THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc.

Vol. 259, No. 21, Issue of November 10, pp. 13344-13346,1984 Printed in U.S.A.

Proteolysis in Eukaryotic Cells PROTEINASE yscE, A NEW YEAST PEPTIDASE*

(Received for publication, February 24, 1984)

Tilman Achstetter, Claudia Ehmann, Akikazu Osaki, and Dieter H. Wolfs From the Biochemisches Institut der Uniuersitat Freiburg, Hermann-Herder-Str. 7, 0-7800 Freiburg i. Br., Federal Republic of Germany

A new peptidase, which we call proteinase yscE, was purified from the yeast Saccharomyces cerevisiae. The enzyme cleaves the synthetic substrates Cbz-Gly-Gly- Leu-4-nitroanilide, Cbz-Ala-Ala-Leu-.l-nitroanilide, and Suc-Phe-Leu-Phe-4-nitroanilide (Cbz and SUC are defined as benzyloxycarbonyl and succinyl, respec- tively) at the 4-nitroanilide bond and exhibits a slight activity against [3H]methylcasein. Optimum pH for cleavage of the chromogenic substrates is found to be in the range of 8.2 to 8.6. The purified enzyme has an apparent Stokes radius of R. = 75.2 di as judged by gel chromatography and is composed of subunits. Mercu- rials were found to be strong inhibitors of the enzyme activity.

Intracellular proteolysis represents an essential mechanism in the regulation of cellular functions at the post-translational level (1-4). The yeast Saccharomyces cereuisiue serves as an excellent organism to study proteinase function as it is easily accessible to a combined biochemical and genetic approach ( 5 ) . Use of these techniques led to the finding that several of the well-known proteolytic enzymes in yeast are involved in general protein degradation (5-8). However, these studies left researchers with crucial questions about more specific proteo- lytic processes in yeast, which were shown or postulated to occur (3,5, 9-14) and most likely many others which are still completely unknown. Mutants devoid of several peptidases led to the discovery of a multitude of new proteolytic activities (15-19). A detailed insight into the actual number of newly discovered peptidases requires purification and partial char- acterization of the enzymes. This approach is also a prereq- uisite for further studies on the cellular function of the en- zymes. Here we describe the purification and some properties of one of these proteinases, proteinase yscE.

MATERIALS AND METHODS

Chemicals-The chromogenic peptide substrate Bzl-Phe-Val-Arg- NA was purchased from Kabi. All other chromogenic peptides used

schaft, Bonn and the Fonds der Chemischen Industrie, Frankfurt. * This work was supported by the Deutsche Forschungsgemein-

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom reprint requests should be addressed. The abbreviations used are: Bz-, benzoyl; Bzl-, benzyl; Cbz-,

benzyloxycarbonyl; E64, [~-3-trans-carboxyoxiran-2-carbonyl]-~- Leu-agmatin; MeO-, methoxy; NA, 4-nitroanilide; PhCH’SOZF, phen- ylmethanesulfonyl fluoride; SUC-, succinyl; Tos-~-LysCh&I, Na-p- tosyl-L-lysine chloromethyl ketone; Tos-L-PheCHzC1, tosylphenylal- any1 chloromethyl. ketone; unless otherwise stated amino acids were of the L-configuration.

(see Table 111) and the carboxypeptidase substrates (amino terminally blocked dipeptides) were obtained from Bachem. Aminopeptidase M from porcine kidney was from Merck. Molecular weight marker proteins, DEAE-Sepharose CL-GB, octyl-Sepharose CL-4B, and Sepharose CL-GB were from Pharmacia. The proteinase inhibitors chymostatin, antipain, pepstatin, leupeptin, elastatinal, phosphor- amidon, and E64 were from the Peptide Institute (Osaka, Japan). Captopril was a generous gift of Dr. E. R. Lucania, Squibb, Princeton, NJ. Other proteinase inhibitors were from Serva. All other chemicals, which were of highest purity available, were purchased from Merck. [3H]Methylcasein was prepared by the procedure of Jentoft and Dearborn (20).

Yeast Strains and Growth Conditions-The haploid yeast strain ABYSl (apral prbl prcl cpsl ode)’ (19) was used for purification of proteinase yscE and studies on regulation of the enzyme under vegetative growth conditions. The diploid yeast strain BYS-D2 (a prbl prcl cpsl ade/a prbl prcl cpsl ade) (16) was used for studies on the regulation of proteinase yscE under sporulation conditions. Media and growth of cells on YPD (1% yeast extract, 2% peptone, 2% glucose), YPEthanoI (1% yeast extract, 2% peptone, 2% ethanol), mineral medium containing 1.5% L-leucine as sole nitrogen source as well as sporulation conditions were as described in Ref. 19.

Preparation of Soluble Extracts-Cells were harvested, crude ex- tracts were prepared, and centrifugation steps were done as outlined in Ref. 19. Buffer was 0.1 M Tris, pH 7.2. For the purification of proteinase yscE, 1 mM EDTA was added.

Enzyme Assays-Unless otherwise stated buffers were adjusted and pH was measured at 25 ‘C. All chromogenic peptide substrates were dissolved in dimethyl sulfoxide at a concentration of 10 mM and stored in the dark at -20 “C. Under these conditions the peptide stock solutions were stable for at least 2 months. Proteinase yscE activity was assayed routinely using the chromogenic peptide Cbz- Gly-Gly-Leu-NA as substrate. The test was performed in 0.167 M Tris, pH 8.2, and the appearance of 4-nitroaniline was measured at 405 nm in an Eppendorf spectrophotometer. Proteolytic activities different from proteinase yscE were tested with chromogenic sub- strates in the absence and presence of microsomal aminopeptidase M (60 milliunits) added to the system. In this way the proteolytic activity on the 4-nitroanilide bond as well as proteolytic activity on a chro- mogenic peptide at some bond different from the 4-nitroanilide bond can be measured (19). The enzyme activity unit is the amount of enzyme that catalyzes the release of 1 @mol of product/min under the test conditions (19). Carboxypeptidase activity was tested using amino terminally blockeddi- or tripeptides. The amino acids liberated were subjected to oxidative deamination by L-amino acid oxidase and the appearance of hydrogen peroxide was followed spectrophotomet- rically by measuring the oxidation of o-dianisidine (21). 13H]Meth- ylcasein activity was measured according to Ref. 22, with the modi- fications described in Ref. 19. Following trichloroacetic acid precipi- tation and centrifugation of the test system, samples of the superna- tants were taken and counted (19) in order to measure soluble fragments.

Electrophoresis-Polyacrylamide gel electrophoresis (4% acrylam- ide (w/v) and 0.107% N,N’-methylenebisacrylamide (w/v) of the native protein was done in rods (0.5 X 7 cm) using the discontinuous

The genetic markers used are: pral, proteinase A deficiency; prbl , proteinase B deficiency; prcl, carboxypeptidase Y deficiency; cpsl, carboxypeptidase S deficiency; ade, adenine auxotrophy; a, a, cell- mating type.

13344

Yeast Proteinase yscE 13345

buffer system according to Davis (23) or, alternatively, the method of Hedrick and Smith (24). A current of 2 mA per rod was applied. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was done according to Laemmli (25). Gels were stained with Coomasaie Bril- liant Blue R-250 according to the method of Weber and Osborn (26) and destained by diffusion.

Protein Determination-Protein was determined by the method of Lowry et d . (27) using crystalline bovine serum albumin as standard.

Purification of the Enzyme-All steps were carried out at 4 “C. 160 g of cells (wet weight) grown into stationary phase on YPD medium were suspended in 0.1 M Tris buffer, pH 7.2, containing 1 mM EDTA and disintegrated by breaking them twice in a French pressure cell at 20,000 p.s.i. as described in Ref. 19 (step 1). The supernatant obtained after the 134,000 X g centrifugation step was brought to 40% saturation using crystalline ammonium sulfate. Final pH of the solution was about 6. After stirring for 1 h at 0 “C the precipitate formed was sedimented at 90,000 X g for 35 min (step 2). The

octyl-Sepharose CL-4B (100 ml), which had been equilibrated with supernatant was loaded onto a column (3 X 14-cm gel bed) containing

40% ammonium sulfate in 0.1 M Tris buffer, pH 7.2, containing 1 mM EDTA. Protein was eluted from the column with a linear gradient of decreasing ammonium sulfate concentration (40 to 15%) in buffer. The gradient was formed by an automatic gradient mixer (Ultrograd, LKB). The flow was 19 ml/h. Fractions of about 1.7 ml were collected and those of highest activity were pooled (step 3). After this hydro- phobic chromatography step, protein was precipitated in the pooled fractions by addition of solid ammonium sulfate until 70% saturation was reached. After 1 h of stirring at 0 “C the precipitate was collected by centrifugation at 90,000 X g for 35 min. The supernatant was discarded. The protein precipitate was dissolved in one-tenth of the original pool volume in 0.1 M Tris buffer, pH 7.2, containing 1 mM EDTA and 150 mM NaCl. The protein solution was dialyzed overnight against 2 X 500 ml of the same buffer (step 4). This solution was then applied to the top of a DEAE-Sepharose CL-GB column (1.6 X 15-cm gel bed, 30 ml of gel) previously equilibrated with the same buffer.

r

-300 - E

-200 f e C

U

-100 5 u z -

- n - 0” 20 LO 60 80 100 120 1LO 160 BO

Froctlon number

FIG. 1. Chromatography of proteinase yscE on DEAE- Sepharose CL-GB. For details see “Purification of the Enzyme.” 0, Cbz-Gly-Gly-Leu-NA activity; 0, absorbance at 280 nm; -, NaCl concentration.

Elution of the protein was carried out with a linear salt gradient of 150 and 300 mM NaCl in buffer. The flow was 16 ml/h. Fractions of about 1.5 ml were collected and tested for activity (step 5). The enzyme emerged from the column in two activity peaks at NaCl concentrations of 270 mM (S.D. * 6.2%, mean of six determinations) and 280 mM (S.D. k 5.5%, mean of six determinations), respectively (Fig. 1). As physical characterization of both activity peaks did not show any differences (see “Results”) active fractions of both peaks were pooled and protein was precipitated by saturating the solution with 70% ammonium sulfate. After centrifugation (90,000 x g) the supernatant was discarded and sedimented protein was dissolved in 20 mM Tris buffer, pH 7.2, containing 150 mM NaCI, 10% glycerol (v/v),and 1mM EDTA (step 6).

RESULTS

A summary of the purification procedure for proteinase yscE is given in Table I. A purification of more than 300-fold with a yield of about 20% was obtained. The ion-exchange chromatography on DEAE-Sepharose CL-GB can be consid- ered as the crucial step in this purification procedure. In the accompanying paper (19) we described the partial separation of new proteinases including proteinase yscE with the aid of ion-exchange chromatography. Under the low salt conditions used in this study to bind proteins on the ion-exchange column, high activity against the proteinase yscE substrate Cbz-Gly-Gly-Leu-NA was not bound. By contrast, application of the protein solution to the column under the high salt conditions described here (0.1 M Tris buffer, 150 mM NaCl, pH 7.2, with or without 1 mM EDTA) results in complete binding of proteinase yscE to the ion-exchange column and, after elution, leads to a 20-fold purification (Table I). The effect of ionic strength on the binding of proteinase yscE to DEAE-Sepharose CL-GB suggests considerable conforma- tional changes of the enzyme under the different conditions. The most likely explanation for the two activity peaks eluting at slightly different NaCl concentrations (Fig. 1) is that there are two forms of the enzyme with different charge and not two different enzymes that cleave the chromogenic substrate Cbz-Gly-Gly-Leu-NA. This view is supported by the following observations. 1) Electrophoresis at pH 7.9 of a sample of the pooled fractions from the DEAE-Sepharose CL-GB column shows one sharp protein band with coinciding Cbz-Gly-Gly- Leu-NA activity (Fig. 2). 2) The separate electrophoresis at pH 8.9 of samples from both activity peaks resulted in a single protein band with Cbz-Gly-Gly-Leu-NA activity of identical hobility (not shown). 3) Gel filtration on Sepharose CL-GB of the pooled fractions from DEAE-Sepharose showed a single symmetrical activity peak (Fig. 3). 4) Sodium dodecyl sulfate-

TABLE I Summary of purification of proteinase yscE from Saccharomyces cerevisiae

For details see “Materials and Methods.” Data were taken from one of three purification procedures all leading to similar values.

Purification SbP

Volume Activity Protein Specific Purification Yield

ml mi&iunits ng -fold %

activity milliunitaj mg protein

1. Crude extract 178 1801 8834 0.2 170 1049

1 100 2. 40% ammonium sulfate 4995 0.2 1 58

fractionation superna- tant

chromatography

precipitation

chromatography

precipitation

3. Octyl-Sepharose CL-GB 122 529 484.3 1.1 5 29

4. 70% ammonium sulfate 14.3 599 556.6 1.1 5 33

5. DEAE-Sepharose CL-GB 19.0 306 15.3 20.0 100 17

6. 70% ammonium sulfate 1.3 357 5.6 63.4 317 20

13346

015 c

r X

E In 0.10 C

0 .l

Yeast Proteinase yscE

Front marker

dye

0 0 20 LO 60

Gel length [mml

I 1 FIG. 2. Polyacrylamide gel electrophoresis of pooled frac-

tions of proteinase yscE after ion-exchange Chromatography. Electrophoresis was done according to Hedrick and Smith (24). 20 pg of protein were applied on top of the gel. One gel was stained for protein. A second gel was cut into 2-mm slices and proteinase yscE activity was measured in the standard test.

L

t E l

W

0 L Mr. 10-5

0 0 10 20 30 LO 50 60 70 Bo 90

Froctm nunkr

FIG. 3. Gel filtration and molecular weight determination of proteinase yscE. Gel filtration was done using Sepharose CL- 6B. 0.85 mg of protein of the pooled and ammonium sulfate-concen- trated fractions of the ion-exchange chromatography step exhibiting proteinase yscE activity were applied on top of the column (1 X 100 cm). Buffer was 20 mM Tris containing 100 mM NaCl and 1 mM EDTA, pH 7.2. Flow rate was 4.5 ml/h. Standard proteins had the indicated molecular weights and Stokes radii (28,29): I , rabbit musle aldolase (M, = 158,000, R, = 48.1 A); 2, bovine liver catalase (M, = 232,000, R, = 52.2 A); 3, horse spleen femtin (M, = 44o,ooO, R. = 61.0 A); 4, bovine thyroglobulin (M, = 669,000, R. = 85.0 A). Arrow marks molecular weight of proteinase yscE. 0, proteinase yscE activ- ity; 0, protein.

polyacrylamide gel electrophoresis of samples of both peaks showed identical protein banding patterns (not shown). 5) Both peaks exhibited identical specificities when tested against Cbz-Gly-Gly-Leu-NA, Cbz-Ala-Ala-Leu-NA, and Suc-Phe-Leu-Phe-NA (not shown). Identical sensitivity to- wards the inhibitory agents 4-hydroxymercuribenzoate and chymostatin was found (not shown).

The pH optimum of proteinase yscE was found in the range of 8.2 to 8.6 (Fig. 4). At optimum pH and 30 "C no decrease in enzyme activity was found over a period of 6 h. After 24 h a 35% loss of activity was detectable. Using gel filtration, a Stokes radius of Rs = 75.2 A (S.D. -C 10.3%; mean of three independent determinations) was calculated for the purified enzyme (not shown; a plot of (-log K.J112 versus R. was used to determine the Stokes radius (28,29)). When elution posi-

FIG. 4. Dependence of proteinase yscE activity on pH. 2 milliunita of purified proteinase E were included in the test. Test was done as outlined under "Materials and Methods." Buffers were ad- justed with HCI and their concentration was 20 mM in the test. 0, Tris; 0, 2-amino-2-methyl-1,3-propanediol.

TABLE I1 Effect of inhibitors on proteinclse yscE activity

Activity was determined as described under "Materials and Meth- ods." 0.25 milliunits of purified enzyme were included in the test. Preincubation was done at 30 "C. 16 h preincubation in the presence of N-ethylmaleimide, iodoacetamide, Tos-L-PheCH&I, and TOS-L- LysCH2Cl was done in 5 mM Tris/maleate/NaOH buffer, pH 7, containing 50 mM NaCl. Prior to test the pH was adjusted to 8.2. All inhibitors except Tos-L-PheCH&I (methanol), PhCH2SOzF (2-pro- panol), chymostatin (dimethyl sulfoxide), pepstatin A (dimethyl sulf- oxide), and elastatinal (dimethyl sulfoxide) were solubilized in water. ResDective controls were run in all cases.

Inhibitor Final Preincubation concentration time

None HgC& 0.01 mM 4-Hydmxymercuribenmate 0.01 mM . ZnC1, 1 mM N-Ethylmaleimide 1 mM N-Ethylmaleimide 1 mM Iodoacetamide 200 mM Iodoacetamide 200 mM Tos-L-PheCH2Cl 2 mM Tos-L-L~sCH~C~ 2 mM

1 mM p-Aminobenzamidine 1 mM EDTA 1 mM o-Phenanthroline 1 mM Chymostatin 0.1 mg/ml Antipain 0.1 mg/ml Pepstatin A 0.1 mg/ml Leupeptin 0.1 mg/ml Elastatinal 0.1 mg/ml E64 0.1 mg/ml Phosphoramidon 0.1 mg/ml Captopril 0.1 mg/ml

PhCHZSOzF

h

0.5 0.5 1.0 0.5

0.5 16

16 16 16 0.5 0.5

16 16 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

% of contml 100

9 7 30

104 18 36 3

87 100 98 96

117 129 38 72

110 85 85 95

104 107

tion of proteinase yscE upon gel filtration was correlated with molecular weight, a value of approximately 600,000 (S.D. * 6.7%; mean of three independent determinations) could be calculated (Fig. 3).

Studies on the subunit structure of the enzyme using so- dium dodecyl sulfate-polyacrylamide gel electrophoresis did not give a clear answer. In all experiments a protein band with M, of about 70,000 appeared, which was accompanied by a series of eight to ten protein bands at M, = 35,000 and less (not shown). This banding pattern did not change under different conditions which included pretreatment of the en- zyme with chymostatin, N-ethylmaleimide, EDTA, and when dithiothreitol was omitted during the dissociation process, Hg2+ ions. There are two possible explanations for this find- ing. 1) The enzyme has a complex structure with multiple subunits of the molecular weights observed. 2) The enzyme becomes artifactually nicked during the purification process by some contaminating proteinase or in a self-digestion proc- ess resulting in subunit fragments.

Yeast Proteinase yscE 13347

The effect of various inhibitors on enzyme activity is sum- marized in Table 11. Mercurials like HgClz and p-hydroxy- mercuribenzoate were found to be very potent inhibitors indicating a sulfhydryl group necessary for activity. ZnCL also showed some inhibition. N-Ethylmaleimide and iodoaceta- mide were found to be inhibitory. Whether inhibition by these agents is due to modification of the same su l fhyhl residue(s) blocked by mercurials or whether some other amino acid side chain necessary for activity is attacked has to be elucidated. Metal chelating agents like o-phenanthroline and EDTA, inhibitors of metalloproteinases, did not influence activity. The serine proteinase inhibitor phenylmethanesulfonyl fluo- ride also had no effect. Among the actinomycete inhibitors (30) only chymostatin showed considerable inhibition when applied at a rather high concentration. None of the other inhibitors were effective. Reducing agents like mercaptoeth- anol or dithiothreitol did not influence activity (not shown).

The substrate specificity of the enzyme was tested against 9 chromogenic substrates. With enzyme preparations of greater than 95% purity as checked by polyacrylamide gel electrophoresis, activities against chromogenic endopro- teinase substrates of very different sequences were obtained. These activities varied from preparation to preparation (Table 111). This indicated the presence of proteinase impurities in the proteinase yscE preparations tested. Therefore, a further purification by electrophoresis of the chromatographically isolated proteinase yscE was made. Gels were sliced into 2- mm pieces, each piece was cut in half and one-half of the gel was tested against the proteinase yscE substrate Cbz-Gly- Gly-Leu-NA. The other half of the gel slice was tested with each of the five other chromogenic peptides which were cleaved in all enzyme preparations tested. As can be seen from Table 111 only the activities against the substrates Cbz- Ala-Ala-Leu-NA and Suc-Phe-Leu-Phe-NA co-migrate on the gel with the Cbz-Gly-Gly-Leu-NA hydrolyzing activity. Activity measured against these three substrates did not in- crease upon aminopeptidase M addition to the test, indicating that proteolytic cleavage occurs at the 4-nitroanilide bond and not at some other position of the peptide. The activities against Cbz-Ala-Ala-Leu-NA and Suc-Phe-Pro-Phe-NA were inhibited by p-hydroxymercuribenzoate (Table 111) as was the case with the activity with Cbz-Gly-Gly-Leu-NA. These ob- servations support the notion that hydrolysis of these three

substrates is due to action of one enzyme. Contaminating activities were either not found in all proteinase yscE prepa- rations or they were found at different positions relative to proteinase yscE in the gel (Table 111) and in most cases showed negligible protein stain. Also, a small [3H]methylcas- ein activity was detectable in purified proteinase yscE prep- arations which only partly co-migrated with the Cbz-Gly-Gly- Leu-NA activity during polyacrylamide gel electrophoresis (not shown).

Proteinase yscE did not show any activity against the carboxypeptidase substrates Cbz-Gly-Leu, Cbz-Phe-Leu, Bz- Gly-His-Leu, Cbz-Leu-Phe, Cbz-Leu-Tyr, Cbz-Leu-Leu, Cbz- Phe-Trp, Cbz-Ser-Phe, and Cbz-Ala-Phe. These studies in- dicate an endoproteolytic specificity for proteinase yscE. The primary specificity of the enzyme as determined by the amino acid after which cleavage of the chromogenic substrates occurs seems to depend on the amino acids L-leucine or L-phenylal- anine. The secondary specificity of the enzyme can be seen by comparing the cleavage of Cbz-Ala-Ala-Leu-NA and Cbz- Gly-Gly-Leu-NA. Cbz-Gly-Gly-Leu-NA undergoes hydrolysis at a considerable higher rate (Table 111) indicating a more effective interaction of this substrate with.the enzyme. All three sustrates have a limited solubility and precipitated from the incubation mixture at concentrations of 0.5 mM or higher. This phenomenon prevented the determination of the Mi- chaelis constant for these substrates. At the substrate concen- trations which could be attained a Lineweaver and Burk plot of the kinetic data was nonlinear, which probably indicates substrate activation of the enzyme.

Proteinase yscE undergoes very little changes in activity during different vegetative growth conditions. Activity in stationary phase YPD-grown cells, YPEthanol-grown cells or logarithmic phase cells in mineral medium containing L: leucine as sole nitrogen source is about 1.5-fold increased over activity found in logarithmically growing cells on YPD. Under the differentiation conditions of sporulation, enzyme activity was found to be increased about 2-fold over cells grown logarithmically on YPD (not shown).

DISCUSSION

A new proteolytic enzyme of the yeast S. cerevkiae was purified. When using the current nomenclature for yeast proteinases the new enzyme should be named with the letter

TABLE I11 Substrate specificity of proteinuse yscE

In one type of experiment two different preparations of purified proteinase yscE (step 6 of the purification procedure) were tested against the substrates indicated. In another type of experiment electrophoresis of purified proteinase yscE (20 pg) (step 6 of the purification procedure) was carried out on polyacrylamide gels (4%) at pH 8.9. Gels were sliced into 2-mm pieces. Each piece was cut in half. In each case, one-half of the piece was tested against the standard substrate Cbz-Gly-Gly-Leu-NA, the other half was tested against the substrate in question. Inhibition of enzyme activity was tested with p-hydroxymercuribenzoate (0.5 mM). Two different experiments were carried out.

Substrate tested Preparation1 Preparation11 Co-migration in

specific specific electrophore- Residual

activity activity sis activity

P5 PI Pa P2 PI milliunitsfmgprotein % of control Bz-Phe-Val-Arg-NA 3 . 9 9 . 9 No N D n

Suc-Ala-Ala-Val-NA 0 ND ND ND Suc-Tyr-Leu-Val-NA ND 6 . 0 No ND

.MeO-Suc-Ala-Ala-Pro-Met-NA 0 ND 0 . 3

ND Suc-Ala-Ala-Pro-Phe-NA a ND

0 . 4 ND

Suc-Phe-Pro-Phe-NA 0 . 2 No 1 4 . 6

ND Suc-Phe-Leu-Phe-NA 9 5 . 8 Yes

8 . 2 4

Cbz-Ala-Ala-Leu-NA 2 1 . 8

10 .1 Yes 0 Cbz-Gly-Gly-Leu-NA 9 5 . 1 Yes 5

0 . 4

'ND, not determined.

13348 Yeast Proteinase yscE

E (for proteinase A, B, and C, see Ref. 3 (review), for protein- ase D, see Ref. 31). However, the nomenclature used for the yeast enzymes has also been used for proteinases of other microorganisms often without paying attention to the fact that enzymes with the same name mostly do not share com- mon characteristics (for review and references, see Refs. 3 and 4). To easily distinguish the yeast proteinases from pro- teinases with identical names from other microorganisms we therefore add the first letters of the organism (yeast), the genus (Saccharomyces), and the species (cerevisiae) to the name of the proteinase. Following such a nomenclature, the new enzyme is named proteinase yscE. The enzyme appears to have an unusually high apparent M, of about 600,000. Among the characterized peptidases of S. cereuisiae only aminopeptidase I has a similar molecular weight (32). The ionic strength of the solution seems to influence the confor- mation of the enzyme as can be inferred from its different binding properties on DEAE-Sepharose CL-GB at pH 7.2. Whereas high-ionic strength leads to complete binding of the enzyme, low-ionic strength prevents binding. Proteinase yscE may be composed of subunits. However, no unequivocal as- signment about the subunit composition can be made. The electrophoretic analysis of highly purified enzyme prepara- tions suggests that conclusions about purity and specificity of any proteolytic enzyme must be made with caution. Varying amounts of proteolytic impurities with different substrate specificities, which were nearly undetectable by Coomassie Blue staining of gels, could be separated from purified pro- teinase yscE in different preparations by polyacrylamide gel- electrophoresis. This result is not at all surprising when one considers the large number of proteinases that can be found in the yeast cell (19). The primary specificity of the enzyme seems to be mainly directed toward peptide bonds in which the carboxyl group is provided by the hydrophobic amino acid t-leucine. L-Phenylalanine at this position also seems to allow hydrolysis. In addition to this primary specificity, the nature of substituents in positions P2, Pa, and Pq (33) greatly influ- ences the specificity. This can be seen from the differences in activity against the two substrates Cbz-Ala-Ala-Leu-NA and Cbz-Gly-Gly-Leu-NA. The strong inhibition of the enzyme by mercurials suggests that thiol residue(s) may be involved in catalysis. However, a conformational change of the enzyme leading to loss of activity brought about by modification of a thiol residue distant from the active site cannot be excluded. The inhibition of the enzyme by iodoacetamide and N-ethyl- maleimide may be due to a blockage of the same reactive thiol group(s); however, modification of some other essential resi- due cannot be excluded (34, 35). A proteinase cleaving Cbz- Gly-Gly-Leu-NA at the PI position which has a similar mo- lecular weight to proteinase yscE and which is inhibited by mercurials as well has been purified recently from bovine pituitaries (36, 37). In contrast to the yeast enzyme the pituitary enzyme seems to contain two distinct additional proteinase specificities (35).

Proteinase yscE shows very little regulation during different growth phases of cells, probably indicating an indispensible activity for the yeast life cycle. The fact that proteinase yscE is not located in the vacuole (38), the lysosome-like organelle of the yeast cell (39), may suggest a more specific function for proteinase yscE, as has been found for the highly unspecific vacuole-located endoproteinases A and B (for review, see Ref. 5 ) . A final answer about proteinase yscE function requires

further biochemical and most likely genetic studies.

Acknowledgments-We thank Dr. J. Phillips for critical reading of the manuscript and H. Gottschalk and W. Fritz for expert help during preparation of the manuscript.

REFERENCES 1. Reich, E., Rifkin, D. B., and Shaw, E. (eds) (1975) Proteases and

Biological Control, pp. 1-987, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

2. Steiner, D. F., Quinn, P. S., Chan, S. J., Marsh, J., and Tager, H. S. (1980) Ann. N. Y. Acad. Sci. 3 4 3 , 1-16

3. Wolf, D. H. (1980) Adu. Microb. Physiol. 21, 267-338 4. North, J. M. (1982) Microbiol. Reu. 4 6 , 308-340 5. Wolf, D. H. (1982) Trends Biochem. Sci. 7,35-37 6. Wolf, D. H., and Ehmann, C. (1979) Eur. J. Biochem. 98 , 375-

7. Zubenko, G. S., and Jones, E. W. (1981) Genetics 97, 45-64 8. Mechler, B., and Wolf, D. H. (1981) Eur. J. Biochem. 121,47-

9. Neupert, W., and Schatz, G. (1981) Trends Biochem. Sci. 6 , 1-4 10. Hasilik, A., and Tanner, W. (1978) Eur. J. Biochem. 85, 599-

608 11. Mechler, B., Muller, M., Muller, H., Meussdoerffer, F., and Wolf,

D. H. (1982) J. Biol. Chem. 267 , 11203-11206 12. Ciejek, E., and Thorner, J. (1979) Cell 18, 623-635 13. Kurjan, J., and Herskowitz, I. (1982) Cell 30,933-943 14. Emter, O., Mechler, B., Achstetter, T., Miiller, M., and Wolf, D.

H. (1983) Biochem. Biophys. Res. Commun. 116,822-829 15. Achstetter, T., Ehmann, C., and Wolf, D. H. (1981) Arch.

Biochem. Biophys. 207,445-454 16. Wolf, D. H., and Ehmann, C. (1981) J. Bacteriol. 147, 418-426 17. Achstetter, T., Ehmann, C., and Wolf, D. H. (1982) Biochem.

18. Achstetter, T., Ehmann, C., and Wolf, D. H. (1983) Arch.

19. Achstetter, T., Emter, O., Ehmann, C., and Wolf, D. H. (1984) J.

20. Jentoft, N., and Dearborn, D. G. (1979) J. Bwl. Chem. 264,

21. Wolf, D. H., and Weiser, U. (1977) Eur. J. Biochem. 73,553-556 22. Swamy, K. H. S., and Goldberg, A. L. (1981) Nature ( L o n d . ) 292,

23. Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 112,404-427 24. Hedrick, J. L., and Smith, A. J. (1968) Arch. Biochem. Biophys.

25. Laemmli, U. K. (1970) Nature (Lond.) 227,680-685 26. Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244,4406-4412 27. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.

28. Siegel, L. M., and Monty, K. J. (1966) Biochim. Biophys. Acta

29. Gel Filtration Calibration Kit Instruction Manual, Pharmacia Fine Chemicals AB, Uppsala, Sweden

30. Aoyagi, T., and Umezawa, H. (1975) in Proteases and Biological Control (Reich, E., Rifkin, D. B., and Shaw, E., eds) pp. 429- 454, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

31. Achstetter, T., Emter, 0.. Ehmann, C., and Wolf, D. H. (1982) Hoppe-Seyler’s 2. Physiol. Chem. 363,976-977

32. Frey, J., and Rohm, K.-H. (1978) Biochim. Biophys. Acta 627 , 31-41

33. Nakajima, K., Powers, J . C., Ashe, B. M., and Zimmerman, M. (1979) J. Biol. Chem. 254,4027-4032

34. Gurd, F. R. N. (1967) Methods Enzymol. 11,532-541 35. Riordan, J. F., and Vallee, B. L. (1967) Methods Enzymol. 11,

36. Wilk, S., Pearce, S., and Orlowski, M. (1979) Life Sci. 2 4 , 457-

37. Orlowski, M., and Wilk, S. (1981) Biochem. Biophys. Res. Com-

38. Emter, O., and Wolf, D. H. (1984) FEBS Lett. 166,321-325 39. Matile, Ph., and Wiemken, A. (1967) Arch. Mikrobiol. 66, 148-

384

52

Biophys. Res. Commun. 109,341-347

Biochem. Biophys. 2 2 6 , 292-305

Biol. Chem. 259 , 13334-13343

4359-4365

652-654

126,155-164

(1951) J. Bid. Chem. 193 , 265-275

112,346-362

541-548

464

mun. 101,814-822

155