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Isolation and Characterization of Schizosaccharomyces pombe Mutants Lacking Aminopeptidase Activity MAJOSE ARBESU, SANTIAGO GASCON AND PAZ SUAREZ-RENDUELES* Dpto Biolgogia Funcional, Area Bioquimica y Biologia Molecular, Facultad de Medicina, Universidad de Oviedo, Spain

Received 3 January 199 1

A mutant strain of the fission yeast Schizosaccharomycespombe defective in aminopeptidase I was isolated by screening for lack of activity against the chromogenic substrate lysine-P-naphthylamide in isolated colonies. Tetrad dissection of sporulated diploids heterozygous for the wild-type and mutant allele resulted in a 2:2 segregation of mutant and wildtype phenotype indicating a single chromosomal gene mutation. Gene dosage experiments indicated that the mutation might reside in the structural gene of aminopeptidase I. No vital consequences of aminopeptidase I deficiency on cell life and sporulation could be detected. However, the enzyme seems to be involved in protein degradation under conditions of nutrient deprivation. KEY WORDS - Fission

yeast; aminopeptidase; mutant; peptidase.

INTRODUCTION The well-studied yeast Saccharomyces cerevisiae has a multiplicity of aminopeptidase activities (Achstetter et al., 1983) ofwhich the best characterized are the vacuolar aminopeptidase ysc I (Matile e t a l . , 1971;Metzand Rohm, 1976; Metzetal., 1977; Cueva et al., 1989; for nomenclature see Achstetter et al., 1984) and the partially external aminopeptidase ysc I1 (Frey and Rohm, 1979; Hirsch et al., 1988). Unlike budding yeast, Schizosaccharomyces pombe has only one aminopeptidase active towards basic residues (Atmanspacher and Rohm, 198l), whose function remains to be established. One approach to assigning a particular enzyme to a specific physiological process involves isolation mutants lacking the enzyme and determining the effect of loss of the enzyme on the process. This approach has been proved to be useful in understanding cellular functions of S . cerevisiae peptidases (for reviews see Suarez-Rendueles and Wolf, 1988; Hirsch et al., 1989). We have undertaken the characterization of the proteolytic system of the fission yeast Schiz. pombe with the final aim of constructing genetic engineered strains with an altered set of proteinases which might be suitable for the expression of heterologous *Addressee for correspondence. 0749 503)3/91/05052547 $05.00 0 I991 by John Wiley & Sons Ltd

proteins. Here we report on the isolation of aminopeptidase mutants and their genetic and biochemical characterization. MATERIALS AND METHODS Chemicals Yeast growth media were manufactured by Difco (U.S.A.). Ethylmethane sulfonate was from Serva (F.R.G.). Lys-P-naphthylamide and fast garnet GBC (70% dye content) were purchased from Sigma (U.S.A.). [3H]Leucine (1 mCi/ml) was obtained from Amersham (U.K.). Amino acids of the L-configuration, peptides Arg-Leu and Leu-Gly were purchased from Bachem (Switzerland). All other chemicals, which were of the highest purity available, were purchased from either Merk (F.R.G.) or Sigma (U.S.A.). Strains used Schizosaccharomyces pombe 972h- (Dr P. Nurse, Microbiology Unit, Department of Biochemistry, University of Oxford, U.K.) was used as starting material to obtain the mutants. For crosses the following strains (Dr A. Duran, Instituto de Microbiologia Bioquimica, CSIC, Universidad de Salamanca, Spain) were used: 975h+, mat 2-P lysl131, h- his3-327, h- leu1 -32, h + argl lysl .

526 Growth conditions

Complete medium (YPD) contained 1940 yeast extract, 2% peptone and 2% glucose. Minimal medium consisted of 0.67% yeast nitrogen base without amino acids and 1YOglucose. When necessary, it was supplemented with 30mg/l of the substances required by auxotrophic strains. Minimal medium without amino acids and ammonium sulfate consisted of 0.17% yeast nitrogen base without amino acids and ammonium sulfate, 1% glucose and 1.5% of the desired peptide as nitrogen source. Sporulation medium contained 1 YOglucose, 0.1 % KH,PO,, 10 pg/l biotine, 1 mg/l calcium pantothenate, 10 mg/l nicotinic acid and 10 mg/l inositol. Strains were grown at 28°C in conical flasks and aerated by shaking. Cells were harvested in stationary phase. M u tagenesis

Cells weremutagenized in 0.1 M-phosphate buffer, pH 8.0 ( lo7 cells/ml) with ethylmethane sulfonate (50 pl/ml) for 60 min at room temperature. Genetic methods

Sporulation was induced as described by Egel ( 197I). Tetrad analysis was performed by micro-

manipulation of the asci (Gutz et al., 1974). After introduction of convenient auxotrophic markers (Kohli et al., 1977), stable diploids were obtained in crosses with strain mat 2-P lysl-131. This mutation blocks the meiotic process at a very early stage (Egel, 1973). Preparation of cell extracts

Crude extracts were prepared from cells grown on YPD plates for 48 h at 30°C. Cell material was suspended in 200 p1 20 mM-Tris/HCl buffer, pH 7.0, and 200 pl of acid-washed glass beads (0.5 mm diameter) were added. The mixture was vigorously shaken for six I-min periods with I-min intervals between them. During the idle periods the tubes were placed in ice. After centrifugation in an Eppendorf microfuge for 5 min, the supernatants were used for enzyme assays. Soluble extracts were prepared as described by Suarez-Rendueles et al. (1981). Enzyme assay Aminopeptidase activity against lysine-Cnitroanilide was determined in 0.1 M-Tris/HCl buffer,

M. J. ARBESU ET AL.

pH 7.0. The liberated 4-nitroaniline was measured in a Shimadzu spectrophotometer at 405 nm and activity was calculated from a standard calibration curve. 1 unit is defined as the amount of enzyme that transforms 1 pmol substrate/min under the assay conditions used. Polyacrylamide gel electrophoresis Non-denaturing polyacrylamide slab gels with discontinuous pH were prepared as described by Hedrick and Smith (1968). The stacking gel was 3.5%, the separating gel was 7.5%. Aminopeptidase activity staining in non-denaturing gels Aminopeptidase activity against lysine-P-naphthylamide was determined in 0.1 M-Tris/HCl buffer, pH 7.0 as described (Hirsch et al., 1988): Selection of aminopeptidase I mutants Mutagenized cells of strain 972h- were plated on YPD agar to yield 100-150 surviving colonies/plate and grown at 23°C until colonies were visible. Cells were then replica-plated onto YPD medium in glass petri dishes and allowed to grow further at 23°C. When the colonies had reached a sufficient size, cells were incubated with 10 ml of chloroform. The solvent was evaporated within about 15 min. The cells were then incubated at 37°C for 2 h to inactivate any thermosensitive enzyme generated by mutagenesis. Aminopeptidase activity in the colonies was monitored by carefully pouring a mixture prepared by adding 13.5 ml of 1YOmelted agar in 0.1 M-potassium phosphate buffer, pH 6.5, to 1.5 ml of a solution containing 10 mM-lysine-p-naphthylamide and 8 mg of fast garnet GBC in 50% dimethyl sulfoxide. The plates were incubated at room temperature until most colonies had acquired a deep red colour (usually 15 min). Colonies displaying reduced staining activity were picked from the master plate, and single colonies were isolated and retested for their staining ability. Mutant candidates were tested biochemically for aminopeptidase activity. Other methods Growth was followed by turbidimetry at 600 nm. Protein was assayed by the method of Lowry et al. (1951). Protein degradation measurements Labeling of yeast protein with [3H]leucine and estimation of protein degradation were carried out

ISOLATION AND CHARACTERIZATION OF SCHIZOSACCHAROMYCESPOMBE MUTANTS

essentially according to Lopez and Gancedo (1979). Strains bearing either the wild-type APE1 allele (strain 972hp) or the mutant ape1 allele (strain JM3 I ) were compared in two different experiments. First, 50 ml minimal medium containing 2 pCi [3H]leucine/ml was inoculated with cells from stationary-phase precultures and grown for 20 h at 28''C. Cells were washed twice and resuspended in the chase medium, consisting of minimal medium without any nitrogen source. Aliquots (2 ml) were withdrawn at various times into 110% trichloroacetic acid (0.2 ml) and allowed to stand overnight at 4 C. The acid-insoluble precipitate was pelleted by centrifugation (20 min, 1OOOOxg) and 2ml samples of the supernatant fluid were taken. After determination of radioactivity by liquid scintillation counting in Triton/toluene scintillation fluid, the trichloroacetic acid was removed from the acidsoluble samples by repeated washes with ether ethylic. The acid-free fractions were lyophilized, resuspended in 0.2 ml of chromatography solution A (0.1YO trifluoroacetic acid), and chromatographed on a reverse-phase column C18 (4.46 x 150mm). Elution was performed with a linear gradient 2-100% in solution B (0.1 YOtrifluoroacetic acid in acetonitrile). A similar experiment was performed but after labeling cells were transferred to a carbon-free medium during the chase. RESULTS Mutants lacking aminopeptidase I activity The isolation of aminopeptidase I mutants was facilitated by the finding that Schiz. pombe has only one aminopeptidase active towards basic residues (Atmanspacher and Rohm, 1981). The application of an overlay agar mixture made of the substrate Lys-P-naphthylamide and fast garnet GBC to cells mutagenized as indicated in Materials and Methods provides a selective screening method for aminopeptidase activity. The reaction of the fast garnet with the P-naphthylamine liberated upon enzymatic hydrolysis of the peptidebond-like lysyl-P-naphthylamide linkage by wildtype colonies produces a red azo dye. Of about 8000 survivors, we selected one non-staining clone. The colony was picked from the master plate, grown and restreaked. Biochemical analysis revealed that a cell extract from the staining-deficient mutant strain (JM31) did not possess activity against the aminopeptidase substrate Lys-4-nitroanilide, when cells grown at 23°C were assayed at either 23 or 37°C. A

527

1 2

Figure 1. Staining of aminopeptidase activity on nondenaturing polyacrylamide gels. Soluble extracts were prepared from wild-type strain 972h and mutant strain JM3 I growing on YPD medium at 28°C and harvested in early stationary growth phase. Equal amounts of protein (600 bg) of each extract were subjected to non-denaturing slab polyacrylamide gel electrophoresis followed by aminopeptidase activity staining as indicated in Materials and Methods. Lane I : 972h wild-type extract: lane 2: JM31 mutant extract.

further analysis was achieved by subjecting equal amounts of protein of soluble extract from wildtype and mutant strains to non-denaturing polyacrylamide gel electrophoresis followed by aminopeptidase activity staining. Figure I shows that the slowly migrating activity band visible in the wild-type strain was completely undetectable in the extract of strain JM31.

Genetic analysis of the mutant Strain JM3 1, devoid of aminopeptidase activity, was crossed with strain 975h' and the resulting diploids were sporulated. Crude extracts of the haploid spore clones from 33 tetrads dissected were analysed

528

M. J. ARBESU ET AL.

Table 1. Aminopeptidase I levels in crude extracts of segregants of the cross of strains 975h+ ( A P E I ) x JM31

Table 2. Gene dosage effect on aminopeptidase I activity

1)

Tetrad

Spore

Strain

AminopeptidaseI activity (mU/mg)

MDI

Genotype

Aminopeptidase activity (mU/ms)

APEl ~

1

4

A B C D

0.00 0.00 4.10

A B C D

3.68 0.00 0.00 3.50

MDII

APEl

10.0

ape I APEI

5.5

~-

3.30 Cells were grown at 28°C in YPD medium for 48 h. Crude extracts were prepared and activity was measured at 37°C as detailed in Materials and Methods.

ation of sporulation frequency was observed in mutant diploids homozygous or heterozygous for aminopeptidase I deficiency (not shown). Evidence has been presented that aminopeptidase I is at least partially located exocellularly in Schiz. biochemically for aminopeptidase activity. We pombe NCYCl32 strain (Atmanspacher and Rohm, found that all ascospores showed a 2+:2- segrega- 1981). Therefore, we wanted to test a possible function of aminopeptidase activity (Table I). This fact tion of the enzyme in the external degradation of indicated that the mutant strain JM31 indeed bore a peptides to provide the cell with amino acids for single chromosomal gene mutation, called apel-1, protein synthesis, as has been shown to be the case with aminopeptidase ysc I1 of S . cerevisiae resulting in the absence of aminopeptidase I. The structural gene for an enzyme should show a (Hirsch et al., 1988). Thus, we tested growth of gene dosage effect on the level of enzyme activity aminopeptidase-deficient strains on several pep(Zimmermann et al., 1969; Hilger, 1973). Thus, dip- tides. All strains contained either a leucine or loids heterozygous for ape1 should show gene arginine auxotrophy marker, and leucine or argidosage if A P E l were the structural gene for amino- nine were offered as amino acid residues in dipeppeptidase I. Because of the genetic peculiarities of tides. Growth of these strains on minimal medium supplemented with peptides required peptidolytic Schiz. pombe, several additional crosses were perliberation of leucine or arginine. Aminopeptidase I formed with strain JM31 in order to give rise to mutant and wild-type strains grow equally well on stable diploids (Kohli et al., 1977; Egel, 1973). All minimal medium supplemented with peptides, indithe constructed diploids, heterozygous for the cating either that a nutritional role for the enzyme is aminopeptidase mutation, showed about 50% of non-existing or that there are additional enzymes the activity found in homozygous wild-type diploids (dipeptidase, carboxypeptidases) that carry out (Table 2). This result is consistent with the expecredundant functions. When these strains were tation that A P E l is the structural gene encoding grown on Arg-Leu or Leu-Gly as sole nitrogen aminopeptidase I. source in the absence of ammonia, no growth alteration of aminopeptidase I mutant strains was Physiological consequences of aminopeptidase 1 observed. dejiciency Drastic changes in growth conditions lead to To reduce the interference of possible back- increased intracellular protein degradation perground markers introduced by mutagenesis in the formed by proteinases. In S. cerevisiae it has been experiments described below, we used segregants of shown that vacuolar endopeptidases are needed for strain JM31 which were back-crossed to the wild- protein degradation under nitrogen starvation contype at least twice. When comparing growth rates on ditions (Teichert et al., 1987). A model for cytoplaseither rich or minimal medium of mutant and wild- mic protein degradation has been proposed in which type strains, no differences were observed. No alter- aminopeptidases might play an important role by ~

~

~

~

~~

~

~

Cells were grown at 28°C in YPD medium for 48 h Crude extracts were prepared and aminopeptidase activity was measured at 37 C as described in Materials and Methods

ISOLATION AND CHARACTERIZATION OF SCHIZOSACCHAROMYCES POMBE MUTANTS

400

300

200

100

B 400

k

V

300

21

c ._

> ._ c 0

::

._

200

'D

0

I Y

100

529

generating amino termini of stabilizing or destabilizing nature (Bachmair etuf.,1986). Ifaminopeptidase I were involved in such a process, differences in protein degradation should be found in aminopeptidase mutant and wild-type cells. Protein degradation under nitrogen starvation conditions was measured in wild-type and aminopeptidase I mutant strains. Even though degradation rates were the same for both strains, the nature of the trichloroacetic acid-soluble degradation products was quite different when analysed by HPLC reverse-phase chromatography (Figure 2). In the wild-type strain essentially all the trichloroacetic acid-soluble material eluted as a single radioactive peak (Figure 2A), which emerged from the column in the same fractions as L-leucine. Chromatography of acid-soluble material from the mutant strain (Figure 2B) revealed a heterogeneous mixture of labeled material. After incubation of this material with a soluble extract of a wild-type strain, much of the radioactivity was converted into free leucine (Figure 2C). These results show that the capacity of the amino-peptidase-deficient strain to produce free amino acids from protein during nitrogen starvation is reduced in comparison with the wild-type. They also show that the product of protein degradation in the mutant strain is a mixture of small leucine peptides. We found similar results when analysing the nature of the acid-soluble products of protein degradation under carbon starvation in wild-type and aminopeptidase mutant strains.

DISCUSSION C 400

300

200

100

0

10

20 Time (rnin)

30

Taking advantage of the fact that Schiz. pombe has only one aminopeptidase active towards basic residues (Atmanspacher and Rohm, 1981), we have isolated a mutant devoid of aminopeptidase activity by searching for colonies unable to cleave the chromogenic substrate lysine-p-naphthylamide. Crude extracts from strain JM31 (upel) lack the only band of electrophoretic activity observed in a wild-type strain (Figure 1). The results presented in this paper show that a mutation in the APE1 gene leads to the absence of Figure 2. Reverse-phase chromatography of trichloroacetic acid-soluble protein breakdown products. Acid-soluble breakdownproductsfrom(A)972h- (wild-type)and(B) JM31 (upel-I) were collected after 6 h of nitrogen starvation and chromatographed as described in Materials and Methods. (C) Products in (B) after 180 min of digestion with an extract of a wild-type strain (972h-).

530 aminopeptidase I activity. The gene dosage effect observed for aminopeptidase I activity in strains carrying combinations of mutant ( a p e ] ) and wildtype genes strongly supports the proposal that APEZ is the structural gene for aminopeptidase I of Schiz. pomhe. The aminopeptidase I deficiency does not seem to affect vegetative growth in either rich or minimal medium. Sporulation is not impaired in diploids homozygous for the absence of the enzyme. Growth of either leucine or arginine auxotrophs carrying the aminopeptidase I mutation is not affected when the leucine-containing peptide Leu-Gly or the argininecontaining peptide Arg-Leu are used either as supplements on minimal medium or as sole nitrogen source. This behaviour might rule out any nutritional role for the enzyme, but it should be kept in mind that additional peptidases do exist (dipeptidase, carboxypeptidases) in the fission yeast able to catalyse cleavage of those peptides. According to the N-end rule (Bachmair et al., 1986), aminopeptidases might be involved in prote in degradation by generating stabilizing or destabilizing N-termini. The results presented here indicate that aminopeptidase I of Schiz. pombe does function in either carbon or nitrogen starvationinduced protein degradation because in its absence, even though the extent of such degradation is not altered, the products are different. The results also show that measurements of the formation of trichloroacetic acid-soluble protein breakdown products do not provide an accurate assessment of the production of free amino acids from protein because the acid-soluble products of protein breakdown in the mutant strain appear to be a heterogeneous mixture of small peptides rather than free amino acids. The most obvious hypothesis is that aminopeptidase I functions late in the degradation pathway, hydrolysing small (trichloroacetic acidsoluble) peptides produced by the attack of other enzymes on larger polypeptides. The finding that aminopeptidase I is involved in intracellular protein degradation might have further implications when using yeast strains as hosts for the production of heterologous proteins. It seems advisable to use strains devoid of aminopeptidase activity in order to increase the stability of the foreign protein. ACKNOWLEDGEMENTS This work was supported by grant PBT87/0030 from the Comision Interministerial de Ciencia y Tecnoiogia ofspain and Fundacibn R a m o n Areces.

M. J . ARBESU E T A L .

The authors are specially grateful to Dr Angel Duran (Instituto de Microbiologia Bioquimica, Salamanca, Spain) for his interest in this work and scientific advice. REFERENCES Achstetter, T., Ehmann, C. and Wolf, D. H. (1983). Proteolysis in eukaryotic cells: Aminopeptidases and dipeptidyl aminopeptidases of yeast revisited. Arch. Biochem. Biophys. 226,292-305. Achstetter, T.. Ehmann, C., Osaki, A. and Wolf, D. H. (1984). Proteolysis in eukaryotic cells. Proteinase yscE, a new yeast peptidase. J . Biol. Chem. 259,13344-13348. Atmanspacher, D. and Rohm, K. (1981). Only one aminopeptidase in Schizosaccharomycespombe. HoppeSyler 's Z.Physiol. Chem. 362,459463. Bachmair, A,, Finley, D. and Varshavsky, A. (1986). In vivo half life of a protein is a function of its amino terminal residue. Science 234, 179-1 86. Cueva, R.. Garcia-Alvarez, N. and Suarez-Rendueles, P. (1989). Yeast vacuolar aminopeptidase yscI. Isolation and regulation of the APEI f L A P 4 ) structural gene. FEBS L,ett. 259, 125-129. Egel, R. (1971). Physiological aspects of conjugation in fission yeast. Planta (Berl.) 98,89-96. Egel, R. (1973). Genes involved in mating type expression of fission yeast. Mol. Gen. Genet. 121,277-284. Frey, J . and Rohm, K . (1979). External and internal forms of yeast aminopeptidase yscII. Eur. J . Biochem. 97, 169-173. Gutz, H.. Neslot, H., Leupold, U. and Loprieno, N. (1974). Schizosaccharornyces pombe. In King, R. G. (ed.), Handbook of Genetics, vol. 1. Plenum Press, New York, pp. 395446. Hedrick, J. L. and Smith, A. J. (1968). Size and charge isomer separation and estimation of molecular weights of protein by disc gel electrophoresis. Arch. Biochem. Biophys. 126, 155-164. Hilger, F. (1973). Construction and analysis of tetraploid yeast sets for gene dosage studies. J. Gen. Microbiol. 75, 23-3 1. Hirsch, H. H., Suarez-Rendueles, P., Achstetter, T. and Wolf, D. H. (1988). Aminopeptidase yscII of yeast. Isolation of mutants and their biochemical and genetic analysis. Eur. J . Biochem. 173, 589-598. Hirsch, H. H., Suarez-Rendueles, P. and Wolf, D. H. (1989). Yeast (Saccharomyces cerevisiae) proteinases: structure, characteristics and function. In Walton, E. F. and Yarranton, G . T. (eds), Molecular and Cell Biology of Yeasts. Blackie and Son Ltd, London, pp. 134-200. Kohli. J., Hottinger, H., Munz, P., Strauss, A. and Thuriaux. P. (1977). Genetic mapping in Schizosaccharomyces pombe by mitotic and meiotic analysis and induced haploidization. Genetics 87,474-489. Lopez, S. and Gancedo, J. M. (1979). Effect of metabolic conditions on protein turnover in yeast. Biochem. J. 178,769-776.

ISOLATION AND CHARACTERIZATION OF SCHIZOSACCHAROMYCES POMBE MUTANTS

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