of membrane.

Mentions: To better characterize the compartment to which Vps35p is localized in wild-type cells, a more detailed fractionation of the light P100 membranes was carried out. Wild-type cells were spheroplasted before labeling for 15 min at 30°C and then chasing for 45 min as before. The spheroplasts were lysed as described in the Materials and Methods section, and a fraction containing the Golgi and endosomal membranes (P100) was generated by centrifugation at 13,000 g which pellets vacuolar membranes, plasma membrane, and endoplasmic reticulum to produce a supernatant that contained only P100 membranes and soluble cytosolic proteins. This was loaded onto the top of a sucrose gradient and spun to equilibrium. Twelve fractions were collected and the distribution of Vps35p, Vps10p, and Kex2p was analyzed by immunoprecipitation of the respective proteins. In Fig. 6, the amount of Vps35p in each fraction is shown by the autoradiogram, while the distribution of Vps10p and Kex2p in shown in the graph. It appears that Vps10p and Kex2p fractionate in two discrete peaks and that Vps35p is associated with both of these peaks. It is possible that these peaks correspond to Golgi membranes (fractions 3–4) and lighter endosomal membranes (fraction 6). In previous studies it has been observed that p2CPY can be separated into two discrete peaks of light membranes (Vida et al., 1993) and that a marker for endosomes, Pep12p, fractionates away from Kex2p on similar gradients (Becherer et al., 1996). Hence, it appears that the distribution of Vps35p mirrors that of the proteins that cycle between the late-Golgi and the endosome.


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)

Vps35p is associated with two discrete pools of light  membranes. Wild-type cells were spheroplasted, labeled with  [35S]methionine for 15 min, and then chased for 45 min. After lysis by dounce homogenization, large membranes were cleared by  centrifugation at 13,000 g for 10 min. The cleared lysate was then  loaded onto a 10–60% sucrose gradient and spun to equilibrium.  12 fractions were collected; Vps35p, Vps10p, and Kex2p were immunoprecipitated from the fractions and subjected to SDSPAGE and fluorography. The autoradiogram shows the distribution of Vps35p in the gradient, and the graph shows the amounts  of Vps10p and Kex2p in each fraction. Vps35p appears to associate with two discrete pools of membrane.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139870&req=5

Figure 6: Vps35p is associated with two discrete pools of light membranes. Wild-type cells were spheroplasted, labeled with [35S]methionine for 15 min, and then chased for 45 min. After lysis by dounce homogenization, large membranes were cleared by centrifugation at 13,000 g for 10 min. The cleared lysate was then loaded onto a 10–60% sucrose gradient and spun to equilibrium. 12 fractions were collected; Vps35p, Vps10p, and Kex2p were immunoprecipitated from the fractions and subjected to SDSPAGE and fluorography. The autoradiogram shows the distribution of Vps35p in the gradient, and the graph shows the amounts of Vps10p and Kex2p in each fraction. Vps35p appears to associate with two discrete pools of membrane.
Mentions: To better characterize the compartment to which Vps35p is localized in wild-type cells, a more detailed fractionation of the light P100 membranes was carried out. Wild-type cells were spheroplasted before labeling for 15 min at 30°C and then chasing for 45 min as before. The spheroplasts were lysed as described in the Materials and Methods section, and a fraction containing the Golgi and endosomal membranes (P100) was generated by centrifugation at 13,000 g which pellets vacuolar membranes, plasma membrane, and endoplasmic reticulum to produce a supernatant that contained only P100 membranes and soluble cytosolic proteins. This was loaded onto the top of a sucrose gradient and spun to equilibrium. Twelve fractions were collected and the distribution of Vps35p, Vps10p, and Kex2p was analyzed by immunoprecipitation of the respective proteins. In Fig. 6, the amount of Vps35p in each fraction is shown by the autoradiogram, while the distribution of Vps10p and Kex2p in shown in the graph. It appears that Vps10p and Kex2p fractionate in two discrete peaks and that Vps35p is associated with both of these peaks. It is possible that these peaks correspond to Golgi membranes (fractions 3–4) and lighter endosomal membranes (fraction 6). In previous studies it has been observed that p2CPY can be separated into two discrete peaks of light membranes (Vida et al., 1993) and that a marker for endosomes, Pep12p, fractionates away from Kex2p on similar gradients (Becherer et al., 1996). Hence, it appears that the distribution of Vps35p mirrors that of the proteins that cycle between the late-Golgi and the endosome.

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