The Intracellular Proteinases and Their Inhibitors in Yeast

THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 255, No. 10, Issue of May 25, pp. 4 8 2 1 4 2 8 , 1980 Printed in U.S.A.

The Intracellular Proteinases and Their Inhibitors in Yeast A MUTANT WITH ALTERED REGULATION OF PROTEINASE A INHIBITOR ACTIVITY* (Received for publication, May 15, 1979)

Ingrid Beck,$ Gerald R.F’inlc,g and Dieter H.Wolf1 From the $Division of Biological Sciences, Cornel1 University, Ithaca, New York 14853, Slnstitut fur Toxikologie und Bwchemie der Gesellschaftfur Strahlen- und Umweltforschung m.b.H., Munich, and 7Biochemische.sZnstitut der Universitat D 7800 Freiburg, West Germany

in this report. Proteinases A and A mutation of yeast p a i l has been isolated which fore, refer only to IA and IB leads to altered regulation of proteinase A inhibitor B, carboxypeptidase Y, and their specific inhibitors are localactivity. Under conditionsof derepression, this mutant ized indifferent compartments of the cell. The proteinases are as compared with wild type has a 70% reduction in localized in the vacuole, while their inhibitors reside in the proteinase A inhibitor activity, but no change in the cytosol of the yeast cell (16, 17). activities of proteinase A or B. The growth of strains From in vitro studies, a variety of in vivo functions have carrying thep a i l mutation is sensitive to temperature been attributed to the proteinases (for reviews, see Refs. 1and and pH. Thealtered physiological and biochemical phe2). Activation of chitin synthetase (18, 19), glucose-induced notypes of the mutant appear to be the consequences inactivation of several enzymes, such as cytoplasmic malate of a single mutation which segregates 2:2 at meiosis. dehydrogenase and fructose 1,6-bisphosphatase (20-23), and Results obtained with this mutant indicate that pro- protein degradation during differentiation (24, 25) are examteinase A and its inhibitor are two independently syn- ples. The recent isolation and characterization of proteinase thesized polypeptide chains rather than two proteins resulting from proteolytic cleavage of a single precur- mutants have opened a route toelucidate the role of proteinsor. Furthermore, proteinase A and its inhibitor appear ases in metabolism. First, results not only answer questions to be regulated independentlyof each other. Studies on about proteinase function but also point to a necessary rethemutant also indicatethattheregulation of the evaluation of mechanisms which had been proposed to be due to action of known proteinases (26-31); for a review see Ref. proteinase A inhibitor is different from that ofthe proteinase B inhibitor. In wild type cells there is an 1). At the present time, little is known about the function of excess of proteinase A inhibitor over proteinase A, whereas inthe mutant cells there is a 2.5-foldexcess of the proteinase inhibitors. It has been hypothesized that they proteinase A over proteinase A inhibitor. This excess function as a safetydevice against unwanted proteinase action proteinase A does not leadto detectable alterationsin in case of vacuolar leakage or during transport of newly overall protein degradation. However, tryptophan syn- synthesized proteinase (17,20).Recent studies on the carboxthase and proteinase B inhibitor proteins which are ypeptidase Y .inhibitor complex led to the hypothesis that sensitive to proteinase A attack in vitro show an en- proteinase inhibitors might also be involved in regulation of hanced loss of activity in the mutant. proteolytic events (1, 32).

Several intracellular proteolytic enzymes have been found in the yeast Saccharomyces cerevisiae (for reviews see Refs. 1 and 2): two endoproteinases, proteinase A and B (3); two carboxypeptidases, carboxypeptidase Y (formerly called proteinase C) (4) and carboxypeptidase S (5);several aminopeptidases (6-8); and onedipeptidase (8).In addition, three proteinase inhibitors, IA specific for proteinase A, IBspecific for proteinase B, and ICspecific for carboxypeptidase Y, have been detected (9-13; for reviews, see Refs. 1 and 2). Two isoinhibitors each of proteinase A(IA2and IA3)and proteinase B (IB1and IB2)have been purified from bakers’s yeast (9, 11, 12). In a haploid S. cereuiszae strain, however, only one inhibitorprotein of eachproteinase (IA3and IB2)can be detected (10, 14, 15). For reasons of simplicity, we will, there-

* This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. T o whom correspondence should be addressed. The abbreviations used are: I*, proteinase A inhibitor; IB, proteinase B inhibitor; CRM,antibody cross-reacting material. The enzymes are: proteinase A (EC 3.4.23.8.); proteinase B (EC 3.4.22.9.).

Also, little is known about the mechanism of s-ynthesis of the proteinases and their inhibitory activities. Are these two activities synthesized as one polypeptide chain and cleaved into proteinase and inhibitor activities? Are they synthesized as two polypeptide chains? Are they co-regulated? Mutants, that specifically lack one or more of the proteinase inhibitors or exhibit an altered regulation of inhibitor activity, should provide answers to these questions. In this paper, we report the isolation and characterization of a temperature andpH-sensitive mutant with altered regulation of the IAactivity. MATERIALS AND METHODS

Chemicals-Azocoll was obtained from Calbiochem (Los Angeles, Calif.); bovine serum albuminwas from Behringwerke A. G. (Marburg, Germany).Yeastextract, peptone, yeast nitrogenbase, agar,and Freund’s complete and incomplete adjuvant were from Difco (Ann Arbor, Mich.). Ethyl methane sulfonate was from Eastman Organic Chemicals (Rochester, N. Y.). ~-[4,5-”H]Leucine(60 Ci/mmol) was obtained from Amersham-Buchler (Braunschweig, Germany). NCS solubilizer was from Amersham/Searle Corp. (Arlington Heights, Ill.). Toluene scintillation mixture was from Roth (Karlsruhe, Germany). Pepstatin was purchased from Protein Research Foundation (Osaka, Japan). Alcohol dehydrogenase, glucose-6-phosphate dehydrogenase, hexokinase, ATP, NAD, and NADP were obtained from Boehringer (Mannheim, Germany). Purified proteinase B inhibitor was a generous gift of Dr. P. Biinning. Hemoglobin, glucose, and all other chem-

4821

4822

Proteinase Yeast

A Inhibitor Regulatory Mutant

icals were from Merck A. G. (Darrnstadt, Germany). Yeast Strains-The haploid yeast strains used in this work were strain S288C (e, mal -), obtained from R. K. Mortimer, and strain 4275-2A (a, mal-), our standard mating type a strain. B7 mutant was derived from S288C. The nuclear petite mutants €3234-805 A (petl, arg4, thrl, trp5,his5, lys2, and ade2) and 1893-11-6C @et2, met2, pha2, ga17, trpl, ura3, tyrl, lys2, and lys7) were obtained from the Yeast Genetic Stock Center, University of California, Berkeley. Media a n d Growth-The minimal medium contained 0.7% yeast nitrogen base and 2% glucose. YPD mediumcontained 1% yeast extract, 2% peptone, and 2% glucose. Ethanol (2%) was used as a nonfermentable carbon sourcein medium containing 1%yeast extract and 2%' peptone. Adjustment of the pH was done with potassium hydroxide to obtain pH 6.5 or sulfuric acid to obtain pH 2.6. When these media were used as solid media, they contained 2% agar. The presporulation medium contained 5% glucose, 1% yeast extract, 3% nutrient broth, and 2% agar. Sporulation of diploids was performed on a medium containing 0.98%potassium acetate, 0.1%glucose, 0.125% yeast extract, and2% agar. Liquid cultures were grown in Erlenmeyer flasks and aerated by shaking on a rotary shaker (Typ LSR, Braun Melsungen, Germany). Growth was followed in N e t t flasks, measuring cell density a t 546 nm in an Eppendorf spectrophotometer (Hamburg, Germany). Cells were counted in a Neubauer hemocytometer. Mutagenesis-Mutagenesis was performedwith ethylmethane sulfonate as outlined by Fink (33). Genetic Analysis-The procedures for sporulation and tetrad dissection have been described by Hawthorne and Mortimer (34). Preparation of Extracts-Cells were harvested by centrifugation, washed once with distilled water, andresuspended in 0.1 M potassium phosphate buffer, pH 7.0. The ratio of cells to buffer was 1:l (w/v). Extracts were prepared for the measurement of enzyme activities by breaking cells twice in a French pressure cell at 20,000 p.s.i. (Aminco, Silver Spring, Md.). Thesuspension was centrifuged 40 min a t 30,000 X g in a Sorvall RC 2B centrifuge. For measuring the proteinase inhibitor activities, the cell suspension washeated for 20 min a t 95°C. The precipitate was removed by centrifugation, and the inhibitors were assayed in the clear supernatant as described in earlier publications (35). Proteinase a n d Inhibitor Activity Assays-Proteinase A activity was measured according to Saheki andHolzer (36),using hemoglobin as a substrate. The trichloroacetic acid-soluble product was determined by the modified Folin colorimetric method of McDonald and Chen (37). Units are expressed as micrograms of tyrosine released per min per mg of protein a t 25°C. Proteinase B activity was measured according to Saheki and Holzer (36) using azocoll as a substrate. Activity is expressed as AA 520 nm/min/mg of protein a t 25°C. I* was determined as described by Saheki et al. (11).'I activity was measured as described by Betz et al. (9). Inhibitory activity is expressed as the amount of enzyme activity inhibited perml of inhibitor solution. Activation of proteolytic activitieswas performed as outlined by Saheki and Holzer (38). Other Assays-Alcohol dehydrogenase,glucose-6-phosphatedehydrogenase, and hexokinase were measured as outlined in Ref. 39. Phosphofructokinase was determined according to the procedure of Afting et al. (40). Pyruvate decarboxylase was tested according to Holzer and Goedde (41). Tryptophansynthase was measured as described (9). Ethanol concentration inmedia was determined as outlined in Ref. 42. ZA a n d ZH Levels under Conditions of Protein Synthesis Znhibition-Mutant and wild type cells were grown in unbuffered minimal medium a t 30°C for 36 h. One-fourth of the culture was harvested. To the remaining culture, 50 pg/ml of cycloheximide was added, and the cells were further incubated at 30°C. After 4, 8, and 12 h, cells wereharvested. Boiled cell extracts were preparedand inhibitor activities were tested. Protein Determination-Protein was determined by the methodof Lowry et al. (43) using crystalline bovine serum albuminas standard. Preparation of Anti-IB Antibodies-Rabbit antibody against purified IB from s. cereuisiae was elicited according to the method of Chan and Schatz (44). Immunochemical Methods-Immunoprecipitation measurements of I" were carried out in boiled cell extracts, in which about IO-fold purification of I" over crude extractsis obtained. Purity of anti-IBwas checked using the double diffusion technique of Ouchterlony (45). A single precipitation line was observed. Quantitative immunoprecipitation using boiled cell extracts andpurified IBwas done according to Schimke (45) and showedidentical amounts of IB necessary for maximum precipitation. Protein found as immunoprecipitate after

reaction with antibody of a known amount of IBin boiled cell extracts never exceeded immunoprecipitate protein generated by the same amount of purified IB reacting with the antiserum. Incubation mixtures for quantitative precipitation of IBcontained 0.5 ml of boiled cell extract (about 3 mg/ml of protein). Antiserum (50 pl) and 15 p1 of potassium phosphate buffer, pH 7.5, containing 0.1 M NaC1, 3% Triton X-100, 3% sodium deoxycholate, and 0.2% NaN:I of antiserum, mixtures were added/2 pgof 1'. Priortoaddition containing boiled cell extracts and buffer were incubated for 30 min a t 37°C and for 8 h a t 4°C to remove unspecifically precipitating protein. Controls using boiled cell extract or antiserum under the same conditions were run in parallel. After additionof antiserum, the mixtures were again incubated 30 min at 37°C and overnight at 4°C. Precipitates were collected by centrifugation for 8 min a t 8000 X g in an Eppendorf centrifuge. Immunoprecipitates were washed three times using 0.1 M potassium phosphate buffer, pH 7.5, containing 0.1 M NaCI, 1%Triton X-100, 1% sodium deoxycholate, and 0.02% NaN:,. Final precipitates were dissolved in 1 ml of 0.1 M NaOH, neutralized with HCl, and the radioactivity was counted in 10 ml of Triton X100-toluene scintillation mixture (1/2, v/v). Measurement of IB DegradationduringStationary Growth Phase-IB degradation was measured essentially as described by Betz (46). Cells were grown a t 30°C in unbuffered minimal medium. Wild type cell cultures contained 1.5 pCi/ml, and pai mutant cultures, because of early cessationof growth under theseconditions, contained 0.75 pCi/ml of [3H]leucine. After 21 h of growth, 1liter of each culture was harvested by centrifugation. T h e remaining cultures were chased with 2 mM nonradioactive leucine. After 33 and 45 h of growth, 1 liter of cells each were harvested. Chase of the remaining cultures was renewed by adding 1.33 m~ and 0.66 m~ nonradioactive leucine, respectively. T h e final cultures were harvested in late stationary phase after 57 h of growth. Boiled cell extracts were prepared in 0.1 M potassium phosphate buffer containing 0.1 M NaCl and 0.02% NaN3. Measurement of IB activity and protein in boiled cell extracts and analysis of IB CRM were done as described above. One microgramof purified IB exhibited an activity of 0.148 unit. Protein Degradation-Cellular proteins were prelabeledwith 1 pCi of [3H]leucine/ml during growth of cells on unbuffered minimal medium a t 30°C. After reaching late stationary phase (48 h), cells were harvested a t room temperature, washed twice with sterile water, and suspended in the supernatant culture fluid from parallel culture grown for the same time without labeled amino acid. Nonradioactive leucine (2 mM) was added. Shaking a t 30°C was continued, and I-ml samples were taken at the timesindicated. The release of trichloroacetic acid-soluble radioactivity from prelabeled protein was followed as described by Betz and Weiser (25). Protein was solubilized with 1 ml of NCS/H20 (9/1, v/v). Radioactivity was counted in 10 ml of a Triton X-l00/toluene (1/2, v/v) scintillation mixture. Quenching of protein-containing samples andtrichloroacetic acid supernatants was corrected by internal standardization. RESULTS

During a search for carboxypeptidase Y and proteinase B mutants, we accidentallyfoundamutantwhichexhibited about 10-fold increasedactivation of proteinase B activity in crude extracts compared to wild type (Fig. 1) and a 2- to 2.5fold enhancedactivity of "free" proteinase These A. properties were seen when the mutant was grown to stationary phasein unbuffered minimal mediumat 3OoC, but there was nodifference in "free" proteinase A activity and proteinase B activation between mutant and wild type crude extracts of cells grown at 23°C. Growthof mutant cells at 37°C did not enhance the differences found in mutantcrude extracts at 30°C. These properties suggested that a temperature-conditionalmutationin some component of the proteinase or proteinase inhibitor system had been isolated and led US to conduct the following characterization. Growth of the Mutant-At 23"C, the mutant, which we called B7, grew at the samerate as wild typeinminimal medium adjusted to pH 6.5, in unbuffered minimal medium, andin YPD. However,growthof the mutant at 30°C was significantly altered. In minimal medium adjustedto pH 6.5, the growth rate was slowerthan the wild type, but the mutant cells finallyreached wild type cell density afterprolonged

Mutant Regulatory Yeast ProteinaseA Inhlibitor growth (Fig. 2). In unbuffered minimal medium, mutant cells grew more slowly during logarithmic phase than wild type 70%of wild type cell density and were only able to reach about (Fig. 2). The lowered maximum cell density compared to wild type was confirmed by hemocytometer count and measurement of wet weight of mutant cells. Growth of mutant and wild type cells in unbuffered minimal medium was accompanied by a change in pH. The pH drops during logarithmic growth from about 4 to a value below pH 3 in the diauxic growth phase. Growth of mutant and wild type cells at 30°C in YPD medium of two different pH values was similar to minimal medium (Fig. 3). In YPD at pH6, mutant cell growth was slower during the logarithmic phase. After prolonged growth, the mutant slowly reached wild type cell density. Growth in YPD medium, which had been adjusted to pH2.6, reduces the logarithmic growth rate of the mutant. Under these conditions the mutant cells were unable to reach wild type cell density, While wild type cells were able to grow at

341

301

/ /

26

0

1

3

I

5

4823

30°C on ethanol-containing medium at pH 2.6, growth of the mutant was barely detectable under these conditions. These studiesindicate that mutation leads to alterations in the temperature sensitivity and pH dependence of growth. The shift of the mutant cells to 37°C had no further effect on the mutant phenotype. It has been shown that proteinaseAandB and their inhibitors are derepressed by severalfold when the cells reach the diauxic and stationary growth phases in glucose-containing medium (38, 47). Therefore, from the properties of the mutant described above, it seemed likely that any effects of the mutation on these components of the proteolytic system would be most readily detected in cells that had been grown into diauxic or stationary phase in unbuffered minimal medium at 30OC. We will refer to these conditions as therestrictive conditions.

A Mutant with Low Proteinase

A InhibitorActiuity-

While the proteinases and theirspecific inhibitors are located in different cell compartments, disruption of the cell and preparation of crude extracts by mechanical methods lead to rupture of the vacuole bearing the proteinases and to formation of the inactive complexes with their specific inhibitors in the cytosol (16, 17, 38). By adjusting the pH of the crude extract to around 5, activation of the proteinase activities is achieved by an autocatalytic digestion of the proteinase inhibitors, which is triggered by a low amount of "free" proteinase A activity (11, 38). Thus, one possible explanation of the phenotype of themutant might be an enhanced level of proteinase A activity or a lowered IAactivity. As shown in Table I, the activities of proteinase A and B and their specific inhibitors were similar in wild type and mutant cells grown to stationary phase in minimal medium (pH 6.5) at 23°C. Growth in unbuffered minimal medium at 23°C also produced no difference between wild typeand

9

Time of activation [h]

FIG. 1. Activation of proteinase B activity in crude extracts of minimal medium at 30°C grownpail mutant and wildtype cells. Proteinase B activity ofwild type (S288C) ( o " 0 ) and mutant (B7) (M cells. )

Grovth tame [hl

FIG.3. Growth of pail mutant and wild type cells in YPD medium of different pH values at 30°C. Growth of wildtype (4468-1A)( O " - O ) and mutant (4468-1B)(c".) at pH 6; growth of wild type (4468-1A) (A-A) and mutant (4468-1B)(A-A) at pH 2.6.

TABLEI Specific activities ofproteinase A and B and their inhibitors of late stationaryphase cells ofpail mutant and wild type grown in minimal medium at p H 6.5 and 23°C Cells were harvested after 60 h of growth. Proteinase activities were measured after activation. GrOWthtlrn

Specific activities

rm

FIG. 2. Growth ofpail mutant and wild type cells in minimal media of different pH values at 30°C. Growth of wildtype (44681-4) (A-A) and mutant (4468-1B) (A-A) at pH 6.5; growth of wild type (4468-1A) ( C F - 0 ) and mutant (4468-1B) (w) in unbuffered minimal medium.

Strain

I"

Proteinase A

I~

Proteinase B

1.9

0.1

1.7

0.1

units/mg

Wild type Mutant

29.5 25.4

5.3 5.7

A Inhibitor Regulatory Mutant

Proteinase Yeast

4824

TARLE I1 Growth parameters and specific activities of proteinases A and B and I* and I” of pail mutant and wild type cells grown under restrictive conditions Cells were grown in unbuffered minimal medium at 30°C. Proteinase activities were measured after activation. Specific activity phase Growth

Cell density

Wild type/mutant

Mutant Wild

t-we

Wild

units/mg

Diauxic Stationary

1.7 1.9

1 .o 1.3

4.9 1.7 7.5

12.4 30.3

TABLE111 I Aactivity and growthbehavior of segregants of six tetrads

derived from cross of mutant B7 with wild type 4275-2A Growth of tetrads was tested on Petri plates containing the indiactivity of the tetrads, grown in liquid media for 60 h, cated media. IA was measured a.described under “Materials and Methods.” ++++, normal growth; +, reduced growth; -, no growth. Tetrad

Growth on glucose minimal medium

Growth on yp medium, pH 2.6

30°C 23°C 30°C 23°C

1-A I-B I-c I-D 2-A 2-B 2-c 2-D 3-A 3-B 3-c 3-D 4-A 4-B 4-c 4-D 5-A 5-B 5-c 5-D 6-A 6-B 6-C 6-D

++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++

++++ + + ++++ ++++

++++ + + ++++ +

+ ++++ + ++++ ++++ + + + ++++ ++++ + ++++ ++++ ++++ ++++ ++++ +

++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++

++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++

++++ -6.7

++++

++++ ++++ -

++++ -

++++ -

++++ ++++ -

-

++++ ++++ -

++++

++++ - 6.6

Wild

Mutant t-we units/mg units/mg

Wild Mutant type

type

I I’

Proteinase A

IA

IA activity (units/ after mg) growth in unbuffered minimal medium 23OC

30°C

29.5 25.4 22.2 22.7

15.3 5.6

18.0 16.1 21.1 19.2

15.1 10.0 4.4 3.2

20.2 15.5 19.6 25.1

17.2 6.8 7.5 16.7

17.1 14.1 19.2 15.0

5.9 16.4 13.4 2.4

25.5 20.4 19.4 15.2

4.5 5.2 11.5 16.5

28.1 21.8 18.4 23.7

3.6 15.0 16.7

2.1 5.7

2.3 8.1

Proteinase €3 Mutant

Wild t.yp

Mutant

0.06

0.08 0.15

unitn/mg

0.15

1.4

1.6 0.8

was analyzed in two independent crosses withwild type strain 4275-2A. Diploids were sporulated, asci were dissected, and the spore clonesweregerminated and analyzed. Table I11 shows a segregationpattern of six tetrads. All tetrads analyzed showed a 212 segregation of low IA activity and alteredgrowth behavior of the clones under restrictive conditions or during growth on ethanol at acidic pH and 30°C. A detailed growth analysis of a representative tetrad (tetrad 1) is shown in Fig. 4. We have called the mutation that leads to the defect in IA activity andat the same time to the altered growth behavior in strain B7 pail-I. The mutationwas shown to be recessive. Heterozygous diploids for the mutant gene (pai+/pai-) ex-

19

mutant proteinase and inhibitor activities. However, IA activity differed significantly in wild type and mutant extracts when the cells were grown under restrictive conditions for mutant growth. Whereas proteinases A and B reached wild type levels throughout, and I” is at wild type levels in diauxic phase, IA activity never reached wild type levels and is about 70% reduced throughout (Table 11). Furthermore, I” activity declinedin stationaryphase in themutant.This will be discussed later. Mutantcells grown in glucose-containing medium a t pH 2.6 a t 30°C had a IA activity of 9.6 units/mg when the cells were harvested in the late stationary phase; this value is comparable to that obtained in unbuffered minimal medium (Table 11). This control experiment shows that the presence of acidicgrowthconditions throughout does not completelyabolish IA activityin mutant cells, nordoes it completely prevent growth (Fig. 3). Segregation Patternof the MutantPhenotype-Strain B7

I

I

II FIG. 4. Growth behavior of four segregants of tetrad 1 derived from the cross of mutantB7 with wild type4275-2A. The two wild type segregants of cros.. 4468 ( A and I)) and the two mutant segregants ( B and C ) were grown on the following media:minimal medium, pH 6.5 at 23°C ( a )and at 30°C ( h ) ;minimal medium, pH 2.6 at 23°C ( c / d ) ;YP ethanol medium, pH 2.6 at 23°C ( e ) and at 30°C (f).

Yeast Proteinase A Inhibitor Regulatory Mutant

4825

TABLEIV

present (Table V). In the mutant, however, an excess of 4.2 X M concentration of proteinase A over IAwas present. Only 1 molecule of IAwas present for about 2 proteinase A molecules. Conditions that partially restore growth of the mutant to wild type cell density in stationary phase, namely adjusting the minimal medium to pH 6.5 (Fig. 2), also led to a surplus of IAover proteinase A (Table V). At acidic pH at 30"C, the mutant was unable to grow on IA activity after heat ethanol as carbon source (Fig. 4f). Therefore, it was interestIA activity after heat treatment + 37°C triStrain treatment chloroacetic acid ing to test whether the pail phenotype (i.e. low IAactivity treatment with unchanged proteinase A) is a phenomenon generally units/mg found in nuclear petite mutants. Table VI shows that this is 29.5 18.7 Wild type not the case. The two petite mutants tested exhibited a decreased IA activity and decreased proteinase A activity. 25.4 19.4 Mutant Both petite mutants showed an excess of IAover proteinase A; a ratioof IA:proteinase A can be calculated which issimilar TABLEV to wild type grown under the same conditions (Table VI). It Molar concentrations ofproteinase A and I A ofpail mutant and is also apparent that thebiochemical defect in petite mutants wild type cells grown under different conditions is different from the defect in the pail mutant because these Mutant (4468-1B) and wild type (4468-1A) cells were grown for 48 strains show different growth behavior. The pail mutant h in unbuffered minimal medium and for 92 h in minimal medium, pH 6.5, at 30°C. For calculation of the molar concentrations and the shows distinct diauxic growth and can use about 30% of the IA/proteinaseA ratio, a molecularweight of 7,600 and a specific ethanol that had been produced during logarithmic growth activity of 1,420 units/mg for I* (47) and the molecular weight of (not shown). Petite mutants do not show diauxie as they 45,000 and a specific activity of 770 units/mg for purified proteinase cannot use ethanol for growth. A (9) were used. Protein Degradation-Since the pail mutation leads to an Minimal medium 30°C Minimal medium pH 6.5, 30°C excess of proteinase A of that which could be complexed by IA, does this excess of free proteinase A cause enhanced Ratio Concentration Ratio Concentration Strain Z A activity of wild type and pail mutant strain of cells grown at 23OC andpH 6.5 after heat and acid treatment Cells of mutant (4468-1B) and wild type (4468-1A) were grown 60 h in minimal medium. Heat treatment at 95°C wasperformed as described under "Materials and Methods." Acid treatment was performed at 37'C with 10%trichloroacetic acid in the mixture. Incubation time after each step was 60 min.

IA/pro-

Proteinase temase A A M

Wild type 8.7 X Mutant 2.6 X

M

6.4 X

lob6 6.8 X

1.35 9.5 X 0.38 5.3 X

2.9 X 3.4 X

3.3 1.56

hibited normal growth and normal IAactivity under restrictive growth conditions (not shown). Regulation of Proteinase A Inhibitor Levels is AffectedThe reduced IAactivity measured in mutant cells after growth under restrictive conditions is not due to a temperature and pH-sensitive IAprotein, as can be seen from Table IV. Heat treatment at 95°C of the mutant protein from cells grown at 23°C and pH 6.5, as well asheattreatment followedby trichloroacetic acid treatment at 37°C (steps which are part of the IAisolation procedure ( l l ) ) ,did not lead to inactivation of mutant proteinase A inhibitory activity. These results suggest that thelower concentration of IAactivity observed in mutant cells after growth under restrictive conditions is due to an altered regulation of IA synthesis or degradation. Which of these twoprocesses is responsible for low IA activity in the mutant? Since antibody against IA is not yet available, we determined degradation of IAby measuring the decay of IA activity after inhibition of protein synthesis by cycloheximide(45,48). In mutant and wild type cells the same, nearly linear decrease in IAactivity of 0.36 unit/mg/h was measured under restrictive conditions. This result excludes the possibility that a mutationin the structuralgene of IA has occurred that leads to enhanced degradation of IA protein. Also, alteration of a proteolytic system that causes accelerated degradation of IAcan be excluded. Since enhanced degradation of IA protein can be ruled out, the most likely explanation for low IA activity in the mutant is decreased synthesis of IA protein. However, low IA activity due to a mutation in some unknown modification reaction necessary for IAactivity cannot be excluded. Proteinase A and Its Inhibitor under Different Growth Conditions-When grown under restrictive conditions, wild type cells exhibited an excess of 2.3 X lo-" M IAover proteinase A. About 1.35 molecules of IA/molecule of proteinase A were

TABLEVI Specific activities ofproteinase A and I A in petite mutants and wild type cells Petite mutants (pet2 andpet2) and wild type cells were grown for 70 h at 30°C in unbufferedminimalmediumsuppliedwith 0.2% casamino acids,1.4 mM adenine, and1.4 m~ uracil. Proteinase activity and IA/proteinwas measured after activation. Molar concentrations ase A ratios were calculated as in the legend of Table V. Concentration

ity Strain

Genotype

Proteinase A

I~

Proteinase A M

units/mg

BZ 34-805A pet1 1893-11-6C pet2 S288C Wild type

0

1.9 2.2

2

8.8 1.4 X 1O"j 1.7 X 8.7 1.9 x 2.2 x 23.8 6.3 4.6X 9.7 x

4

Molar ratio I"/proteinase A

6 Time [h]

1.3 1.2 1.5

e

FIG. 5. Protein degradation in pai mutant and in wild type cells under restrictive growth conditions. Per cent cell protein broken down in wild type (4468-1A) (o"-o) andpail mutant (44681B) (M).

Yeast Proteinase A Inhibitor Regulatory Mutant

4826

protein degradation in mutant cells grown under restrictive conditions? As can be seen from Fig.5, no accelerated general protein degradation was visible in thepail mutant. In Vivo Activity Levels of Proteins That are Sensitive to Proteinase A in Vitro-Determination of general protein degradation might be too insensitive to detect enhanced proteolytic inactivation of proteins in thepail mutant since only a very limited number of proteins might be affected. Therea

TABLEVI1 Activities ofproteins in p a i l mutant cells grown under restrictive conditions Mutant and wild type cells were grown for 62 h in unbuffered minimal medium a t 30°C. Activities are expressed as per cent of the specific activity of wild type cells. Specific activity relative to wild t.we .. 70

Protein tested

Hexokinase Phosphofructokinase Pyruvate decarboxylase Alcohol dehydrogenase Glucose-6-phosphate dehydrogenase Tryptophan Proteinase B inhibitor

synthase

158 127 86 165 96 20 25

-

I

”.

10

io

30

50

40

60

0

70

Growth time [hl

FIG. 6 . Tryptophan synthase activity duringgrowth of pail mutant and wild type cells under restrictive growth conditions. Mutant (4468-1B) and wild type (4468-1A) cells,grownin unbuffered minimal medium at 30°C, were harvested at the times indicated. Prior to preparation of crude extracts, pepstatin (4 mg/ml) was added to cell suspensions to prevent artifactual in vitro degradation by proteinase A. Tryptophan synthase activity of wild type (M and ) mutant (M).

m L

4 r c

m

2oi 04 20

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LO

50

60

Growth time ( h )

FIG. 8. Immunoprecipitation of IB of pail mutant and wild type cells grown under restrictive conditions.Degradation of Is was measured as described under “Materials and Methods.” Per cent counts per minute per pg of Is of wild type (4468-1A) (o”-o) and mutant (4468-1B)(M).

fore, we tested two yeast proteins, tryptophan synthase and IB,in the mutant under restrictive conditions. These proteins have been shown to be rapidy inactivated by proteinase A in vitro (9, 49). As a control, we tested several other enzymes that are known to be unaffected by proteinase A in vitro (49). As can be seen from Table VII, tryptophan synthase and IB showed activities relative to wild type of 20% and 25%, respectively, in mutant cells. In contrast, the enzymes that are not inactivated in vitro by proteinase A showedno decreased activity in the mutant. The idea that “free” proteinase A in the mutant is responsiblefor the low activities of tryptophan synthase and IB implies that a decrease of these activities should beonly visible under those growth conditions where the surplus of proteinase A appears, i.e. diauxic or stationary growth phase. Figs. 6 and 7 show that, as predicted, tryptophan synthase and IB activities, after reaching nearly wild type levels, disappeared more rapidly in the mutant whencells entered stationary phase under restrictive growth conditions (Fig. 2). Degradation of Proteinase B Inhibitor-To determine whether accelerated disappearance of IB in the mutant is due to enhanced proteolytic degradation, two experiments were undertaken. ( a )Disappearance of IBactivity was measured in stationary phase cells after inhibition of protein synthesis by cycloheximide. ( b ) Degradation of protein was followedin cells growing under restrictive conditions by use of specific antibodies. Disappearance of IBactivity under conditions abolishing protein synthesis was found to be nearly linear with time inwild type and pail mutant cells. In wild type, a units/mg/h was measured. A 2-fold decrease of 9.6 X enhanced decrease (19.2 X units/mg/h) of IB activity was found in mutant cells. This accelerated decrease of Is activity was accompanied by a 2- to 2.5-fold increase in the rate of disappearance of IB CRM in mutant cells (Fig. 8). Only Experiment a, measurement of disappearance of IBactivity, was done by using cycloheximideto stopprotein synthesis. Experiment b, measurement of degradation of IB protein, was done without using the drug. As both experiments essentially lead to the same results, a different effect of cycloheximide on Is degradation in mutant and wild type cells seems unlikely.

1

e

P

O 10

M

30

50

40 Growth time

(hl

60

70

DISCUSSION

The results presented in this paper show that a mutationin FIG. 7. IB activity during growth of pail mutant and wild a gene, which we will call pail (standing for proteinase A type cells under restrictive growth conditions. IB activity of inhibitor), leads to a temperatureand pH-sensitive reduction mutant (4468-1B) and wild type (4468-1A) cells,grown in unbuffered of IA activity, paralleled by altered growth behavior and minimal medium at 30°C, was measured. Pepstatin (4 mg/ml) was accelerated inactivation of tryptophan synthaseand IB in vivo. added to the cell suspensions to prevent artifactual invitro degradation of IB by proteinase A. IB activity ofwild type (M and ) The pail -1 mutation could be( a )a mutationin the structural gene coding for I*, ( b )a mutation affecting post-translational mutant (e-.).

Yeast Proteinase A Inhibitor Regulatory Mutant modification of IA, or (c) a mutation affecting the regulation of IA activity. The levels of IA activity in the mutant are sensitive to high temperature and low pH, yet the IAprotein is insensitive to heat and acid treatment. The stability of IA from the mutant makes it unlikely that pail is a mutation in the structural gene for the inhibitor. Our studies cannot distinguish between possibilities ( b ) and (c). As no modification reaction affecting IA activity is known as yet, we currently favor the idea that pail is a defect affecting the regulation of synthesis or degradation. Previous studies onthe regulation of the proteinases A and B and their specific inhibitors have shown that both proteinases and inhibitors respond in parallel to the presence of glucose in the medium as do such catabolite-repressible enzymes as malate dehydrogenase and fructose bisphosphatase (38, 50). Since proteinase A and proteinase B reach wild type levels in the mutant under conditions that are derepressing activity inthe mutantunder for wild type andsince the low IA those conditions is not due to accelerated degradation of IA, our results might indicate that thepail-1 mutation affects the Malate dehydrogenase and fructose bisphossynthesis of IA. phatase activities, which were measured as a control,’ show wild type levels in the pail-1 mutant. If this conclusion is correct, our studieswould suggest that the simultaneous increase of proteinase A and its inhibitor activity is not a result of their derivation from a common precursor. It could be imagined that joint control over proteinase and inhibitor resultsfrom synthesis of one polypeptide chain consisting of inactive proteinase A and its inhibitor, which is cleaved into the two active componentsimmediately before or during the entry of proteinase A into the vacuole. The generation of a pepsin-inhibiting peptide and active pepsin from inactive pepsinogen is an example for cleavage of proteinase and inhibitor from a common precursor (51). Such a mechanism could avoid unwanted proteolytic damage at the origin of proteinase synthesisand during proteinase transport into the vacuole, a function that has been attributed to the inhibitors (17,20).However, if our assumptionholds true that synthesis of IA activity in the pail strain is altered independently from proteinase A by mutation,this model can be excluded. The results with the pail mutant also tend to exclude the hypothesis that synthesis of I* activity and proteinase A is stringently coupled by sharing common regulatory elements. The ability to regulateproteinase and inhibitor levels independently might enable the cell to regulate proteolysis under different environmental conditions (1). The studies on the pail mutant revealed a strong relationship between growth as defined by the ability to reach wild type cell density a t stationary phase and the capacity of the mutant toproduce maximal IAactivity. Growth and IA activity were normal in mutant cells grown at 23”C, but both mutant cell density (Figs. 2 and 3) and IAactivity (Tables I1 and V) were reduced after growth at 30°C and acidic pH. When the pH of the growth medium was raised toward neutrality, the cell density of the mutant culture grown at 30°C reached nearly that of wild type cells grown in the same medium (Fig. 2), and the IA activity was nearly normal (Table V). Wild type cells grown on ethanol show extensive derepression of proteinase A inhibitor activity (50), but the mutant fails to grow on ethanol under the restrictive conditions of 30°C and acidic pH. LOWactivity and altered growth result from a defect in a single nuclear gene. Is the lowered ability of the mutant to synthesize IAthe sole cause of the altered growth behavior of the mutant?Or, alternatively, does the mutationlead to some I. Beck, unpublished results.

4827

unknown general cellular disorder which in turn results in altered growth behavior and lowered derepression of IAactivity? Our results do not provide a clear choice between these alternatives. If IAplays a crucial role in intracellularregulation of proteinase A activity or in protection from unwanted proteolytic damage, the pail mutation should lead to proteolytic disorder only under conditions of proteinase and inhibitor derepression, that is, in late diauxic and in stationary growth phase. Calculation of inhibitor and proteinase levels under these growth conditions leads to the conclusion that in wild type proteinase A theoretically can always be complexed by IA (Table V). However, under the same conditions, a more than 2.5-fold excess of proteinase A over its inhibitor can be calculated for the mutant (Table V). We do not know how the cellular proteins come into contact with the vacuolar proteinases. During the cell cycle, fragmentation of big vacuoles into many small vacuoles has been observed prior to bud emergence. This fragmentation was correlated with mass liberation of amino acids and other storage metabolites. Liberation of enzymes as well has been proposed (52). During starvation conditions of sporulation, a weakening of the vacuolar membrane has been observed, which probably leads to liberation of vacuolar enzymes (52).If such processes actually take place, one may put forward the idea that the gradually increasing in amount of proteinase A, which cannot be complexed by IA the mutant, leads to damage of proteins necessary for cell growth. The observation that glucose-grown mutant cells are initially able to consume ethanol in the diauxic phase, but rapidly lose this ability in early stationary phase, can be rationalized by proposing such proteolytic damage. From in vitro studies, two proteins are known to be highly sensitive to IB (9).Both proteinase A, tryptophan synthase(49),and proteins exhibit nearly wild type activity levels in the mutant at the end of logarithmic growth phase (Figs. 6 and 7). However, activities of both proteins decrease much more rapidly in mutant cells during stationary phase (Figs. 6 and 7). Recently, it has been shown that only a 60,000-dalton fragment of the 95,000-dalton protein normally coded for by the his4 region of yeast can be isolated from the pail mutant (53). It was demonstrated that appearance of the low molecular weight form of the protein is due to high proteolytic activity in the mutant (53). In this case, the low molecular weight fragment of the enzyme is generated during the isolation procedure. Proteins that are insensitive to proteinase A hydrolysis in vitro do not show decreased activity levels in vivo in the mutant (Table VII). In the case of IB,the accelerated decrease in activity could be shown to be accompanied by an enhanced loss of IB-CRM,most likely due to proteolysis. One might imagine that other proteins that are sensitive to proteinase A and vital for growth might undergo unwanted proteolytic attack in the mutant. As yet, we do not know how proteinase and inhibitor action is regulated in the cell. Is proteinase A action restricted to the vacuolar compartment, or does proteinase A also act in the cytoplasm? Is IA always located in the cytoplasm or does it also serve functions in the vacuole? The experiments on the pail mutant give a first indication that IA is a vital cellular component which is necessary to regulate proteolytic activity. However, since the pail mutation does not appear to affect the structuralgene of IA,one must recognize that thestriking relationship of low IAactivity, excess of “free” proteinase A, early cessation of growth, accelerated inactivation of proteins, and enhanced proteolysis of IB does not prove the validity of the model described above. For instance, IAmight also be an inhibitor of another, as yet undetected, cytoplasmic proteinase. Furthermore, the connections between the phenotypic events detectable in the mutant might have a very different

Proteinase Yeast

4828

A Inhibitor Regulatory Mutant

basis. Answers to these questions may be possible when mutants harboring mutations in the structuralgene of IA or double mutants lacking IA and proteinase A are available. Acknowledgments-We are grateful to Dr. P. Bunning for supplying pure IB. The excellent assistance of Mrs. Claudia Ehmann during part of this work is gratefully acknowledged. Thanks are due to Dr. R. L. Switzer for critical reading of the manuscript. We thank Mrs. L. Kotschau and Mrs. H. Gottschalk for their expert help during preparation of this manuscript. REFERENCES 1. Wolf, D. H. (1980) in Advances in Microbial Physiology (Rose, A.H., and Morris, J. G., e&) Vol. 21, pp. 267-338, Academic Press, New York 2. Wolf, D. H., and Holzer, H. (1980) in Transport and Utilization

Amino Acids, Peptides and Proteins by Microorganisms (Payne, J . W., ed) pp. 431-458, John Wiley, Chichester Hata, T., Hayashi, R., and Doi, E. (1967) Agr. Bwl. Chem. 31, 150-159 Doi, E., Hayashi, R., and Hata, T. (1967) Agr. Biol. Chem. 31, 160-169 Wolf, D. H., and Weiser, U. (1977) Eur. J. Biochem. 73, 553-556 Matile, P., Wiemken, A., and Guyer, W. (1971) PZanta 96,43-53 Masuda, T., Hayashi, R., and Hata, T. (1975) Agr. Biol. Chem. 39,499-505 Frey, J., and Rohm, K.-H. (1978) Biochim. Biophys. Acta 527, 31-41 Betz, H., Hinze, H., and Holzer, H. (1974) J. Biol. Chem. 249, 4515-4521 Ulane, R. E., and Cabib, E. (1974) J. Biol. Chem. 249,3418-3422 Saheki, T., Matsuda, Y., and Holzer, H. (1974) Eur. J. Biochem. 47,325-332 Nufiez de Castro, I., and Holzer, H. (1976) Hoppe-Seyler’s 2. Physiol. Chem. 357, 727-734 Matern, H., Hoffmann, M., and Holzer, H. (1974) Proc. Nutl. Acad. Sci. U. S. A . 71,4874-4878 Bunning, P., Meussdoerffer, F., and Holzer,H. (1977) HoppeSeyler’s 2. Physiol. Chem. 358,1186 Biinning, P., and Holzer, H. (1977) J. Biol. Chem. 252,5316-5323 Matern, H., Betz, H., and Holzer, H. (1974) Biochem. Biophys. Res. Commun. 60,1051-1057 Lenney, J . F., Matile, P., Wiemken, A., Schellenberg, M., and Meyer, J . (1974) Biochem. Biophys. Res. Commun. 60, 13781383 Cabib, E., and Ulane, R. (1973) Biochem. Biophys.Res. Commun. 50, 186-191 Cabib, E. (1976) Trends Biochem. Sci. (TIBS) 1,275-277 Saheki, T., and Holzer, H. (1976) Tokaj J. Erp. Clin. Med. 1, 115-125 Neeff, J., Hagele, E., Nauhaus, J., Heer, U., and Mecke, D. (1978) Eur. J. Biochem. 87,489-495 Gancedo, C. (1971) J. Bacteriol. 107,401-405 Molano, J., and Gancedo, C. (1974) Eur. J. Biochem. 44,213-217 of

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