induction of a proteinase by heat-shock in yeast

FEMS Microbiology Letters 33 (1986) 199-203 199 Published by Elsevier

FEM 02337

Induction of a proteinase by heat-shock in yeast

(Proteinase activity; heat-shock; Saccharomyces cereoisiae)

T h o m a s Gross and Bernd Schu lz -Harde r *

Freie Unioersiti~t Berlin, Institut f ~ Biochemie und Molekularbiologie, Ehrenbergstrasse 26- 28, D- 1000 Berlin 33, F.R.G.

Received 9 September 1985 Revision received 14 October 1985

Accepted 17 October 1985

1. SUMMARY

In Saccharomyces cerevisiae heat-shock induces an increase in proteinase activity. The induction is probably due to newly synthesized enzyme molecules, since the increase in proteinase activity can be inhibited by cycloheximide. Degradation of endogenous proteins is enhanced by EDTA, while the azocasein assay is not affected by MnCI 2, MgCI 2, or E D T A . The proteinase has a pH optimum of 8, and phenylmethylsulfonyl fluoride (PMSF) as well as chymostatin are strong inhibitors. We infer that the induced proteinase is probably identical with proteinase B of yeast.

2. INTRODUCTION

Most prokaryotic and eukaryotic cells respond to heat shock with the induction of a small num- ber of 'stress'-proteins [1]. However, the regulation of the rapid changes in protein synthesis in different organisms is due to various control mecha-

* Present address: RWHT Aachen, Anatomie I, Melatener Strasse 211, I)-5100 Aachen, F.R.G.

nisms of gene expression. In Xenopus oocytes the heat-shock response depends on translational control of preformed and masked hsp 70 mRNAs [2], whereas in Drosophila cells heat-shock mRNAs are newly transcribed, and the translation of most pre-existing mRNAs is repressed [3,4].

In contrast, yeast cells possess only a mecha- nism of transcriptional control, and pre-existing mgNAs are rapidly degraded [5]. Moreover, heat stress of yeast cells leads to a reduction of the ribosome [6] and RNA content [71, and to an increase in ribonuclease activity [8]. Similar changes in the metabolism occur during the stationary growth phase when yeast cells suffer a lack of nutrition [9]. Recently we have shown that a ribonuclease is inducible by glucose starvation, heat-shock or acrylonitrile treatment [10,11]. Be- cause protein degradation activities are also enhanced during the stationary growth phase [12], we assumed that yeast cells react to different kinds of 'stress' by the induction of catabolic enzymes.

In this publication, we report on the heat-shock induction of a proteinase in S. cerevisiae, and present data demonstrating that this proteinase is likely to be the same enzyme which has been described by Saheki and Holzer [13], Ulane and Cabib [14] and Burlini et al. [15] as proteinase B of yeast.

0378-1097/86/$03.50 © 1986 Federation of European Microbiological Societies

200

3. MATERIALS AND METHODS

3.1. Strains and growth conditions Cells of the tetraploid strain 22130 of S. cerevisiae

[16] were grown in medium containing 0.5% (w/v) peptone, 1% (w/v) Difco yeast extract and 4% (w/v) glucose at 28°C, and harvested in the fermentative growth phase. To induce a heat shock the cells were transferred to the same fresh medium and incubated at 39°C for 3 h.

3.2. Preparation of proteinase 15 g (wet weight) of yeast cells were suspended

in buffer consisting of 30 mM Tris-HCl, pH 7.5, 500 mM KCI, 5 mM MgC12, 0.25 mM EDTA, 6 mM 2-mercaptoethanol, and 20% glycerol (final volume 40 ml), incubated at 0°C for 30 min, and poured into a French Pressure Cell (American Instrument Co). The cell suspension was frozen in an ethanol/solid CO 2 bath and homogenized at a pressure of 20000 lb/square inch. Following dis- ruption of the cells, the homogenate was centrifuged at 18000 x g at 4°C for 15 min in a Sorvall (ss-34 rotor) centrifuge. The supernatant was centrifuged again in a Beckman Ti60 rotor at 50000 rev./min for 2 h. A crude fraction of the proteinase was prepared from the postribosomal supernatant by ammonium sulfate precipitation (80%) and dialysis against 30 mM Tris-HCl, pH 7.5, 50 mM KCI and 6 mM 2-mercaptoethanol. The protein concentration was determined accord- ing to the method of Layne [17] and the samples were stored at -20°C.

3.2. Proteinase assays For determination of proteinase activity the

degradation of endogenous yeast cell proteins or azocasein was assayed. The degradation of endogenous proteins was carded out in a volume of 1 ml containing 30 mM Tris-HCl, pH 7.5, 50 mM KCI, 5 mM EDTA, 6 mM 2-mercaptoethanol and 0.4 to 0.5 mg protein of the proteinase solution. After incubation at 37°C for 1 h the proteins were precipitated with 5 vols. of acetone and centrifuged at 18000 × g for 20 min. The pellets were dried, redissolved in electrophoresis buffer, and loaded on a sodium dodecyl sulfate (SDS)-polyacrylamide gel (10-18% acrylamide). Electro-

phoresis was carried out as described by Laemmli [18]. Proteinase activity was also assayed by degradation of azocasein in 3.3 ml of a mixture containing 100 mM Tris-HCl, pH 8, 50 mM KCI, 6 mM 2-mercaptoethanol, 10 mg/ml azocasein, and 0.5 mg/ml protein of the proteinase sample. To reduce spontaneous degradation of azocasein, samples were preincubated at 37°C for 1 h. The reaction was stopped after various times by adding 0.5 ml TCA (10%) to 0.6 ml of the mixture. After centrifugation at 5000 rev./min for 10 min (Heraeus-Christ Minifuge II) the absorbances of the supernatants were measured against a blank at 335 nm (proteinase activity (U): 1 unit = 0.1 /1335 nm/h/ml) .

4. RESULTS

During preparation of heat-shock-inducible RNase from yeast ribosomes a protein-degrading activity hampered purification of the enzyme. Be- cause this proteolytic activity was inhibited by PMSF we infer that it is due to a proteinase. In contrast to RNase which is tightly bound to the 40S ribosomal subunit [19], the proteinase could be washed from the ribosomes by 500 mM KCI (results not shown). For further experiments we used the postribosomal supernatant obtained after extraction of the homogenate with high salt buffer. Fig. 1 shows the degradation of proteins of the postribosomal supernatant of exponentially growing yeast cultivated at 28°C and 39°C.

Cells grown at the elevated temperature possess a markedly higher proteinase activity than control cells. In addition, the proteolytic activity is enhanced by EDTA, whereas PMSF acts as an inhibitor. The increase in proteinase activity during heat treatment of yeast cells could be due to the inactivation of proteinase inhibitor, an activation of proteinase already present in the cell or newly synthesized enzyme. The inhibition of the induction of the proteolytic activity by cycloheximide (Fig. 1) demonstrates that the proteinase is newly synthesized after the elevation of cultivation temperature.

To characterize the proteinase we have used the azocasein assay. When crude extracts of heat-

201

A B C D E F G H I Fig. 1. Analysis of the degradation products of the protcinase assay by SDS-polyacrylamide gel electrophoresis. The postribosomal supernatants of: (A, B, C) exponential growth phase cells (cultivation temperature 28°C); (D, E, F) heat-shocked cells (exponential growth phase cells incubated at 39°C for 3 h), and (G, H, I) heat-shocked cells incubated in the presence of 1 mM cycloheximide were incubated for 1 h at 37°C as described in MATERIALS AND METHODS. Samples (A, D, G) were incubated with 5 mM EDTA, (B, E, H) with 2 mM MgCI2, and in the presence of 5 mM EDTA and 0.17o PMSF (C, F, I).

0,2-

.! 0.1.

®

j/ I I I

1 2 3

®

I 2 Tirr, e (h)

-0.3

-0.2 8

-0.1

Fig. 2. Protcinase activity determined by the azocasein assay. (A) No pre-incubation, and (B) preAncubation for 2 h at 37°C. The azocasein assay was performed as described in MATERI- ALS AND METHODS. Protcinas¢ was prepared from exponential-growth-phase cells (cultivation temperature 28°C; A); heat-shocked cells (exponential-growth-phase cells incubated at 39°C for 3 h; O), and heat-shocked cells incubated in the presence of I mM cyclobeximide (D).

with the results of Fig. 1, Figs. 2b and 3a, b also demonstrate that cycloheximide diminishes the increase of proteinasc activity after heat treatment.

When the proteinasc activities of the crude extracts of control and heat-shocked cells were tested at different pH, a pH optimum of 8 was found for

s h o c k e d and cont ro l cells were tested with the pro te inase assay a lag-phase of 30 rain to 1 h was recognisable (Fig. 2a), which is p robab ly due to the presence of a pro te inase inhibi tor . Because yeast cells possess endogenous pro te inase inhibitors [20], which can influence the result of the enzyme assay dur ing t ime-course exper iments , it is necessary to inact ivate the inhibi tors by an ap- p ropr i a t e t rea tment . Therefore, several methods for the inact iva t ion of pro te inase inhibi tors were examined. Pre- incubat ion at 37°C for 2 h (Fig. 2b), p re - t r ea tment with 0.26~ SDS f o r 6 h [21] (Fig. 3a) or dialysis agains t p H 5 buffer for 8 h [22] (Fig. 3b) were successful in act ivat ing the enzyme. Pre- incubat ion at 37°C and SDS treatment gave comparab l e results, while after act iva- t ion at p H 5 the pro te inase act ivi ty was reduced. In la ter exper iments , the fastest procedure , pre- in- cuba t ion at 37°C for 2 h was used. In agreement

0.2-

o.1-

o /

1 2

®

1 2

-0.2

-0.1

d~

Time (h)

Fig. 3. Proteinase activity determined by the azocasein assay as described in MATERIALS AND METHODS. Samples were pre-incubated for 6 h with 0.267O SDS (A), or dialysed a~aingt a buffer consisting of 30 mM Na-succinate, pH 5, 50 mM KC1 and 6 m_M 2-mercaptoethanol (B). Proteinase was prepared from exponential-growth-phase cells (cultivation temperature 28oC; A); heat-shocked cells (exponential-growth-phase cells incubated at 39°C for 3 h; O), and heat-shocked cells grown in the presence of 1 mM cycloheximide (O).

202

_0+4 1

~ 0 2 -

~601-

+ /

i "

1 2 3 4

+ /

I 2 3 4 T,rne {hl

~0.4

o3 ,~ 2"

-02

-01 ct

Fig. 4. Effect of PMSF and chymostatin on proteinas¢ activity. Degradation of azocascin was measured with 0.1% PMSF (A) or 60 /~g/ml chymostatin (B). Proteinase activity of extracts derived from exponential-growth-phase cells (cultivation temperature 28°C; A; zx with inhibitor), heat-shocked cells (exponential-growth-phase cells incubated at 39°C for 3 h; e; O with inhibitor), and heat-shocked cells grown in the presence of 1 mM cycloheximide (I); [] with inhibitor).

both preparations (results not shown). The similarity of the pH dependence of both extracts suggests that the heat-shock-inducible proteinase is also present in exponentially growing yeast at a normal temperature.

For further characterization of the proteinase the enzyme assay was carried out in the presence of proteinase inhibitors. PMSF and chymostatin diminished the proteolytic activity of the crude extracts of control cells, heat-shocked cells, and cells which were incubated at elevated temperature in the presence of cycloheximide (Fig. 4, a and b). The similarity of the remaining proteinase activities in these experiments further supports the speculation that the heat-inducible proteinase is active in heat-shocked cells and also in control cells at a lower level.

5. DISCUSSION

In yeast, heat shock leads to an increase in proteolytic activity, which can be inhibited by cycloheximide. Therefore we conclude that this activity is most probably due to newly synthesized proteinase molecules. However, our results show that proteolytic activity is also present to a lesser extent in cells at normal temperature. Thus, it is

possible that the heat-inducible proteinase is identical with one of the known yeast proteinases. Since the lag-phase during the time-course assay suggests the presence of an endogenous inhibitor, proteinase A and B as well as carboxypeptidase Y which possess intracellular inhibitors [23] are possible candidates for the heat-shock-inducible proteinase. None of the three proteinases is affected by divalent cations or EDTA [23], which is in accordance with our observation that the proteolytic activity measured with the azocasein assay is not influenced by MgCI 2, MnCI 2 or EDTA (re- suits not shown). The enhanced degradation of endogenous proteins in the presence of EDTA, as shown in Fig. 1, could be due to a conformational change of the substrate.

To characterize the heat-inducible proteinase, the enzyme has been tested at different pH ranges and in the presence of proteinase inhibitors. The pH optimum of the proteinase has been found to be 8, which correlates with the pH optimum of proteinase B (pH 6-10) [23]. Proteinase A pos- sesses pH optima of 2 -4 [23] and 6-8 [24], while carboxypeptidase Y exhibits an optimum in the range of pH 5.5-6.5 [23].

Specific inhibitors are also useful tools for the characterization of enzymes. Chymostatin which is a strong inhibitor of proteinase B has no effect on proteinase A and carboxypeptidase Y, while PMSF inhibits proteinase B and carboxypeptidase Y. As shown in Fig. 4, both inhibitors reduce the proteolytic activity of the extract of yeast cells grown at elevated temperature.

These results imply that the heat-shock proteinase is probably identical with proteinase B. However, ubiquitin, which plays a role in intracellular protein breakdown [25,26], has been found by Bond and Schlesinger [27] to be a heat-shock protein in chicken embryo fibroblasts. Since Ozkaynak et al. [28] have shown that a homolo- gous protein to ubiquitin exists in S. cereoisiae, the proteinase activity described in this publication may also be a component of the ubiquitin system.

The response of yeast cells to heat shock by inducing a proteinase and RNase [10] is similar to the changes that occur when yeast cells enter the stationary-growth phase. Moreover, ribonuclease activity is also increased by glucose starvation [29]

or treatment with acrylonitrile [11]. The induction of catabolic enzymes could, however, be a response of yeast to 'stress'. Therefore, it would be of interest to investigate whether different kinds of 'stress' which vitiate growth of yeast are also capa- ble of inducing an increase in proteinase activity.

REFERENCES

[1] Heat Shock: From Bacteria to Man (Schlesinger, M.J., Ashbumer, M. and Tissi*res, A., Eds.) (1982) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

[2] Bienz, M. and Gurdon, J.B. (1982) Cell 29, 811-819. [3] Storti, R.V., Scott, M.P., Rich, A. and Pardue, M.L. (1980)

Cell 22, 825-834. [4] Scott, M.P. and Pardue, M.L. (1981) Pro(:. Natl. Acad. Sci.

USA 78, 3353-3357. [5] Lindquist, S. (1981) Nature 293, 311-314. [6] Wessel, J. and Lochmann, E.-R. (1980) Z. Naturforsch.

35c, 171-172. [7] Maul, S.B., Sinskey, A.J. and Tannenbaum, S.R. (1970)

Nature 228, 181. [8] Bueno, G.E., Otero, M.A. and Gonzalcz, A.C. (1982)

Biotechnol. Lett. 4, 145-148. [9] Swida, U., Schulz-Harder, B., Ki~cherer, C. and K~ufer, N.

(1981) Biochim. Biophys. Acta 652, 129-138. [10] Schulz-Harder, B. (1983) FEMS Microbiol. Lett. 17, 23-26. [11] Pfeffer, U. and Schulz-Harder, B. (1985) Z. Naturforsch.

40c, 26-28.

203

[12] Katsunuma, T., SchStt, E., Elsisscr, S. and Holzer, H. (1972) Eur. J. Biochem. 27, 520-526.

[13] Saheki, T. and Holzer, H. (1974) Eur. J. Biochem. 42, 621-626.

[14] Ulane, R.E. and Cabib, E. (1976) J. Biol. Chem. 251, 3367-3374.

[15] Burhni, N., Tortora, P., Hanozet, G.M., Vincenzini, M.T., Vanm, P. and Guerritorc, A. (1982) Biochim. Biophys. Acta 708, 225-232.

[16] Reichert, U. (1967) Zbl. Bakteriol. Parasitenk. Infektionsk. Hyg. Abt. 1, Orig. 205, 63-68.

[17] Layne, E.C. (1957) Methods Enzymol. 3, 447-454. [18] Laemmli, U.K. (1970) Nature 227, 680-685, [19] Schulz-Harder, B., Swida, U. and K~lufer, N. (1979) Bio-

chim. Biophys. Acta 465, 173-182. [20] Lenney, J.F. (1975) J. Bacteriol. 122, 1265-1273. [21] Jones, E.W. (1977) Genetics 85, 23-33. [22] Lenney, J.F. and Delbec, J.M. (1967) Arch. Biochem.

Biophys. 120, 42-48. [23] Wolf, D.H. (1980) Adv. Microbial Physiol. 21,267-338. [24] Meussdorfer, F., Tortora, P. and Holzcr, H. (1980) J. Biol.

Chem. 255, 12087-12093. [25] Hershko, A. (1983) C¢11 34, 11-12. [26] Ciechanova, A., Finley, D. and Varshavsky, A. (1984) J.

Cell. Biochem. 24, 27-53. [27] Bond, U. and Schlesinger, M. (1985) Mol. Cell. Biol. 5,

949-956. [28] Ozkaynak, E., Finley, D. and Varshavsky, A. (1984) Na-

ture 312, 663-666. [29] Schulz-Harder, B. and Kl~cherer, C. (1980) Z. Naturforsch.

35c, 168-170.

induction of a proteinase by heat-shock in yeast

Documents