Biogenesis of the Yeast Vacuole (Lysosome) THE PRECURSOR FORMS OF THE SOLUBLE HYDROLASE CARBOXYPEPTIDASE yscS ARE ASSOCIATED WITH THE VACUOLAR MEMBRANE*

We have studied the structure, biosynthesis, intra- cellular routing, and vacuolar localization of carboxypeptidase yscS in the yeast Saccharomyces cerevis- iae. Nondenaturing polyacrylamide gel electrophoresis revealed two forms of carboxypeptidase yscS with dif- ferent electrophoretic mobility. Antibodies specific for carboxypeptidase yscS recognized two glycoproteins of 77- and 74-kDa apparent molecular mass which differ by one N-linked carbohydrate residue. Both observations suggest that carboxypeptidase yscS exists in two catalytically active forms. The enzyme was found to be synthesized as two active high molecular mass precursor forms which are converted to the mature forms with a half-time of 20 min. The mature forms of carboxypeptidase yscS appeared soluble in the vacuolar lumen, while the precursor proteins ac- cumulated tightly associated with the vacuolar membrane. The single hydrophobic domain present at the N terminus is believed to be responsible for the membrane association of the precursor molecules. Double mutants defective in proteinase yscA and proteinase yscB synthesize solely the carboxypeptidase yscS pre- cursor forms. Correct proteolytic cleavage of the precursor

We have studied the structure, biosynthesis, intracellular routing, and vacuolar localization of carboxypeptidase yscS in the yeast Saccharomyces cerevisiae. Nondenaturing polyacrylamide gel electrophoresis revealed two forms of carboxypeptidase yscS with different electrophoretic mobility. Antibodies specific for carboxypeptidase yscS recognized two glycoproteins of 77and 74-kDa apparent molecular mass which differ by one N-linked carbohydrate residue. Both observations suggest that carboxypeptidase yscS exists in two catalytically active forms. The enzyme was found to be synthesized as two active high molecular mass precursor forms which are converted to the mature forms with a half-time of 20 min. The mature forms of carboxypeptidase yscS appeared soluble in the vacuolar lumen, while the precursor proteins accumulated tightly associated with the vacuolar membrane. The single hydrophobic domain present at the N terminus is believed to be responsible for the membrane association of the precursor molecules. Double mutants defective in proteinase yscA and proteinase yscB synthesize solely the carboxypeptidase yscS precursor forms. Correct proteolytic cleavage of the precursor forms was performed using purified proteinase yscB in vitro. Sec61, secl8, and sec7 mutants, conditionally defective in the secretory pathway, accumulate carboxypeptidase yscS precursor protein. Thus the carboxypeptidase yscS precursor molecules are delivered to the vacuole in a membrane bound form via the secretory pathway. After assembly into the vacuolar membrane, proteinase yscB presumably cleaves the precursor molecules to release soluble carboxypeptidase yscS forms into the lumen of the vacuole. The proposed mechanism is different from the delivery mechanism found for the other soluble vacuolar hydrolases in yeast.
The vacuole of the yeast Saccharomyces cereuisiae is a cellular compartment analogous to the mammalian lysosome since both organelles have an acidic pH and contain a multitude of hydrolases (see Ref. 1-6, for reviews). In addition, the vacuolar membrane encloses numerous metabolically important compounds and ions (for review, see Ref. 7). The major * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I TO whom correspondence should be addressed Institut fur Biochemie der Universitat Stuttgart, Pfaffenwaldring 55, D-7000 Stuttgart 80, Federal Republic of Germany. Tel.: 0-711-685-4390; Fax: 0-711- 685-4392. function of the vacuole is protein degradation, a process that has been demonstrated to be very important during the differentiation event of sporulation and essential for cell survival under nutritional stress (4, 6, 8).
Among the best characterized vacuolar hydrolases are two soluble endopeptidases, proteinase yscA and proteinase yscB, two soluble exopeptidases, carboxypeptidase yscY and aminopeptidase I, and two integral membrane proteins, dipeptidyl aminopeptidase yscV (dipeptidyl aminopeptidase B) and the repressible alkaline phosphatase (Refs. 9-21, for reviews, see Refs. 4 and 6). With the exception of dipeptidyl aminopeptidase yscV (B), these hydrolases are synthesized as inactive high molecular mass precursor molecules, which are activated by proteolytic cleavage prior or upon delivery to the vacuole. All of the proteolytic maturation events have been shown to be self-catalyzed or catalyzed by proteinase yscA or proteinase With one exception (28), all vacuolar hydrolases studied so far are targeted to the vacuole via part of the secretory pathway (12, 13, 15-19, 29, 30), as has been found for lysosomal proteins in higher eukaryotic cells (31). During transit through the endoplasmic reticulum (ER)' and the Golgi complex, the vacuolar hydrolases undergo several modification events, like the addition of N-linked oligosaccharides and subsequent carbohydrate trimming (29). After synthesis on ribosomes associated with the ER, secretory proteins are translocated across the ER membrane. In yeast, the soluble vacuolar proteins are released into the lumen of the ER after translocation and cleavage of the signal sequence (30,32), while the vacuolar membrane proteins remain membraneassociated after translocation across the ER membrane (7, 18,19). Thus the final location of both classes of proteins within the vacuole, lumenal or membrane bound, appear to be determined during the first step of the secretory pathway. In contrast, the soluble lysosomal enzymes a-mannosidase and &glucosidase of Dictyostelium discoideum or the soluble human lysosomal acid phosphatase, have been found to be transported through the secretory pathway as membraneassociated precursors which are subsequently processed and solubilized (33)(34)(35)(36).
Carboxypeptidase yscS of S. cerevisiae has been shown to be a vacuolar enzyme essential for cellular utilization of certain peptides as nitrogen source and has been found to be involved in the differentiation process of sporulation (37)(38)(39).
Recently we (40) and others (41) reported the cloning of the CPSI gene by complementation of the cpsl-3 mutation which was shown to reside in the structural gene of carboxy-peptidase yscS (38). The cloning procedure we used clearly demonstrated that carboxypeptidase yscS activity, unlike aminopeptidase I, proteinase yscA, proteinase yscB, and carboxypeptidase yscY activities, was completely independent of both proteinase yscA and proteinase yscB activity (40). This observation did not rule out the possibility, however, that a catalytically active precursor molecule of carboxypeptidase yscS might exist. The sequence of the CPSl gene was found to encode a putative protein of 576 amino acids with five potential N-glycosylation sites. Analysis of the hydrophobicity profile and the deduced primary structure revealed a single hydrophobic domain between amino acid positions 20 and 40 and predicted a signal sequence cleavage site between glycine at position 39 and leucine at position 40. A comparison between the N-terminal sequence of carboxypeptidase yscS and other vacuolar proteins did not allow any predictions concerning location or topology of carboxypeptidase yscS within the vacuole or its surrounding membrane (40).
Here we report on the biosynthesis, transport, and vacuolar location of carboxypeptidase yscS. We show that carboxypeptidase yscS is synthesized as one polypeptide chain precursor which after carbohydrate modification in the secretory pathway yields two active precursor molecules. The proteolytically unprocessed forms of carboxypeptidase yscS are associated with the vacuolar membrane, whereas the mature forms of the enzyme are soluble. We identified proteinase yscB to be the major processing activity for carboxypeptidase yscS which liberates the proteins from the membrane to yield the soluble vacuolar enzymes.

EXPERIMENTAL PROCEDURES
Materials-DNA restriction-and modifying enzymes, as well as endoglycosidase F, were obtained from Boehringer Mannheim (Mannheim, Federal Republic of Germany), [35S]methionine was from Du Pont-New England Nuclear products, and chymostatin and pepstatin were from the Peptide Institute (Osaka, Japan). Low range prestained SDS-PAGE molecular weight standards, used to determine the apparent molecular mass of carboxypeptidase yscs, were obtained from Bio-Rad. Purified proteinase yscB (42) was a gift from U. Weiser, Universitat Freiburg, Federal Republic of Germany, and proteinase A was from Sigma.
Strains and Media-All strains used in this study are listed in Table I. Yeast strains were grown in YPD complete medium (1% yeast extract, 2% peptone, and 2% glucose) or in MV mineral medium (0.67% yeast nitrogen base without amino acids, 2% glucose, and supplements required for auxotrophic strains). Escherichia coli J M 109 was grown in LB medium (43) with or without ampicillin (50 mg/ liter) and used as host strain for amplification, subcloning, and sequencing of plasmid DNA. E. coli strain SG936 served for the expression of the MS2 replicase-carboxypeptidase yscS fusion protein and was grown in LB medium (42) with or without kanamycin (40 mg/liter) and ampicillin (50 mg/liter).

Plasmid Constructions and Molecular Biological Techniques-Has-
mid DNA was isolated from E. coli by alkaline lysis (44). Purification, restriction, ligation, analysis of DNA on agarose gels, and preparation of competent E. coli cells were performed as described (43). Deletion and disruption of the chromosomal CPSl gene was done in strain YB18 by the one-step gene disruption method (45). A 2.7kb KpnI-EcoRI fragment carrying the CPSl gene was subcloned into a EcoRI-KpnI linearized Bluescript KS' M13 vector (Stratagene) as described (40). Digestion with NcoI and religation of this vector deleted a unique 0.7-kb NcoI fragment of the CPSl coding region 157 base pairs downstream of the putative start codon of the CPSl gene. The single HindIII site within the remaining Acpsl coding region was used to insert a 1.1 kb HindIII URA3 fragment as previously reported (40). The resulting vector, pBSAcpsl::URA3, was double digested with EcoRI-KpnI and used for transformation of strain YB18 (46). Transformants with a stably inherited URA3 phenotype were analyzed as described (40).
For the production of antibodies specific to carboxypeptidase yscS, a MS2 replicase-carboxypeptidase yscS fusion protein was constructed using the expression vector pPLc24 which contains the 5'coding region of the MS2 replicase gene under the control of the P L promoter (47). A 1.7-kb HindIII fragment encoding amino acids 374-576 of the deduced carboxypeptidase yscS primary structure was isolated (40) and subcloned into the single HindIII site of vector pPLc24 which is located downstream of the MS2 replicase gene. The correct orientation of the subcloned HindIII fragment was determined by restriction analysis and the continuous reading frame from the MS2 replicase gene to the CPSl gene was confirmed by dideoxysequencing of the junction (48). The gene fusion, pPLc24CPS1, encodes 100 amino acids of the MS2 replicase and 203 amino acids of carboxypeptidase yscS, separated by 1 amino acid encoded by pPLc24 vector sequences. Plasmid pcZ857 carrying the temperature-sensitive X-repressor cZ857 and the kanamycin resistance gene has been described (49).
Production of Carboxypeptidase yscS Antigen and Generation of Antiserum to Carboxypeptidase yscS-E. coli cells SG936 harboring pcZ857 were transformed with the gene fusion pPLc24CPSl. Transformants were grown in 25 ml of LB medium containing ampicillin (50 mg/liter) and kanamycin (40 mg/liter) at 30 "C to saturation, then diluted 1:4 in fresh medium and shifted to 42 "C for 4 h. Cells were collected by centrifugation, resuspended in 20 ml of 10 mM EDTA, 50 mM Tris, pH 6.8, and lysed by sonication. The cell lysate was centrifuged at 12,000 X g for 10 min and the pellet was resuspended in 20 ml of 10 mM EDTA, 50 mM Tris, pH 6.8, and the lysis procedure was repeated. After centrifugation, the pellet was resuspended in 20 ml of SDS gel sample buffer and subjected to discontinuous SDS-PAGE (15% acrylamide). A predominant protein band of the expected size was visible after incubation of the gel in ice-cold 0.25 M KC1 for 10 min. A gel slice containing the fusion protein was isolated and used for electroelution (50 mM NH~HCOS, 0.1% SDS). The eluate was lyophilized, resuspended in H20, and the fusion protein was precipitated by adding 4 volumes of methanol/acetone (5050). The precipitate was incubated for 4 h a t -20 "C and thereafter isolated by centrifugation a t 15,000 X g for 10 min. The fusion protein (300 pg) was mixed with complete Freund's adjuvants and injected into rabbits. After 4 weeks two secondary injections were done every second week with the fusion protein (200 pg) mixed with incomplete Freund's adjuvants. I n Vivo Radiolabeling and Immunoprecipitation-Yeast cells were grown in YPD medium to 2 Am/ml, collected by centrifugation, and concentrated to 20 Am/ml in sodium citrate buffer (40 mM sodium citrate, 2% glucose, pH 6.0, containing supplements for auxotrophic strains). Cells were incubated for 15 min a t 30 "C and labeled with 200 pCi/ml of [35S]methionine for 7 min at 30 "C. Chase of radioactivity was done by the addition of nonradioactive methionine to a final concentration of 20 mM.
Sec mutant cells were grown in YPD medium at the permissive temperature (23 "C) to 2 Am/ml, shifted to the restrictive temperature for 20 min, collected by centrifugation, and resuspended in sodium citrate buffer (20 Am/ml). Cells were incubated for 20 min at the restrictive temperature and labeled for 1 h with 200 pCi/ml of [:':'S]methionine. For cell lysis, 200 pl of the labeled cell suspension were incubated with 120 pl of 1.85 M NaOH, 7.5% P-mercaptoethanol for 10 min on ice. Labeled protein was precipitated by adding 120 pl of 50% trichloroacetic acid, incubated for 10 min on ice, and centrifugated a t 15,000 X g for 10 min. The pellet was washed in 1 M Tris and resuspended in 400 pl of 25 mM imidazol, 2.5 mM EDTA, 2% SDS, pH 6.8. The samples were boiled for 5 min and diluted in 1 ml of INET (50 mM imidazol, 140 mM NaCI, 5 mM EDTA, 1% Triton X-100, pH 8.0). After centrifugation a t 15,000 X g for 10 min, the supernatant was isolated and diluted in INET to a final volume of 5 ml. Antiserum (5 pl) to carboxypeptidase yscS was added and the mixture was shaken overnight at 4 "C. Samples (200 p1) of 5% (g/ml) protein A-Sepharose CL-4B (Pharmacia, Sweden) were added and shaken at room temperature for 2 h. After the precipitate was washed three times with INET and two times with 62.5 mM Tris, pH 6.8, 30 pl of SDS gel sample buffer were added for solubilization. The mixture was boiled for 5 min and centrifuged for 2 min. The supernatant was isolated and analyzed by discontinuous SDS-PAGE (10% acrylamide). Gels were fixed with isopropyl alcohol, H20, acetate (25:65:10) for 30'min and treated with Amplify (Amersham Corp., United Kingdom) for 40 min before exposure to X-Omat K film (Kodak-Pathe, France).
For endoglycosidase F digestion, immunoprecipitated samples bound to protein A-Sepharose were solubilized by boiling for 5 min in 30 pl of 50 mM Tris, 10 mM EDTA, 1% SDS, pH 6.8. The mixtures were centrifuged for 2 min and the supernatants were transferred to a new tube. Four-fold concentrated endoglycosidase F incubation buffer (10 p l ) (200 mM KPi, 200 mM EDTA, 8% Triton X-100,4% 8mercaptoethanol, pH 6.8) and 50 milliunits of endoglycosidase F were added and the samples were incubated for 1 h at 37 "C. A time course of endoglycosidase F digestion was performed by collecting labeled carboxypeptidase yscS from 14 Am units of cells using the method described above, adding 50 milliunits of endoglycosidase F and withdrawing aliquots at the times indicated in Fig.

1B.
After treatment with endoglycosidase F, SDS gel sample buffer was added and the samples were analyzed on SDS-PAGE as described above.
Isolation of Vacuoles, Proteinase K Protection Assay, and Immunoblotting-Spheroplast formation and isolation of vacuoles was done as described (2) with modifications as outlined by Mechler et al. (12). Enrichment of vacuoles was 15-20-fold as determined by Lu-mannosidase enzyme activity (vacuolar membrane marker) (50) and immunoblotting with carboxypeptidase yscS-specific antiserum.
Proteinase K protection assays were performed a t a concentration of 1 mg of proteinase K/15 mg of vacuolar protein in the absence or presence of 1% Triton X-100. To ensure osmotic stability of the intact vacuoles, proteinase K (1 mg/ml) was resuspended in 0.2 M sorbitol, pH 6.8. The mixtures were incubated on ice and aliquots were withdrawn at the times indicated in Fig. 7. The reaction was terminated by adding SDS gel sample buffer containing phenylmethylsulfonyl fluoride and boiling for 5 min.
SDS-denatured samples were subjected to SDS-PAGE (10% acrylamide) and electroblotted onto nitrocellulose (51) using a semi-dry electroblotter A (Ancons, Denmark) following the instructions provided by the manufacturer. The transferred proteins were detected by probing the nitrocellulose with carboxypeptidase yscS-specific antiserum and a color reaction of alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin with nitrotetrazolin blue chloride and 5-bromo-4-chloro-3-indolyl phosphate (52).
Sodium Carbonate Fractionation of Vacuoles-Vacuoles (100 pg) were diluted 10-fold in ice-cold 100 mM sodium carbonate, pH 11.5, vortexed vigorously three times for 2 min, and after 30 min on ice the lysate was centrifuged for 3 h at 100,000 X g. The supernatant was neutralized with 1 N acetic acid and precipitated in 20% trichloroacetic acid for 30 min on ice. The precipitated protein was pelleted by centrifugation at 15,000 X g for 10 min, washed twice in diethyl ether, dried, and resuspended in 60 pl of SDS gel sample buffer at 70 "C. The pellet fraction was resuspended in 60 p1 of SDS gel sample buffer at 70 "C. To extract integral membrane proteins from the pellet fraction, the membrane fraction was resuspended in 1 ml of 100 mM sodium carbonate, 2% Triton X-100, pH 11.5, and the fractionation procedure was performed as described above. All samples were analyzed by SDS-PAGE and subsequent immunoblotting with antiserum to the appropriate antigen. The membrane fraction was resuspended in 0.1 M Napi buffer, pH 7.4, and analyzed for carboxypeptidase yscS enzyme activity (37). I n Vitro Processing of the Carboxypeptidase yscS Precursor Form-Vacuoles (6 pg) prepared from strain YHH32 containing the carboxypeptidase yscS precursor proteins were lysed by 10-fold dilution in 0.1 M Tris maleate, pH 5.0, and incubated as described in the legend to Fig. 5. Samples were analyzed by immunoblotting with antiserum to carboxypeptidase yscS.

Identification of the Carboxypeptidase yscS Glycoprotein-
In order to characterize biosynthesis, structure, intracellular routing, and location of the carboxypeptidase yscS protein, we prepared antibodies specific to carboxypeptidase yscS using a MS2 replicase-carboxypeptidase yscS fusion protein containing 203 amino acids of the C terminus of the deduced amino acid sequence of carboxypeptidase yscS (for details, see "Experimental Procedures").
To test the specificity of the carboxypeptidase yscS antiserum, we constructed a Acpsl mutant by deleting a 700-base pair NcoI fragment located 156 base pairs downstream of the putative start codon of the CPSl gene. In addition, the URA3 gene was inserted into the single Hind111 site of the deleted gene as described (40). The resulting yeast strain YD19 (Acps1::URAS) showed the same phenotype as strain YD18 (cpsl::URA3) solely disrupted in the CPSI locus (40).
The specificity of the antiserum was tested by precipitating pulse-labeled immunoreactive material from lysed cells of the carboxypeptidase yscS wild type strain, YB18, and the isogenic strain, YD19, carrying a deleted and disrupted CPSl gene. As can be seen in Fig conclude that two distinct proteins of 77-and 74-kDa recognized by the antiserum are specific for the CPSl gene product.
In addition, treatment of carboxypeptidase yscS wild type cell extracts with the antiserum specifically inhibited carboxypeptidase yscS enzyme activity in vitro, whereas the second vacuolar carboxypeptidase, carboxypeptidase yscY, remained unaffected (data not shown). This behavior provides additional evidence that the CPSI gene represents the structural gene of carboxypeptidase yscS or a protein component of the enzyme.
Nondenaturing PAGE of cell extracts with subsequent staining of carboxypeptidase yscS activity (37) also revealed two distinct activity bands in wild type cells. As expected, these activity bands were absent in Acpsl mutant extracts (data not shown). Taken together these observations suggest that carboxypeptidase yscS exists in two catalytically active forms which differ in their electrophoretic mobility.
The deduced amino acid sequence of carboxypeptidase yscS contains five potential N-glycosylation sites (40). T o determine how many of these sites are actually modified on the two carboxypeptidase yscS species in vivo, limited endoglycosidase F digestion was performed (Fig. 1B). Wild type cells for CPSl were pulse labeled and chased for 60 min and the carboxypeptidase yscS proteins were subsequently immunoprecipitated. The carboxypeptidase yscS antigens were incubated with endoglycosidase F over a time course of 60 min and samples were withdrawn at the times indicated (Fig. 1B). After 60 min treatment with endoglycosidase F, the 77-and 74-kDa protein bands present a t time point 0 min (lane 1 ) had completely disappeared, while a new single protein band of 69 kDa apparent molecular mass appeared (lane 6). This form is believed to represent carboxypeptidase yscS completely devoid of N-linked carbohydrates, since longer incubation in the presence of endoglycosidase F did not alter the electrophoretic mobility of the protein which also appears with the identical apparent molecular mass after pulse labeling in the presence of tunicamycin (data not shown). Assuming an average molecular mass of 2.5-3 kDa per N-linked oligosaccharide and analysis of the partially deglycosylated intermediate forms of carboxypeptidase yscS during endoglycosidase F digestion (Fig. lB, lanes 2-5) suggests that the high molecular mass form of carboxypeptidase yscS of 77 kDa corresponds to a protein with three carbohydrate residues, whereas the low molecular mass form of 74 kDa represents a carboxypeptidase yscS molecule modified with only two carbohydrate residues.
The discrepancy between the calculated molecular mass of 64.6 kDa for carboxypeptidase yscS on the basis of the nucleotide sequence (40) and the observed apparent molecular mass of 69 kDa for the protein might be explained by additional modification, e.g. 0-linked glycans, as suggested for proteinase yscB (11) which increase the apparent molecular mass.
Biosynthesis and Processing of Carboxypeptidase yscS-To study the biosynthesis of carboxypeptidase yscS, cells of wild type yeast strain YB18 were pulse labeled for 7 min with [35S] methionine and chased with nonradioactive amino acid for different times as indicated in Fig. 2A. After a 7-min pulse (lane 1 ), carboxypeptidase yscS appears as two distinct forms of 81 and 78 kDa apparent molecular mass, which after 60 min are chased to two distinct forms of 77 and 74 kDa (lane 7). A direct comparison by Western hybridization of the precursor and mature forms of carboxypeptidase yscS was used to determine the relative molecular masses, as shown in Fig. 5, lanes 1 and 2. After treatment of the immunoprecipitated samples with endoglycosidase F (Fig. 2B), which re- moves the N-linked carbohydrate residues, the two distinct forms of 81 and 78 kDa (Fig. 2 A , lane 1 ) migrate as a single band of 73 kDa apparent molecular mass (Fig. 2B, lane 1 ). This band is converted to a 69-kDa form after the 60-min chase period (Fig. 2B, lane 7). This indicates that the decrease in apparent molecular mass during biosynthesis found for the two differently glycosylated forms (Fig. 2 A ) must be due to a proteolytic processing event of carboxypeptidase yscS. Analysis of the deduced carboxypeptidase yscS amino acid sequence revealed a single hydrophobic domain between amino acid positions 20 and 40 and predicted a signal sequence cleavage site at position 40 (40). The observed difference in size of 4 kDa between the unprocessed deglycosylated precursor of 73 kDa (Fig. 2B, lane 1 ), as compared to the processed deglycosylated mature form of 69 kDa (Fig. 2B, lane 7), is consistent with the possible cleavage of the N-terminal 39 amino acids of carboxypeptidase yscS. The kinetics for the processing event is unusual. Carboxypeptidase yscS is converted to its mature forms with a half-time of approximately 20 min, a value which is very different to the half-time of around 6 min that has been observed for the processing of procarboxypeptidase yscY (16), proproteinase yscA (30), and proalkaline phosphatase (19).
Proteinase yscA and yscB have been shown to be major processing enzymes of vacuolar hydrolase precursors (4,6,12, In order to identify the enzymatic activity responsible for the processing of the carboxypeptidase yscS precursor forms, we followed the biosynthesis of the enzyme in isogenic yeast strains carrying different combinations of wild type and mutant alleles for proteinase yscA and yscB. Despite the different genotype, all strains exhibited identical specific activity for carboxypeptidase yscS in vitro (data not shown).
Immunoblot analysis of cell extracts prepared from these isogenic yeast strains confirmed these results, although strain YHH19, deficient in proteinase yscB but containing active proteinase yscA, showed mainly unprocessed but also some processed carboxypeptidase yscS (data not shown). This led us to extend the chase time in a pulse labeling experiment with this strain to 4 h. As shown in Fig. 4A, lane 5, after a 4h chase approximately half of the labeled carboxypeptidase yscS molecules appeared in the processed glycosylated forms of 77 and 74 kDa and, after endoglycosidase F treatment in the 69-kDa form (Fig. 4B, lane 5 ) , which is consistent with our finding from immunoblotting of YHH19 cell extracts.
These results indicate that proteinase yscB is an essential enzyme for the processing of the carboxypeptidase yscS precursor forms in wild type cells. In the absence of proteinase yscB, proteinase yscA itself or a protein that is dependent on proteinase yscA activity or maturation can also mature some carboxypeptidase yscS. The maturation of proteinase yscB has been found to depend on proteinase yscA activity (13,15). This explains the inability of strain YHH65 carrying the proteinase yscB wild type allele, but lacking proteinase yscA activity, to process the carboxypeptidase yscS precursor forms. It is not clear, however, whether proteinase yscB itself is the actual carboxypeptidase yscS maturing enzyme or if it activates another peptidase responsible for the production of mature carboxypeptidase yscS.
In vitro experiments also point to proteinase yscB as the major enzyme activity involved in carboxypeptidase yscS maturation: vacuoles of the double mutant strain deficient in proteinase yscA and yscB (YHH32 pral::URA3 prblAAV CPSl ) were isolated and used as source of carboxypeptidase yscS precursor forms. To convert the precursors to their mature forms, we incubated osmotically lysed YHH32 vacuoles in the absence and presence of purified proteinase yscA, purified proteinase yscB, and vacuole preparations from Acpsl mutant strain YD19 ( P R A I PRBl Acpsl::URA3), which contains mature proteinases yscA and yscB (Fig. 5 ) . Addition of purified proteinase yscA to unprocessed carboxypeptidase yscS forms caused dramatic degradation of the enzyme (compare lanes 2 and 3). This result was not unexpected and reflects the fact that carboxypeptidase yscS is rapidly degraded in proteinase yscA wild type cell extracts? Presence of the proteinase yscA-specific inhibitor pepstatin completely inhibited proteinase yscA-dependent degradation activity (lane 4 ) . Incubation with purified proteinase yscB generated mature carboxypeptidase yscS forms (compare lanes 1 and 5 ) . As expected, the proteinase yscB-specific inhibitor chymostatin completely prevented processing (lane 6). In the presence of vacuole preparations of yeast strain YD19 containing no carboxypeptidase yscS protein but wild type levels of proteinases yscA and yscB activities, carboxypeptidase yscS is processed (compare lanes 1 and 7). Addition of the proteinase yscA inhibitor pepstatin did not prevent the processing event (lane 8 ) , whereas the proteinase yscB inhibitor chymostatin blocked the processing of carboxypeptidase yscS (lane 9).
As was found in vivo, the in vitro processing system iden- tifies proteinase yscB to be the major enzyme triggering the maturation of carboxypeptidase yscS. Vacuolar Location of Carboxypeptidase yscS-In wild type cells for proteinase yscA and proteinase yscB, most of the carboxypeptidase yscS enzyme activity has been found to reside within the vacuole (39), however, the precise location of the enzyme within the vacuole remained unknown.
We therefore isolated vacuoles from wild type strain YB18, which contains the mature forms of carboxypeptidase yscS. Intact vacuoles were osmotically lysed in 0.1 M sodium carbonate, pH 11.5, and subjected to centrifugation a t 100,000 X g. Treatment with alkali buffer releases soluble and extrinsic membrane proteins, while integral proteins remain membrane associated (53). After centrifugation, the soluble and membrane fractions were analyzed for carboxypeptidase yscS by immunoblotting (Fig. 6 A ) . As can be seen in Fig. 6A, lane 3, the mature forms of carboxypeptidase yscS are only present in the soluble fraction. Previous experiments using the proteinases yscA-and yscB-deficient strain, YHH32, showed that the carboxypeptidase yscS precursor forms are also present in the vacuole (Fig. 5, lane 2). We repeated the above fractionation procedure with the vacuoles of strain YHH32 and found the carboxypeptidase yscS precursor forms tightly associated with the vacuolar membrane (Fig. 6A, lane 5). In addition, this membrane fraction exhibited high levels of activity in the carboxypeptidase yscS enzyme assay (data not shown). Only the use of detergent (Triton X-100) was able to solubilize the carboxypeptidase precursor forms (Fig. 6A, lane  8). To check the sodium carbonate fractionation procedure, we tested the fractions for other vacuolar proteins. Most of carboxypeptidase yscY, a marker for a soluble vacuolar enzyme (54), was recovered in the supernatant of fractionated vacuoles from wild type strain YB18 (Fig. 6B, lane 3 ) , whereas the pro-form of alkaline phosphatase, known to be an integral vacuolar membrane protein (19), remained associated with the membrane pellet after fractionation of proteinase yscAand yscB-deficient vacuoles from strain YHH32 (Fig. 6C, lane 2).
In summary, it is clear that the precursor forms of carboxypeptidase yscS are located in the vacuolar membrane, whereas the mature forms of the enzyme are in the lumen of the vacuole.
Those findings might be explained by a mechanism where the carboxypeptidase yscS precursor forms are membrane associated during transport to the vacuole and, upon arrival, are proteolytically cleaved to produce the mature soluble forms of carboxypeptidase yscS. Based on this assumption, that a single N-terminal hydrophobic domain functions in anchoring the carboxypeptidase yscS precursor forms in the membrane, we hypothesize that this N-terminal sequence represents the segment which is cleaved off by proteinase yscB a t or near the predicted signal sequence cleavage site. Such a mechanism would generate the mature carboxypeptidase yscS forms which are subsequently released into the lumen of the vacuole, as has been observed. The proposed mode of generation of soluble carboxypeptidase yscS would require an orientation of the carboxypeptidase yscS precursor forms in the membrane, with a short N-terminal tail facing the cytosol and the C-terminal part of the protein exposed to the lumen of the vacuole. We examined the predicted orientation performing proteinase K protection assays. Vacuoles from wild type strain YB18 (soluble carboxypeptidase yscS forms), and the proteinase yscA-and proteinase yscB-deficient strain YHH32 (membrane-associated carboxypeptidase yscS forms) were treated with proteinase K in the absence and presence of detergent and subsequently analyzed for carboxypeptidase yscS by immunoblotting (Fig. 7). As expected, the mature and soluble forms of carboxypeptidase yscS were fully protected against degradation by proteinase Vacuoles of strain YB18 (soluble mature carboxypeptidase yscS forms) (panel A ) and strain YHH32 (membrane-associated carboxypeptidase yscS precursor forms) (panel B ) were treated with proteinase K in the absence or presence of 1% Trion X-100 as described under "Experimental Procedures." Aliquots were withdrawn from the mixtures at the times indicated and analyzed by immunoblotting with carboxypeptidase yscS-specific antiserum. K in the absence of detergent (Fig. 7A). Similarly, no change in the electrophoretic mobility of the membrane-associated precursor forms were detectable after incubation with proteinase K, whereas addition of detergent resulted in degradation of carboxypeptidase yscS by proteinase K (Fig. 7B). The complete resistance of the membrane-associated forms against degradation in the absence of detergent can be explained as the long C-terminal segments facing the vacuolar lumen and the N-terminal 20 amino acids preceeding the hydrophobic domains being exposed to the cytoplasm, but due t o their partly hydrophobic characters are buried in the lipid bilayer (40). All these data support our hypothesis that the carboxypeptidase yscS precursor forms adopt the orientation of a vacuolar type I1 integral membrane protein.
Modification of Carboxypeptidase yscS in the Secretory Pathway-As shown above, the membrane-associated precursor forms as well as the soluble mature forms of carboxypeptidase yscS exist as two differentially glycosylated proteins in the vacuole.
In order to determine whether carboxypeptidase yscS passes through the secretory pathway, we followed the biosynthesis of carboxypeptidase yscS in several conditionally defective sec mutants (see Ref. 55, for review): s e d 1 which is defective in protein translocation across the ER membrane (56); sec59 which accumulates inactive and incompletely glycosylated forms of secretory proteins in the lumen of the ER (57,58); secl8 which is defective in vesicular transport from ER to the Golgi (17,29); and sec7 which is blocked in translocation from the Golgi complex (17,59).
After shifting to the restrictive temperature, wild type cells were labeled with ["Slmethionine for 7 min and chased with nonradioactive methionine for 60 min, and mutant cells were labeled with ["S]methionine as described under "Experimental Procedures." Carboxypeptidase yscS-specific protein was precipitated with the antiserum. Immunoprecipitated carboxypeptidase yscS proteins were analyzed before and after digestion with endoglycosidase F to determine the glycosylation state of the accumulating intermediates (Fig. 8, A and B ) .
When wild type strain YB18 was shifted to 37 "C, two differently glycosylated forms of carboxypeptidase yscS appeared (Fig. 8A, lane 1 ). Treatment with endoglycosidase F resulted in a carboxypeptidase yscS protein with the apparent molecular mass of the processed form (Fig. 8B, lane 1 ), as expected from wild type cells. The sec61 mutation resulted in the accumulation of the unglycosylated as well as the fully glycosylated precursor which must have escaped the sec61 block (Fig. 8A, lane 2). As this strain carries, in addition to the sec61 mutation, a mutation in the PRAl gene (allelpep4-3 ) the carboxypeptidase yscS precursor cannot be processed (see previous section), and thus only the protein precursor is visible after treatment with endoglycosidase F (see also Fig.  8B, lane 2). In sec59 mutant cells an unglycosylated and an incompletely glycosylated form of carboxypeptidase yscS is detected (Fig. 8A, lane 3). After endoglycosidase F treatment, both bands show the apparent molecular mass corresponding to the mature form of the enzyme (Fig. 8B, lane 3 ) . This result suggests that the unglycosylated and the partially glycosylated carboxypeptidase yscS precursors do not accumulate in the ER of sec59 cells but transit to the vacuole where processing takes place. As found for the precursor escaping the block in sec61 cells, in secl8 and sec7 cells mainly carboxypeptidase yscS protein of the apparent molecular mass of the precursor carrying three carbohydrate chains appeared under restrictive conditions (Fig. 8A, lanes 2,4, and 5 ) . Under permissive conditions the two glycosylated carboxypeptidase yscS proteins appeared in all secretory mutants (Fig. 8C, lanes 2-5). The accumulation of unglycosylated, partially and fully glycosylated forms of carboxypeptidase yscS show that the enzyme traverses the secretory pathway to the vacuole. Upon translocation into the ER, the carboxypeptidase yscS is core glycosylated, a phenomenon which has also been described for other vacuolar enzymes (17)(18)(19)30). The identical apparent molecular mass of the accumulating carboxypeptidase yscS forms under restrictive conditions in the ER-blocked secl8 and the Golgi-blocked sec7 strains suggests that either no addition of oligosaccharides occur during transit through the Golgi, or that carbohydrate modifications in the Golgi do not lead to a grossly altered apparent molecular mass of the carboxypeptidase yscS precursor forms. This observation is consistent with the finding also that in wild type cells, no increase in apparent molecular mass of carboxypeptidase yscS was detectable during the course of biosynthesis of carboxypeptidase yscS (Fig. 2 A ) . DISCUSSION Biochemical analysis of carboxypeptidase yscS had indicated that most of the enzyme activity resides within the vacuole (39). We have studied the biosynthesis of carboxypeptidase yscS, the vacuolar location of the enzyme, and the mechanism of its delivery to the vacuole.
Nondenaturing electrophoresis of wild type cell extracts with subsequent staining for carboxypeptidase yscS activity revealed two bands with different electrophoretic mobility. SDS-PAGE of immunoprecipitated wild type carboxypeptidase yscS led to the appearance of two distinct glycosylated forms of the enzyme with apparent molecular masses of 77 and 74 kDa. Deglycosylation studies with endoglycosidase F indicate that two carboxypeptidase yscS proteins exist, which differ in their carbohydrate content, one protein containing two, the other containing three N-linked carbohydrate residues. In this respect carboxypeptidase yscS is different from other vacuolar hydrolases which exist only in one distinct glycosylated catalytically active form (for review, see Ref. 7).
Pulse-chase labeling of wild type cells showed that carboxypeptidase yscS is synthesized as a single polypeptide chain which is differentially glycosylated giving species of 81 and 78 kDa. Proteolytic cleavage of the precursor polypeptide chains during biosynthesis leads to the mature carboxypeptidase yscS forms of 77 and 74 kDa. I n vivo analysis of isogenic yeast strains carrying various combinations of wild type and mutant alleles of the PRA13 and PRBl locus predicted that proteinase yscB might be the major processing enzyme of carboxypeptidase yscS. In the absence of proteinase yscB, proteinase yscA seems to be able to weakly process carboxypeptidase yscS. Whether both endopeptidases possess the same cleavage specificity for carboxypeptidase yscS in vivo remains unknown. The in vivo processing function of proteinase yscB was clearly demonstrated also in vitro, where only mature proteinase yscB was able to convert the carboxypeptidase yscS precursor forms into the processed forms. Studies on the vacuolar location of the carboxypeptidase yscS precursor forms and the mature forms of carboxypeptidase yscS using sodium carbonate fractionation showed that the mature carboxypeptidase yscS forms are located in the vacuolar lumen in a soluble form, while the precursor forms are tightly associated with the vacuolar membrane. The resistance of the carboxypeptidase yscS protein precursors against proteinase K degradation suggests a topology similar to type I1 integral membrane proteins. The analysis of the deduced primary structure of carboxypeptidase yscS had revealed a single hydrophobic domain which is located between amino acid positions 20 and 40, and a predicted signal sequence cleavage site at amino acid position 40 (40). We believe that the hydrophobic core is responsible for anchoring the precursor proteins in the membrane and that the predicted signal sequence cleavage site is not cleaved by signal peptidase, but it or some site near to it is actually cleaved in the vacuole to produce the soluble and mature forms of carboxypeptidase yscS. This explanation is in agreement with the observed difference in electrophoretic mobility of approximately 4 kDa on SDS-PAGE between the protein precursor forms of carboxypeptidase yscS and the mature forms of carboxypeptidase yscS.
Delivery of carboxypeptidase yscS to the vacuole has been found to depend on an intact secretory pathway. The mature forms of carboxypeptidase yscS consist of two glycoproteins with two or three N-glycosyl residues, respectively. The predominant form of carboxypeptidase yscS which accumulated after a block in the transport from the ER to the Golgi complex (secl8 cells) or after a block of the transit from the Golgi to the vacuole (sec7 cells) appeared to be the protein precursor modified with three oligosaccharides. We believe that the presence of only one glycosylated form reflects the disturbed physiological situation caused by the accumulation of secretory proteins in sec mutant strains under restrictive conditions. One cannot exclude the possibility that under restrictive conditions (37/39 "C), the carboxypeptidase yscS precursor form modified with only two oligosaccharides is degraded due to incomplete folding.
All data observed can be summarized in a model for the biosynthesis and delivery of carboxypeptidase yscS to the vacuole (Fig. 9). The carboxypeptidase yscS precursor protein molecule is synthesized and translocated into the ER. Here the protein precursor receives two or three carbohydrate chains. The two differently glycosylated precursor forms remain membrane associated and are transferred through the a PRAl designates the structural gene of proteinase yscA PEP4 is allelic to PRAl (12).

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GOLGI VACUOLE P r O t C O l Y t l C mat"ratI0" FIG. 9. Proposed model for the biosynthesis of carboxypeptidase yscS. The model suggests that carboxypeptidase yscS goes via the secretory pathway to the vacuole. The appearance of two differently glycosylated precursor forms (81 and 78 kDa) and mature forms (77 and 74 kDa) of carboxypeptidase yscS is indicated. It is proposed that the carboxypeptidase yscS precursors remain membrane associated via the single N-terminal hydrophobic domain (st+pled box) until the delivery to the vacuole. Proteinase yscB is assumed to be the major processing enzyme for the carboxypeptidase yscS precursors in the vacuole. The processed forms of carboxypeptidase yscS are soluble in the vacuolar lumen.
Golgi to the vacuole, where they are proteolytically cleaved by proteinase yscB to release the mature forms of carboxypeptidase yscS of about 4 kDa lower molecular mass into the lumen of the vacuole (Fig. 9).
This mechanism is similar to the mechanism observed for the soluble lysosomal enzymes a-mannosidase and p-glucosidase in D. discoideum, which are also synthesized as membrane-associated precursor molecules. However, the physical nature of the precursor-membrane interaction has not been defined (60). In case of the soluble human lysosomal acid phosphatase, the C-terminal hydrophobic domain of the precursor is believed to be responsible for its membrane association during transport. Cleavage of the C-terminal segment upon arrival at the lysosome results in the soluble form. The N terminus of the human lysosomal acid phosphatase precursor molecule with its hydrophobic core is postulated to function as cleavable signal sequence during translocation across the ER membrane (36,61). This targeting and transport mechanism is different from carboxypeptidase yscS, where the N termini of the precursors seem to function in translocation and anchoring of the precursor proteins in the ER membrane, as the N-terminal hydrophobic domains of vacuolar integral membrane proteins have been proposed to function (18, 19).
The proposed biosynthetic pathway for carboxypeptidase yscS is clearly different from other soluble vacuolar hydrolases which have never been found to be membrane associated during transport. A different mechanism is also reflected in the half-time of about 20 min for the processing of carboxypeptidase yscS in comparison to about 6 min for the processing of other soluble hydrolases (16,19,30). It is also important to note that in contrast to what has been found for other soluble hydrolases (9-13, 22, 23), the proteolytic processing of the carboxypeptidase yscS precursor forms has no influence on the activity of the enzyme. Thus, the exclusive function of the processing event seems to reside in the correct localization of carboxypeptidase yscS in the vacuolar lumen.
It seems, to our surprise, that the presumptive signal sequence cleavage site is not recognized by the signal peptidase during translocation. This can be explained by the unusual length of 39 amino acid residues for the putative signal sequence and its 20-amino acid residue long N-terminal hydrophobic domain. The role of signal peptidase seems to have been taken over by proteinase yscB, for proteinase yscB most probably removes the N termini of the carboxypeptidase yscS precursor proteins responsible for targeting the protein to the vacuole.
Construction and analysis of hybrid proteins containing the N-terminal sequence of carboxypeptidase yscS will provide further information on the signals responsible for the membrane association and processing of the carboxypeptidase yscS precursor forms.