Limits...
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

Related in: MedlinePlus

Proposed role of Vps29p, Vps30p, and Vps35p. (A) In  wild-type cells, the VPS29, VPS30, and VPS35 gene products all  play a role in the recycling of Vps10p and other late-Golgi proteins (e.g., Kex2p and DPAP A) from the endosome back to the  TGN. Vps10p is efficiently sorted and retrieved from the prevacuolar endosomal compartment and returned to the late-Golgi.  (B) In the absence of Vps29p, Vps30p, or Vps35p, Vps10p (and  Kex2p and DPAP A) is transported by default to the vacuole and  becomes limiting in the late-Golgi resulting in the secretion of  p2CPY. Disruption of VPS35 does not block arrival of Vps10p in  the class E compartment/endosome in a vps4 mutant; however,  transport of Vps10p to the vacuole in a vps29, vps30, or vps35  mutant requires a functional endosomal t-SNARE, Pep12p, but  does not require Sec1p-dependent arrival at the plasma membrane and subsequent endocytosis.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139870&req=5

Figure 10: Proposed role of Vps29p, Vps30p, and Vps35p. (A) In wild-type cells, the VPS29, VPS30, and VPS35 gene products all play a role in the recycling of Vps10p and other late-Golgi proteins (e.g., Kex2p and DPAP A) from the endosome back to the TGN. Vps10p is efficiently sorted and retrieved from the prevacuolar endosomal compartment and returned to the late-Golgi. (B) In the absence of Vps29p, Vps30p, or Vps35p, Vps10p (and Kex2p and DPAP A) is transported by default to the vacuole and becomes limiting in the late-Golgi resulting in the secretion of p2CPY. Disruption of VPS35 does not block arrival of Vps10p in the class E compartment/endosome in a vps4 mutant; however, transport of Vps10p to the vacuole in a vps29, vps30, or vps35 mutant requires a functional endosomal t-SNARE, Pep12p, but does not require Sec1p-dependent arrival at the plasma membrane and subsequent endocytosis.

Mentions: Each of these mutants change the subcellular localization of the Vps10 sorting receptor. The receptor is shifted from a high speed pellet (P100) fraction which is enriched in Golgi, endosomal, and vesicular membranes, to a low speed pellet (P13) fraction which contains membranes from several organelles including the vacuole (Fig. 5 A). The shift of Vps10p from a Golgi/endosome-enriched membrane fraction to a low speed pellet fraction also occurs in the vps35ts mutant cells at the nonpermissive temperature, resulting in very little (∼20%) Vps10p remaining in the Golgi/endosome P100 fraction. Immunofluorescence experiments with a Myc-tagged version of Vps10p demonstrated that Vps10p was mislocalized to the vacuolar membrane in vps29, vps30, and vps35 mutants, but not in wildtype cells or other vps mutants that also were tested (Fig. 7). Furthermore, Vps10p appears to follow a direct route to the vacuole. It does not arrive at the vacuole via endocytosis from the plasma membrane as a double sec1 Δvps35 mutant failed to block Vps10p delivery; however, inactivation of a t-SNARE required for Golgi to endosome transport (Pep12p) did prevent the mislocalization of Vps10p to a P13 membrane fraction in a pep12ts Δvps35 double mutant (see Fig. 8). These data show that VPS29, VPS30, and VPS35 play a role in controlling the intracellular location of Vps10p and probably act at the prevacuolar endosome to direct the recycling of Vps10p to the Golgi (see Fig. 10).


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)

Proposed role of Vps29p, Vps30p, and Vps35p. (A) In  wild-type cells, the VPS29, VPS30, and VPS35 gene products all  play a role in the recycling of Vps10p and other late-Golgi proteins (e.g., Kex2p and DPAP A) from the endosome back to the  TGN. Vps10p is efficiently sorted and retrieved from the prevacuolar endosomal compartment and returned to the late-Golgi.  (B) In the absence of Vps29p, Vps30p, or Vps35p, Vps10p (and  Kex2p and DPAP A) is transported by default to the vacuole and  becomes limiting in the late-Golgi resulting in the secretion of  p2CPY. Disruption of VPS35 does not block arrival of Vps10p in  the class E compartment/endosome in a vps4 mutant; however,  transport of Vps10p to the vacuole in a vps29, vps30, or vps35  mutant requires a functional endosomal t-SNARE, Pep12p, but  does not require Sec1p-dependent arrival at the plasma membrane and subsequent endocytosis.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: Proposed role of Vps29p, Vps30p, and Vps35p. (A) In wild-type cells, the VPS29, VPS30, and VPS35 gene products all play a role in the recycling of Vps10p and other late-Golgi proteins (e.g., Kex2p and DPAP A) from the endosome back to the TGN. Vps10p is efficiently sorted and retrieved from the prevacuolar endosomal compartment and returned to the late-Golgi. (B) In the absence of Vps29p, Vps30p, or Vps35p, Vps10p (and Kex2p and DPAP A) is transported by default to the vacuole and becomes limiting in the late-Golgi resulting in the secretion of p2CPY. Disruption of VPS35 does not block arrival of Vps10p in the class E compartment/endosome in a vps4 mutant; however, transport of Vps10p to the vacuole in a vps29, vps30, or vps35 mutant requires a functional endosomal t-SNARE, Pep12p, but does not require Sec1p-dependent arrival at the plasma membrane and subsequent endocytosis.
Mentions: Each of these mutants change the subcellular localization of the Vps10 sorting receptor. The receptor is shifted from a high speed pellet (P100) fraction which is enriched in Golgi, endosomal, and vesicular membranes, to a low speed pellet (P13) fraction which contains membranes from several organelles including the vacuole (Fig. 5 A). The shift of Vps10p from a Golgi/endosome-enriched membrane fraction to a low speed pellet fraction also occurs in the vps35ts mutant cells at the nonpermissive temperature, resulting in very little (∼20%) Vps10p remaining in the Golgi/endosome P100 fraction. Immunofluorescence experiments with a Myc-tagged version of Vps10p demonstrated that Vps10p was mislocalized to the vacuolar membrane in vps29, vps30, and vps35 mutants, but not in wildtype cells or other vps mutants that also were tested (Fig. 7). Furthermore, Vps10p appears to follow a direct route to the vacuole. It does not arrive at the vacuole via endocytosis from the plasma membrane as a double sec1 Δvps35 mutant failed to block Vps10p delivery; however, inactivation of a t-SNARE required for Golgi to endosome transport (Pep12p) did prevent the mislocalization of Vps10p to a P13 membrane fraction in a pep12ts Δvps35 double mutant (see Fig. 8). These data show that VPS29, VPS30, and VPS35 play a role in controlling the intracellular location of Vps10p and probably act at the prevacuolar endosome to direct the recycling of Vps10p to the Golgi (see Fig. 10).

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