Vpsl0p Cycles between the Late-Golgi and Prevacuolar Compartments in Its Function as the Sorting Receptor for Multiple Yeast Vacuolar Hydrolases

VPSIO (Vacuolar Protein Sorting) encodes a large type I transmembrane protein (Vpsl0p), involved in the sorting of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) to the Saccharomyces cerevisiae lysosome-like vacuole. Cells lacking Vpsl0p 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 Vpsl0p binds CPY precursor in a 1:1 stoichiometry, further supporting the assignment of Vpsl0p as the CPY sorting receptor. Vpsl0p has been immunolocalized to the late-Golgi compartment where CPY is sorted away from the secretory pathway. Vpsl0p is synthesized at a rate 20-fold lower than that of its ligand CPY, which in light of the 1:1 binding stoichiometry, requires that Vpsl0p must recycle and perform multiple rounds of CPY sorting. The 164--amino acid Vpsl0p cytosolic domain is involved in receptor trafficking, as deletion of this domain resulted in delivery of the mutant Vpsl0p to the vacuole, the default destination for membrane proteins in yeast. A tyrosine-based signal (YSSLs0) within the cytosolic domain enables Vpsl0p 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 Vpsl0p which, when overproduced, is capable of suppressing the CPY and PrA missorting defects of a vpslOA strain. These results indicate that a family of related receptors act to target soluble hydrolases to the vacuole. ENETIC screens have identified a large number of Vacuolar Protein Sorting (VPS) 1 genes involved in the sorting and delivery of carboxypeptidase Y (CPY) to the vacuole (Jones, 1977; Banta et al., 1988; Robinson et al., 1988; Rothman et al., 1989; Raymond et al., 1992). Recently, it has become clear (Stack et al., 1995) that these genes define a post-Golgi protein sorting pathway very similar to what has been described for lysosomal hydrolases in mammalian cells (Kornfeld, 1992). The targeting of mammalian lysosomal proteins involves the addition of a mannose-6-phosphate moiety to soluble hydrolases following their translocation into the endoplasmic reticulum (Kornfeld, 1992). This sorting signal is recogPlease address all correspondence to T.H. Stevens, Institute of Molecular Biology, University of Oregon, Eugene, OR 97403-1229. Tel.: (503) 3465884. Fax: (503) 364-4854. e-mail: [email protected] The current address of A.A. Cooper is Divison of Cell Biology & Biophysics, School of Biological Sciences, University of Missouri-Kansas City, 5100 Rockhill Road, Kansas City, MO 64110-2499. 1. Abbreviations used in this paper: CPY, carboxypeptidase Y; M6PR, mannose-6-phosphate/IGF II receptor; mCPY, mature CPY; mPrA, mature PrA; PrA, proteinase A; proCPY, CPY precursor; proPrA, PrA precursor; VPS, vacuolar protein sorting. nized by the cation-independent mannose-6-phosphate/ IGF II receptor (M6PR), which binds newly synthesized proteins in the Golgi and delivers them to the prelysosomal/endosomal compartment. Here the protein dissociates from the receptor in the lower pH environment of the endosome and is subsequently delivered to the lysosome. The receptor is then recycled to the trans-Golgi network (TGN) or plasma membrane to sort additional ligand. In the yeast Saccharomyces cerevisiae the vacuolar hydrolase CPY is sorted away from secreted proteins in a late-Golgi compartment, which is likely analogous to the mammalian TGN (Graham and Emr, 1991; Wilsbach and Payne, 1993; Nothwehr et al., 1995; Stack et al., 1995). There is growing evidence that S. cerevisiae also has a post-Golgi, prevacuolar compartment analogous to a mammalian prelysosome/late endosome (Raymond et al., 1992; Vida et al., 1993; Piper et al., 1995; Stack et al., 1995). In contrast to the specific recognition of mannose-6-phosphate by the M6PR, yeast vacuolar hydrolases are sorted via a signal within their propeptides (Vails et al., 1987; Johnson et al., 1987; Klionsky et al., 1988). These propeptides are proteolytically cleaved once the hydrolase precursors reach the vacuole (Stevens et al., 1982; Jones, 1991). The best characterized of these sorting signals is © The Rockefeller University Press, 0021-9525/96/05/529/13 $2.00 The Journal of Cell Biology, Volume 133, Number 3, May 1996 529-541 529 on A uust 0, 2017 jcb.rress.org D ow nladed fom within the propeptide of CPY, and is minimally defined by the amino acids QRPL (Vails et al., 1990). Evidence that the sorting of yeast vacuolar hydrolases is receptor mediated is provided by the observations that the sorting capacity of yeast can be saturated by overexpression of either CPY (Stevens et al., 1986) or proteinase A (PrA; Rothman et al., 1986). The identification and characterization of the CPY receptor is important for several reasons. An analysis of the binding association between the receptor and CPY might reveal in vivo conditions important in regulating binding and dissociation of ligand. Secondly, the receptor is likely to recycle, as does the M6PR, and an investigation of the recycling signals on the receptor should provide important insights into membrane trafficking in yeast. Finally, an analysis of the sorting receptor in vps mutant strains should help reveal the molecular basis for the CPY sorting defect in those strains. Several lines of investigation have indicated that the VPSIO gene is likely to encode the CPY sorting receptor. The yeast genome sequencing project identified PEP1~ VPSIO as a gene on chromosome II predicted to encode a 178-kD type I transmembrane protein of 1579 residues (van Dyck et al., 1992). The predicted protein, Vpsl0p, contains a signal sequence at its amino terminus, a large lumenal domain of 1,380 amino acids, a 17-amino acid transmembrane domain and a carboxy-terminal domain of 164 amino acids. Of the many VPS genes sequenced, VPSIO was the first gene identified to encode a transmembrane protein and therefore was a likely candidate for the CPY receptor (Marcusson et al., 1994). These investigators found that Vpsl0p could be cross-linked to proCPY, but not to a sorting defective mutant form of proCPY. Additional support that Vpsl0p is the CPY sorting receptor comes from phenotypic studies indicating that vpslOA cells secrete >90% of newly synthesized CPY yet vpslOA cells exhibit normal vacuolar morphology (Banta et al., 1988; Raymond et al., 1992; Marcusson et al., 1994). Because normal vacuole membranes are assembled in vpslOA cells, overall membrane traffic to the vacuole appears to be unaffected. A recent analysis of Vpsl0p has found the cytosolic domain to influence the membrane trafficking of the protein, and Vpsl0p lacking this domain is delivered to the vacuole (Cereghino et al., 1995). Here we report that the lumenal domain of Vpsl0p binds proCPY stoichiometrically in vitro. In contrast to a previous report (Marcusson et al., 1994), we found that vpslOA cells missort PrA as well as CPY, indicating that Vpsl0p sorts at least two vacuolar hydrolases. The 1:1 stoichiometry of ligand binding, taken together with the expression levels of receptor and ligand, indicate that Vpsl0p must recycle to sort the excess of newly synthesized CPY. Vpsl0p cycles between the late-Golgi and prevacuolar compartments and this recycling is dependent on a tyrosine-based signal within its cytosolic domain. This signal is similar to the recycling signal in the cytosolic domain of the M6PR. Finally, VTH2 (Vps Ten Homologue) encodes a homologue of Vpsl0p that, when overexpressed, suppresses the missorting phenotype of vpslOA cells. This suggests that a family of receptors participate to varying degrees in the sorting of soluble hydrolases to the yeast vacuole. Materials and Methods Strains, Media, and Microbiological Techniques Yeast strains used in this study are listed in Table I. Strains were construtted by standard genetic techniques and grown at 30°C in rich media (1% yeast extract, 1% peptone, 2% dextrose; YEPD) or standard minimal medium (SD) with appropriate supplements as described by Sherman et al. (1986). Strain TSY108 was constructed by transforming SF838-5A (MA Ta ura3-52, leu2-3,112 ader) with linearized plasmid pCAV3, which contains both the LEU2 and the CPY encoding gene, PRC1. pCAV3 was linearized with either XbaI (cleaves in PRC1) or ClaI (cleaves in LEU2) and the mixture transformed to direct integration of the plasmid borne PRC1 to both the LEU2 and PRC1 loci. Leu ÷ transformants were selected and screened by CPY overlay blot (Roberts et al., 1991; Piper et al., 1994) to identify colonies overproducing and therefore secreting CPY. TSY108 overproduced CPY ~6-8-fold of which 40% was secreted. DNA manipulations and DNA-mediated transformation of E. coli strains MC1061 and CJ236 were performed by routine procedures.