[31] Vacuolar proteases in yeast Saccharomyces cerevisiae

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Finally, we have analyzed the termination region of 131 yeast genes. In essence, there is no clear consensus that emerges that would suggest that a specific sequence element is required for termination of translation in yeast genes other than a termination codon of which there is no particular preference for UAA, UAG, or UGA at this position. [31] Vacuolar Proteases

i n Y e a s t Saccharomyces

cerevisiae By ELIZABETH W. JONES

Interest in vacuolar proteases of the yeast Saccharomycescerevisiae, for those involved in gene expression technology, takes the form of trying to eliminate the proteases and being sure that one has done so. The former task is accomplished genetically; the latter through a combination of genetic and biochemical tests. The complement of proteases found in the lumen of the vacuole includes two endoproteinases, protease A (PrA) and protease B (PrB); two carboxypeptidases, carboxypeptidase Y (CpY) and carboxypeptidase S (CpS); and two aminopeptidases, the 600K aminopeptidase I (API) and the Co2+-dependent aminopeptidase.l.2 Most of the genetic analysis has concentrated on the two endoproteinases, PrA and PrB, and the two carboxypeptidases, CpY and CpS. Plate tests or microtiter well tests have been developed that allow one to test directly for activity of PrB, CpY, and CpS and indirectly for activity of PrA in colonies. In this article, I first present the plate and well tests for assaying protease activities of colonies, followed by assays that allow quantitation of activity levels in cell-free extracts, and end with guidelines for designing useful protease-deficient strains and a list of strains that we have sent to the Yeast Genetics Stock Center (MCB/Biophysics and Cell Physiology, University of California, Berkeley, CA 94720). Genetic Analyses

Requirements of Tests Several requirements must be met if one is to succeed in assaying activities of particular intraceUular enzymes in colonies. The first condition is that the enzyme gain access to the externally supplied substrate. i E. W. Jones, Annu. Rev. Genet. 18, 233 (1984). 2 T. Achstetter a n d D. H. Wolf, Yeast 1, 139 (1985).

METHODS IN ENZYMOLOGY, VOL. 185

Copyright© 1990by AcademicPress, Inc. All rightsof reproduction in any form reserved.

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This is accomplished either by permeabilizing the cells in a colony with a solvent or by establishing conditions that result in lysis of cells. The second requirement is that, where lysis is employed, conditions be established that free the enzyme from its naturally occurring, intracellular, polypeptide inhibitor) ,2 This is accomplished, for PrB, by including sodium lauryl sulfate (SDS) in the overlay. The third requirement is to find a substrate or condition that tests for one enzyme activity only. How this is accomplished for each enzyme will be given in the procedure for that enzyme.

Protease B Protease B activity in colonies can be assayed using an overlay test. Initially, we developed a procedure for cells grown on YEPD plates [20 g Difco (Detroit, MI) Bacto-peptone, 10 g Difco yeast extract, 20 g dextrose, 13-20 g agar, according to brand, per liter] that necessitated use of a lysis mutation to cause release of intracellular PrB from cells. We have since realized that growth of cells on YEPG plates (20 g Difco Bacto-peptone, 10 g Difco yeast extract, 50 g glycerol, 13-20 g agar, according to brand, per liter) obviates use of a lysis mutation, since some lysis occurs when cells are grown on this medium. The substrate is particulate Hide Powder Azure (HPA), for PrB is the only protease in S. cerevisiae that catalyzes cleavage and solubilization of this substrate. 3 HPA Overlay Test for PrB Activity. 4,5 Principle. Protease B, which is freed from cells by lysis and from its inhibitor by the SDS present in the overlay, solubilizes the particles of Hide Powder Azure in the overlay, uncovering the colony and surrounding it with a clear halo. Mutant colonies remain covered.

Reagents Sodium lauryl sulfate (SDS): 20% (w/v) in 0.1 M Tris-HC1 pH 7.6 Cycloheximide: Sterile solution at 5 mg/ml Penicillin G-streptomycin: Use a sterile solution containing 5000 Units/ml penicillin base and 5000 #g/ml streptomycin base 0.6% agar, molten, held at 50* Hide Powder Azure (Calbiochem, San Diego, CA): Hide Powder Azure is pulverized by homogenization (VirTis homogenizer) of 200 ml of a slurry (100 mg/ml 95% ethanol) in a 500-ml flask for 3 R. E. Ulane and E. Cabib, ,L Biol. Chem. 249, 3418 (1974). 4 G. S. Zubenko, A. P. Mitchell, and E. W. Jones, Proc. Natl. Acad. Sci. U.S.A. 76, 2395 (1979). s C. M. Moehle, M. W. Aynardi, M. R. Kolodny, F. J. Park, and E. W. Jones, Genetics 115, 255 (1987).

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5 min at 40,000 rpm or by sonication of a slurry of the same proportions. Aliquots containing 50-100 mg are transferred to sterile 13 × 100 m m tubes, centrifuged (5 min at 1650 g), and the supernatants are discarded. The pellets are washed with 2.5 - 3 ml of sterile water, repelleted, and the supernatants discarded.

Procedure. Add 0.2 ml of cycloheximide solution, 0.2 ml of penicillinstreptomycin solution, and 0.1 ml of 20% SDS to a HPA pellet, vortex, then add 4 ml of molten agar. Vortex to mix and resuspend particles. Streaks or replica plates of cells grown for 2 days at 30 ° on YEPG agar are overlaid with the molten agar cocktail, with pouring along the length of the stripes rather than across. After the agar solidifies, the plates are incubated at 34-36 ° for 8 hr to 2 days, depending on the properties of the strains. (Some strains lyse well and are difficult to score at later times.) Plates can (and should) be incubated upside down, so long as the medium will absorb the moisture in the overlay. It is important that a seal does not form between the lid and base of the petri dish. Utility. This test works very well for following mutations in the PrB structural gene PRB1, and less well for pleiotropic mutations like the pep mutations. In using prbl mutations or in constructing strains, be aware that, although prbl homozygotes sporulate, the asci may be very small (the size of a normal spore) and that superimposition of heterozygosity for pep4 in such prbl homozygotes may prevent sporulation. 6 Carboxypeptidase Y Carboxypeptidase Y activity in colonies can be assessed by using an overlay test that relies on the esterolytic activity of the enzyme. The substrate is N-acetyl-oL-phenylalanine fl-naphthyl ester (APE), whose cleavage, in colonies anyway, is catalyzed only by CpY. APE Overlay Test for CpY Activity. 7 Principle. Dimethylformamide present in the initial overlay permeabilizes cells on the surface of colonies. CpY within cells catalyzes cleavage of the ester. The product fl-naphthol reacts nonenzymatically with the diazonium salt Fast Garnet GBC to give an insoluble red dye; Cpy + colonies are red; Cpy- colonies are yellow or pink.

Reagents N-Acetyl-oL-phenylalanine fl-naphthyl ester (Sigma, St. Louis, MO): Make a solution of 1 mggml dimethylformamide 0.6% agar, molten, held at 50 ° 6 G. S. Zubenko and E. W. Jones, Genetics 97, 45 (1981). 7 E. W. Jones, Genetics 85, 23 (1977).

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VACUOLAR PROTEASES IN YEAST S. c e r e v i s i a e

375

Fast Garnet GBC (Sigma) 0.1 MTris-HC1, pH 7.3-7.5

Procedure. Replica plate strains to thick YEPD plates (40-45 ml/100-mm plate). Grow for 3 days at 30 °. To form the overlay mix, add 2.5 ml of the ester solution to 4 ml of molten agar in a 13 × 100 mm tube. Vortex or cover with Parafilm and invert 3 or 4 times until the schlieren pattern disappears. After the bubbles exit, pour the contents over the surface of colonies or stripes (along, not across, stripes; colonies must be covered). After 10 min (or after the agar is hard), carefully flood the surface of the agar with 4 . 5 - 5 ml of a solution of Fast Garnet GBC (5 mg/ml 0.1 M Tris-HC1, pH 7.3-7.5). Do not tear the agar overlay during the flooding. The Fast Garnet GBC solution must be made immediately before use, for diazonium salts are very unstable in solution. We use Fast Garnet GBC from Sigma and store it in the freezer. Watch the color develop and pour offthe fluid when Cpy + colonies turn red (several to a few minutes). If color development takes longer than 5 - 10 min, replace your bottle of Fast Garnet GBC. Do the test at room temperature. The color is not stable, but is more stable if the plates are placed in the cold in the dark. Utility. The APE test has wide utility for following many mutations that reduce protease activity so long as CpY activity is among the activities reduced as a consequence of the mutation. The APE test can be used for following mutations in the CpY structural gene PRC1, and for following the pleiotropic pep mutations, including pep4-3. 7 If an adel or ade2 mutation is segregating in the cross, the red pigmentation problem can be circumvented by growing cells on YEPG or on YEPD supplemented with 100/tg/ml adenine sulfate. Petites give aberrant phenotypes in the test (but see well test below). PEP4 is the structural gene for the PrA precursor, s,9 PrA activity is essential for proper maturation of several vacuolar hydrolases, including PrB, CpY, one or more RNase species, the 600K vacuolar aminopeptidase API, and the repressible species of alkaline phosphatase, t°-12 We have cloned and sequenced several of the pleiotropic pep4 mutations (including

8 C. A. Woolford, L. B. Daniels, F. J. Park, E. W. Jones, J. N. Van Arsdell, and M. A. lnnis, MoL Cell. Biol. 6, 2500 (1986). 9 G. Ammerer, C. Hunter, J. Rothman, G. Saari, L. Vails, and T. Stevens, MoL Cell. Biol. 6, 2490 (1986). L0B. A. Hemmings, G. S. Zubenko, A. Hasilik, and E. W. Jones, Proc. NatL Acad. Sci. U.S.A. 78, 435 (1981). H E. W. Jones, G. S. Zubenko, and R. R. Parker, Genetics 102, 665 (1982). 12 B. Mechler, M. M011er, H. Miiller, and D. H. Wolf, Biochem. Biophys. Res. Commun. 107, 770 (1982).

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pep4-3) as well as the allelic pra mutations '3 that are not fully pleiotropic.'4 Pleiotropic pep4 mutations like pep4-3 usually prove to be nonsense mutations that totally eliminate PrA activity and greatly reduce levels of all hydrolase activities, including CpY, in whose maturation PrA participates.~3 For this reason, one can follow many mutations that eliminate PrA activity (the pleiotropic pep4 mutations) by means of this APE test. Indeed, the more devastating the effect of the pep4 mutation on PrA activity, the easier it is to follow by the APE test. And, of course, utilization of pep4 mutations, because of their effects on hydrolase maturation, results in reduction or elimination of several protease activities simultaneously, including PrA, PrB, CpY, and API (but not CpS; the Co2+-dependent aminopeptidase was not tested) as well as RNase activity. ~ In working with pleiotropic pep4 mutations (in crosses or in constructing alleles), be aware that pep4 homozygotes do not sporulate6 and that the mutations show phenotypic lag) 5 To circumvent phenotypic lag, streak spore clones for single colonies on YEPD plates (quadrants suffice). Do the APE test. Stab negative colonies through the agar overlay and make a new master plate. Then carry out the usual analyses for other markers. If this procedure is followed, the overlays should be made with sterile solutions and the colonies should be stabbed soon after the test, for the cocktail will kill cells. Well Test for CpY Activity. s Principle. Dimethylformamide present in the solution permeabilizes cells. Cleavage of the amide bond in N-benzoylL-tyrosine p-nitroanilide (BTPNA) is catalyzed only by CpY to give the yellow product p-nitroaniline. This test works for petites as well as for grandes, and is unaffected by adel and ade2 mutations.

Reagents N-Benzoyl-L-tyrosine p-nitroanilide (Sigma): Make a solution of 2.5 mg/ml dimethylformamide 0.1 M Tris-HC1, pH 7.5: Use a sterile solution

Procedure. Mix 4 volumes of buffer to l volume BTPNA solution. Distribute 0.2 ml into wells in a 96-well microtiter test plate. Cells are transferred into the solution by rotating an applicator stick in the fluid after dipping the sterile stick into a colony grown on a YEPD plate. (Alterna13 E. W. Jones, C. A. Woolford, C. M. Moehle, J. A. Noble, and M. A. Innis, in "Cellular Proteases and Control Mechanisms" (Proceedings UCLA Symposium), p. 141. Alan R. Liss, New York, 1989. t4 E. W. Jones, G. S. Zubenko, R. R. Parker, B. A. Hemmings, and A. Hasilik, in "Molecular Genetics in Yeast" (D. von Wettstein, J. Friis, M. Kielland-Brandt, and A. Stenderup, eds.), p. 182 (Alfred Benzon Symposium, Vol. 16). Munksgaard, Copenhagen, 1981. t5 G. S. Zubenko, F. J. Park, and E. W. Jones, Genetics 102, 679 (1982).

VACUOLAR PROTEASESIN YEAST S. cerevisiae

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377

tively, a 48-prong replicator can be used, taking care to adjust individual wells for colonies that may not transfer effectively with the technique.) Cover the wells and incubate overnight at 34-37*. Cpy ÷ cells give yellow fluid in the wells; Cpy- cells produce no color. Utility. This test can be used to follow prcl mutations as well as pleiotropic pep mutations, like pep4, that result in CpY deficiency.

Carboxypeptidase S Carboxypeptidase S activity in colonies can be assessed by using a well test that incorporates a coupled assay that detects release of free leucine from the blocked dipeptide carbobenzoxyglycyl-L-leucine (Cbz-Gly-Leu). Carboxypeptidase Y, which also catalyzes this cleavage, is inactivated by preincubation with phenylmethylsulfonyl fluoride (PMSF), which reacts covalently to inactivate serine proteases like CpY. Principle. The principle of the coupled assay, devised by Lewis and Harris 16 and adapted by Wolf and Weiser, 17 is shown below. N-Cbz-Gly-Leu + H20

carboxYlaeptidase

, Leucine L-amino-acidoxidase Leucine + 02 , Keto acid + NH 3 + H202 peroxidase H202 + o-dianisidine , Oxidized dianisidine

Oxidized dianisidine is dark brown in color. The amino acid generated must be a substrate for the L-amino-acid oxidase employed. Typically, snake venoms serve as sources. For Crotalus adamanteus (Eastern Diamondback rattlesnake) venom, leucine, isoleucine, phenylalanine, tyrosine, methionine, and tryptophan are good substrates; arginine, valine, and histidine are poor substrates and the other amino acids are not oxidized. We have successfully used venoms from Crotalus atrox (Western Diamondback rattlesnake) and Bothrops atrox (a viper). Cps ÷ colonies give brown fluid in the wells; Cps- cells do not. Cells are permeabilized and CpY is inactivated by preincubation of cells in a solution containing Triton X-100 and PMSF.

Reagents 0.1% (v/v) Triton X-100 made 1 mg/ml in phenylmethylsulfonyl fluoride 0.2 M KPO4, pH 7.0 50 m M MnC12 N-Cbz-Gly-Leu Horseradish peroxidase, type I (Sigma) 16 W. H. P. Lewis and H. Harris, Nature (London) 215, 351 (1967). 17 D. H. Wolf and U. Weiser, Eur. J. Biochem. 73, 553 (1977).

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L-Amino-acid oxidase, type VI (Sigma; actually crude dried v e n o m f r o m Crotalus atrox) or type II (Sigma; dried v e n o m f r o m Bothrops

atrox) o-Dianisidine dihydrochloride

Procedure. Cells are transferred into 50/zl/well o f the Triton X-100/ P M S F solution in a 96-well microtiter test plate by applicator stick or m u l t i p r o n g replicator (see C p Y procedure) after growth on a Y E P D plate. Cover a n d let sit for 2 hr at r o o m temperature. Add to each well 150/zl o f the substrate mix m a d e in the proportions: 1 ml o f buffer, 0.01 m l o f MnC12, 3.22 m g o f Cbz-Gly-Leu (final concentration, l0 m M ) , 0.2 m g o f peroxidase, 0.4 m g o f a m i n o - a c i d oxidase type VI or 1.2 m g o f type II, 0.4 m g o f dianisidine dihydrochloride. Cover and incubate at 37* for 1718 hr. ( W e have not investigated other concentrations o f v e n o m s b u t k n o w that these work.) An example o f this test for 20 tetrads f r o m a cross is shown in Fig. 1. One parent o f this cross c a r d e d m u t a t i o n s in PRC1, the structural gene for CpY, and DUT1, a gene required for production o f CpS activity. T h e prcl-407 dutl-1 strain was isolated based on its inability to use Cbz-Gly-

ii~ii~Ti!! !i!iiiii!!ii~iii~ii!i~ii!!iiii!ii:ilqi~iii!ii3il L~ ? /

13iiii~i ¸ i3ii !t3iSi~==ii~ixli

::iili

;

i xdi!S!

i

~:x

= ~ U ~ % t

~-~¸

FIG. I. Scoring for the presence or absence of carboxypeptidase S (CpS) activity in meiotic segregants using the well test for CpS activity. Parents of the cross were of genotype a leu2 trpl prcl-407 (well 9E) and a ura3-52 leu2 prcl-407 dutl-1 (well 9F). The dutl-1 mutation results in failure to produce CpS activity. Spores of a given tetrad are in columns: 1A-D, 2A-D, 1E-H, 2E-H, etc. The scoring for CpS activity for tetrads 1 and 2 is, respectively, - + - + and + - +. Blank wells such as 3C correspond to segregantsthat did not grow. Well 9H contains the reaction mixture but no cells. The Cps+ phenotype and ability to use Cbz-Gly-Leu as a nitrogen source cosegregated in all 20 tetrads.

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VACUOLAR PROTEASES IN YEAST S. cerevisiae

379

Leu as a nitrogen source. ~s (Either CpY or CpS activity alone is sufficient to allow utilization of the dipeptide as a nitrogen source.) The diploid was homozygous for prcl-407 and heterozygous for dutl-1. In this cross, segregants possessing CpS activity should retain the ability to use Cbz-Gly-Leu as a nitrogen source and give oxidation of o-dianisidine; those lacking CpS activity should do neither. The two characteristics cosegregated in all tetrads, as expected. Utility. This test appears to work well for following mutations that result in CpS deficiency, whether or not the strain lacks CpY activity. We have used it particularly to follow dutl-1,19 a mutation we suspect to be allelic to cpsl.~S

Protease A We have found no general plate test to directly assess PrA activity that is satisfactory, although preliminary tests indicate that it may be possible to adapt the fluorescence-based assay given in the Biochemistry section of this article for a well test. (A plate test that necessitates that strains carry a lysis mutation has been described.2° However, since total loss of function for PrA results in failure to activate a set of vacuolar hydrolase precursors, including that of CpY, many mutations in the PrA structural gene PEP4 can be followed using the indirect test (APE test, see section Carboxypeptidase Y above) that detects the esterolytic activity of CpY, the processing and activation of which is dependent on PrA. This latter test is satisfactory for following the pleiotropic pep4 mutations (Pra- Prb- Cpy- . . .) that are of most utility in biotechnological applications. Biochemical Analyses

Growth of Cells and Preparation of Extracts For most of our assays of cell-free extracts for protease activities we employ extracts prepared according to the following protocol. Cells are grown to stationary phase in YEPD at 30* with vigorous shaking (usually 48-52 hr for our conditions of inoculation), harvested by centrifugation, washed once with distilled water and resuspended in 2 ml of 0.1 M TrisHC1, pH 7.6, per gram of cells. The cells are broken (3 min) with 0.45-mm glass beads [40: 60 to 50: 50 (v/v) glass beads: cell suspension] in a Braun homogenizer (Braun-Melsungen, West Germany) without CO2 cooling. ,8 D. H. Wolf and C. Ehmann, J. Bacteriol. 147, 418 (1981). ~9G. S. Zubenko, Ph.D. Thesis, Carnegie Mellon University, Pittsburgh, Pennsylvania, 198 I. 2o B. Mechler and D. H. Wolf, Eur. J. Biochem. 121, 47 (1981).

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After centrifugation for 30 min at 35,000 g in the cold, the supernatant is removed to a fresh tube on ice. Enzymatic Assays for Protease A Two assays for protease A activity will be described, the first based on release ofpeptides from hemoglobin, the second a fluorescence assay based on cleavage of a peptide. Principle. The most commonly used assay for PrA activity measures the release of tyrosine-containing acid-soluble peptides from acid-denatured hemoglobin. Protease A is apparently the only protease to catalyze the reaction at acid pH. The procedure is based on that of Lenney et al. 2' Reagents 2% (w/v) acid-denatured hemoglobin: Dissolve 2.5 g of hemoglobin (Sigma) in 100 ml of distilled water, and dialyze against three changes of 3 liters of water in the cold. Bring the pH to 1.8 with 1 N HCI. After 1 hr of incubation with stirring at 35", bring the pH to 3.2 with 1 M N a O H and adjust the volume to 125 ml. Aliquots can be stored frozen for years 0.2 M glycine-HC1, pH 3.2 1 N perchloric acid 0.5 M NaOH 2% (w/v) Na2CO3 in 0.1 M NaOH 1% (w/v) CuSO4.5H20 2% (w/v) Sodium or potassium tartrate Folin and Ciocalteu's phenol reagent (diluted 1 : 1 with water) Procedure. The reaction mixture consists of 2 ml of a hemoglobin solution (prepared by mixing equal volumes of the 2% hemoglobin, pH 3.2, described above and 0.2 M glycine, pH 3.2) and 0.1 ml of cell-free extract ( 1 - 2 mg protein/incubation), with incubation at 37*. At 0, 15, and 30 min, 0.4-ml samples are removed to 0.2 ml of 1 Nperchloric acid on ice and the tubes are shaken briefly. After centrifugation at 1650 g for 5 min, 0.1 ml of each sample is removed to 0.1 ml 0.5 M NaOH. Tyrosine-containing peptides in the neutralized 0.2-ml sample are determined with the Folin reagent according to Lowry et al. 22 To each 0.2-ml sample add 1 ml of a reagent consisting of 20/0 Na2CO3 in 0.1 M NaOH, 1% CuSO4.5H20, 2% sodium or potassium tartrate in the proportion of 100:1 : 1 (mix just before use). Incubate at room temperature for at least 10 min. Add 0.1 ml 2, j. Lenney, P. Matil¢, A. Wiemken, M. Sch¢llenbcrg, and J. Meyer, Biochem. Biophys. Res. Commun. 60, 1378 (1974). 22 O. Lowry, N. Rosebrough, A. Farr, and R. Randall, J. Biol. Chem. 193, 265 (1951).

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of the diluted phenol reagent and vortex immediately. After 30 min, determine absorbance at 750 nm. Definition of Unit and Specific Activity. One unit corresponds to l #g of tyrosine per minute. The A75o of 1/tg of tyrosine is 0.058 in the Lowry assay performed as described. Using the change in absorbance for a 30-min incubation, the conversion to /~g Tyr/min/mg protein is made by the following calculation: AA75o 1 2.1 6 1 30 × ~ × 0-~ × 4 × 0.1 (mg protein/ml extract) = ( 181)(AA75o)/(mg protein/ml extract) An abbreviated PrA assay can be used for segregants of crosses known to be segregating a pra mutation, where distinction between + and - is all that is sought. It can also be used for mutant screens if a 0 point is added. Extract (0.05 ml) is added to 1 ml of the hemoglobin solution (equal volumes of 2% hemoglobin and 0.2 Mglycine, pH 3.2). Incubate at 37* for 30 min. Remove a sample to PCA and work up as described above. An alternative, much more sensitive assay for PrA is available. We find it more satisfactory than the hemoglobin assay with respect to linearity, reproducibility, etc., but have much less experience with it. It was developed for assaying renin 23 but has been used for assaying PrA. 24 We have adapted it for use in crude extracts. Principle. Protease A will catalyze cleavage at the Leu-Val bond of the octapeptide N-succinyl-L-arginyl-L-prolyl-L-phenylalanyl-L-histidyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine 7-amido-4-methylcoumarin. After removal of the valine and tyrosine residues by aminopeptidase M, the fluorescence of 7-amino-4-methylcoumarin can be determined at 460 nm after excitation at 380 nm. PMSF is included to react covalently with and inactivate PrB, a serine protease that could also catalyze cleavage of the peptide.

Reagents McIlvaine's buffer: 0.2 M Na2HPO4, 0.1 M citric acid. Adjust pH to 6.0 with NaOH or HC1 N-Succinyl-L-arginyl-L-prolyl-L-phenylalanyl-L-histidyl-L-leucyl-Lleucyl-L-valyl-L-tyrosine 7-amido-4-methylcoumarin (Sigma; MW 1300): Make a solution of 0.325 mg/ml dimethylformamide (0.25 mM) 7-Amino-4-methylcoumarin (Sigma; MW 175): Make a 0.4 mg/ml solution. 23 K. Murakami, T. Ohsawa, S. Hirose, K. Takada, and S. Sakakibara, AnaL Biochem. 110, 232 (1981). 24 H. Yokosawa, H. Ito, S. Murata, and S.-I. Ishii, Anal. Biochem. 134, 210 (1983).

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Serially dilute this 1/100 × 1/40 (1/4000) to make a stock solution for the standard curve PMSF (Sigma): 0.2 M in 95% ethanol (34.8 mg/ml) Aminopeptidase M (Sigma, L-0632): 0.5 mg/ml in McIlvaine's buffer. (We have not explored whether cheaper and apparently more active preparations like L-9876 or L-1503 will work, as they should, since the original protocol for renin calls for only 50 mU. 23)

Procedure. Mix 90 or 85/~1 of McIlvaine's buffer, pH 6.0, l0 #1 of peptide solution, and 10 or 15 ]zl of crude extract (pretreat 1 ml of extract with 5/~1 of 0.2 M PMSF for 2 hr at room temperature). Incubate for 15 min at room temperature. Immerse the tube in boiling water for 5 min to stop the reaction. Cool. Add 5 ]zl of aminopeptidase M. After a 90-min incubation at room temperature, add 1.61 ml of buffer (to dilute the reaction 15-fold) and centrifuge the tubes at 1650 g for 5 min. Remove the supernatant and determine the fluorescence in a fluorimeter, with excitation at 380 nm and emission at 460 nm. A standard curve is constructed for 7-amino-4-methylcoumarin. The diluted stock (0.1 ag/ml) is mixed with buffer as given below and the fluorescence is determined. Stock (gl)

Buffer (ml)

Total nanomoles

350 175 88 44 22 11

1.375 1.550 1.637 1.681 1.703 1.714

0.2 0.1 0.05 0.025 0.0125 0.00625

For experimental samples, read nanomoles in the sample from the standard curve (the volumes for the standard and the experimental sample are the same). The samples and standards must be read in exactly the same way (same slit width, etc.). Prepare the standard curve the same day. Definition of Unit and Specific Activity. We have been using a unit to equal l nmol/min and the specific activity to be units/milligram protein. Crude extracts for this assay procedure are made as follows. The volume of cells sampled is 2500 ml/Klett. Pellet the cells, wash with water, and freeze the pellet. Resuspend the pellet in 1.5 ml of 0.1 M Tris-HCl, pH 7.6, and transfer to a small Braun homogenizer tube (40-50% full of glass beads). Add buffer to the top (fill tube completely) to prevent foaming. Homogenize for 3 min at room temperature. Transfer to a long centrifuge tube and centrifuge for 20 min at 25,000 g in the cold. Transfer the supernatant to a 1.5-ml microfuge tube. (The small homogenizer tubes are about 35 m m high and are cut down from 12 X 75 m m tubes.)

VACUOLAR PROTEASES IN YEAST S. cerevisiae

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383

We have preliminary evidence that suggests that this assay can be adapted for use in a well test for genetic analyses.

Protease B Protease B is an endoproteinase that will solubilize particulate substrates like HPA and Azocoll. It is apparently the only enzyme in the yeast cell that can do so. 25 Principle. Protease B catalyzes hydrolysis of the peptide bonds in Azocoil, resulting in release of the trapped red dye. Absorbance of the dye is read at 520 nm. 4

Reagents Azocoll (Calbiochem) 1% Triton X- 100 0.1 M Tris-HC1, pH 7.6 20% SDS in 0.1 M Tris-HC1, pH 7.6

Procedure. Use 20 mg of Azocoll for each 0.54 ml of solution made by mixing the three listed solutions in the proportion 0.125 ml of Triton X-100, 0.375 ml of buffer, 0.040 ml of SDS. Once the Azocoll is thoroughly wetted, 0.54 ml of the suspension is transferred with a wide-bore pipetter (cut ¼inch off of a 1-ml pipette tip) to a tube. Extract (0.2 ml) of a suitable dilution is added and the tube is placed in a 37 ° constant temperature block. At 1-min intervals, each tube is removed and shaken gently (do not vortex) to resuspend the Azocoll, and replaced in the block. Avoid leaving Azocoll on the tube walls. At the end of the 15-min incubation, the tubes are plunged into ice and 3.5 ml or 2 ml of ice-cold distilled water is added. Tubes are immediately centrifuged for 3 - 5 min at 1650 g and the supernatants are removed to fresh tubes. The absorbance of these supernatants is relatively stable. Absorbance is read at 520 nm. Dilutions are chosen such that the kinetics are linear with time and protein concentration. Best results are obtained if the AA520is less than 0.3. When comparing different strains, the extracts should be diluted such that the protein concentrations are similar for all strains. For extracts made from stationary phase wild-type cells, about 0.1 mg of extract protein/assay is appropriate. Correction is made for a blank lacking extract. A 0-min time point is needed for ade2 mutant strains. Definition of Unit and Specific Activity. One unit of PrB activity is defined as a change in absorbance at 520 nm of 1.0 per minute for the 0.74 ml of reaction mixture as assayed at 37*. For a reaction stopped with 25 E. Juni and G. Heym, Arch. Biochem. Biophys. 127, 89 (1968).

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3.5 ml of water and run for 15 min, the conversion to units/milligram protein is AA520 4.24 1 15 × ~ - ~ X 0.2 (mg protein/ml extract) = (1.9 l)(AA520)/(mg protein/ml extract)

Carboxypeptidase Y Carboxypeptidase Y will catalyze cleavage of esters, amides, and peptides. An assay based on its amidase activity can be employed for kinetic analyses26 or modified to a fixed time point a s s a y . 7 Principle. Carboxypeptidase Y will catalyze the hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide to give the yellow p-nitroaniline. Production can be followed by absorbance at 410 nm.

Reagents N-Benzoyl-L-tyrosine p-nitroanilide (BTPNA) (Sigma) (6 mM): Dissolve 2.43 mg in 1 ml of dimethylformamide (DMF) 0. l M Tris-HC1, pH 7.6 l rnM HgCl 2 20% SDS in 0.1 M Tris-HC1, pH 7.6

Procedure. To a tube containing 0.40 ml of 0.1 M Tris-HC1, pH 7.6, and 0.1 ml of extract at 37 ° is added 0.1 ml of 6 m M B T P N A in dimethylformamide. After 30 min, 1.5 ml of 1 m M HgCl2 is added to stop the reaction. If the extract being assayed has low activity (as is typical for our wild-type strains), 0.2 ml of 20% SDS, pH 7.6, is added and, after vortexing, the tubes are incubated at 70 ° until solubilization of the protein, as evidenced by clearing, ensues. Absorbance at 4 l0 nm is determined. Permeabilized cells can be used as an enzyme source in this assay. We have used cells permeabilized with 0.1-0.2% Triton X-100 or with 10-20% DMF. Definition of Unit and Specific Activity. One unit of activity corresponds to 1/zmol ofp-nitroaniline produced per minute, assuming a molar absorbance of 8800. Corrections for absorbance due to substrate and protein are made. The conversion to units/milligram protein is AA4~0 2.3 10a 30 X ~ X 0.1 (mg protein/ml extract) = (0.087)(AA410)/(mg protein/ml extract) Carboxypeptidase Y levels can also be determined using the kinetic assay described below for CpS, but using Cbz-Phe-Leu as the substrate, 26 S. Aibara, R. Hayashi, and T. Hata, Agric. Biol. Chem. 35, 658 (1971).

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VACUOLAR PROTEASES IN YEAST

S. cerevisiae

385

since 95% of the hydrolytic activity toward this peptide is apparently due to CpY. 17

Carboxypeptidase S Carboxypeptidase S will catalyze hydrolysis of dipeptides that are blocked at the amino terminus. Principle. Free leucine released from a peptide by CpS catalysis is oxidized by amino-acid oxidase. Reduction of the product hydrogen peroxide by horseradish peroxidase is coupled to oxidation of o-dianisidine, yielding brown oxidized dianisidine. The absorbance at 405 nm is followed. The kinetic assay was developed by Wolf and Weiser.17

Reagents 0.2 M KPO4 buffer, pH 7.0, containing 0.5 m M MnCI2 L-Amino-acid oxidase type I (Sigma) Horseradish peroxidase type I (Sigma) Cbz-Gly-Leu (20 raM): 6.44 mg/ml 0.2 M KPO4 buffer, pH 7.0 [or carbobenzoxy-L-phenylalanyl-L-leucine (15 raM) in 0.2 M KPO4 buffer, pH 7.0, for CpY] o-dianisidine dihydrochloride: 2 mg/ml water 0.2 M PMSF in 95% ethanol

Procedure. A solution is made in the proportion 1 ml of phosphate buffer-MnC12, 0.25 mg of amino-acid oxidase, 0.4 mg of peroxidase. To 0.5 ml of this is added 0.5 ml of the peptide solution followed by 0.05 ml of the o-dianisidine solution and 0.05 ml of dialyzed extract. The mixture is incubated at 25 ° and the absorbance followed at 405 nm. To render the assay specific for CpS when Cbz-Gly-Leu is the substrate, extracts are preincubated for 2 hr at 25 ° with 0.1 m M PMSF to inactivate CpY. Use Cbz-Phe-Leu as the substrate if CpY activity is to be measured with this assay. Definition of Unit and Specific Activity. One unit corresponds to production of 1 nmol of L-leucine per rain and 0.1 /zmol of leueine corresponds to a change in absorbance of 0.725 for this procedure. Specific activity is expressed as nmol L-leucine/min/mg extract protein. Designing the Most Useful Protease-Deficient Strain A great deal of evidence, both published (see Ref. 27 for compilation) and anecdotal, suggests that a major source of in vitro proteolytic artifacts is PrB, and that elimination of this protease by mutation results in stabilization of numerous proteins and, indeed, even changes the gross protein signature revealed by Coomassie blue staining of SDS-polyacrylamide gels

386

EXPRESSION IN YEAST

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after electrophoresis of extracts. It is worth remembering that PrB activity in extracts is activated both by heating 27,28 and by treatment with SDS, 4'27 two treatments generally employed in preparing samples for S D S polyacrylamide gel electrophoresis. The usual approach taken to reduce proteolytic degradation of a useful protein is to start with a strain that carries a pep4 mutation. Because pep4 mutations are pleiotropic and greatly reduce or eliminate activity for PrA, PrB, CpY, and API, they have proved to be useful starting points. However, while attempting to purify the 40K precursor to PrB from a pep4bearing strain, C. M. Moehle found that the precursor became activated to PrB of mature size during one column purification step. 29 We have no information on whether the activation was autocatalytic or due to the activity of another protease. Nonetheless, it seems clear that strains that carry the precursor to PrB may not be ideal. A better starting point may well be a pep4 prbl double mutant that carries no PrB precursor. We have constructed and lodged the following strains in the Yeast Genetics Stock Center, MCB/Biophysics and Cell Physiology University of California, Berkeley, CA 94720 for the use of interested parties. BJ926 a trpl + prcl-126 pep4-3 prbl-l122~canl gal2

a + hisl prcl-126 pep4-3 prbl-l122 canl gal2 BJ1984 - 20B-12 a trpl pep4-3 gal2 BJ 1991 cz leu2 trpl ura3-52 prbl-1122 pep4-3 gal2 BJ2168 a leu2 trpl ura3-52 prbl-l122 pep4-3 prcl-407 gal2 BJ2407a leu2 trpl ura3-52 prbl-l122 prcl-407 pep4-3 gal2

a leu2 trpl ura3-52 prbl-l122 prcl-407 pep4-3 gal2 BJ3501 a pep4: :HIS3 prbl-A1.6R his3-A200 ura3-52 canl BJ3505 a pep4: :HIS3 prbl-A1.6R his3-A200 lys2-801 trpl-

AIO1 ura3-52 gal2 canl The prbl-l122 and pep4-3 alleles are nonsense mutations (UAA 3° and UGA t3, respectively). The pep4::HIS3 mutation is an insertion of a BamHI fragment bearing HIS3 into the HindIII site in PEP4.8 The prbl-A1.6R mutation is a deletion of a 1.6-kb EcoRI fragment internal to the PRB1 gene. 3t

2~j. R. Pringle, in "Methods in Cell Biology" (D. B. Prescott, ed.), Vol. 12, p. 149. Academic Press, New York, 1975. 2s R. E. Ulane and E. Cabib, J. Biol. Chem. 251, 3367 (1976). 29 C. M. Moehle, Ph.D. Thesis, Carnegie Mellon University, Pittsburgh, Pennsylvania, 1988. 3o G. S. Zubenko, A. P. Mitchell, and E. W. Jones, Genetics 96, 137 0980). 3~ C. M. Moehle, M. W. Aynardi, M. R. Kolodny, F. J. Park, and E. W. Jones, Genetics 115, 255 (1987).