We previously reported the initial characterization of the S. cerevisiae
protein Vps10p, the sorting receptor for the vacuolar hydrolase CPY (Marcusson et al., 1994
; Cereghino et al., 1995
). Mutations in VPS10
lead to a selective vacuolar missorting phenotype in which CPY is missorted and secreted from the cell while the majority of PrA is properly sorted and matured. In this paper and previously (Paravicini et al., 1992
), we show that mutations in three other VPS
) lead to the same selective CPY missorting phenotype (Fig. A
). A temperature conditional allele of vps35
missorts and secretes progressively more CPY with increasing incubation at the nonpermissive temperature. The relatively slow onset (~15 min) of the CPY missorting phenotype (relative to either the vps34ts
[Stack et al., 1995
] or the pep12ts
[Cowles et al., 1997
]) suggests that either the Vps35 protein is inactivated slowly at the nonpermissive temperature, or that inactivation of Vps35p results in the gradual depletion of a factor(s) required for CPY sorting. Analysis of the other vps35ts
alleles isolated at the same time indicated that they all exhibited a slow onset of CPY secretion, hence we favor the latter explanation.
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. 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. ). 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. ). 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. ).
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 (more ...)
In mammalian cells, the MPRs sort lysosomal enzymes away from secreted proteins in the TGN. MPRs plus cargo are then concentrated in clathrin-coated vesicles and subsequently delivered to a prelysosomal endocytic compartment (Kornfeld, 1992
). The pathway taken by the Vps10 receptor in the sorting of yeast vacuolar hydrolases is very similar to the MPRs. Vps10p binds vacuolar hydrolases in the late-Golgi/TGN (Graham and Emr, 1991
) and transports them to the prevacuolar endosome in a process that is altered shortly after shifting a clathrin heavy chain (CHC) temperature sensitive mutant to the nonpermissive temperature, thus suggesting a role for CHC in this process (Seeger and Payne, 1992
). In the endosome, the hydrolases must be released and the unoccupied Vps10 receptor is then recycled back to the Golgi for further rounds of sorting. The results of our analysis suggest that this recycling step is dependent on VPS29
, and VPS35
. When any of these genes are mutated, Vps10p is not able to recycle back to the TGN and instead is mislocalized to the vacuole, presumably via default flow of membrane proteins in the yeast secretory pathway which leads to the vacuole (Roberts et al., 1992
; Wilcox et al., 1992
). With the recycling pathway inoperative, the Vps10 receptor becomes limiting for CPY sorting, causing CPY to be secreted from the cell by the default pathway for soluble proteins. It has been recently demonstrated that Vps10p is also responsible for sorting some PrA (Cooper and Stevens, 1996
; Westphal et al., 1996
); however, PrA can be delivered to the vacuole in a Vps10p-independent manner. This Vps10p-independent PrA sorting is very sensitive to media pH, (which presumably affects the pH of the lumen of intracellular organelles) as acidic media was required for efficient PrA sorting. It seems possible that PrA may be delivered to the vacuole by “piggybacking” on a membrane protein(s) destined for the vacuole. This interaction may be very sensitive to pH, and hence efficient PrA sorting requires low pH. In this way, PrA could arrive at the vacuole in a manner independent of a sorting receptor that requires efficient retrieval from the endosome, a process that would be defective in vps29
Our experiments suggest a role for the Vps29, Vps30, and Vps35 proteins not only for the trafficking of Vps10p, but also for other proteins that cycle between the TGN and the endosome, such as Kex2p. The subcellular location of Kex2p is also changed in vps29, vps30, and vps35 mutants resulting in a reduction in the half life of Kex2p (Table ). Consistent with this conclusion, a hybrid protein in which the cytoplasmic tail domain of Vps10p was replaced by the cytoplasmic tail of Kex2p resulted in efficient sorting of CPY to the vacuole when expressed in a Δvps10 mutant strain (Marcusson, E.G., and S.D. Emr, unpublished observations). However, this hybrid protein cannot suppress either Δvps10/Δvps29 or Δvps10/Δvps35 double mutants. This indicates that the Vps10 Kex2 hybrid protein can replace Vps10p, but its transport still requires Vps29p, Vps30p, and Vps35p function.
Vps35p is a peripheral membrane protein which probably associates with a transmembrane protein(s) via an ionic interaction as Vps35p can be removed from the membrane pellet fraction by washing with 1 M NaCl or 1% Triton X-100 (Paravicini et al., 1992
). The subcellular fractionation pattern of Vps35p mimics that of Vps10p, both in wildtype cells and vps29
(and partially vps30
) mutant cells. The distribution of Vps35p mimics that of Vps10p to the extent that sucrose gradient fractionation of a Golgi/endosome membrane-enriched fraction which was able to resolve Vps10p distribution into two discrete peaks indicated that Vps35p was associated with both these pools of Vps10p containing membranes. It appears therefore that the localization of Vps10p (along with proteins such as Kex2p and DPAP A) is the crucial factor in determining Vps35p localization.
What role might Vps35p, Vps30p, and Vps29p play in retrieval of Vps10p? One possibility is that these proteins act at the late-Golgi to restrain Vps10p and delay its exit in order to allow for cargo binding by Vps10p. Thus, in the absence of Vps35p, Vps30p, or Vps29p, Vps10p exits the late- Golgi before it has bound CPY and subsequently accumulates in the prevacuolar endosome resulting in saturation of the retrieval machinery. Vps10p would then become limiting in the late-Golgi and CPY would be secreted. While this possibility cannot be formally excluded, it seems unlikely because in a class E vps35ts double mutant, the kinetics of the clipping of Vps10p are unaltered by the inactivation of Vps35p (Fig. ). Furthermore, inactivation of Pep12p in a class E pep12ts double mutant completely prevented clipping of Vps10p (Fig. ). Thus, inactivation of Vps35p has no effect upon exit from the Golgi and arrival at the endosome/class E compartment, and PEP12 is epistatic to VPS35 which is consistent with a role for Vps35p in the recycling of Vps10p from the endosome back to the Golgi.
The data presented here are consistent with a model in which Vps35p, Vps30p, and Vps29p act in the retrieval of Vps10p from the prevacuolar endosome (see Fig. ). The finding that the localization of Vps35p appears to be very significantly affected by the distribution of Vps10p raises the possibility that Vps35p interacts with the cytoplasmic tail of Vps10p (and other proteins such as Kex2p and dipeptidylaminopeptidase A [DPAP A]). One possibility is that Vps35p is required for cargo selection at the endosome during formation of recycling vesicles destined for the Golgi. Vps29p might act in a similar fashion, possibly in a complex with Vps35p and other proteins. As Vps30p is predominantly a soluble protein, of which only a small fraction is membrane associated, it seems likely that Vps30p acts at a slightly different step. It is interesting to note that in vps30
cells Kex2p has a longer half-life compared to the half-life of Kex2p in vps29
cells (Table ), suggesting that it is not mislocalized to the vacuole as quickly as it is in vps29
cells. Thus, significant amounts of Kex2p and possibly other proteins such as DPAP A will remain in the late-Golgi and endosomes. This could account for the reduced shift of Vps35p to the vacuolar fraction in vps30
mutants. Consistent with this, a vps35
mutant was recently identified by a completely different genetic approach designed to identify mutants that were unable to properly localize another late-Golgi protein, DPAP A (Nothwehr et al., 1996
). DPAP A is a type II transmembrane protein which, like Vps10p and Kex2p, contains an aromatic motif which has been shown to be important for maintaining the protein in the Golgi/endosome pathway (Wilcox et al., 1992
; Nothwehr and Stevens, 1994
; Cereghino et al., 1995
; Cooper and Stevens, 1996
; Bryant and Stevens, 1996
). This observation, combined with the data presented in this paper, indicates that Vps35p may play a general role in recycling transmembrane proteins (both type I and type II) back to the late Golgi apparatus, possibly by interaction with the aromatic residue-containing sorting signals present in these proteins. Consistent with this hypothesis is the finding that the localization of the type II resident Golgi membrane protein, Mnn1p, is not affected in vps29
mutants. Mnn1p does not have an aromatic residue-containing sorting signal in its cytoplasmic tail domain (Graham et al., 1994
While the precise molecular mechanisms involved in Vps35p, Vps30p, and Vps29p function remain to be elucidated, it seems likely that these proteins act together to retrieve not only Vps10p, but also Kex2p and DPAP A from a prevacuolar endosome to the late-Golgi. Conservation between yeast and higher eukaryotes of the proteins involved in membrane trafficking has recently been shown to occur at virtually every membrane traffic step in the secretory and endocytic pathways (reviewed in Rothman, 1994
; Schekman and Orci, 1996
; Pfeffer, 1996
). The fact that Vps35p, Vps30p, and Vps29p are also conserved in higher eukaryotes underscores their important roles in membrane trafficking, and it is likely that these proteins will have similar functions in all eukaryotes.