Alternative pathways for the sorting of soluble vacuolar proteins in yeast: a vps35 null mutant missorts and secretes only a subset of vacuolar hydrolases.

vps35 mutants of Saccharomyces cerevisiae exhibit severe defects in the localization of carboxypeptidase Y, a soluble vacuolar hydrolase. We have cloned the wild-type VPS35 gene by complementation of the vacuolar protein sorting defect exhibited by the vps35-17 mutant. Sequence analysis revealed an open reading frame predicted to encode a protein of 937 amino acids that lacks any obvious hydrophobic domains. Subcellular fractionation studies indicated that 80% of Vps35p peripherally associates with a membranous particulate cell fraction. The association of Vps35p with this fraction appears to be saturable; when overproduced, the vast majority of Vps35p remains in a soluble fraction. Disruption of the VPS35 gene demonstrated that it is not essential for yeast cell growth. However, the null allele of VPS35 results in a differential defect in the sorting of vacuolar carboxypeptidase Y (CPY), proteinase A (PrA), proteinase B (PrB), and alkaline phosphatase (ALP). proCPY was quantitatively missorted and secreted by delta vps35 cells, whereas almost all of proPrA, proPrB, and proALP were retained within the cell and converted to their mature forms, indicating delivery to the vacuole. Based on these observations, we propose that alternative pathways exist for the sorting and/or delivery of proteins to the vacuole.     

Vps10p cycles between the late-Golgi and prevacuolar compartments in its function as the sorting receptor for multiple yeast vacuolar hydrolases

VPS10 (Vacuolar Protein Sorting) encodes a large type I transmembrane protein (Vps10p), involved in the sorting of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) to the Saccharomyces cerevisiae lysosome-like vacuole. Cells lacking Vps10p missorted greater than 90% CPY and 50% of another vacuolar hydrolase, PrA, to the cell surface. In vitro equilibrium binding studies established that the 1,380-amino acid lumenal domain of Vps10p binds CPY precursor in a 1:1 stoichiometry, further supporting the assignment of Vps10p as the CPY sorting receptor. Vps10p has been immunolocalized to the late-Golgi compartment where CPY is sorted away from the secretory pathway. Vps10p is synthesized at a rate 20-fold lower that that of its ligand CPY, which in light of the 1:1 binding stoichiometry, requires that Vps10p must recycle and perform multiple rounds of CPY sorting. The 164-amino acid Vps10p cytosolic domain is involved in receptor trafficking, as deletion of this domain resulted in delivery of the mutant Vps10p to the vacuole, the default destination for membrane proteins in yeast. A tyrosine-based signal (YSSL80) within the cytosolic domain enables Vps10p to cycle between the late-Golgi and prevacuolar/endosomal compartments. This tyrosine-based signal is homologous to the recycling signal of the mammalian mannose-6-phosphate receptor. A second yeast gene, VTH2, encodes a protein highly homologous to Vps10p which, when over-produced, is capable of suppressing the CPY and PrA missorting defects of a vps10 delta strain. These results indicate that a family of related receptors act to target soluble hydrolases to the vacuole.     

Distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar protease

We have mapped a sequence determinant in the vacuolar glycoprotein carboxypeptidase Y (CPY) that directs intracellular sorting of this enzyme. Through the study of hybrid proteins, consisting of amino-terminal segments of CPY fused to the secretory enzyme invertase, we have found that the N-terminal 50 amino acids of CPY are sufficient to direct delivery of a CPY-Inv hybrid protein to the yeast vacuole. Our data suggest that this 50 amino acid segment of CPY contains two distinct functional domains; an N-terminal signal peptide followed by a segment of 30 amino acids that contains the vacuolar sorting signal. Deletion of this putative vacuole sorting signal from an otherwise wild-type CPY protein leads to missorting of CPY. Furthermore, examination of the Asn-linked oligosaccharides present on CPY and CPY-Inv hybrid proteins suggests that an additional determinant in CPY specifies the extent to which these proteins are glycosylated in the Golgi complex.     

Yeast vacuolar proenzymes are sorted in the late Golgi complex and transported to the vacuole via a prevacuolar endosome-like compartment

We are studying intercompartmental protein transport to the yeast lysosome-like vacuole with a reconstitution assay using permeabilized spheroplasts that measures, in an ATP and cytosol dependent reaction, vacuolar delivery and proteolytic maturation of the Golgi-modified precursor forms of vacuolar hydrolases like carboxypeptidase Y (CPY). To identify the potential donor compartment in this assay, we used subcellular fractionation procedures that have uncovered a novel membrane-enclosed prevacuolar transport intermediate. Differential centrifugation was used to separate permeabilized spheroplasts into 15K and 150K g membrane pellets. Centrifugation of these pellets to equilibrium on sucrose density gradients separated vacuolar and Golgi complex marker enzymes into light and dense fractions, respectively. When the Golgi-modified precursor form of CPY (p2CPY) was examined (after a 5-min pulse, 30-s chase), as much as 30-40% fractionated with an intermediate density between both the vacuole and the Golgi complex. Pulse-chase labeling and fractionation of membranes indicated that p2CPY in this gradient region had already passed through the Golgi complex, which kinetically ordered it between the Golgi and the vacuole. A mutant CPY protein that lacks a functional vacuolar sorting signal was detected in Golgi fractions but not in the intermediate compartment indicating that this corresponds to a post-sorting compartment. Based on the low transport efficiency of the mutant CPY protein in vitro (decreased by sevenfold), this intermediate organelle most likely represents the donor compartment in our reconstitution assay. This organelle is not likely to be a transport vesicle intermediate because EM analysis indicates enrichment of 250-400 nm compartments and internalization of surface-bound 35S-alpha-factor at 15 degrees C resulted in its apparent cofractionation with wild-type p2CPY, indicating an endosome-like compartment (Singer, B., and H. Reizman. 1990. J. Cell Biol. 110:1911-1922). Fractionation of p2CPY accumulated in the temperature sensitive vps15 mutant revealed that the vps15 transport block did not occur in the endosome-like compartment but rather in the late Golgi complex, presumably the site of CPY sorting. Therefore, as seen in mammalian cells, yeast CPY is sorted away from secretory proteins in the late Golgi and transits to the vacuole via a distinct endosome-like intermediate.     

Novel Golgi to vacuole delivery pathway in yeast: identification of a sorting determinant and required transport component.

More than 40 vacuolar protein sorting (vps) mutants have been identified which secrete proenzyme forms of soluble vacuolar hydrolases to the cell surface. A subset of these mutants has been found to show selective defects in the sorting of two vacuolar membrane proteins. Under non-permissive conditions, vps45tsf (SEC1 homolog) and pep12/vps6tsf (endosomal t-SNARE) mutants efficiently sort alkaline phosphatase (ALP) to the vacuole while multiple soluble vacuolar proteins and the membrane protein carboxypeptidase yscS (CPS) are no longer delivered to the vacuole. Vacuolar localization of ALP in these mutants does not require transport to the plasma membrane followed by endocytic uptake, as double mutants of pep12tsf and vps45tsf with sec1 and end3 sort and mature ALP at the non-permissive temperature. Given the demonstrated role of t-SNAREs such as Pep12p in transport vesicle recognition, our results indicate that ALP and CPS are packaged into distinct transport intermediates. Consistent with ALP following an alternative route to the vacuole, isolation of a vps41tsf mutant revealed that at non-permissive temperature ALP is mislocalized while vacuolar delivery of CPS and CPY is maintained. A series of domain-swapping experiments was used to define the sorting signal that directs selective packaging and transport of ALP. Our data demonstrate that the amino-terminal 16 amino acid portion of the ALP cytoplasmic tail domain contains a vacuolar sorting signal which is responsible for the active recognition, packaging and transport of ALP from the Golgi to the vacuole via a novel delivery pathway.     

Intracellular sorting and processing of a yeast vacuolar hydrolase: proteinase A propeptide contains vacuolar targeting information.

An inactive precursor form of proteinase A (PrA) transits through the early secretory pathway before final vacuolar delivery. We used gene fusions between the gene coding for PrA (PEP4) and the gene coding for the secretory enzyme invertase (SUC2) to identify vacuolar protein-sorting information in the PrA precursor. We found that the 76-amino-acid preprosegment of PrA contains at least two sorting signals: an amino-terminal signal peptide that is cleaved from the protein at the level of the endoplasmic reticulum followed by the prosegment which functions as a vacuolar protein-sorting signal. PrA-invertase hybrid proteins that carried this sequence information were accurately sorted to the yeast vacuole as determined by cell fractionation and immunolocalization studies. Hybrid proteins lacking all or a portion of the PrA prosegment were secreted from the cell. Our gene fusion data together with an analysis of the wild-type PrA protein indicated that N-linked carbohydrate modifications are not required for vacuolar sorting of this protein. Furthermore, results obtained with a set of deletion mutations constructed in the PrA prosegment indicated that this sequence also contributes to proper folding of this polypeptide into a stable transit-competent molecule.     

Gene dosage-dependent secretion of yeast vacuolar carboxypeptidase Y

The structural gene for yeast vacuolar carboxypeptidase Y (PRC1) has been cloned by complementation of the prc1-1 mutation. As much as an eightfold elevation in the level of carboxypeptidase Y (CPY) results when a multiple-copy plasmid containing the PRC1 gene is introduced into yeast. Unlike the situation with a single copy of PRC1 in which newly synthesized CPY is efficiently localized to the vacuole, plasmid-directed overproduction results in secretion of greater than 50% of the protein as the precursor form. Secretion is blocked in a mutant that is defective at a late stage in the transport of periplasmic proteins. Unlike normal cell surface glycoproteins, secreted CPY precursor acquires no additional oligosaccharide modifications beyond those that accompany normal transport to the vacuole. In the periplasm, the CPY precursor is proteolytically activated to an enzymatically active form by an enzyme that is unrelated to the vacuolar processing enzyme. These findings suggest that proper sorting and transport of CPY is saturable. This may reflect limiting amounts of a CPY-sorting receptor, or of CPY-modifying machinery that is essential for recognition by such a receptor.     

A role for clathrin in the sorting of vacuolar proteins in the Golgi complex of yeast.

We have investigated the role of clathrin in vacuolar protein sorting using yeast strains harboring a temperature-sensitive allele of clathrin heavy chain (chc1-ts). After a 5 min incubation at the non-permissive temperature (37 degrees C), the chc1-ts strains displayed a severe defect in the sorting of lumenal vacuolar proteins. Sorting of a vacuolar membrane protein, alkaline phosphatase, and transport to the surface of a cell wall protein, was not affected at 37 degrees C. In chc1-ts cells incubated at 37 degrees C, secretion of the missorted lumenal vacuolar protein carboxypeptidase Y (CPY) was blocked by the sec1 mutation which prevents fusion of secretory vesicles to the plasma membrane. Unexpectedly, chc1-ts cells incubated for extended periods at 37 degrees C regained the ability to sort CPY. Cells carrying deletions of the CHC1 gene (chc1 delta) also sorted CPY to the vacuole even when subjected to temperature shifts. Vacuolar delivery of CPY in chc1 delta cells was not blocked by sec1 suggesting that transport does not occur by secretion and endocytosis. These results provide in vivo evidence that clathrin plays a role in the Golgi complex in sorting of vacuolar proteins from the secretory pathway. With time, however, yeast cells lacking functional clathrin heavy chains are able to adapt in a way that allows restoration of vacuolar protein sorting in the Golgi complex. These conclusions clarify previous studies of chc1 delta cells which raised the possibility that clathrin is not involved in vacuolar protein sorting.     

Retrograde Traffic Out of the Yeast Vacuole to the TGN Occurs via the Prevacuolar/Endosomal Compartment

A large number of trafficking steps occur between the last compartment of the Golgi apparatus (TGN) and the vacuole of the yeast Saccharomyces cerevisiae. To date, two intracellular routes from the TGN to the vacuole have been identified. Carboxypeptidase Y (CPY) travels through a prevacuolar/endosomal compartment (PVC), and subsequently on to the vacuole, while alkaline phosphatase (ALP) bypasses this compartment to reach the same organelle. Proteins resident to the TGN achieve their localization despite a continuous flux of traffic by continually being retrieved from the distal PVC by virtue of an aromatic amino acid–containing sorting motif. In this study we report that a hybrid protein based on ALP and containing this retrieval motif reaches the PVC not by following the CPY sorting pathway, but instead by signal-dependent retrograde transport from the vacuole, an organelle previously thought of as a terminal compartment. In addition, we show that a mutation in VAC7, a gene previously identified as being required for vacuolar inheritance, blocks this trafficking step. Finally we show that Vti1p, a v-SNARE required for the delivery of both CPY and ALP to the vacuole, uses retrograde transport out of the vacuole as part of its normal cellular itinerary.     

Protein sorting in yeast: mutants defective in vacuole biogenesis mislocalize vacuolar proteins into the late secretory pathway

We have devised a genetic selection for mutant yeast cells that fail to properly deliver the vacuolar glycoprotein CPY to the lysosome-like vacuole. This has allowed us to identify mutations in eight VPL complementation groups that result in aberrant secretion of up to approximately 90% of the immunoreactive CPY. Other soluble vacuolar proteins are also affected by each vpl mutation, demonstrating that a sorting system for multiple vacuolar proteins exists in yeast. Mislocalized CPY apparently traverses late stages of the secretory pathway, since a vesicle-accumulating sec1 mutation prevents secretion of this protein. Despite the presence of abnormal membrane-enclosed organelles in some of the vpl mutants, maturation and secretion of invertase are not substantially perturbed. Thus vpl mutations define a new class of genes that encode products required for sorting of newly synthesized vacuolar proteins from secretory proteins during their transit through the yeast secretory pathway.     

vac2: a yeast mutant which distinguishes vacuole segregation from Golgi-to-vacuole protein targeting.

We have isolated four yeast mutants that are unable to partition maternal vacuoles into growing buds. Three of these vacuole segregation (vac) mutants also mislocalize the vacuolar protease carboxypeptidase Y (CPY) to the cell surface, a phenotype previously reported for vac strains. A fourth mutant, vac2-1, exhibits a temperature-sensitive defect in vacuole segregation but does not show a defect in protein targeting from the Golgi apparatus to the vacuole. Haploid vac2-1 cells grown at the non-permissive temperature do not secrete CPY or a second vacuolar protease, proteinase A (PrA). Furthermore, newly synthesized precursors of CPY are converted to mature forms with similar kinetics in both vac2-1 and wild-type cells. In addition, invertase is secreted normally from vac2-1 cells, indicating that post-Golgi steps in the secretory pathway are not blocked in this mutant. These results suggest that VAC2 function is necessary for vacuole division and segregation in yeast but is not involved in vacuole protein sorting events at the Golgi apparatus.     

Acidic Di-leucine Motif Essential for AP-3–dependent Sorting and Restriction of the Functional Specificity of the Vam3p Vacuolar t-SNARE

The transport of newly synthesized proteins through the vacuolar protein sorting pathway in the budding yeast Saccharomyces cerevisiae requires two distinct target SNAP receptor (t-SNARE) proteins, Pep12p and Vam3p. Pep12p is localized to the pre-vacuolar endosome and its activity is required for transport of proteins from the Golgi to the vacuole through a well defined route, the carboxypeptidase Y (CPY) pathway. Vam3p is localized to the vacuole where it mediates delivery of cargoes from both the CPY and the recently described alkaline phosphatase (ALP) pathways. Surprisingly, despite their organelle-specific functions in sorting of vacuolar proteins, overexpression of VAM3 can suppress the protein sorting defects of pep12Δ cells. Based on this observation, we developed a genetic screen to identify domains in Vam3p (e.g., localization and/or specific protein–protein interaction domains) that allow it to efficiently substitute for Pep12p. Using this screen, we identified mutations in a 7–amino acid sequence in Vam3p that lead to missorting of Vam3p from the ALP pathway into the CPY pathway where it can substitute for Pep12p at the pre-vacuolar endosome. This region contains an acidic di-leucine sequence that is closely related to sorting signals required for AP-3 adaptor–dependent transport in both yeast and mammalian systems. Furthermore, disruption of AP-3 function also results in the ability of wild-type Vam3p to compensate for pep12 mutants, suggesting that AP-3 mediates the sorting of Vam3p via the di-leucine signal. Together, these data provide the first identification of an adaptor protein–specific sorting signal in a t-SNARE protein, and suggest that AP-3–dependent sorting of Vam3p acts to restrict its interaction with compartment-specific accessory proteins, thereby regulating its function. Regulated transport of cargoes such as Vam3p through the AP-3–dependent pathway may play an important role in maintaining the unique composition, function, and morphology of the vacuole.     

Analysis of a processing system for proteases using yeast cell surface engineering: conversion of precursor of proteinase A to active proteinase A

The display of a protease, carboxypeptidase Y (CPY) or procarboxypeptidase Y (proCPY), which is the vacuolar protease, on the yeast-cell surface was successfully performed using yeast-cell-surface engineering for the first time. Through that we could confirm the processing of vacuolar proteases containing proteinase A (PrA) and proteinase B (PrB) which are related to the maturation of proCPY, using a novel cell-surface engineering technique. Various protease-knockout strains of Saccharomyces cerevisiae with the CPY-displaying system were constructed to evaluate the operation of the activation process of CPY. The display of CPY (CPY-agg, which is a fusion protein of CPY with C-terminal half of α-agglutinin) on the cell surface was confirmed by immunofluorescence staining. The activity of the CPY-agg was determined after the conversion of proCPY to active CPY by treatment of whole cells with proteinase K. In the proCPY-displaying CPY-knockout strain and PrB-knockout strain, CPY was displayed as an active (mature) form, but in the proCPY-displaying PrA-knockout strain, CPY was present as an inactive form (proCPY). These facts indicate that PrA had been already activated before its transport to the vacuole and that active mature PrA might convert proCPY to CPY before the transport of proCPY to the vacuole. From these results, it was suggested that by using the yeast-cell-surface engineering at the location of the initial step, the autocatalytic activation from proPrA to PrA might occur before the vacuolar branch separates from the main secretory pathway.     

Protein sorting in yeast: the localization determinant of yeast vacuolar carboxypeptidase Y resides in the propeptide

We have isolated cis-acting mutations in the gene encoding the yeast vacuolar protein carboxypeptidase Y (CPY) that result in missorting and aberrant secretion of up to 95% of newly synthesized CPY. The CPY polypeptides synthesized by these mutants use the late secretory pathway to exit the cell, since the late-acting sec1 mutation prevents their secretion. The mutant versions of CPY are secreted as the proCPY zymogen and are enzymatically activatable in vivo and in vitro. All the mutations, including small deletions and an amino acid substitution, map to the amino-terminal propeptide region and define a discrete yeast vacuolar localization domain whose integrity is required for efficient sorting of the CPY zymogen. Thus, the N-terminal propeptide of CPY carries out at least three functions: it mediates translocation across the endoplasmic reticulum, renders the enzyme inactive during transit, and targets the molecule to the vacuole.     

In vitro reconstitution of intercompartmental protein transport to the yeast vacuole

Toward a detailed understanding of protein sorting in the late secretory pathway, we have reconstituted intercompartmental transfer and proteolytic maturation of a yeast vacuolar protease, carboxypeptidase Y (CPY). This in vitro reconstitution uses permeabilized yeast spheroplasts that are first radiolabeled in vivo under conditions that kinetically trap ER and Golgi apparatus-modified precursor forms of CPY (p1 and p2, respectively). After incubation at 25 degrees C, up to 45% of the p2CPY that is retained in the perforated cells can be proteolytically converted to mature CPY (mCPY). This maturation is specific for p2CPY, requires exogenously added ATP, an ATP regeneration system, and is stimulated by cytosolic protein extracts. The p2CPY processing shows a 5-min lag period and is then linear for 15-60 min, with a sharp temperature optimum of 25-30 degrees C. After hypotonic extraction, the compartments that contain p2 and mCPY show different osmotic stability characteristics as p2 and mCPY can be separated with centrifugation into a pellet and supernatant, respectively. Like CPY maturation in vivo, the observed in vitro reaction is dependent on the PEP4 gene product, proteinase A, which is the principle processing enzyme. After incubation with ATP and cytosol, mCPY was recovered in a vacuole-enriched fraction from perforated spheroplasts using Ficoll step-gradient centrifugation. The p2CPY precursor was not recovered in this fraction indicating that intercompartmental transport to the vacuole takes place. In addition, intracompartmental processing of p2CPY with autoactivated, prevacuolar zymogen pools of proteinase A cannot account for this reconstitution. Stimulation of in vitro processing with energy and cytosol took place efficiently when the expression of PEP4, under control of the GAL1 promoter, was induced then completely repressed before radiolabeling spheroplasts. Finally, reconstitution of p2CPY maturation was not possible with vps mutant perforated cells suggesting that VPS gene product function is necessary for intercompartmental transport to the vacuole in vitro.     

Vacuole biogenesis and protein transport to the plant vacuole: A comparison with the yeast vacuole and the mammalian lysosome

Summary The vacuole is often termed the lytic compartment of the plant cell. The yeast cell also possesses a vacuole containing acid hydrolases. In animal cells these enzymes are localized in the lysosome. Recent research suggests that there is good reason to regard these organelles as homologous in terms of protein transport. Although sorting motifs for the recognition of vacuolar proteins within the endomembrane system differ between the three organelles, there is an underlying similarity in targeting determinants in the cytoplasmic tails of Golgi-based receptors. In all three cases these determinants appear to interact with adaptins of clathrin-coated vesicles which ferry their cargo first of all to an endosomal compartment. The situation in sorting and targeting of plant vacuolar proteins is complicated by the fact that storage and lytic vacuoles may exist together in the same cell. The origin of these two types of vacuole is also a matter of some uncertanity.Abbrevations AP assembly protein - ALP alkaline phosphatase - ARF adenosine diphosphate ribosylation factor - BiP immunoglobulin binding protein - CCV clathrin coated vesicle - CPY carboxypeptidase-Y - DPAP dipeptidyl aminopeptidase - ER endoplasmic reticulum - GApp Golgi apparatus - LAMPs lysosomal associated membrane protein(s) - LAP lysosomal acid phosphatase - LIMPs lysosomal integral membrane protein(s) - MPRs mannosyl 6-phosphate receptors - MVB multivesicular bodies - NSF N-ethylmaleimide sensitive fusion (protein) - PAT phosphinotricine acetyltransferase - PB protein body - PHA phytohemagglutinin - PM plasma membrane - PSV protein storage vacuole - SNAPs soluble NSF attachment protein(s) - SNAREs SNAP receptor(s) - TGN trans Golgi network - TIP tonoplast integral protein - VPS vacuolar protein sorting - ZIO zinc iodide/osmium     

Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe.

PCR was used to isolate a carboxypeptidase Y (CPY) homolog gene from the fission yeast Schizosaccharomyces pombe. The cloned S. pombe cpy1+ gene has a single open reading frame, which encodes 950 amino acids with one potential N-glycosylation site. It appears to be synthesized as an inactive pre-pro protein that likely undergoes processing following translocation into appropriate intracellular organelles. The C-terminal mature region is highly conserved in other serine carboxypeptidases. In contrast, the N-terminal pro region containing the vacuolar sorting signal in CPY from Saccharomyces cerevisiae shows fewer identical residues. The pro region contains two unusual repeating sequences; repeating sequence I consists of seven contiguous repeating segments of 13 amino acids each, and repeating sequence II consists of seven contiguous repeating segments of 9 amino acids each. Pulse-chase radiolabeling analysis revealed that Cpy1p was initially synthesized in a 110-kDa pro-precursor form and via the 51-kDa single-polypeptide-chain intermediate form which has had its pro segment removed is finally converted to a heterodimer, the mature form, which is detected as a 32-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Like S. cerevisiae CPY, S. pombe Cpy1p does not require the N-linked oligosaccharide moiety for vacuolar delivery. To investigate the vacuolar sorting signal of S. pombe Cpy1p, we have constructed cpy1+-SUC2 gene fusions that direct the synthesis of hybrid proteins consisting of N-terminal segments of various lengths of S. pombe Cpy1p fused to the secreted enzyme S. cerevisiae invertase. The N-terminal 478 amino acids of Cpy1 are sufficient to direct delivery of a Cpy1-Inv hybrid protein to the vacuole. These results showed that the pro peptide of Cpy1 contains the putative vacuolar sorting signal.     

Endosome to Golgi Retrieval of the Vacuolar Protein Sorting Receptor, Vps10p, Requires the Function of the VPS29, VPS30, and VPS35 Gene Products

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.     

Vps10p cycles between the TGN and the late endosome via the plasma membrane in clathrin mutants

Clathrin-coated vesicles mediate the transport of the soluble vacuolar protein CPY from the TGN to the endosomal/prevacuolar compartment. Surprisingly, CPY sorting is not affected in clathrin deletion mutant cells. Here, we have investigated the clathrin-independent pathway that allows CPY transport to the vacuole. We find that CPY transport is mediated by the endosome and requires normal trafficking of its sorting receptor, Vps10p, the steady state distribution of which is not altered in chc1 cells. In contrast, Vps10p accumulates at the cell surface in a chc1/end3 double mutant, suggesting that Vps10p is rerouted to the cell surface in the absence of clathrin. We used a chimeric protein containing the first 50 amino acids of CPY fused to a green fluorescent protein (CPY-GFP) to mimic CPY transport in chc1. In the absence of clathrin, CPY-GFP resides in the lumen of the vacuole as in wild-type cells. However, in chc1/sec6 double mutants, CPY-GFP is present in internal structures, possibly endosomal membranes, that do not colocalize with the vacuole. We propose that Vps10p must be transported to and retrieved from the plasma membrane to mediate CPY sorting to the vacuole in the absence of clathrin-coated vesicles. In this circumstance, precursor CPY may be captured by retrieved Vps10p in an early or late endosome, rather than as it normally is in the trans-Golgi, and delivered to the vacuole by the normal VPS gene-dependent process. Once relieved of cargo protein, Vps10p would be recycled to the trans-Golgi and then to the cell surface for further rounds of sorting.     

Heterologous expression of syntaxin 6 in Saccharomyces cerevisiae

The molecular mechanisms of vesicular protein transport in eukaryotic cells are highly conserved. Members of the syntaxin family play a pivotal role in the membrane fusion process. We have expressed rat syntaxin 6 and its cytoplasmic domain in wild-type and pep12 mutant strains of Saccharomyces cerevisiae to elucidate the role of the syntaxin 6-dependent vesicular trafficking step in yeast. Immunofluorescence microscopy revealed a punctate, Golgi-like staining pattern for syntaxin 6, which only partially overlapped with Pep12p in wild-type yeast cells. In contrast to Pep12p, syntaxin 6 was not mislocalized to the vacuole upon expression from 2 micron vectors, which might be attributed to conserved sorting and retention signals. Syntaxin 6 was not capable of complementing the sorting and maturation defects of the vacuolar hydrolase CPY in pep12 null mutants. No dominant negative effects of either syntaxin 6 or syntaxin 6 delta C overexpression on CPY sorting and maturation were observed in wild-type yeast cells. We conclude that syntaxin 6 and Pep12p do not act at the same vesicular trafficking step(s) in yeast and higher eukaryotes.