Endosome to Golgi retrieval of the vacuolar protein sorting receptor, Vps10p, requires the function of the VPS29, VPS30, and VPS35 gene products

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.

Figure 1

Selective missorting of vacuolar proteins. (A) Yeast cells were pulse-labeled with Tran³⁵S for 10 min and chased for 30 min. The cells were then spheroplasted and separated into intracellular (I) and extracellular (E) fractions. CPY or PrA was immunoprecipitated from both fractions and resolved by SDSPAGE and fluorography. The strains used were SEY6210 (WT), EMY3 (Δvps10), PSY129 (vps29), and JCY3000 (Δvps30). The position and molecular weights of both precursor and mature forms of CPY and PrA are indicated. (B) vps35ts cells (MSY354ts) were incubated at either 26°C for 0 min before labeling or 38°C for the specified time. The cells were labeled for 5 min and chased as above, and then spheroplasted and separated into intracellular (I) and extracellular (E) fractions. CPY was immunoprecipitated from both fractions and resolved by SDS-PAGE and fluorography.

Figure 2

Complementation of the vps29 and vps30 mutants. Yeast cells were pulse-labeled with Tran³⁵S for 10 min and chased for 30 min. The cells were then spheroplasted and separated into intracellular (I) and extracellular (E) fractions. CPY or PrA was immunoprecipitated from both fractions and resolved by SDSPAGE and fluorography. The strains used were PSY129 (vps29), PSY129 carrying a centromeric plasmid containing the VPS29 gene (PSY129 + pVPS29), JCY3000 (Δvps30), and JCY3000 carrying a centromeric plasmid containing the VPS30 gene (JCY3000 + pVPS30).

Figure 3

VPS35, VPS30, and VPS29 are highly conserved in eukaryotes. (A) Schematic view of homology between Vps35p and proteins found in the C. elegans and M. musculus proteomes. The numbers between the proteins are percent identities between Vps35p and the other two proteins. In the lower section, the two most highly conserved domains (designated domains I and III) are aligned to show the regions of greatest homology between the yeast, mouse, nematode, and also human homologues. The black boxes indicate completely conserved residues. (B) Alignments between Vps29p and homologues present in the proteomes of C. elegans and H. sapiens, and also alignments between Vps30p and homologues present in the proteomes of C. elegans and H. sapiens. Black shaded regions indicate areas that are completely conserved.

Figure 4

Detection of Vps30p. (A) Yeast cells were pulselabeled with Tran³⁵S for 20 min and chased for either 0 or 60 min. The position of Vps30p is indicated. The strains used were JCY3000 (Δvps30), SEY6210 (WT), and SEY6210 carrying a 2-μ plasmid containing the VPS30 gene (WT + 2 μ VPS30). (B) Wildtype spheroplasts (SEY6210) were pulse-labeled with Tran³⁵S for 20 min and chased for 60 min. The spheroplasts were then gently lysed by dounce homogenization in a hypotonic buffer and then washed in either 10 mM Tris/1 mM EDTA, pH 8.0 (Buffer), 1 M NaCl, or 3 M urea. The homogenates were then spun in an ultracentrifuge at 100,000 g. The pellet (P) and supernatant (S) fractions were then immunoprecipitated with antiserum specific to Vps30p and resolved by SDS-PAGE.

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).

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.

Figure 7

Vps10p is localized to the vacuole in vps29, vps30, and vps35 mutants. Using an epitope-tagged version of Vps10p expressed at centromeric levels, Vps10p protein was localized in wild-type cells, vps29, Δvps30 and Δvps35 cells, and also Δpep12/ vps6 cells. In wild-type cells the labeling pattern appeared punctate and characteristic of a Golgi and endosomal localization. In the vps29, Δvps30, and Δvps35 cells, Vps10p was localized to the vacuolar membrane. The redistribution of Vps10p to the vacuolar membrane is not a general phenomenon associated with disruption of the VPS pathway as in Δpep12/vps6 cells; Vps10p has a punctate distribution indistinguishable from wild-type cells.

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.

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 26^oC, before being shifted to the specified temperature for 10 min before labeling. The cells were labeled and chased as described above at either 26^oC or 38^oC, 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.

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.