Aminopeptidase Y, a new aminopeptidase from Saccharomyces cerevisiae. Purification, properties, localization, and processing by protease B.

A novel aminopeptidase, termed aminopeptidase Y, was purified from yeast, Saccharomyces cerevisiae, as two molecular forms of 70 and 75 kDa, which were identical immunologically, catalytically and in N-terminal sequence up to 14 residues. They contained 0.81 and 0.84 mol of zinc atom/mol of protein, respectively. N-Gly-canase generated a single 53-kDa species from both forms, and endoglycosidase H gave a 54-kDa species. Immunoblotting after subcellular fractionation showed that aminopeptidase Y is localized in the vacuole/lysosome-soluble compartment as the 70-kDa form. Aminopeptidase Y hydrolyzed all amino acid-4-methylcoumaryl-7-amides (MCA) examined, especially Lys- and Arg-MCA. Dipeptidyl-MCAs were far more rapidly hydrolyzed (Lys-Ala-MCA/Lys-MCA = 350-fold, Arg-Arg-MCA/Arg-MCA = 150-fold). Hydrolysis of amino acid-MCAs or dipeptides was markedly enhanced by Co2+, whereas that of dipeptidyl-MCAs, tripeptides, and larger peptides was inhibited. Rabbit anti-aminopeptidase Y antiserum adsorbed over half the aminopeptidase activities in vacuolar extract from wild-type yeast cells. ABYS1 mutant cells from which the genes of four vacuolar proteases had been deleted contained aminopeptidase Y as a 74-kDa proform. This was converted to the mature form (70 kDa) by vacuolar extract of wild-type cells and by purified yeast vacuolar protease B.

PrA, PrB, and CPY are imported as proforms and processed on the way to or after reaching the vacuoles; they function as the mature proteases (9). Of these proteases, PrA seems primarily responsible for the processing of a number of vacuolar zymogens, since the pep4 mutant lacking the activity of PrA also lacks the activities of PrB, CPY, and alkaline phosphatase (101, and at least CPY and PrB were accumulated as larger precursors (11, 12). Conversion of pro-CPY to mature CPY is catalyzed by PrB, which might be activated both by PrA and autocatalytically (13, 14), or by an alternate activation cascade in which active intermediates are formed by the direct action of PrA (15).
Aminopeptidases exist ubiquitously in organisms (16) and play a role in catabolizing peptides and processing bioactive peptides, a s well as functioning as leukotriene A4 hydrolase (17). Despite many studies on aminopeptidases, nothing is known about their processing for activation or sorting. Several yeast aminopeptidases have been reported as vacuolar enzymes, but many of them correspond to A P I (9). We describe here a new and highly potent vacuolar aminopeptidase, termed aminopeptidase Y (APY). This paper deals with the purification, enzymological properties, localization, and processing of this major yeast vacuolar aminopeptidase. The accompanying paper (36) describes the molecular cloning of the APY gene and the construction of an APY-deleted mutant.

General Methods
Protein concentration was determined with the BCA protein assay reagent (Pierce Chemical Co.). SDS-polyacrylamide gel electrophoresis was run according to Laemmli (18). The anti-APY IgG fraction was prepared from antiserum raised in rabbit against APY with protein G-agarose (Calbiochem). Atomic absorption analysis was carried out by using a polarized Zeeman atomic absorption spectrophotometer (Hitachi 2-7000).
Yeast Cells and Growth Yeast strains used were D273-1B (ATCC25657) as wild-type and ABYSl (MATa, pral, prbl, prcl, cpsl, ade) lacking vacuolar proteases A and B and carboxypeptidases Y and S. Cells were grown as described to stabilize the fluorescence. Hydrolysis of the substrate was calculated (19).
from the slope of the linear decrease of fluorescence using the ratios Subfractionation of Yeast Cells The yeast cells were grown to the late logarithmic stage and converted to spheroplasts with zymolyase (20). After precipitation of the spheroplasts by centrifugation at 1,500 x g for 5 min, the supernatant was centrifuged at 200,000 x g for 1 h, and the supernatant thus obtained was recovered as the periplasmic fraction. The spheroplasts were homogenized in a Dounce homogenizer in 0.6 M sorbitol, 10 m~ Tris-HC1 (pH 7.5) (homogenate l), layered on 0.4 M sucrose solution and centrifuged at 700 x g for 10 min. The supernatant was centrifuged at 7,000 x g for 10 min. The supernatant thus obtained was successively centrifuged at 105,000 x g for 100 min, and the precipitate was recovered as microsomal fraction. The precipitate of the 7,000 x g centrifugation was suspended in 0.25 M sucrose and the precipitate from centrifugation at 24,000 x g for 10 min was recovered as the crude mitochondrial fraction.
This was subjected to 0.8-1.2 M sucrose gradient centrifugation at 53,000 x g for 2 h, and the lower fraction was recovered as the mitochondrial fraction. To subfractionate the vacuolar fraction, spheroplasts were homogenized in 12% Ficoll, 0.1 m~ MgCI,, 10 m~ HEPES (pH 7) (homogenate 2) and processed as described (21). The prepared vacuolar fraction was diluted with 3 volumes of water and sonicated. The soluble and membrane fractions were separated by centrifugation at 200,000 x g for 1 h. Published methods were employed for assaying NADPHcytochrome c reductase (22), cytochrome c oxidase (23), and carboxypeptidase Y with N-(2-furylacryloyl)-~-Phe-~-Phe as the substrate (24).

Enzyme Assay
Enzyme assays for aminopeptidase were carried out by the following three methods.
Method A-With amino acid-MCA as the substrate, fluorescence due to the 7-amino-4-methylcoumarin produced was measured at A, 380 nm and A,, 460 nm. During the purification steps, 20 p Arg-MCA was used as the substrate in 0.5 ml of 50 m~ MOPS (pH 6.5). For characterization of the enzyme, 0.1 m~ amino acid-MCAs in 0.5 ml of MOPS (pH 7.5) were used, and enzyme reactions were performed at 30 "C.
Method B-Taking advantage of our finding that dipeptidyl-MCAs are more strongly fluorescent than amino acid-MCAs at A,, 325 nm and A,, 390 nm, hydrolysis of dipeptidyl-MCAs to amino acid and amino acid-MCAs was estimated by the following three methods. In method B-1, dipeptidyl-MCAs (0.1 m~) were hydrolyzed by the enzyme in 0.1 ml of 50 m~ MOPS (pH 7.5) at 30 "C. After the reaction was stopped by addition of 0.5 ml of 50% ethanol, 10 m~ EDTA, 0.1 M Tris-HC1 (pH 7.5), hydrolyzed dipeptidyl-MCA was quantified from the decrease of fluorescence at A,, 325 nm and A, , 390 nm. The fluorescence ratios of Lys-Ala-MCNAla-MCA and Arg-Arg-MCNArg-MCA were taken to be 1.7 and 1.8, respectively. Further hydrolysis of dipeptidyl-MCA, via amino acid-MCA, to 7-amino-4-methylcoumarin could be estimated at A,, 380 nm and A,, 460 nm as in method A. In method B-2, assayed solutions prepared as in method B-1 were subjected to high-performance liquid chromatography (HPLC) with a reverse-phase column (Superspher RP-8, Merck Co.), using 11% acetonitrile, 0.1% trifluoroacetic acid for Lys-Ala-MCA and Arg-Arg-MCA or 14% acetonitrile, 0.1% trifluoroacetic acid for Pro-Phe-Arg-MCA. Hydrolysis of each substrate was estimated from the diminution of peak area of the substrate as detected at A, , 325 nm and A,, 390 nm with a Shimadzu fluorescence HPLC monitor FtF-535 and quantified with a Shimadzu Chromatopac C-R5A data processor. In method B-3, hydrolysis of dipeptidyl-MCA to amino acid-MCA was continuously monitored in the cuvette of a spectrofluorophotometer at A, , 325 nm and A,, 390 nm. The enzyme reaction was performed in 0.5 ml of 50 m~ MOPS (pH 7.5) containing 50 p of each dipeptidyl-MCA. A narrow excitation slit width, 2 nm, was selected mentioned in method B-1.
Method C-Hydrolysis of peptides by the aminopeptidase was assayed in 25 pl of 10 n m MOPS (pH 7.5) containing 0.1 m~ peptide at 30 "C. After the reaction was stopped by heating at 100 "C for 4 min, the amino acids released from the N-terminal ends of peptides were analyzed as their dansyl derivatives by HPLC with a reverse-phase column (25). Hydrolysis of peptides was estimated from the diminution of peak area of dansylated substrate peptide or from the new peak of dansylated N-terminal amino acid. One unit was defined as the amount of enzyme required to hydrolyze 1 pmol of substratdmin in each assay system.
To quantify the antigen-antibody complexes, lZ5I-protein G (Amersham) was added, and autoradiography was conducted with a Bio-image analyzer BAS 2000 (Fuji Film Co.).

Enzyme Purification
Purification of Aminopeptidase Y-Three kg of commercial bakers' yeast cells (Saccharomyces cereuisiae, Oriental Yeast Co., Japan) was homogenized in a Dynomill in isotonic buffer, 0.6 M sorbitol, 10 m~ Tris-HC1 (pH 7.9, and a crude membrane-rich fraction was prepared by differential centrifugation (26). This fraction was sonicated in 10 m~ Tris-HC1 (pH 7.9, and the extract was obtained by centrifugation at 15,000 x g for 45 min followed by 150,000 x g for 45 min. The extract (1850 ml) was treated with 1 m~ PMSF, adjusted to pH 7.5 with NaOH, and applied to a DEAE-cellulose column (6 x 17 cm) equilibrated with 10 m~ Tris-HC1 (pH 7.5). After washing of the column with 2 liters of equilibration buffer, absorbed materials were eluted with a linear gradient to 0.5 M NaCl, 10% glycerol, 10 m~ Tris-HC1 (pH 7.5) (1.4 liters total in 65 fractions). Fraction numbers 2 0 4 4 were collected, and NaCl was added to give a concentration of 0.5 M. Next, zinc-chelating Sepharose column chromatography was camed out in 0.5 M NaCI, 10% glycerol, 20 m~ Tris-HC1 (pH 7.5). After application of the active fractions from DEAE-cellulose chromatography, the column (1.6 x 25 cm) was washed with 1 liter of the buffer. Absorbed materials were eluted with a linear pH gradient from the above buffer to 0.5 M NaCI, 10% glycerol, 10 m~ NaH,PO, (pH 3.3) (400 ml total in 60 fractions). The active fractions eluted at around pH 6.5 (fraction numbers 3-2, 165 ml) were collected. After the dialysis against 10% glycerol, 20 m~ Tris-HC1 (pH 7.5), the active fractions were applied to a DEAE-cellulose column (1 x 44 cm) equilibrated with the buffer. The absorbed materials were eluted with a 0-0.3 M linear gradient of NaCl in the buffer. The active fractions eluted around 0.1 M NaCl were collected and concentrated to 1.6 ml by ultrafiltration with a PM-10 membrane (Amicon). The concentrate was applied to a Sephadex G-200 column (2 x 92 cm) and developed with 0.2 M NaCl, 10% glycerol, 10 m~ MOPS (pH 7.5). The combined active fractions (29 ml) were dialyzed against 1 liter of 10% glycerol, 20 m~ HEPES (pH 7.5) and applied to a protamine-Sepharose column (1 x 27 cm) equilibrated with the same buffer. Elution was camed out with a linear gradient to 4 M NaCI, 10% glycerol, 10 m~ NaH,PO, (pH 3.3) (150 ml total in 45 fractions). The column was chilled with ice, and the flow rate was 1 mumin. The active fractions which eluted around 2 M NaCl (fraction numbers 37-48) were collected and applied to a hydroxylapatite column (1 x 27 cm) equilibrated with the starting buffer, 10% glycerol, 20 m~ HEPES (pH 7.5). The adsorbed enzymes were eluted with a linear gradient to 10% glycerol, 0.2 M potassium phosphate (pH 7.5) (400 ml total in 100 fractions). Two active peaks appeared. The first part of the first peak, which eluted around 0.1 Y potassium phosphate (fraction numbers 40-55), was collected as APY,,. The latter half of the second peak, which eluted around 0.13 M potassium phosphate (fraction numbers 70-90) was collected as APY,,. The overlapping part (fraction numbers 56-69) was rechromatographed on the hydroxylapatite column under the same conditions as before. The active fractions were collected as before and combined with those previously obtained.

Purification of Protease B-Preparation of crude extract of bakers'
yeast (1 kg) and protein precipitation with ammonium sulfate were camed out as described (27). The purification was carried out with five steps of column chromatography: DEAE-cellulose, hydroxylapatite, DEAE-cellulose (second), Sephadex G-200, and HPLC with hydroxylapatite (HCA column, Mitsui Toatsu Chemicals). Activity of protease B was measured according to Saheki and Holzer (28) with Azocoll as the substrate. The purified protease B (72 mg) showed a specific activity of 16.8AS,, n,.min".mg".

RESULTS
Purification of Yeast Aminopeptidase-The final purification step on a hydroxylapatite column is shown in Fig. 1. The two peaks of activity coincided with the peaks of absorbance at 280 nm, and each of the two purified components showed a single band on SDS-polyacrylamide gel electrophoresis (Fig. 2). The enzyme from the first peak fractions showed a molecular mass value of 70 kDa and the other a value of 75 kDa. These values were the same under nonreducing conditions (data not shown). Thus, aminopeptidase Y was distinguished as APY,, and APY,,, respectively. Estimation of molecular mass by gel filtration gave values of 70 kDa for APY,, and 80 kDa for MY,, (data not shown). MY,, and APY,, were obtained in amounts of 3.8 and 3.5 mg, respectively, from 3 kg of cells (Table I).
Molecular Difference between MY,, and MY7,5-To establish whether APY,, and APY,, are the same enzyme or not, partial N-terminal amino acid sequence determination was conducted in a gas-phase protein sequenator. Both molecules had the same N-terminal sequence up to 14 amino acids: Ile-Pro-Lys-

Pro-His-Ile-Pro-Tyr-Phe-Met-Lys-Pro-His-Val.
The possible difference in sugar chain content between APY,, and MY,, was examined by treating them with endoglycosidase H and N-glycanase (Fig. 3). Treatment of both MY,, and APY,, with N-glycanase resulted in their conversion to a single product of molecular mass 53 kDa. Treatment with endoglycosidase H also converted both molecules into a single species of molecular mass 54 kDa, showing that the conjugated sugar chains are of the high mannose type. These results indicate that APY,, and M Y , , consist of a n identical core protein with different sugar contents.
Atomic Absorption Analysis-Many aminopeptidases contain a zinc atom in the active site, and atomic absorption anal- AMCImin under the assay condition.  (Table 11). These results, together with the inhibition by the aminopeptidase-specific inhibitors bestatin and amastatin and the presence of a zinc atom, show that APY is a typical aminopeptidase.
In regard to metal ions, 0.5 mM Co2+ enhanced the activities &9-fold, whereas Ca2+ and Mg2' had little effect, and Mn2+, Cu2+ and Zn2+ were inhibitory. The inhibition of APY by o-phenanthroline was restored only by Co2+ (data not shown).
Substrate Specificity of M Y a n d Effects of Co2+ on the Activity-The substrate specificity of APY toward MCA substrates was examined in the presence of 0 and 0.25 mM Co2+ (Table 111). Peptidyl-MCAs were far more rapidly hydrolyzed than amino acid-MCAs, and ratios of the release rates of same N-terminal amino acids from those substrates in the absence of cobalt were as follows; Lys-Ala-MCA/Lys-MCA = 350, Arg-Arg-MCAfArg-MCA = 150, Pro-Phe-Arg-MCAPro-MCA = 750. Co2+ had different effects, depending on the substrate used.
Hydrolysis of amino acid-MCAs was enhanced by Co". In contrast, hydrolysis of peptidyl-MCAs was inhibited.
The concentration dependence of the Co2+ effect on the activities of APY was examined (Fig. 4). With Arg-MCA as the substrate, Co2+ enhanced the activity of APY concentrationdependently. On the other hand, the activity toward Arg-Arg-MCA was progressively inhibited. There was no essential difference between MY,, and APY,,.
Various peptides were hydrolyzed by APY, and the release rates of the N-terminal amino acids were measured (Table IV). APY hydrolyzed the peptides, except Gly-Gly-Gly, at various    rates with different effects of Go2+ on the activity. Hydrolysis of Leu-Leu and kg-Val was activated 3.7-and 1.9-fold by Co2+, respectively. Esterification of both dipeptides resulted in greater susceptibility to APY, although the effect of Go2+ then became inhibitory. Localization of Aminopeptidase Y-Localization of APY was examined by subfractionation of the yeast cells followed by immunoblot analysis (Fig. 5). Enrichment of individual fractions was ascertained in terms of the enhanced specific activities of marker enzymes: carboxypeptidase Y for vacuoles, cytochrome c oxidase for mitochondria, and NADPH-oxidase for microsomes. Antisera raised against APY,, and MY,, crossreacted completely, and here we used antiserum against APY,,. The similar distribution profile of APY to that of carboxypeptidase Y confirmed the vacuolar localization of this enzyme.
The enzyme was localized in the soluble fraction of vacuoles in the molecular form of APY,,. By comparing the intensity of the  Gly-Gly-Gly 0 0 band in the vacuole fraction with that of purified APY, we estimated that APY accounts for about 1% of vacuolar proteins.

Absorption of Vacuolar Aminopeptidase Activity by Anti-APY
ZgG-The contribution of APY to total aminopeptidase activities in the vacuoles was estimated by immunoabsorption. As shown in Fig. 6, absorption with 31 pg of anti-APY IgG left only 12% of Lys-Ala-MCA-hydrolyzing activity, 32% of Lys-MCAhydrolyzing activity (18% when assayed in the presence of Co"), and 47% of Leu-MCA-hydrolyzing activity. Western blot analysis showed that a little APY still remained after the absorption. These results show that APY accounts for the major part of the aminopeptidase activities at least for these examined substrates in this organelle.
Conversion ofAminopeptidase Y Precursor to Mature Form-ABYS1 mutant cells are mutants from which the genes of vacuolar proteases (proteases A and B, carboxypeptidases Y and S ) have been deleted. In the mutant cells, it was expected that APY might exist as a precursor form, since protease A and protease B are responsible for processing or activation of vacuolar zymogens (10). We therefore attempted to detect a precursor form of APY in vacuolar extract of ABYSl mutant cells and examined its conversion to the mature form (Fig. 7). Since the soluble fraction of wild-type cell vacuoles was used in small amount, 1/10 of the protein ofABYS1, the Lys-Ala-MCA-hydro- lyzing activity of the wild-type extract was as low as that of ABYS1. Neither soluble fraction showed much change in aminopeptidase activities during incubation. However, when the two vacuolar extracts were mixed, the hydrolytic activity toward Lys-Ala-MCA gradually increased during incubation (Fig.  7A). This increase was inhibited by PMSF but not by pepstatin. Immunoblot analysis revealed a 74-kDa precursor form of APY (pro-APY) in the vacuoles ofABYSl cells. During incubation for 30 min, about half the APY precursor was converted to mature APY, and conversion was complete at 3 h (Fig. 7B). Inhibition of the conversion of 74-kDa pro-APY to mature 70-kDa APY by PMSF strongly suggested that vacuolar serine protease, protease B, is responsible for processing of the pro-APY. To confirm this, we prepared 31-kDa protease B of high purity, as shown by SDS-polyacrylamide gel electrophoresis (Fig. 8A) About half of the pro-APY was converted to 70-kDa APY in 8 min, although at this point, the Lys-Ala-MCA-hydrolyzing activity was still only 35% of that at 30 min. This apparent disagreement between the conversion of the mature form and activity was also seen when pro-APY was activated by extract of wild-type cell vacuoles; about half of pro-APY was converted to 70-kDa form at 30 min, although the increase of activity was only about 20% of that at 3 h (Fig. 7). DISCUSSION A number of yeast aminopeptidases have been described (for review, see Ref. 9), but the best characterized are methionine aminopeptidase and API. The methionine aminopeptidase is a cytosolic aminopeptidase which removes the N-terminal methionine of newly synthesized proteins (29). API is a vacuolar aminopeptidase with a molecular mass of 640 kDa, composed of 12 subunits. API is typical leucine-aminopeptidase which is highly active toward Leu-p-nitroanilide (PNA), but weakly toward Lys-PNA (30). Aminopeptidase I1 preferentially hydrolyzes Lys-PNA and Leu-PNA, but its periplasmic location (31) distinguishes it from APY. The other known aminopeptidases also differ from APY in location, substrate specificities, or molecular weight, except APCo. APCo has not been purified yet, but the activating effect of Co2+ on its hydrolysis of amino acid-PNA (32) is similar to that in the case of APY. Since the source strain of APCo was the ABYS1 mutant (32), there is a possibility that the reported activity of APCo may be derived from the 74-kDa precursor of APY, although the reported molecular mass of APCo is 100 kDa.
The immunoabsorption experiment with anti-APY I& indicated that APY is responsible for most of the hydrolyzing activity to release N-terminal basic amino acids and over half the activity for Leu, indicating that a major aminopeptidase activity in yeast vacuoles is that ofAPY. Thus, aminopeptidase Y, in cooperation with carboxypeptidase Y, must play a major role in the complete degradation of proteolytic intermediates of proteins or peptides to amino acids.
The activating effect of Co" on the APY activity was observed only when the substrates were amino acid-MCAs or dipeptides (Table 111). whereas Co" inhibited the hydrolysis of dipeptidyl-MCAs, tripeptides, and larger peptides, except Met-Ala-Ser and Met-Gly-Gly (Tables 111 and IV). In view of the nonhydrolyzability of Gly-Gly-Gly, small residues may be unfavorable at the P'], P', subsites of M Y . Opposite effects of metals on the activity of aminoprptidasr depending on the N-terminal amino acid of suhstratrs haw been reported ( 3 3 ) . In the case of APY. however. the kry factor is not the nature of the N-terminal amino acid hut seems to hr the length of the peptides. Esterification of thr dipeptidrs Leu-Leu and Arg-Val altered the effrct of Co'. from activator to inhibitor. Further studies are needed on the action of Co", hut binding site occupancies on APY could he an important factor.
Aminopeptidases have been assayed convrnirntly by using amino acid-conjugated chromophorcs, such as p-nitroanilinr. a-naphthylamide, and MCA. However, such dipeptidr-likr chromogenic or fluorogenic substrates occupy onlv the PI site (subsite where the N-terminal amino acid of thr suhstratr is bound), whereas the P1' site (subsite where the penultimatc. amino acid of the substrate is hound) of methioninr aminnpeptidase is known to function critically in t h r removal of N-trrminal methionine (34). We therefore devrioprd a convrnirnt method which can cover the P I ' site as wrll as t h r PI sitr of thc enzyme. The method utilizes thr lowrr fluorrsccncr of amino acid-MCA than dipeptidyl-MCA a t A,., 325 nm. A, , 390 nm (Arg-Arg-MCNArg-MCA = 1.8. Lvs-Aln-~ICNAla-MCA = 1.7 I. Estimation of the activity o f APY hy this method showrd that Lys-Ala-MCA and Arg-Arg-MCA werr far morr rapidly hydrolyzed than Lys-MCA and Arg-MCA (350-and 150-fold. resprc-tively) in the absence of Co2+. The great susceptibility of dipeptidyl-MCAs or oligopeptides demonstrates the importance of the correct choice of substrates for the estimation of aminopeptidase activity, i.e. not only the variety of amino acid chromophores, but also longer substrates. This convenient method can also be applied to the estimation of the activity of endopeptidase, when the hydrolysis products of peptidyl-MCA are peptide and amino acid-MCA.
APY was purified in two forms, APY,, and APY7,, from commercial yeast cells. The results from N-terminal amino acid sequencing, catalytic properties, immunological cross-reactivity, and identical molecular mass, 53 kDa, of the protein left after removal of sugar chains show that the difference between the two molecules is one of sugar content. Cultured yeast cell vacuoles contained only MY,, (Fig. 5). Thus, APY exists predominantly or exclusively as the 70-kDa form with 24% carbohydrate in yeast vacuoles. The heterogeneity in the extent of glycosylation or its processing appeared under some growth conditions when whole cells were subjected to Western blot analysis, but the details are unknown at present (data not shown).
APY exists as a 74-kDa precursor form in the vacuolar protease-deleted mutant ABYS1. The pro-APY was converted to mature 70-kDa APY by incubation with vacuolar extract from wild-type cells (Fig. 7B). The conversion was inhibited by PMSF but not by pepstatin. Incubation with purified protease B (Fig. 8) confirmed that the vacuolar serine protease, protease B, is responsible for the conversion. There was disagreement between the conversion ratio of pro-APY and the increase of activity (Figs. 7 and 8). The reason for this is unknown, but it may take a certain time to develop stable activity of the enzyme after the processing or some other type of processing which cannot be detected by SDS-polyacrylamide gel electrophoresis may occur.
In yeast vacuoles, the PEP4 gene product PrA is primarily responsible for the processing of vacuolar protein precursors to their mature forms. PrA either processes those precursors directly or is essential for the activation of processing proteases (10). Pro-PrB is one of the precursors processed by PrA, and after subsequent autocatalysis (13, 141, activated PrB successively processes pro-CPY (15) and pro-CPS (35) to the mature forms. The present study showed that maturation of pro-APY is also involved in the vacuolar proteolytic processing system. Although the precise mechanism remains to be elucidated, aminopeptidase Y is the first aminopeptidase known to be activated by proteolytic processing.