Eur. J. Biochem. 207,867-876 (1992) 0FEBS 1992

Biogenesis of the yeast vacuole (lysosome) Proteinase yscB contributes molecularly and kinetically to vacuolar hydrolase-precursor maturation Hans H. HIRSCH', Hans H. SCHIFFER' and Dieter H. WOLF' Institut fur Medizinische Mikrobiologie der Universitat Basel, Switzerland

' Institut fur Biochemie der Universitat Stuttgart, Federal Republic of Germany (Received January 29/May 11, 1992) - EJB 920118

The vacuolar proteinase yscB (PrB) has been implicated in the final maturation of procarboxypeptidase yscY (pro-CpY) to the mature wild-type form CpYb of 61 kDa. In PrB-deficient mutants, only the proteinase yscA processed form CpY" of 62 kDa is found [Mechler, B., Miiller, H. & Wolf, D. H. (1987) EMBO J. 6, 21.57-21631. We report now that, akin to CpY, two forms of mature proteinase yscA (PrA) can be distinguished. In PrB-deficient mutant cells, PrA", migrating at about 43 kDa in SDS/PAGE, is found, whereas PrAb, found in wild-type cells, had the known molecular mass of 42 kDa. In the PrB-deficient strain, pro-PrA and pro-CpY matured only to the higher-molecular-mass forms, PrA" and CpY", and the maturation of both precursors was slower than in the isogenic wild-type strain. Pulse-labeling experiments indicated that the mature forms, PrAb or CpYb,are generated directly in the PrB-containing wild-type strain in vivo. In vitro experiments showed that PrB is able to trigger maturation of its 42-kDa pro-PrB precursor to mature PrB in the absence of PrA. Mature PrB and its proteolytic activity, however, shows a higher stability in the presence of mature PrA. The data indicate a molecular and kinetic participation of proteinase yscB in vacuolar hydrolase precursor maturation.

Due to the presence of a high number of hydrolytic enzymes and a relatively acidic pH, the vacuole of the eukaryote yeast Saccharomyces cerevisiae is regarded as a cellular compartment analogous to the mammalian lysosome [l -41. The proteolytic activities in the vacuole are induced by nutrient starvation and increase several fold during the transition of cells from the exponential to the stationary growth phases [3-81. Studies on mutant strains deficient in the enzymatic activity of vacuolar proteinases have indicated the essential role of proteinase yscA (PrA) for survival under nutrientstress conditions [9] and the necessity of PrA and proteinase yscB (PrB) for the sporulation of a/. diploid cells [9 - 121. Besides their role in the rather unspecific process of protein degradation [9, 101, both PrA and PrB also mediate limited proteolytic action, as shown by the specific maturation of procarboxypeptidase yscY (pro-CpY) in the vacuole [13, 141. Pulse-chase experiments indicated that the soluble vacuolar enzymes PrA, PrB, CpY, the membrane-bound alkaline phosphatase (Alp) and carboxypeptidase yscS (CpS) are present in the endoplasmatic reticulum as N-glycosylated precursors of a higher molecular mass of 52 kDa for pro-PrA, 41.5 kDa for pro-PrB generated by processing of the short-lived 73-kDa super-pro-PrB precursor, 67 kDa for the p l precursor of proCpY, 76 kDa for pro-A1P and 78 kDa and 81 kDa for proCorrespondence to D. H. Wolf, Institut fur Biochemie, Universitat Stuttgart, Pfaffenwaldring 55, W-7000 Stuttgart 80, Federal Republic of Germany Abbreviations. AIP, vacuolar alkaline phosphatase; CpS, carboxypeptidase yscS ; CpY, carboxypeptidase y s c Y ; PrA, proteinase yscA; PrB, proteinase yscB. Enzymes. Carboxypeptidase yscY (EC 3.4.16.1); proteinase yscA (EC 3.4.23.6); proteinase yscB (EC 3.4.21.48).

CpS [I5 - 241. Studies, using conditionally secretion-deficient sec mutants [25], have shown that these precursors reach the vacuole from the endoplasmic reticulum via the Golgi apparatus [17,18,21-241. In the wild-typevacuole, these precursors are proteolytically processed to the mature and active enzymes of 42 kDa for PrA [19,21], 33 kDa for PrB [I7 - 191, 51 kDa for CpY [I 5,22],72 kDa for AlP [23] and 74 kDa and 77 kDa for CpS [24]. The structural gene of PrA had first been defined by a mutation resulting in the sole loss in PrA activity and had been called PRAl [26]. A mutation (pep4-3) leading to a pleiotropic defect in the activities of the vacuolar enzymes PrA, PrB, CpY, and A1P [27, 281 had only later been shown to reside in the structural gene of PrA [29]. In this paper, PRAl is used to designate the wild-type allele of PrA. Activation and maturation of other vacuolar hydrolase precursors is dependent on PrA, since the pep4-3 mutation or the disruption of the structural gene of PrA leads to the accumulation of proPrB of 42 kDa, pro-CpY of 69 kDa and pro-A1P of 76 kDa in the vacuole [13, 14, 17,20,29]. The final vacuolar maturation process is initiated by autocatalytic maturation of the pepsinogen-like pro-PrA upon entering the vacuolar compartment [30-321. Mature PrA seems to be required for the vacuolar activation process, as pro-PrB and pro-CpY also accumulate in PrA-mutant cells bearing the active-site mutant [Ala294]pro-PrA protein which is unable to undergo selfmaturation [32]. Precursor organization and maturation events for PrB differ substantially from the ones found for CpY and PrA. The structural gene for PrB, PRBl,gives rise to a high-molecular-mass precursor of 73 kDa, called pre-super-pro-PrB [17, 18, 331. Maturation to the 33-kDa vacuolar PrB includes several (auto-)proteolytic processing steps [17, 18, 33, 341.

868 Upon translocation into the endoplasmic reticulum and subsequent signal-sequencecleavage, rapid processing to the core N-glycosylated pro-PrB of 41.5 kDa [17, 18, 33, 341 and a 41kDa superpeptide portion takes place [33], most likely in an autocatalytic fashion [33, 341. Both, the 41 kDa-superpeptide and pro-PrB reach the vacuole [33]. There, mature PrA appears to be required for processing of the 42-kDa pro-PrB molecule to a smaller 40-kDa pro-PrB intermediate [17, 18, 33, 341. Subsequently, the 40-kDa pro-PrB intermediate undergoes final maturation to the mature and active PrB enzyme of 33 kDa, a process which seems to require PrB activity in vitro, as it can be inhibited by the PrB-specific inhibitors I! or chymostatin [18,33]. This two-step maturation of the vacuolar pro-PrB has been detected in vitro in a kinetic analysis of PrA-initiated pro-PrB self-processing [33]. Concomittant with the final processing step, PrB activity is released and the superpeptide is degraded [33]. Thus, PrA activity is necessary for the vacuolar pro-PrB maturation, but not sufficient; pro-PrB activity is also required. In-vivo evidence for the vacuolar two-step maturation of pro-PrB has been described in a PrB-activity mutant in which the PrAdependent processing to the intermediate pro-PrB occurs at a normal rate, but the subsequent PrA-independent, presumably autocatalytic step, is severely slowed down [17, 341. An as-yet unresolved dependence of PrB activity on PrA is observed in diploid yeast strains of the genotype (pep#-31 PRAI ) ; these cells, bearing only one wild-type copy of the structural gene for PrA but two wild-type alleles of the structural gene for PrB showed not only a gene-dosage effect for PrA, but also for PrB activity [28]. In contrast, the phenotype of diploid yeast strains of the genotype (prbl-I/PRBI), bearing two wild-type alleles of PRAI but only one wild-type copy of PRBI, displayed no gene-dosage effect for PrB activity [12]. Molecular evidence for a role of PrB in the vacuolar activation of other hydrolase precursors was obtained by studying the maturation of pro-CpY [14]. Addition of purified PrB in Y i t r D allowed for direct activation of pro-CpY to the mature vacuolar enzyme, CpY, of 61 kDa, called CpYb, the molecular form detected in vivo in wild-type cells [14]. In contrast, activation of pro-CpY by addition of purified PrA in vitro was only observed in the presence of an unknown vacuolar factor which could be mimicked by addition of polyphosphates [14]. The PrA-matured form, called CpY", showed a higher molecular mass of about 62 kDa. CpY" was also detected in vivo in PrB-deficient prbl-I mutant strains [14]. The PrA-matured CpY" of 62 kDa could be processed further in vitro to the mature wild-type form, CpYb, of 61 kDa by addition of mature PrB. Thus, PrB activity appeared to be responsible either directly or sequentially for the maturation of pro-CpY in the wild-type vacuole [14]. A role for PrB activity in the maturation of vacuolar-hydrolase precursors was also indicated by the observation of the phenotypic lag of the PrA-deficient pep4-3 genotype in spores derived from heterozygous pep4-3/ PRAI diploids. Thepep4-3 spores retained a wild-type phenotype with respect to CpY activity for some generations, which appeared to depend on the presence of an intact PrB gene [35]. In plasmid-loss experiments, PrA-independent activation of vacuolar hydrolases was observed only after the initial presence of P RA I, when PrB was expressed from a high-copynumber plasmid [8]. In this study, we present further proof on the central role of PrB in the maturation of vacuolar-hydrolase precursors in wild-type yeast cells. We show that, although initially dependent on autoactivated PrA, PrB is involved molecularly and kinetically in the maturation of pro-PrA and of pro-CpY in

vivo. In the absence of PrA, PrB alone can also trigger proPrB maturation in vitro. However, the presence of PrA appears to be required for extended stability of the mature PrB enzyme.

MATERIALS AND METHODS Enzymes, inhibitors and chemicals Proteinase yscA and bovine serum albumin were purchased from Sigma, Deisenhofen (FRG). Proteinase yscB, purified according to [36] or [37] to near homogeneity as judged by Coomassie blue staining after SDS/PAGE, was a gift of H. Hoffschulte and U. Weiser. Proteinase inhibitors, such as pepstatin and chymostatin, were obtained from the Peptide Institute, Osaka (Japan). %-Labeled methionine was obtained from Amersham, Braunschweig (FRG). All other chemicals were obtained from Roth, Karlsruhe (FRG) or Pharmacia, Freiburg (FRG). Media Yeast growth media were 1% yeast extract, 2% peptone and 2% glucose (YPD complete medium) and mineral medium (0.67% yeast nitrogen base without amino acids, 2% or 5% glucose and supplements required for auxotrophic strains). When yeast-cell protein was labeled with [35S]methionine, mineral medium without ammonium sulfate (0.18% yeast nitrogen base without amino acids, 0.1YOyeast extract, 5% or 0.3% glucose, adjusted to pH 6.0 with 0.1 M sodium phosphate and required supplements) was used. Immunesera Antisera against the mature proteinase yscB, proteinase yscA, or against epitopes of the N-terminal superpeptide of the 73-kDa precursor of proteinase yscB, have been described previously [19, 331. Antiserum against carboxypeptidase yscY has been generously provided by Andreas Finger, Stuttgart (FRG). Yeast strains The yeast strains used in this work are listed in Table 1. Transformation of S. cerevisiae was carried out according to standard procedures [38]. YS18 [39] was used as proteinase wild-type strain providing convenient auxotrophic markers for DNA transformation and the generation of the following isogenic mutant derivatives: YHH19, YHH32, YHH45, YHH65 and YHH3/1 (Hirsch, H. H., unpublished results). The isogenic diploids were obtained by mating YS18 or its proteinase mutant derivatives YHH19 or YHH65 with X2180- 1ALlU1 or its isogenic dpep4 derivative. Pulse-chase experiments Cellular protein was labeled using [35S]methionineupon shifting the cells from glucose (5%) minimal medium to minimal medium without ammonium sulfate, containing 0.3% glucose and [35S]methionine. The wild-type YS18 and the isogenic proteinase-yscB-deficient derivative YHH19 were incubated for 30 min at 30°C, followed by incubation in the presence of 300 - 600 pCi/ml [35S]methioninefor 15 min. The chase period was initiated upon addition of non-radioactive methionine up to 1 mM and 50 pg/ml cycloheximide. Preparation of cell extracts at the times indicated, immunoprecipi-

869 Table 1. Yeast strains used. Strain

Genotype

Source

YS18 YHH19 YHH32 YHH45 YHH65 YHH3/1 X2180-1ALIU1 X2180-1AL1Ul/pep4 YHHIOO

MATa his3-11,15 leu2-3,112 ura3A5 MATaprblAAV his3-11,15 leu2-3,112 ura3A5 MATcl pral::URA3 prblAAV his3-11,15 leu2-3,112 ura3A.5 MATcl pral::URA3 his3-11,15 leu2-3,112 ura3A5 MATa pralAEN::HlS3 his3-11,15 leu2-3,112 ura3A5 MATclpral-Ala294 his3-11,15 leu2-3,112 ura3A.5 MATamal me1 gal2 lys2 ura3 MATa ma1 mel gal2 lys2 ura3 Apep4::URA3 MATaIa MALlmal MELlmel GAL2/ga12 LYS2/lys2 ura3A5/ura3 hid-1 1,15/HIS3 leu2-3,112/LEU2 MATala pral AEN::HIS3/PRAI MALImal MELlmel GAL2/ga12 LYS2/lys2 ura3A5/ura3 his3-1I , 15/HIS3 leu2-3,l I2/LEU2 MATala pral A EN:: HIS3lApep4:: URA3 M A Llmal MELlmel GAL2/ga12 LYS2/lys2 ura3A5/ura3 his3-I I , 151HIS3, leu2-3,112/L E U2 MATa/a prblAAVIPRB1 his3-11,15/HIS3 leu2-3,112/LEU2 ura3-5lura3 LYS2/lys2

P91 1331

YHHlOl YHH103 YHH104

tation of proteins, SDSjPAGE and fluorography were performed as outlined previously [19, 201. Yeast-cell lysate and isolation of vacuoles Yeast cells were harvested in early stationary growth for preparation of total cell lysates. Cells were disrupted (0.5 ml of a 30% cell suspension) with glass beads (lg) in 0.1 M sodium phosphate, pH 7.0, or 0.1 M Tris/HCl, pH 7.0, and kept on ice at all times. Spheroplast formation and two-step isolation of intact vacuoles on density gradients were performed according to [40] as described in detail in [33]. The pellet, containing the vacuoles, was resuspended in 0.2 M sorbitol, pH 6.8, and stored frozen as 50-p1 aliquots at -2O'C. Enrichment of vacuoles was eightfold over the spheroplast lysate, as calculated from the increase in the specific vacuolar a-mannosidase activity [41]. In-vitvo experiments

The in-vitro experiments investigating processing of [Ala294]pro-PrA or pro-PrB by mature PrB were performed as described previously [18,33] with the following modifications. Purified vacuoles of strain YHH3/1 @rul-AZu294),containing [Ala294]pro-PrA, were incubated in 0.1 M Tris/maleate, pH 5.0, for 2 h at 30°C after addition of 5 pg mature isolated PrB/5O pg vacuoles and the PrA-specific inhibitor pepstatin (5 pg/l). After incubation, samples (representing 50 pg purified vacuoles) were analysed by SDSjPAGE and immunoblotting using anti-PrA serum. Purified vacuoles of strain YHH65 (pralAEN: :HIS3), containing pro-PrB, were incubated in 0.1 M Tris/maleate, pH 5.0, for 300 min after addition of mature isolated PrB (0.3 pg PrB/40 pg vacuoles) and the PrA-specific inhibitor pepstatin (5 pg/pl). After 15, 45, 150 and 300 min of incubation, aliquots of the incubation mixture (representing 40 pg vacuoles) were tested for PrB activity and subjected to immunoblotting using anti-PrB and anti-superpeptide sera. The protein concentration of purified vacuoles during the incubation was 5 pg/l. The in-vifro experiment investigating the PrB stabilization by PrA was performed as follows. 20 pg mature isolated PrB was incubated in a volume of 50 p1 with 20 pg bovine serum albumin or 20 pg PrA in 0.1 M Tris/maleate, pH 5.0, for at least 8 h at 30°C. After 0, 1, 3, and 8 h of incubation, 5-pl aliquots were tested for PrB or PrA activity, as well as being

Wl

[331 1331 this work T. Stevens T. Stevens this work this work this work this work

subjected to immunoblotting using anti-PrB serum or antiPrA serum. In addition, the stabilization of PrB by PrA was analyzed in more detail. 20 pg mature isolated PrB was incubated under the same conditions as above for 3 h with 0,2.5, 5, 10 or 20 pg purified PrA. The final amount of protein in the incubation mixture was adjusted to 40 pg by addition of bovine serum albumin. After 3 h of incubation, 5 pl-aliquots were tested for PrB activity. Immunoblotting procedures Western blots of yeast-cell lysates or vacuolar preparations onto nitrocellulose were carried out as described [42] with the following modifications. Transfer buffer contained 20% methanol, 27 mM Tris and 190 mM glycine. Precoating and antibody decoration were performed in 1 O/O non-fat dry milk [43]. Visualization of rabbit antibodies to superpeptide, proteinase yscA, or proteinase yscB was achieved by color reaction of peroxidase-coated goat-(anti-rabbit) IgG with 4-chloro-1-naphthol [44]. Enzyme assays Proteinase yscB was tested, using azocoll as a substrate, and as described [45]. Proteinase yscA was tested according to [46] using acid-denatured hemoglobin as substrate. The protein concentration was determined as in [47]. RESULTS Maturation of pro-CpY is slowed down in PrB-deficientmutants Previous work has shown that the vacuolar serine proteinase yscB is required for the generation of the wild-type molecular form of mature CpYb of 61 kDa [14]. In PrB-deficient mutant strains, where PrB activity is lacking, the larger molecular 62-kDa CpY" is detected. In-vitro experiments indicated that pro-CpY can be processed either directly to the mature wild-type form, CpYb, by PrB, or sequentially, first by PrA to CpY", followed by PrB, yielding CpYb [14]. To analyse the influence of PrB on the maturation process of pro-CpY in vivo, the wild-type strain YS18 and the isogenic PrB-deficient derivative YHH19 (prblAAV) were compared for pro-CpY maturation in a pulse-chase experiment using [35S]methionine. Both yeast strains were grown exponentially for 18 h, contain-

870 1

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89101112

Fig. 1. Comparison of the maturation of pro-CpY in wild-type cells and in Pr Bdeficient cells. Radioactive labeling of yeast cell protein using t3 5S]methionineand subsequent immunoprecipitation using anti-CpY serum is outlined in Materials and Methods. Lane 1, 15-min pulse of Y S18 (wild-type parental); lanes 2 -6,lO min, 20 min, 30 min, 40 rnin and 60 rnin of non-radioactive chase. Lane 7,15-min pulse of YHH19 (prhldAV); lanes 8- 12, 10 min, 20 min, 30 min, 40 rnin and 60 rnin of non-radioactive chase. The p l and p2 precursors of pro-CpY, the Pr A-processed molecular form CpY" and the PrB-processed molecular form CpYb are indicated.

ing still more than 1% glucose before being shifted to a lowglucose minimal medium without ammonium sulfate for maximal induction of vacuolar proteinases [3 - 81. After the 15rnin pulse period, mostly pro-CpY of 67 kDa, corresponding to the core glycosylated p l form, is immunoprecipitated from both the wild-type strain YS18 and the PrB-deficient derivativeYHHl9 (Fig. 1, lanes 1 and 7, respectively). After a 10-min chase with non-radioactive methionine, pro-CpY of 69 kDa, corresponding to the Golgi-modified p2 precursor, is detected in both strains (Fig. 1, lanes 2 and 8). However, in the wildtype strain YS18, about half of the radiolabeled CpY antigenic material is already present as mature CpY of 61 kDa, corresponding to the PrB-processed CpYb form (Fig. 1, lane 2). The intensity of the radiolabeled mature CpYbincreases during the chase, leaving only small amounts of the 69-kDa p2 pro-CpY after 60 rnin of chase in the wild-type strain YS18 (Fig. 1, lane 6). In contrast, the maturation of pro-CpY to equivalent levels of mature CpY is delayed in the PrB-deficient strain by up to 30 min, and proceeds only to a larger CpY" form of around 62 kDa (Fig. 1, lanes 10 - 12). An alteration of the N-glycosyl modification, located on the mature CpY protein, cannot account for the difference in molecular mass of the mature CpY" and CpYb, as has been shown previously by endoglycosidase H and tunicamycin treatment [14], and by the identical molecular mass of the pro-CpY precursor forms pl and p2 in both strains (Fig. 1). The rather sharp band observed in every lane above CpY" (Fig. 1) proved to be unspecific as it was also detected in CpY-deficient cells, and appeared to result from the dissociated chains of the antiserum. We conclude that the maturation of pro-CpY in the PrB-deficient strain YHH19 is slowed down and proceeds only to CpY". On the contrary, the PrB activity present in the wild-type cells appears to generate the wild-type CpYb of 61 kDa and to accelerate the vacuolar maturation process of pro-CpY in the wild-type vacuole.

PrB is also required for rapid maturation of pro-PrA to the molecular wild-type form PrAb When the isogenic proteinase mutant derivatives were analysed by immunoblotting using anti-PrA serum, mature wildtype PrA of 42 kDa was detected in the parental wild-type strain YS18 (Fig. 2 , lane 1). However, a mature PrA form of slightly higher molecular mass of about 43 kDa was detected in the PrB-deficient strain YHH19 (prbldAV) (Fig. 2, lane 2 ) . As expected, no PrA-specific antigenic material was detected in the isogenic derivatives YHH32 ( p r a l : :URA3 prblAAV) and YHH45 @ral::URA3), causing disruption of the PrA

3

4

- 46kDa PrA

- 3OkDa

Fig. 2. PrA in wild-type yeast cells and in PrB-deficient mutant cells. Equal amounts of yeast-cell extracts (50 pg) were subjected to SDS/ PAGE and immunoblotting using anti-PrA serum as described in Materials and Methods. Lane 1, YS18 (wild-type parental); lane 2, YHH19 (prbldAV); lane 3, YHH32 @ral: :URA3prblAAV); lane 4, YHH45 (pral ::URA3).

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

p r o - PrAPrAb-

Fig. 3. Comparison of the maturation of pro-PrA in wild-type and PrBdeficient mutant cells. Radioactive labeling of yeast cell protein using [35S]methionineand subsequent immunoprecipitation using anti-PrA serum is outlined in Materials and Methods. Lane 1,15-min pulse of YS18 (wild-type parental); lanes 2 - 6,lO min, 20 min, 30 min, 40 rnin and 60 min of non-radioactive chase. Lane 7,15-min pulse of YHH19 ( p r b l d A V ) ;lanes 8- 12, 10 min, 20 min, 30 min, 40 rnin and 60 rnin of non-radioactive chase. Pro-PrA and the self-processed molecular form PrA" and the PrB-processed molecular form PrAb are indicated.

structural gene (Fig. 2, lanes 3 and 4, respectively). Thus, in the absence of PrB activity, the maturation of pro-PrA leads to a novel form of 43 kDa, called PrA". As has been shown for CpY [14], the wild-type maturation of pro-PrA to the molecular wild-type form, PrAb of 42 kDa, requires the presence of PrB activity. The maturation of pro-PrA in the wild-type strain, YS18, and the PrB-deficient derivative, YHH19, was also analysed by pulse labeling with [3sS]methionine for 15 rnin and during the subsequent chase period started by addition of non-radioactive methionine. Immunoprecipitation using anti-PrA serum and subsequent analysis by SDSjPAGE and autoradiography were carried out after the pulse period and at the indicated times during the chase. After the pulse period of 15 min, mature PrAb of 42 kDa is already detected in addition to the radiolabeled pro-PrA of 52 kDa in the wild-type strain YS18 (Fig. 3, lane 1). During the first 60 min of the chase period, (Fig. 3, lanes 2-6), the intensity of the 52 kDa proPrA immunoprecipitated from the wild-type, fades, while the amount of mature 42-kDa PrAbincreases. In the PrB-deficient strain, YHH19, only 52-kDa pro-PrA is detected after the pulse period (Fig. 3, lane 7). The processing of the 52-kDa pro-PrA is delayed in the isogenic PrB-deficent strain, YHH19, and a matured PrA" of 43 kDa appears after 20 rnin of the chase period (Fig. 3, lane 9). Pro-PrA of 52 kDa remains the major molecular form, even after 60 rnin chase (Fig. 3, lane 12). Importantly, the mobility of the pro-PrA of 52-kDa,

871 1

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A 1

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pro-PrB --c

rAla2941 pro-PrA I A l a2 9 4 1 PrA

-

PrB

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incubation ( m i n ) PrB a c t i v i t y (10 AE/h)

Fig. 4. Mature PrB can process the self-maturation incompetent mutant molecule IAla294jpro-PrA to a mature-like [Ala294jPrAb form. Vacuoles of strain YMH3/1 (pral-Alu294) were prepared and equal amounts (50 pg/lane) were incubated either with or without purified mature PrB ( 5 pg) for 2 h a t 30°C in 0.1 M Tris/maleate, pH 5.0, and analysed by SDSjPAGE and immunoblotting using anti-PrA serum as described in Materials and Methods. Lane 1, vacuoles of strain YHH3/1 without additions; lane 2, same as lane 1, after addition of PrB and the PrA inhibitor pepstatin.

detected in the wild-type strain YS18, is identical to the one detected in the isogenic PrB-deficient derivative. Thus, since both sites for N-glycosylation are located on the mature enzyme [19,23,30,31], unequal N-glycosylation in these isogenic derivatives can be ruled out as a reason for the observed difference in molecular mass between PrA" and PrAb. Instead, we must conclude that PrB activity is also involved kinetically and molecularly in the wild-type maturation of pro-PrA to PrAb. Furthermore, significant amounts of the self-matured PrA" are detected only in the absence (Fig. 3, lanes 9 - 12), but not in the presence of PrB activity. To examine the role of PrB in processing of pro-PrA in vitro more directly, intact vacuoles were prepared from the isogenic derivative YHH3/1 (prul-Alu294) in which the codon of the catalytic aspartyl residue in amino acid position 294 of PrA was changed to alanine. The mutant gene is expressed as [Ala294]pro-PrA, but is unable to undergo self-maturation in the vacuole [32]. The vacuolar preparations were analysed by SUSjPAGE and immunoblotting using anti-PrA serum. Without additions, the unprocessed [Ala294]pro-PrA of about 52 kDa is detected in vacuoles of strain YHH3/1 (Fig. 4, lane 1). We consider the small amount of [Ala294]PrAbdetectable to be due to the action of the pro-PrB present in the vacuolar preparation [14, 331. Addition of purified PrB to YHH3/1 vacuoles is sufficient for processing of the self-maturation incompetent mutant protein [Ala294]pro-PrA to a maturelike PrAb form (Fig.4, lane 2). Thus, direct processing of pro-PrA to PrA by PrB should be considered as a relevant maturation event in wild-type yeast cells. Mature PrB can initiate processing and activation of its own precursor pro-PrB in vitro

It was of interest to determine whether mature PrB is able to process and mature the 42-kDa pro-PrB in vitro, since this activation mode could contribute to an efficient and rapid activation of precursor proteins in the wild-type vacuole. Such a process may also be relevant for the phenotypic lag in PrAdeficient pep4-3 mutant spores, derived from heterozygous (pep4-3/PRAI) diploid cells [35] and for the PrA-independent maintenance of hydrolase activation by high level-expression of PrB [8]. As a source of pro-PrB, yeast vacuoles were

C 36.5

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Fig. 5. Proteolytic processing and activation of pro-PrB, triggered by addition of mature PrB. Vacuolar preparations of the strain YHH65 @raIdEN: :HIS3) (40 pg/lane) were incubated with mature isolated PrB (0.3 pg/lane) in 0.1 M Tris/maleate, pH 5.0, a t 30°C as indicated and analysed by immunoblotting using (A) anti-PrB serum or (B) antisuperpeptide serum. Lane 1, vacuoles at 0 min; lanes 2 - 5 , vacuoles incubated for 15, 45, 150 and 300 min after addition of purified PrB and the PrA inhibitor pepstatin; lane 6 , 0.3 pg purified PrB; lane 7, 5 pg purified PrB. The azocoll splitting activity of a sole PrB (0.3 pg) aliquot was determined and is compared to the activities exhibited by 40 pg vacuolar preparations after incubation with PrB (activities are given as 10-fold dE/h.). n.d., not determined.

obtained from the PrA-deficient strain YHH65 @ d d E N : :HIs3). The azocoll splitting activity in the incubation mixture at the time points indicated, and of the activity of PrB added, was determined and the incubation reactions were analysed by immunoblotting using anti-PrB serum (Fig. 5A). In order to be able to follow any increase in PrB activity presumably generated by pro-PrB activation, only a small amount of PrB (0.3 pg), corresponding to 0.02 AE/h activity (Fig. 5A, lane 6), was added to 40 pg protein of purified vacuoles. The PrB added was purified to near-homogeneity as judged by Coomassie-blue staining after SDS/PAGE. In order to inhibit any PrA-like activity, the aspartic acid proteinase inhibitor pepstatin ( 5 pg/ml) was included in the incubation mixture. The result indicates that mature PrB, purified from wild-type cells, can induce the conversion of pro-PrB to a matured PrB form in vitro (Fig. 5A, lanes 1-5). The proPrB maturation is inhibited by addition of the PrB-specific inhibitor chymostatin, as well as by the cytoplasmic PrB inhibitor I! (data not shown). The maturation proceeds via an approximately 40-kDa intermediate form. The matured PrB generated shows a slightly higher molecular mass in SDS/ PAGE than the purified PrB added (Fig. 5A, compare lanes 4 and 5 with lanes 6 and 7). Since the purified PrB and the vacuole preparation used were derived from different yeast strains, the significance of this difference is at present unclear. The kinetics and intermediate forms of pro-PrB maturation,

872 1

Table 2. Dependence of the proteinase yscB activity on the number of structural genes of the proteinase yscA in isogenic diploid strains. Cell extracts of the indicated yeast strains were prepared, the protein content measured and the specific PrA and PrB activities determined as described in Materials and Methods. Diploid strain (relevant genotype)

Number of wild type alleles

Specific Specific PrA PrB activity activity

PrB

PRAI

PRBI

YHHlOO PRAIIPRAI PRBIIPRBI

2

2

YHHlOl pral AEN:: HlS3/PRAI PRBljPRBl

1

2

0.158 (48.0)

0.112 (48.0)

YHH103 0 p r a l A E N : : HIS3jApep4:: URA3 PRBljPRBl

2

0.008 (2.5)

0.036 (15.6)

YHH104 PRAIIPRAI prhlAAVjPRB1

1

0.306 (94.0)

0.210 (91 .O)

2

following the addition of purified mature PrB (Fig. 5A) are reminiscent of the maturation of the 42-kDa pro-PrB after addition of mature PrA, also proceeding via a smaller 40-kDa pro-PrB form [18, 331. As shown for PrA addition [IS, 331 concomittant with pro-PrB maturation, the PrB superpeptide is degraded (Fig. 5B), and an increase in PrB activity is detected (Fig. 5A). Thus, PrB can substitute for PrA in vitro, and also trigger pro-PrB maturation and activation. The gene dosage of PRAl on the specific PrB activity acts downstream of pro-PrB maturation

In the wild-type vacuole, the amount of PrB activity generated appears to depend largely on intact mature PrA. This conclusion was based on the observed inhibition of the maturation of pro-PrB and a deficiency in biochemical PrB activity in strains bearing particular mutations in the structural gene of PrA, like the pep4-3 mutation [I91 or like the pral-AZa294 mutation in the active site, preventing self-maturation of the mutant [Ala294]pro-PrA [32], or like the deletion of the structural gene for PrA [29-311 as in thepralAEN::HZS3 mutant allele [33]. Furthermore, the PrB activity appeared to be dependent on the number of functional gene copies of the structural gene for PrA. Heterozygous pep4-3IPRAl diploid cells showed not only a gene-dosage effect for PrA activity, but also for PrB activity, although bearing two wild-type copies of PRBl 1281. The puzzling gene-dosage effect of PrA on PrB activity has been suggested to come about either by a requirement for the formation of a stoichiometric complex between mature PrA and the 42-kDa pro-PrB during pro-PrB maturation, or by a stable complex between mature PrA and PrB [ 14, 481. We re-examined isogenic diploid derivatives for the specific PrA and PrB activity (Table 2). Indeed, corresponding to the gene dosage of PrA in these isogenic diploid derivatives, crude-cell extracts of the heterozygous pralAEN::HIS3IPRAI strain YHH101, carrying only one wild-type copy of the structural gene for PrA, but two wild-

-

p r o - P r B --+

2

3

4

-36

5 kDa

-26

6 kDa

Fig. 6. Heterozygous p r a l d EN::HIS3IPRAl mutant diploids contain no 42-kDa pro-PrB. Immunoblotting total cell extracts (70 pg) of different diploid yeast strains using anti-PrB serum. Lane 1, YHHIOO ( P R A l I P R A l ) ; lane 2, YHHlOl @rulAEN::HIS3]PRA1); lane 3, YHH103 (pralAEN::HZS3/Apep4::URA3); lane 4, YHH104 (prblAAVIPRB1).

type copies of PRBI, had around 50% of the specific PrA activity, as well as 50% of the specific PrB activity compared to the enzyme levels in the wild-type diploid strain, YHH100. The PrA-deficient diploid YHH103 (pralAEN::HZS3/ Apep4: :URA3) showed minor activity on the PrA substrate and a low specific PrB activity (2.5% and 15.6% of the wildtype levels, respectively; Table 2). lmmunoblotting of cell extracts (Fig. 6) revealed only mature PrB antigen in the heterozygous pralAEN: :HZS3IPRA1 diploid strain YHHlOl (Fig. 6, lane 2). Pro-PrB of 42 kDa was detected in the PrAdeficient pralAEN::HZS3lApep4: :URA3 diploid strain (Fig. 6, lane 3), whereas mature PrB of 33 kDa was found in the proteinase wild-type strain YHHIOO (Fig. 6, lane 1). Since only mature PrB antigen was detected in the diploid strain YHHlOl (pralAEN::HZS3/PRAI)and given the role of PrB in the wild-type maturation of pro-PrA to PrAb (Figs 2-4) and in the maturation of the 42-kDa pro-PrB precursor (Fig. 5), the gene-dosage effect of the PrA structural gene on PrB activity is unlikely to result from a 1 : 1 stoichiometric maturation of proPrB by PrA. No gene-dosage effect of azocoll splitting activity was observed in the isogenic diploid strain YHH104 (PRAIIPRAI prblAAVIPRB1) carrying only one wild-type copy of the structural gene for PrB and two wild-type copies of the PrA structural gene, but showing wildtype activity for either enzyme (Table 2) as described previously [12]. As expected, mature PrB antigen was detected in the strain YHH104 (Fig. 6, lane 4). In-vitvo stability of PrB is increased in the presence of PrA

Although difficult to quantify, the gene dosage of PRAl on the specific PrB activity detected in the steady state of derepressed yeast cells might result from a reduced amount of mature PrB enzyme following complete maturation of proPrB. We hypothesize that an increased turnover of those mature PrB molecules might occur which are present in a stoichiometric excess over PrA molecules. Chromatographic evidence demonstrated that the purified mature enzymes PrA and PrB are capable of forming a stoichometric complex i~zvitro [48]. Furthermore, it was known from purification of the mature PrB enzyme that PrB is rather unstable, most likely due to autodegradation [36]. Therefore, complex formation between PrA and PrB molecules might then lead to protection of PrB molecules. In order to reduce contributions of other vacuolar proteases, the stability of purified PrB was examined in incu-

873

C 1

PrA

2

0

4

6

8

time (h)

2

3

4

5

6

7

4

5

6

7

8

3

8

-

i n c u b a t i o n (h)

0

1

3

8

+

+

+

+

+

+

+

BSA

1

3

PrA

PrB

B

2

_

-

-

+

-

0

1

+

+

-

-

+

+

+ -

+

+

-

+

8

PrB -+

incubation (h)

0

1

3

8

PrB

+

0 +

-

+ -

+

PrA BSA

+ -

+

-

+

+

+

+

-

1

3

8

+ +

+ +

+ +

-

-

-

Fig. 7.Effect of the purified PrA on the stability of the purified PrB enzyme. Mature purified PrB or mature purified PrA were incubated either together, or with bovine serum albumin, in 0.1 M Tris/maleate, pH 5.0, at 30°C for the times indicated and as described in Materials and Methods. (A) Percent of specific PrB and PrA activity detected after the indicated time of incubation. (a) PrB activity after incubation of 2 pg PrB in the presence of equal amounts of bovine serum albumin; (0) PrB activity after incubation of 2 pg PrB in the presence of equal amounts of PrA; (A)PrA activity after incubation of 2 pg PrA in the presence of equal amounts of bovine serum albumin; ( A ) PrA activity after incubation of 2 pg PrA in the presence of equal amount of PrB. (B) Immunoblot of aliquots using anti-PrB serum, or (C) using antiPrA serum. Lanes 1-4, PrB (2 pg/lane) after 0, 1, 3 and 8 h of incubation with equal amount of bovine serum albumin (BSA); lanes 5-8. PrB (2 pg/lane) after 0, 1 , 3 and 8 h incubation with equal amount of PrA. (D) Percent of specific PrB activities after 3 h incubation of 2 pg PrB in the presence of various amounts of PrA.

bations with or without purified PrA (Fig. 7). In agreement with this hypothesis, we find that PrB activity (Fig. 7A), and corresponding amounts of the 33-kDa PrB antigen detectable by immunoblotting using anti-PrB serum (Fig. 7B), are more stable in the presence rather than in the absence of PrA. Already after 1 h of incubation in the absence of PrA, around 50% of the initial PrB activity is lost and a corresponding decrease in PrB antigen is observed (Fig. 7B, compare lanes 1-4 and lanes 5-8). In the presence of PrA, around 50% of the initial activity is still detected after 8 h of incubation (Fig. 7A). In contrast, neither the PrA activity nor the PrA antigen is significantly altered by these incubation protocols (Fig. 7A and C). To examine the dependence of the PrB activity on the amount of PrA added, increasing amounts of PrA were added to the incubation mixture and PrB activity was assayed after 3 h incubation. As can be seen in Fig. 7D, the PrB activity detected correlated with the amounts of PrA added.

DISCUSSION The aim of this study was to identify contributions of the vacuolar serine proteinase PrB to the maturation and activation of hydrolase precursors in the vacuolar organelle. Previous studies on pro-CpY maturation and activation in vilro have shown that PrB is able to convert pro-CpY directly to the active wild-type mature form, CpYb,of 61 kDa, whereas

PrA addition resulted in maturation to the CpY" form of 62 kDa. This latter form, CpY", was also found in vivo in PrBdeficient mutant cells [14]. Here, we show that PrB processing is also involved in the molecular phenotype of the mature PrA (Figs 2 - 4). In wild-type cells, the PrB processed form, PrAb of 42 kDa, is found, whereas mature PrA" of 43 kDa is detected in the isogenic PrB-deficient derivative. As has been shown for pro-CpY in vitro, mature PrB is able to directly catalyse processing of the self-maturation incompetent [Ala294]proPrA mutant molecule to a mature-like PrA form (Fig. 4). The in-vitro experiments suggest that mature PrB is also able to trigger a PrA-independent maturation of its own 42-kDa precursor pro-PrB (Fig. 5). As the PrB-mediated maturation of the 42-kDa pro-PrB also proceeds via a smaller 40-kDa intermediate [18] and coincides with superpeptide degradation, we suspect that autocatalytic processing of the smaller pro-PrB intermediate is also required, as shown previously for the PrAtriggered maturation of the 42-kDa pro-PrB [33]. Thus, PrAtriggered or PrB-triggered maturation of pro-PrB might not be distinguishable in a pulse-chase experiment. The comparison of the pro-PrA and pro-CpY biosynthesis and maturation after pulse-labeling the proteinase wild-type strain YS18 and the isogenic PrB-deficient mutant derivative YHH19 (Figs 1 and 3) indicates that the presence or absence of functional PrB activity strongly influences not only the molecular species generated, but also the time course of the maturation process of pro-PrA and pro-CpY. The [35S]methioninelabeling was performed after maximal induction of hydrolases from mini-

874 ma1 expression [3 - 81 by shifting the cells from exponential growth in the presence of high glucose to a minimal medium in which the low glucose content is rapidly exhausted and the nitrogen source is limited. Thereby, differences in the acute activation and maturation phase of abruptly expressed vacuolar zymogens are examined, rather than the steady-state events. Thus, the maturation of pro-PrA and pro-CpY in the PrB-deficient strain to the respective PrA" and CpY" form is clearly delayed. In the presence of PrB, pro-PrA and pro-CpY are processed to PrAb and CpYb, respectively. No intermediate PrA" or CpY" form is observed in the PrB-containing wildtype strain, suggesting that direct processing by PrB occurs for the vast majority of precursor molecules. Although the activation and maturation process is slowed down in PrBdeficient mutant cells as shown in the pulse-chase experiment, most of the PrA and CpY antigenic material eventually accumulates as the more slowly processed PrA" and CpY" forms in the steady state of early stationary growth as detected by immunoblotting (Fig. 2) and [14]. Autoactivation of the pepsinogen-like pro-PrA to mature PrA has been suggested to be the first step required for activation of the other vacuolar hydrolases [30, 311. Indeed, the replacement of the distal catalytic aspartyl residue with alanine at position 294 prevents autoactivation of the mutant [Ala294]pro-PrA, as well as efficient activation of pro-PrB or pro-CpY [32]. However, pro-PrA maturation to PrA" by autoactivation, examined during the chase period in the PrBdeficient strain, appears to be a slow process compared to the PrB-involving wild-type maturation (Fig. 3). Therefore, only a small fraction of PrA" might be generated early in the course of the non-radioactive chase in the wild-type vacuole. Consequently, the activation events in the wild-type vacuole might be triggered by only small amounts of autoactivated PrA" which escaped detection [19,23] but allow maturation of proPrB to PrB to occur. In turn, PrB may directly catalyse the maturation of more pro-PrA to PrAb and of pro-PrB to PrB. In this model (Fig. 8, step 3), pro-PrA self-maturation provides the initial vacuolar activation signal picked up by proPrB. Pro-PrB matured to PrB (Fig. 8, steps 4-6) then acts as a key amplifier in a positive feed-back loop (Fig. 8, step 7) leading to substantial amounts of mature PrB, PrAb and other hydrolases in the wild-type vacuole (Fig. 8, steps 7 - 10). The in-vitro data on maturation and activation of pro-CpY [14], [Ala294]pro-PrA (Fig. 4), pro-PrB (Fig. 5), pro-CpS [24] and pro-A1P ([50]and unpublished data) support the notion that, once matured, PrB can fully substitute for PrA function. By PrB-triggered pro-PrB activation (Fig. 8, steps 7) and PrAPrB cross-activation (Fig. 8, steps 8 and 9), a burst of hydrolase activity could be generated in the vacuole (Fig. 8, step 10). What may be the physiological relevance of this type of activation organization? The controlled activation of the hydrolase precursors, transported from the endoplasmic reticulum via the Golgi apparatus, should not occur in the secretory pathway prior to reaching the vacuole. The pepsinogen-like pro-PrA appears to be able to recognize the vacuole as the correct target organelle and signals, by self-maturation, the start of hydrolase activation. At present, it is unclear which vacuole-specific signal triggers the self-maturation of proPrA, since the loss of the overall acidification of the vacuole following the disruption of the H -pumping ATPase in vat2 or vmn3 mutants does not appear to be sufficient for preventing self-maturation of pro-PrA [51, 521. Possibly, other factors which are unique to the vacuole may be sensed, such as the microenvironment around polyphosphates, as suggested in [30]. Self-maturation, triggered in a particular vacuolar +

I

I

I

VAC

.......,...............................

. . :=

@

Fig. 8. Role of proteinase yscA and proteinase yscB in the acute activation phase of hydrolase precursors in the vacuole. The model presents the maturation of PrA and PrB and their role in activation of vacuolar hydrolases, as outlined and referred to in the introduction and discussion. 1. Translocation into the endoplasmic reticulum (ER), signal sequence cleavage, folding and N-glycosylation. 2. Autocatalytic processing of the 73-kDa super-pro-PrB generates the superpeptide and pro-PrB. 3. Reaching the vacuole (VAC), PrA" of 43 kDa is generated slowly by autocatalytic activation from the 52-kDa pro-PrA. 4. In the vacuole, small amounts of PrA" are required for processing of the 42kDa pro-PrB to a 40-kDa pro-PrB form. 5. and 6. Superpeptide degradation coincides with autocatalytic maturation of the 40-kDa pro-PrB form to the mature and active PrB of 33 kDa. 7. Mature PrB also triggers the maturation steps 4, 5 and 6 acting in a positive 'feedback' cycle. 8. Mature PrB catalyzes maturation of the 52-kDa proPrA generating large amounts of the wild-type PrAb of 42 kDa. 9. Mature PrAb may contribute to pro-PrB maturation in a positive feed-back. 10. Increasing amounts of mature PrB rapidly process other hydrolase precursors. 3 1. Complex formation between mature PrA and PrB stabilizes PrB. 12. PrB molecules, present in excess of PrA, are unstable due to autodigestion. 13. In the absence of PrB, the slow autocatalytic maturation of pro-PrA to PrA" can substitute for PrB in processing of pro-CpY to CpY", of pro-AIP to AIP" [50], and pro-CpS to CpS".

875 microenvironment, would also explain the relatively inefficient processing kinetics of pro-PrA to PrA" in the absence of PrB. Then, with the higher pH range of its enzymatic activity [3], PrB may be advantageous as an efficient amplifier of an initially minute PrA" signal. It appears likely that rapid activation of proteinases enhances the ability of the yeast cell to respond to an abrupt deprivation of nutrients [9]. In a/a diploid cells, a physiological response to nutrient deprivation is sporulation. Indeed, in the absence of PrA as the initiator the vacuolar activation process of sporulation is almost completely abolished [lo]. In the presence of PrA, but in the absence of PrB as amplifier of the vacuolar activation process, sporulation still occurs, but at a greatly reduced frequency, with a prolonged time of appearance and altered morphology [lo, 121. Hence, the PrB deficiency (prbl) may adversely affect the efficacy of the sporulation process, though less dramatically than the PrA deficiency as in pep4-3 mutant strains [lo], or in double-mutant pral-1 prbl-1 strains [9]. The molecular details of how PrB and PrA influence the sporulation process are still unresolved, but a role in structural reorganization, protein turn over, or even in the maturation of some as yet undefined precursor might be envisaged. The amount of specific PrB activity is affected by the gene dosage of the structural gene of PrA as reported previously [28] and shown in the isogenic diploids used here (Table 2). No gene dosage for PrB is observed in heterozygous prbll PRBl diploid strains as compared to the wild-type diploid, YHH100. A reasonable model reconciling the two phenomena is still lacking. We show here that the PRAl gene dosage acts at a step beyond the maturation of pro-PrB: In heterozygous PRAl-deleted mutant diploids (pralAEN: :HIS3/PRAI), no pro-PrB antigen was detected but mature PrB antigen of 33 kDa was detected (Fig. 6). Like PrA, mature PrB is able to trigger maturation of its own precursor pro-PrB rather than degrading it (Fig. 5). Accordingly, maturation or degradation of pro-PrB in a quantitative manner are unlikely to account for the gene dosage effect. Therefore, we suggest that the genedosage effect of PrA on PrB activity may affect the mature PrB molecule. We speculate that the trans-acting gene-dosage effect of PRAl might arise through rapid turn over of the mature PrB in the absence of sufficient PrA. In our model, PrB activity might be suicidal to the mature PrB molecule. PrB degradation should not occur when complexing with mature PrA. In fact, biochemical characterization of complex formation between mature PrA and PrB has been described previously [48]. We show, in Fig. 7, that the PrB activity of a purified PrB preparation is lost rapidly within 3 h of incubation unless purified PrA is added. In contrast, the PrA activity is not affected. Loss or stability of PrB activity correlates well with the level of PrB antigen detectable by immunoblotting. Furthermore, the amount of PrB activity detectable after 3 h was proportional to the amount of PrA added to the incubation mixture, thereby mimicking a certain gene-dosage effect in vitro. The activity of PrA is irrelevant for the stabilization of PrB, as the same result was obtained in the presence of the PrA inhibitor, pepstatin (data not shown). No degradation was observed in the presence of the PrB inhibitor, chymostatin, indicating the dependence on PrB activity. However, the bovine serum albumin added in order to equalize the protein concentrations in the incubation mixtures was not responsible for PrB degradation as degradation was also observed in the absence of bovine serum albumin (data not shown). Reportedly, the onset of PrB activity lags behind PRBl mRNA expression [7, 81. The vacuole might be considered as

being loaded with the proteolytically latent pro-PrB molecules. Thus, we propose that expression levels of pro-PrB in the vacuole exceed pro-PrA expression and that mature PrB molecules, present in excess over PrA, are more rapidly degraded following the acute activation phase. In the absence of PrA, small amounts of PrB activity, which might be generated over time even by slow accidental (self-) activation of proPrB [33], are eliminated by autodigestion of PrB, thereby preventing full activation of the vacuolar activation events (Fig. 8, step 12). In the presence of even small amounts of selfmatured PrA" molecules (Fig. 8, step 3), more PrB molecules could be generated (Fig. 8, steps 4 - 6). By positive feed-back activation (Fig. 8, step 7), the amount of PrB activity in the vacuole is pushed above a threshold and rapid activation of vacuolar zymogens ensues. An initially high level of PrB activity would be likely to process pro-CpY and pro-PrA directly to CpYband PrAb, respectively (Fig. 8, steps 8 and lo), reducing the occurence of sequential processing via CpY" and PrA" for the majority of precursors (Figs 1 and 3). In the steadystate phase following the activation burst, PrB molecules, not protected by complexing with PrA (Fig. 8, step l l ) , are degraded by autodigestion (Fig. 8, step 12). The phenotypic lag of pep4-3 mutant spores generated from pep4-3/PRAI diploid yeast cells [28] and the PRAl-independent maturation of vacuolar hydrolases observed during high copy-number expression of PRBI, when activated by the initial presence of PrA [S], are well compatible with this model. Above the threshold, high-level expression of PrB, most dramatically from multiple-gene copies, might allow maturation of proPrB at about the same rate as PrB is being degraded by PrB. Concerning the gene-dosage effect of PRAl gene copies on PrB activity in heterozygous diploid yeast cells of the genotype (pralAEN::HIS3/PRAI) (Table 2), a similar excess of mature PrB should be generated by small amounts of PrA" and subsequent PrB amplification. As the heterozygous diploid cells of the genotype (pralAEN::HIS3IPRAI) express less PrA, corresponding to only one wild-type allele of PRAl (Table 2), even less PrB can be stabilized. Therefore, the activity of only half of the PrB molecules should be detectable in the steady state phase. In contrast, in the heterozygous prbllPRB1 diploid cells, nearly all the PrB molecules expressed from one wild-type allele of PRBl could be stabilized by PrA molecules expressed from two gene copies of PRAI, thereby, masking the prbllPRBI gene dosage. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, (FRG) and the Fonds der Chemisehen Industrie, Frankfurt/Main (FRG).

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