158 Biochimica et Blophvsica Acta, 716 (1982) 158 168 Elsevier Biomedical Press

BBA 21114

REGULATION OF ~ - G A L A C T O S I D A S E S Y N T H E S I S IN S A C C H A R O M Y C E S CEREVISIAE AND EFFECT OF CERULENIN ON T H E S E C R E T I O N OF T H I S ENZYME

JOSE P. MARTINEZ, M. VICTORIA ELORZA, DANIEL GOZALBO and RAFAEL SENTANDREU

Departamento de Microbiologia. Facultad de Farmacia. Uni~ersidad de Valencia At~da. Blasco lbahe: 13. Valencia 10 (Spain)

(Received July 24th, 1981) (Revised manuscript received January 22nd, 1982)

Key words: c~.Galactosidase regulation; Cerulenin," En=vme secretion; (S. cerevisiae)

Saccharono'ces cerevisiae 136ts MEL10 (a thermosensitive mutant whose RNA synthesis is inhibited at 37°C but is normal at 23°C), when grown at 23°C in the presence of galactose, melibiose or L-arabinose, these cells synthesize ~-galactosidase mRNA. in the simultaneous presence of both galactose and glucose the transcription of a-galactosidase mRNA is blocked. Glucose also interferes with mRNA translation, but the degree of inhibition depends on concentration and time of addition of the hexose to induced cells. It has been found that the final concentration of ~-galactosidase produced by induced cells when transferred at the non-permissive temperature (37°C) is inversely proportional to the incubation time in glucose. Cerulenin inhibits lipid formation on growing yeasts, but protein synthesis and selective permeabili~ are not affected. The antibiotic partially inhibits secretion of ~-galactosidase with a parallel accumulation of this enzyme in membranous structures, specially at the level of the plasma membrane. Induction of a-galactosidase or cerulenin addition to growing cells, results in changes in the polypetide composition of the plasma membrane.

Introduction

Several enzymes are found associated with the cell wall of Saccharomvces cerevisiae and consider- able attention has been devoted to the problem of the secretion of these enzymes across the cell mem- brane. In order to understand better the mecha- nism which regulates wall formation we have un- dertaken the study of the processes involved in the synthesis of a-galactosidase (~- D-galactoside galactohydrolase, EC 3.2.1.22). This enzyme was initially described an an adaptative one [1] whose synthesis was dependent on the presence of melibiose in the medium. Subsequently, Lazo et al. [2] showed that galactose, which is part of the melibiose molecule, is able to induce the synthesis of the enzyme in Saccharomvces carlsbergensis. The cellular location, the kinetics and the struct-

0304-4165/82/0000 0000/$02.75 1982 Elsevier Biomedical Press

ural characterization of the purified a-galactosidase was also described [2,3].

Our group has recently studied the regulatory mechanisms involved on the biosynthesis of some extracellular catalytic glycoproteins and found that in the case of invertase and acid phosphatase the presence of glucose or inorganic phosphate re- presses their synthesis, respectively. The effect of repression by glucose on invertase synthesis occurs at the level of both transcription and translation [4], though recently it has been suggested that it takes place at the latter level [5]. Glucose also produces a decrease in the stability of mRNA but has no effect on the secretion of invertase. Regard- ing the regulation of acid phosphatase synthesis by Pi it seems that high concentrations of Pi inhibit enzyme formation at the level of translation. P~ does not block transcription instantaneously until

a certain concentration is reached in the cytoplasm [6].

In the present work we have investigated the regulation of the biosynthesis of a-galactosidase, an inducible enzyme whose synthesis is regulated by catabolite repression, in the mutant S. cerevisia

136tS MEL10, whose RNA synthesis is inhibited at 37°C but is normal at 23°C.

The enzyme is induced by several sugars. It is present in two forms and evidence is also reported which suggests that inhibition of enzyme synthesis by glucose takes place at the level of transcription but not at the level of translation.

On the other hand, there is evidence that in fungi different membranous structures are in- volved in the transport and secretion of wall materials and glycoproteins [7-11], similar to that which occurs in mammalian and plant cells. As in these systems, glycosylation of fungal glycopro- teins takes place through lipid intermediaries [12- 17]. Several studies have confirmed that the glyco- sylation process starts at the level of the nascent peptide chain [ 18-21 ] and since the enzymes trans- ferring the glycosyl groups are also associated to membranes, it can be assumed that yeast exocellu- lar enzymes are glycosylated at this level. Finally, for the release of proteins from the cell, a fusion process between the carrier vesicles and the cyto- plasmic membrane is required, probably at specific sites of the plasmalemma for each type of vesicle. From all these facts the fundamental role of mem- branes in these processes may be deduced.

In an other sense, it has been suggested that there is a relationship between lipid synthesis (es- sential components of cellular membranes) and protein secretion as can be deduced from studies carriers out On bacteria. With respect to this hy- pothesis, it has been demonstrated that antibiotic cerulenin, which inhibits synthesis of lipids [22-24], also blocks production of exocellular enzymes in bacteria [25-29] and mitochondrial inner mem- brane enzymes is S. cerevisiae [30].

In the present work we have studied the effect of cerulenin on the synthesis and secretion of a-galactosidase in S. cerevisiae - 136ts MEL10, and have found that the antibiotic partially inhibits the secretion of enzyme as a consequence of the accu- mulation of enzyme at the level of the cell mem- branous structures, particularly that in the plasma

159

membrane. We also report that cerulenin produces changes in the plasma membrane polypeptide composition.

Material and Methods

Chemicals. Lyophylized concanavalin A, a- methyl-D-mannoside, deoxyribonuclease (DN 25), bovine serum albumin, Coomassie brilliant blue R, mercap toe thano l , N-methy l -N-n i t ro-N-n i t ro- soguanidine, dithiothreitol, p-nitrophenyl-a-D- galactoside, p-nitrophenyl-a-D-glucoside, reduced glutation and cycloheximide were purchased from Sigma Chemical Co. (St. Louis, USA). Zymoliase was from Kirin Brewery (Gumma, Japan). Glusu- lase was purchased from Endo Laboratories Inc. (USA). Cerulenin was obtained through Makor Chemicals (Jerusalem, Israel). Acrylamide, N,N' - methylenebisacrylamide, N,N, N', N'-tetramethyl- ethylenediamine and sodium dodecyl sulphate were purchased from Bio-Rad(Richmond, USA). Am- monium persulphate was from Merck (Darmstadt, F.R.G.). [U-14C]Sodium acetate (spec. act. 58.6 mCi /mmol) and U-14C labeled protein hydro- lysate (spec. act. 59 mCi /mmol) were obtained from Amersham International (Amersham, U.K.). Standard proteins electrophoresis calibration kit was from Pharmacia Fine Chem. (Uppsala, Sweden). All other reagents were of analytical grade.

Yeast strains. The organism used was Sac- charomvces cerevisiae 136Is MELI0. This strain was obtained by crossing of S. cerevisiae 136ts and S. cerevisiae X14. S. cerevisiae 136ts (a ther- mosensitive mutant in which m R N A synthesis is blocked when incubated at 37°C derives from strain A364A by treatment with N-methyl-N-nitro- N-nitrosoguanidine. Both strains, S. cerevisiae A364A and S. cerevisiae X I4 were kindly supplied by Dr. L.M. Hartwell (University of Washington, Seatle, USA).

Genetic procedures. The above two strains were mixed to allow mating, and then plated on Wickerham medium [31]. From this medium di- ploids were isolated and inoculated in the pre- sporulation medium described by McCusker and Haber [32]. After 24h of growth the cells were washed twice with sterile water and then resus- pended in the sporulation medium (only contain-

160

ing 1% potassium acetate as carbon source in distilled water). After sporulation, the cell walls of asci were digested with Glusulase. Tetrads were then dissected onto plates containing medium 1 (1% yeast extract, 1% dextrose, 1.5% agar per liter) as described by Mortimer and Hawthorne [33]. After germination and growing, segregants were picked onto master plates and scored on solid medium containing per liter of distilled water, 1% melibiose as carbon source, 1% yeast extract and 1.5% agar. Incubation was carried out at 23 and

37°C. Growth conditions. Cells were grown in liquid

medium 1 in a rotatory shaker (New Brunswick Scientific Co., USA) at 250 rev. /min. Cells (1.6 mg dry wt.) were used to inoculate 500 ml Erlenmeyer flasks with 150 ml medium and the crop was harvested after about 18-20 h incubation at 23°C (early exponential phase). Induction of a-galactosidase was carried out in a medium con- taining per liter of water, 1% yeast extract and 1% galactose as carbon source. S. cerevisiae ~36ts MEL10 was maintained on slants of medium 1 and subcultured periodically.

Enzyme assays. (A) a-Galactosidase activity: This assay was based on the hydrolysis of p- nitrophenyl-a-D-galactoside according to the pro- cedure described by Lazo et al. [3]. One unit (U) of a-galactosidase was defined as the amount of en- zyme which hydrolyses 1/~mol of substrate in 1 min at 30°C in 0.25 M acetate buffer, pH 4.5, contain- ing 25 mM substrate. (B) a-Glucosidase activity: In this case, activity was assayed by measuring the p-nitrophenol released from p-nitrophenyl-a-D- glucoside by the enzyme activity according basi- cally to themethodology of Halvorson and Ellias [34]. In the case of a-glucosidase (intracellular enzyme), or when necessary in the case of a- galactosidase (in order to determine the intracellu- lar levels of activity of this exocellular enzyme), determinations were carried out in cells permeabi- lized with toluene/ethanol. The procedure is as follows: cells, previously washed twice with dis- tilled water, were resuspended in a small volume of the adequate buffer depending on the enzymatic assay to be performed afterwards, and then 1% (in volume) of a mixture toluene/ethanol (1:1 v /v ) was added. After 5 min vigorous shaking in an Omnimixer (Lab-Line, USA) cells were washed 3

times with 2ml each of chilled buffer (65 mM phosphate buffer, pH 7 for a-glucosidase assay, or 0.5 M acetate buffer, pH 4.5 for a-galactosidase

assay). Preparation ofprotoplasts. Protoplasts were pre-

pared according to Santos et al. [35] with minor modifications. Cells (10 mg, dry wt.) were col- lected in the early stationary phase, washed twice with chilled sterile water and resuspended in 1 ml of 5 mM dithiothreitol in 0.1 M Tr is -HCl /5 mM EDTA, pH 8, for 60 min at 28°C. Cells were then washed twice with chilled water and 2 ml of 1 M sorbitol solution containing 1 m g / m l of Zymoliase added to the suspension. Cells were converted into protoplasts within 20 30 min and after centrifuga- tion at 2000 × g for 1 min they were washed twice with 50 mM Tris-HCl, pH 7.4, containing 0.9 M sorbitol and 10 mM MgSO 4.

Isolation of the plasmalemma. The isolation of plasma membrane was carried out according to the low speed centrifugation procedure described by Santos et al. [35]. All operations were carried out at 0°C. Internal membranes were obtained, after plasmalemma isolation, by centrifugation of supernatant at 40000 × g for 40 rain in a Beckman J2-21 Refrigerated Centrifuge. The plasmalemma and internal membranes fractions were resus- pended in 0.2 ml of 0.5 M acetate buffer, pH 4.5, and stored frozen at - 3 0 ° C .

Protein determination. Protein content of the plasma and internal membranes was determined by using the Folin-Ciocalteau reagent [36] with bovine serum albumin as a standard. Proteins were solubilized in sodium deoxycholate and ab- sorbance was measured at 660 nm in a Perkin- Elmer 55B spectrophotometer.

Analytical polyaco'lamide gel electrophoresis. Analytical gel electrophoresis in sodium dodecyl sulphate (SDS) was performed on 8 and 9% acrylamide gels by the system described by Laem- mli [37]. Samples containing approx. 100 /Lg pro- tein were treated with a solution of 0.8 g SDS, 4 g glycerol and 2ml fl-mercaptoethanol in 10 ml 0.25 M Tris-HCl (pH 6.8). The ratio between sam- ple volume : solubilizing solution volume added was 2 : 1. Bromophenol blue was used as a tracking dye and the proteins were detected as described by Fairbanks [38].

Gels were scanned at 540 nm in a Vitatron

T L D 100 densitometer, and the relative areas un- der the peaks corresponding to the proteins mea- sured. In all experiments a plot relating molecular weight versus relative mobility was charted by using proteins of known molecular weight. The following molecular weight standards were run in parallel: phosphorylase b (94000), bovine serum albumin (67000), ovalbumin (43000), carbonic anhydrase (30000), trypsin inhibitor (20 100) and a-lactalbumin (14400).

Other experimental procedures. (A) Protein synthesis: Protein synthesis was monitored by means of UJ4C-labeled protein hydrolysate (spec. act. 59 m C i / m m o l ) incorporated into the growing cells. Samples (1 mi) were taken from the cultures and mixed with an equal volume of 10% ice-cold trichloroacetic acid. The suspensions were filtered through glass fiber discs (Whatman G F / C , What- man, UK), filters were dried at 80°C, and after- wards the radioactivity retained was determined. (B) Lipid extractions: Cells growing in medium 1 were transferred (400 ~ g / m l dry wt,) to fresh medium I containing cerulenin (cerulenin was added from a solution in 96% ethanol containing 1 mg cerulenin/ml. This stock solution must be stored below 0°C). After 1 h incubation at 23°C, 10 ffCi of sodium [U-14C]acetate (spec. act. 58.6 m C i / m m o l ) were added. Cells were incubated at 23°C for 3 h and then protoplasted. Protoplasts were suspended in 0.1 M Tris-HCl (pH 7.4) con- taining 50 mM MgSO 4 and DNAase (1 mg/ml ) , and homogenized in a Lab-Line Omnimixer. The lysate was centrifuged at 2000 X g for 5 min to remove unbroken cells and then centrifuged at 40000 X g for 40 min in a Beckman JA-20 rotor of a Beckman J2-21 Refrigerated Centrifuge. Pellets (crude membranes) were resuspended in 0.5 ml cold distilled water and protein was determined from one aliquot (0.3 ml). Lipids in the residual suspension were extracted according to Rouslin [30]. The extractions were carried out under nitrogen at 40°C in the scintillation bottles di- rectly. After evaporating the organic solvents, 8 ml of scintillation liquid (containing 3.5 g 2,5-diphen- yloxazole and 50 mg 1,4-bis(2,4-methyl-5-phenyl- oxazolylbenzene) in 1000 ml toluene) were added to bottles and the radioactivity was determined in an Isocap/300 Nuclear Chicago liquid scintilla- tion spectrophotometer.

m

, j

ETJ

E t t 3

> "b 0

> .

L U

161

Synthesis of c~-galactosidase Influence of the carbon source. Synthesis of a-

galactosidase takes place in cells grown in glucose (not induced when transferred to media containing different carbon sources (Fig. 1). Cells incubated in galactose showed the highest activity but E- arabinose also has an important inductor effect (though the enzymatic activity detected in this case is only 50% of that observed with galactose) in spite of the fact that it is a non-metabolizable sugar. Finally, the activity found in melibiose (the natural substrate of the enzyme) is lower than in galactose or L-arabinose, perhaps due to the catabolite inhibition of glucose released by the hydrolysis of melibiose by the enzyme.

The lag period between the transfer of cells to the different media, and the appearance of activity represents the time needed to synthesize and trans- late the specific mRNA, and to export the enzyme, from the cytoplasm to the periplasmic space or to the cell wall. This period was from 2.5 to 3 h.

o.5

0.3

o.1

O{ o 2 4 6 8

Results

I ncuba t ion t ime (h) Fig. 1. Effect of carbon sources on external a-galactosidase synthesis. Cells grown up to early exponential phase in medium 1 were transferred to the same medium but containing the following sugars as carbon source: galactose (O), L-arabinose and glycerol (A), melibiose and glycerol (11), galactose plus cycloheximide (O), galactose but incubating at 37°C (O) and glucose (O). Samples were taken and the enzymatic activity measured.

162

~ o~ 0'5 f

E v~

m 0.3 >.

D O

OJ

0 0 2 4 6

Incubation time (h )

Fig. 2. Induction of intra and extracellular ,~-galactosidase. Cells were grown up to the early exponential phase in medium containing glucose and then resuspended in galactose medium. Samples were removed at the indicated times and the activity in buffer-washed cells (O, extracellular enzyme) and toluenized cells (O, intracellular and extracellular enzyme) was measured. Intraeellular enzyme levels are also indicated (A).

Effect of non-permissive temperature and of cyclo- heximide upon the ~vnthesis o f enzyme in induced cells

In order to establish the turnover of the mRNA that coded for the synthesis of a-galactosidase, the kinetics of enzyme synthesis was determined in conditions in which the synthesis of new molecules of mRNA was blocked. A culture of S. cerevisiae

~36ts MELI0 was incubated in a medium contain- ing galactose, at 23°C for 4h to induce enzyme synthesis. Afterwards, an aliquot of this culture was incubated at 37°C in order to block synthesis of new mRNA molecules and then the a-galac- tosidase activity was measured in these conditions. The enzyme synthesis continued at a decaying rate for 30 - 45 min (Fig. 3), and from this moment the activity reached a plateau. In order to ascertain the origin of this activity and to determine if it corre- sponded in part to the activation of a proenzyme, an aliquot of the original culture was incubated at 23°C but in presence of cycloheximide; that is to say, in conditions of absence of protein synthesis. Addition of cycloheximide causes a quick stabili-

, The relationship between mRNA formation and enzyme synthesis was established by incubating cells in medium with galactose at 37°C (the non- permissive temperature). In these conditions en- zyme activity was not detected. A similar effect was observed when cells were incubated at the permissive temperature (23°C) but in presence of cycloheximide (Fig. 1), though in this case, the lack of activity is due to the inhibition of protein synthesis.

Cellular location of enzyme during the induction process

During the induction process of a-galactosidase the appearance of intra and extracellular activities were followed. Initially, (1.5 h), only intracellular activity was detected, while extracellular activity was detected 1 h later. Intracellular activity in- creased for a period of 60-90 min, and then remained constant. This moment corresponded to the apperance of extracellular enzyme whose activ- ity increased constantly under the experimental conditions (Fig. 2).

o41 k ~

>- 0.2 >

> .

N c

ua 01 I

0 30 60 90 120 Incubation time ( rain )

Fig. 3. Effect of RNA and protein inhibition on a-galactosidase ~ynthesis. Cells grown in medium 1 were collected by ccntrifu- gation, resuspended in galactose medium and incubated for 4 h at 23°C. The culture was then divided in three aliquots and one of them was incubated at 23°C (0), the second one at 37°C ([13) and the third one at 23°C in presence of cycloheximide (A).

163

zation of activity. The antibiotic, at the concentra- tion used, inhibits about 97% of protein synthesis in S. cerevisiae -136tS MEL10 (unpublished data).

Synthesis of a-galactosidase Effect of glucose. Addition of glucose to a cul-

ture of previously induced cells inhibited synthesis of c~-galactosidase. In order to establish the level of this effect, the following experiment was carried out: cells grown in medium containing both galactose and glucose, were transferred to a medium containing galactose alone. Under these conditions, enzyme formation at 23°C followed the kinetics of normal induction, i.e., activity ap- pears after 2.5 h. This result suggests that c~- galactosidase mRNA was not formed during the period that the cells were incubated simulta- neously with galactose and glucose.

When cells were incubated in the presence of galactose plus glucose, and then transferred to a medium with galactose, but at 37°C, no enzymatic activity was detected. These results suggest that if the mRNA that coded for the o~-galactosidase had

0.3 E

d

~ 0.2

0.1

>..

j

.n

A

k l J

0 i I I I 0 30 6 0 9 0 120

I n c u b a t i o n t i m e (nn in ) Fig. 4. Effect of glucose on a-galactosidase synthesis at the level of translation. A culture of previously induced cells was divided into three aliquots and glucose (E3, • ) or cycloheximide ( A , • ) were added in two of them. Controls without addition of glucose or cycloheximide are represented (O, O). Samples were taken at the indicated times and the activity in buffer washed cells (O , ~ . E~) and in toluenized cells (O, • , I ) was de- termined.

been synthesized during incubation in the presence of the two sugars, it would have been translated when incubated in galactose at 23 as well as 37°C. for while transcription is blocked at the non-per- missive temperature, translation of the already present mRNA is not blocked. These results sug- gest that glucose could interfere in the process of DNA transcription, either at the level of c~-galac- tosidase synthesis, or at the level of the galactose transport protein, but further work is needed to verify these hypotheses.

The effect of glucose at the level of mRNA translation process, has been studied in cells that have previously synthesized mRNA, and conse- quently they are actively expressing them. A cul- ture of induced cells was divided into four aliquots and to each of them different concentrations of glucose or cycloheximide were added. The activity

q) (J

E

c~

E U

W

0.4

02

OQ 0 1 3

Incubation time (h )

Fig. 5. Secretion of ~-galactosidase. Effect of ccrulenin. Cells grown in glucose were inoculated into flasks containing galac- rose (A), and galactose plus cerulenin, 10 (O), 25 ( • ) and 50 ( , ) ~g /ml . Samples were taken at the indicated times and the external a-galactosidase activity was determined in cells. 50/~1 96% ethanol were added to the control culture.

164

was determined both in whole cells as well as in toluenized ones. In the presence of glucose there is a decrease in the activity detected externally after some 45 min, while the effect of cycloheximide is detected more quickly (Fig. 4). In a similar way, the increase in the internal activity is inhibited almost immediately, while the effect produced by glucose is detected later on. The levels of basal activity (the Cells have been previously induced) was higher in the cells growing in the presence of glucose, than in those treated with the antibiotic (Fig. 4).

Effect of cerulenin upon secretion of a-galactosidase Cerulenin inhibits lipid synthesis on growing

cells, so it was of interest to test the action of this antibiotic on the synthesis and secretion of a- galactosidase in S. cerevisiae 136ts MELI0.

Fig. 5 shows that cells growing in galactose in the presence of different concentrations of the antibiotic show normal enzyme secretion, since external activity is detected after 2.5 h of induction process, though the activity detected in the pres- ence of cerulenin is lower than in cells growing in its absence. In the presence of i0 ~g /ml of anti- biotic, the activity detected was about 30 40% less than in the control while higher concentrations of cerulenin (25 and 50/tg/ml, respectively) cause a decrease of almost 60% in enzyme activity (Fig. 5).

Effect of cerulenin on protein synthesis, lipid synthe- sis and selective permeability of plasma membrane

In order to determine the mechanism responsi-

ble for the inhibition in external activity, a test of the effect of antibiotic on some physiological activ- ities of the cells was carried out. First, the effect on protein synthesis was studied and the radioac- tivity incorporated from a protein hydrolysate, uniformly labelled with ~4C, by cells in the pres- ence of the drug was the same as that incorporated by the control cells (not shown).

On the other hand, lipid synthesis, as de- termined by incorporation of [U-14C]acetate, was severely affected by the antibiotic (Table I). In fact, concentrations of 25 and 50 ~g /ml of cerulenin reduce the incorporation of [U- t4C]acetate between 92-93%.

Finally, an assay was made to see whether the antibiotic was affecting the selective permeability of the plasma membrane. For this purpose, a-glu- cosidase (an intracellular enzyme) activity was de- termined in cells treated with cerulenin. The activ- ity was only found after the cells were treated with small amounts of a toluene/ethanol mixture that destroys the permeability barrier of the cell, though a-glucosidase activity detected in cells incubated in the presence of cerulenin was remarkably lower.

Internal activity of c~-galactosidase Effect of cerulenin. After it was found that

cerulenin produces a decrease in the secretion of a-galactosidase and that this effect takes place in the absence of any detectable changes or altera- tions on protein synthesis and upon plasma mem- brane selective permeability, it was of interest to determine the effect of the drug on the internal form of the enzyme.

TABLE |

INCORPORATION OF [U)4C]SODIUM ACETATE BY SACCHAROMYCES CEREVISIAE CERULENIN

Values are 1 ml samples.

136ts MELI0 IN PRESENCE OF

Cerulenin (~g/ml) Lipids a (cpm) Protein ~ (p.g) cpm/p.g, prote in Incorporated radioactivitv (%)

-- 876 025 3 500 250 I (X) 25 65025 3 500 18.75 7.42 50 55600 3 500 15.88 6.34

cpm and protein values determined in 0.2 and 0.3 ml of a 0.5 ml suspension in distilled water of a crude membranes preparation respectively.

%"

E I_O

> .

>

E > . N c-

u.J

08--

i 04"--

O

0"_~ ~ = ~ ' ~ - - - - ~ z I 0 1 3 5

Incubat ion t ime (h )

Fig. 6. Induction of internal a-galactosidase. Effect of cerulenin. Cells were grown on medium I to early exponential phase and then transferred into flasks containing medium with galactose without (A) and with 25 (U) and 50 (O) # g / m l of cerulenin. Samples were taken at the indicated times and the enzyme activity was determined in toluenized cells.

As shown in Fig. 6, when enzymatic activity was assayed in toluenized cells, it was found that a-galactosidase formation reached a plateau after

165

about 4 h. In the presence of antibiotic, the kinet- ics of the intracellular enzyme were different: it increases linearly for the time the experiment was carried out (Fig. 6).

Subcellular locafization of a-galactosidase Effect of cerulenin. The results reported above

did not indicate in which subcellular localization accumulation of enzyme appeared.

In order to elucidate this, many cells were in- duced to synthesize the enzyme in both the pres- ence and absence of the drug, and after 6 h, the cells were protoplasted and subcellular fractions were obtained as described in Methods. Significant increases in activity were detected both in the plasma membrane (5-fold) and internal mem- branes (3-fold). On the other hand, no activity was detected in the cell sap (Table II).

From these results it appears that the inhibition of a-galactosidase secretion due to the antibiotic is coupled with its accumulation both in plasma and internal membranes.

Effect of cerulenin on protein composition of cell membranes

As shown before, the antibiotic produces the accumulation of a-galactosidase on cell mem- branes. Thus, it was of interest to find out if it was also responsible for changes at the level of the polypeptides found in the cell membranes.

The electrophoretic profiles of the protein com- ponents of the plasmalemma are shown in Fig. 7.

TABLE II

SUBCELLULAR LOCALIZATION OF a-GALACTOSIDASE. EFFECT OF CERULENIN

Samples containing 25 mg cells (dry wt.) were taken at the indicated times from cultures with or without cerulenin. Proteins are expressed in ~g/ml . Activities are expressed in/Lmol p-nitrophenyl released//~g protein. Cytosol activity was determined using 0.2 ml of supernatant from internal membranes isolation as enzymatic sample are found to be null. Cytosol protein was not determined. Internal membranes in the present context refer to all yeast membrane systems with the exception of the plasma membrane.

Cerulenin Induction time Plasmalemma (~g /ml ) (h)

Internal membranes

Protein '~ Activity b Protein ~ Activity b

- - 0 2200 0.7.10 4 2000 0 6 2000 O.g.lO -3 2660 1.42. I0 4

25 6 1700 4.7.10-3 IS(X) 4.53.10 4 50 6 2200 4.4.10 3 1 500 5.73.10 4

166

c

B

a~

gg I

D

Fig. 7. Densitometer tracings on SDS-8% polyacrylamide gels showing the polypeptide electrophoretic pattern of the compo- nents of the yeast plasma membrane of cells grown in glucose (A), after 6 h in galactose (B), and after 6 h in galactose in presence of cerulenin, 25 (C) and 50 (D) / tg /ml . The amount loaded in each slot was obtained from a similar number of cells.

The changes detected were the result of both the induction of a-galactosidase and the presence of cerulenin.

The apparent molecular weight of the detected bands ranged from 28000 to 180000 (28-180 kDa bands) with a predominance of the bands between 32 and 64 kDa.

After induction (6 h) either in the presence or absence of cerulenin an important increase in the intensity (75-100%) of the 45 and 48 kDa bands and the almost total disappearance of the 63 kDa band was evident (Fig. 7B, C and D). When cells were induced to synthesize the enzyme in the presence of cerulenin, changes in protein composi- tion of the plasma membrane, such as an intensifi- cation of the 32, 38, 47, 49 and 52 kDa poly- peptide bands, were also found (Fig. 7C and D).

Discussion

Cultivation of S. cereeisiae ~36ts MELI0 at 23°C in the presence of galactose, melibiose or L-arabinose stimulates synthesis of the mRNA coding for induced a-galactosidase. This is con-

eluded from the fact that the enzyme is not synthe- sized if the cells are transferred from a glucose medium to a galactose at the precise moment of incubation at 37°C. If a pool of mRNA was present and activated after the addition of galac- tose, this process would have also taken place at the non-permissive temperature. If the enzymic activity were the result of the activation of a proenzyme it would have appeared both at 37 or 23°C in the presence of cycloheximide.

The molecular mechanism of glucose repression is unknown but it could take place as we have already indicated for invertase [4,6] at several levels: (i) by inhibition of DNA transcription of ~-galactosidase mRNA, (ii) by blockage of the c~-galactosidase m R N A translation into the en- zymic protein or (iii) by allosteric inhibition of enzyme activity.

Little is known about the molecular mechanism which regulates this enzyme, though in the case of Saccharomyces carlsbergensis glucose produces a competitive inhibition that seems to be nonspecific since very high concentrations of the effector is needed [3],

Galactose induces synthesis of the enzyme as the result of translation of the specific mRNA but in the presence of both glucose and galactose neither mRNA nor the enzyme are formed. In the presence of galactose and glucose the transcription of a-galactosidase mRNA is blocked. This may be deduced from the fact that if translation had been the step inhibited after the removal of glucose, enzyme synthesis would have taken place at 37°C (Fig. 3). Though at the permissive temperature, synthesis of enzyme did occur, the kinetics of enzyme formation followed those expected of an induction process and not of a translation of m R N A already present in the cells (Fig. 3).

Another point at which higher concentrations of glucose could act is on the ribosomes, at the level of translation. In induced cells incubated at 23°C, the addition of glucose produces a decrease in the rate of synthesis (Fig. 4). When the activity formed by the cells was measured in toluenized cells (intra and extracellular enzyme) the effect of the addition of glucose halting enzyme synthesis was detected earlier than in whole cells (extracellu- lar enzyme); this effect of glucose is different from that produced by cycloheximide (Fig. 4) which took

place after only 30 min. This result indicates that glucose repression perhaps does not interfere with translation of the already present mRNA, or needs a higher concentration or a longer period of time.

The effect of cerulenin on a-galactosidase secre- tion also needs some consideration. This antibiotic inhibits the secretion of exocellular enzymes in bacteria [21-25]. In this work, S. cerevisiae has been taken as a model of secretion for eukaryotic cells, and we have found that a partial inhibition of a-galactosidase secretion is detected (Fig. 6), in parallel with an accumulation of the enzyme (Fig. 6) in the plasma membrane (Table II), in cells in which a-galactosidase has been induced in the presence of cerulenin. Since protein synthesis con- tinues and the total amount of activity detected (intra plus exocellular activity) is similar, both in the absence of presence of the drug (results not shown), the effect of antibiotic may be on the secretion mechanism either at the level of trans- port or at the level of fusion of carrier vesicles with the plasma membrane. In the latter case, this could be due to an alteration on the signals and recep- tors implicated in the secretory process. Cerulenin would also cause an uncoupling effect between energy formation and its utilization, this resulting in a lower rate of the secretory process.

In any case, the effect of cerulenine on c~- galactosidase formation in S. cerevisiae does not seem to be at the level of enzyme synthesis, as suggested in the case of bacteria.

Though the synthesis of lipids in the presence of cerulenin is dramatically reduced (Table I), the plasma membrane retains its selective permeabil- ity. This may be due to the low turnover of the lipids in the membrane. Therefore, it is to be expected that the more recently synthesized ones should be the more altered ones, as may be the case of lipids in membranes of vesicles involved in the transport and secretion of macromolecules.

On the other hand, as it involves a glycoprotein, a-galactosidase secretion may also be affected at the level of glycosylation. If the lipid carrier of glycosyi groups concentration drops, as a conse- quence of the action of cerulenin, glycosylation of proteins may be affected. Glycosylation is proba- bly a limiting step in the secretion process, existing in order to avoid degradation of the secretion proteins [5], at the level of the rough endoplasmic

167

reticulum. In this context, it has been found [9,39] that tunicamycin, an antibiotic that inhibits glyco- sylation at the level of the lipid carrier, also in- hibits, totally or partially, the appearance of dif- ferent enzyme activities.

Finally, it is important to point out that induc- tion of enzyme results in a significant increase in the 45 and 48 kDa polypeptide bands, and in disappearance of the 63 kDa band (Fig. 7B, C and D), bands corresponding to peptides found in the plasma membrane whose physiological function is unknown but might be related to galactose metabolism (further work in this line is at present being carried out in our department). Changes in protein composition found in the plasma mem- brane due to antibiotic action are manifested as an intensification of the 32, 38, 47, 49 and 52 kDa bands (Fig. 7C and D) which may correspond to secretion polypeptides suffering an accumulation phenomenon similar to that described in the case of a-galactosidase.

Acknowledgemenl

This work was supported in part by a grant from the Comisi6n Asesora de Investigacibn Cientifica y Tecnica of the Spanish Ministerio de Educacibn y Ciencia (Project No. 4593).

References

1 Lindegren, C.C., Spiegelman, S. and Lindegren, G. (1944) Proc. Natl. Acad. Sci. U.S.A. 30, 346-350

2 Lazo, P.S., Ochoa, A.G. and Gascbn, S. (1977) Eur. J. Biochem. 77, 375-382

3 Lazo, P.S., Ochoa, A.G. and Gasc6n, S. (1978) Arch. Bio- chem. Biophys. 191, 316-324

4 Elorza, M.V., Villanueva, J.R. and Sentandreu, R. (1977) Biochim. Biophys. Acta 475, 103-112

5 Chu, F.K. and Maley, F. (1980). J. Biol. Chem. 255, 6392- 6397

6 Elorza, M.V., Rodriguez, L., Villanueva, J.R. and Sentandreu, R. (1978) Biochim. Biophys. Acta 521,342-351

7 Van Rijn, H.J.M., Linnemans, W.A.M. and Boer, P. (1975) J. Bacteriol. 123, 1144-1149

8 Linnemans, W.A.M., Boer, P. and Elbers, P.F. (1977) J. Bacteriol. 131,638-644

9 Babczinski, P. and Tanner, W. (1978) Biochim. Biophys. Acta 538, 426-436

l0 Rodriguez, L., Ruiz, T., Villanueva, J.R. and Sentandreu. R. (1978) Curt. Microbiol. I, 41-44

I1 Esmon, B., Novick, P. and Schekman, R. (1981) Cell 25, 451-460

168

12 Babczinski, P. and Tanner, W. (1973). Biochem. Biophys. Res. Commun. 54, 1119 1124

13 Lehle, L. and Tanner, W. (1978) Eur. J. Biochem. 83, 563-570

14 Lehle, L. (1980) Eur. J. Biochem. 109, 589-601 t5 Trimble, R.B., Byrd, J.C. and Maley, F. (198(/) J. Biol.

Chem. 255, 11892-11895 16 Parodi, A.J. (1978) Eur. J. Biochem. 83,253-259 17 Parodi, A.J. (1979) J. Biol. Chem. 254, 10051-10060 18 Ruiz-Herrera, J. and Sentandreu, R. (1975) J. Bacteriol.

124, 127-133 19 Larriba, G., Elorza, M.V., Villanueva, J.R. and Sentandreu,

R. (1976) FEBS Lett. 71,316 320 20 Lehle, L., Bauer, F. and Tanner, W. (1977) Arch. Microbiol.

114, 77-81 21 Marriot, M. and Tanner, W. (1979) J. Bacteriol. 139, 565-

572 22 D'Agnolo, G., Rosenfeld, I.S., Awaya, J., Omura, S. and

Vagelos, P.R. (1973) Biochim. Biophys. Acta 326, 155 166 23 Vance, D., Goldberg, I., Mitsuhashi, O., Bloch, K., Omura,

S. and Nonmra, S. (1972) Biochem. Biophys. Res. Commun. 48, 649-656

24 Omura, S. (1976) Bacteriol. Rev. 40, 681 697 25 Caulfield, M., Chopra, I., Melling, J. and Berkeley, R.C.W.

(1976) Proc. Soc. Gen. Microb. 3, 91-92 26 Fishman, Y., Rottem, S. and Cirri, N. (1978) J. Bacteriol.

134, 434-439

27 Altenbern, R.A. (1977) Antimicrob. Agents Chemother. 11, 906 908

28 O'Connor, R., Elliot, W.H. and May, B.K. (1978) J. Bacteriol. 136, 24 34

29 Sanders, R.L. and May, B.K. (1975) J. Bacteriol. 123, 806-814

30 Rouslin, W. (1979) J. Bacteriol. 139, 502 506 31 Wickerham, L.J. (1946) J. Bacteriol. 52, 293-301 32 McCusker, J.H. and Haber, J.E. (1977) J. Bacteriol. 132,

180-185 33 Mortimer, R.K. and Hawthorne, D.C. (1969) Yeast Genet-

ics pp. 386-460 (Rose, A.H. and Harrison, J.S. eds.) in The Yeast, vol. I. Academic Press, New' York

34 Halvorson, H.O. and Ellias, L. (1958) Biochim. Biophys. Acta 30, 28-34

35 Santos, E., Villanueva, J.R. and Sentandreu, R. (1978) Biochim. Biophys. Acta 508, 39 54

36 Lowry, O.H., Rosebrough, N.J., Farr, A.I~. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275

37 Laemmli, U.K. (1970) Nature (gondon) 227, 680-685 38 Fairbanks, G., Steck, T .L and Wallach, D.F.H. (1971)

Biochemist~ 10, 2606-2617 39 Tkacz, J.S. and Lampen, J.O. (1975) Biochem. Biophys.

Res. Commun. 65, 248-257