BIOCHIMIE, ]984. 66, 71-74 Br/ ves communications

Glutathione Metabolism in Yeast Saccharomyces cerevisiae. Evidence that y-glutamyltranspeptidase is a Vacuolar Enzyme.

Charles J. JASPERS and Michel J. P E N N I N C K X .

Laboratoire de Microbiologie. Facultd des Sciences, Universitd Libre de Bruxelles. c /o Institut de Recherches du CERIA, 1, avenue E. Gyson. B-I070 Bruxelles, Belgique.

(Recu le 21-11-1983. accept~e le 13-1-1984~.

R6sum6 - - Une premi&e expdrience a montr~ que la ?-glutamyhranspeptidase de S. cerevisiae est associde ~ une fraction particulaire obtenue par centrifugation diffdrentieile. Uhdrieurement. nous avons montrd que cette activit( enzymatique suivait fid~iement la distribu:ion de marqueurs vacuolaires. La y-glutamvltranspepndase de ;evure pourrait ~tre impliqude dans les dchanges d'amino-acides entre le cytoplasme et le milieu vacuolaire.

Mots-cl~s : glutathion / y-glutamyltranspeptidase / vacuoles / levure.

Summary - - In a first experiment we have shown that S. cerevisiae [3-glutamyltranspeptidase is associated with a particulate fraction obtained by differential centrifugation. We have subsequently shown that this enzyme activity followed accurately the distribution o f vacuolar mar.k_prs, liberal.ion o f vacuoles was carried out b;, mechanical dis,'~aption of spherop!ast under isotonic conditions and the vacuoles were purified by centrifugation o f Ficoli gradients. Yeast [3-glutamyitran~peptidase could be implicated in the exchanges o f amino acids between the cytoplasm and the vacuolar sap.

Key-words : Glutathione / y-glutamyitranspeptidase / Vacuoles / Y~s t .

I n t r o d u c t i o n

[3-Glutamyltranspeptidase (E.C 2.3.2.2) is the only well characterized enzyme able to degrade glutathione in vivo [l]. The intracellular level of the enzyme in S. cerevisiae depends on the supply of amino acids as nitrogen sources [2]. The role of this enzyme in the translocation of amino acids is currently debated for animal cells [l, 3] and

0 To whom all correspondence should be addressed.

yeast [4,5]. Recently, we have shown that S. cerevio siae ~-glutamyltranspeptidase is a glycoprotein that can promote in v~vo transpeptidation ~i~aspers et al., submitted). One of the question~ raised during this investigation was the cellular !oc~c,n of this enzyme in yeast.

in this paper evidence is presented indicating that [I-glutamyltranspeptidase is associated ,~:ith a cellular fraction which has the characteristics of vacuoles.

72 C.J. Jaspers and M.J. Penninckx

M~.teria!s an¢ m e t l o d s

2.1. Organism and growth conditions

Sacharomyces cerevisiae, El 278b [6] was grown with :_-glutamate as sole nitrogen source [7]. The cells in the logarithmic phase of growth were harvested by centri- fugation (4 000 × g).

2.2. Enzyme assays

S. cerevisiae ¥-glutamyitranspeptidase was estimated as previously described [2]. The following vacuolar markers [7]: were used a-mannosidase [8], leucine aminopeptidase [9], alkaline phosphatase [10] and protease B [Il l Citrate synthase [12] and malate dehydrogenase [13l were chosen as mitochondrial markers and glucose-6-phosphate dehydrogenase [14] as cytosolic enzyme. The samples of the cellular frac- tions described below were subjected to ultrasonic waves (10 min. at 15 kHz) before testing the enzyme activities. Proteins were determined by Lowry's proce- dure [ ! 51.

2.3. Cell fractionation and preparation of vacuoles

2.3.1. Spheroplastsformation. Unless otherwise sta- ted, all c.perations were performed at 4oC. About 1 g of wet weight yeast cells, obtained as described above, were washed twice with distilled water and resuspen- ded in 3.5 volumes (vol/wt of wet cells) of a 0.1 M Tris(hydroxymethyl)aminomethane-hydrochloride buf- fer (pH 8.0) containing 0.5 M 2-mercaptoethanol and 0.1 M Na ethylene diamine tetraacetate. The cell sus- pension wa_~ incubated for 30 min. under slow aaitation and further harvested by centrifugation (~5min, 4,500 x g). The pellet was washed twice with distilled water and once with a 20 mM KH2PO, buffer (pH 6.5) containing !.1 M sorbitol and 0.5 mM CaCI2 (buffer S), centrifuging each time at 4,500 x g between the was- hing operations. The recovered cells were resuspended in four volumes of buffer S supplemented with 0.5 mM phenylmethane sulfonyl fluoride and 6 mg zymolyase (type 5000 -- Seikagaku Kogyo Co., Ltd) / g of wet cells. This preparation was incubated at 30oC under gentle agitation and the formation of spheroplasts was followed by phase contrast microscopy with a green filter. As judged by an hematocytometric count, al',o':t 15 rain are necessary for a 85 per cent conversion of yeast cells into spheroplasts. The spheroplasts were recovered by a l0 min centrifugation at 2,0(;0 x g and washed once with buffer S.

2.3.2. Spheroplasts disruption and fractionation. The pelleted spheroplasts were resuspended in 3 volumes (voi/wt) of a 20 mM KH2PO4 solution (pH 6.5) sup- plemented with 18 per cent Ficoll (type 400 -- Sigma) and ! mM CaCl2 (buffer L). Disruption of the sphero- plasts was performed by hand in a Potter-Elvehjem Teflon-glass homogenizer (type 1561 ! 54 -- Belgolabo,

S.A.) with ten strokes of the pestle. Unbroken cells and heavy debris were pelleted by centrifugation at 750 x g for 10rain. The supernatant was centrifuged at 40,000 x g for 60 min. in order to separate fractions referred to as "soluble fraction" and "particulate fraction".

2.3.3. Preparation of vacuoles. The "'particulate fraction" was gently resu.~pended in 5 volumes (vol/wt) of buffer L contaihir, g only I 1 per cent Ficoll. This suspension was layered on the top of 15 volumes of buffer L containing 14 per cent Ficoli and centrifuged for 70 rain. at 3,000 x g A vacuole enriched fraction was found at the interfa~-e between the two layers and was recovered with a curved tip Pasteur pipette. Vacuoles were also observed in the pellet formed at the bottom of the centrifugation tube but appeared as entangled with membranous elements.

Other methods of isolating vacuoles [7, 10, 191 failed to give results with our S. cerevisiae strain.

The vacuole enriched fraction obtained as described above was diluted with 10 volumes of buffer L contai- ning 13 per cent Ficoll and centrifuged for 60 min. at 6,000 x g. The pellet obtained consist essentially of vacuoles which appeared as darkish rose-violet round objects, as seen under phase contract microscopy with the green light of an interference filter.

Results

TABLE I

Enzyme activities in isolated vacuoles as compared with the enzyme activities in the total spheroplast lysates ~°J.

Enzyme Specific relative • " - ( a )

actwtty

"/-glutamyltranspeptidase 5.5

Indicative of vacuoles ct-mannosidase 4.8 alkaline phosphatase 6.1 leucine aminopeptidase 6.2 protease B 12.9

Indicative of mitochondria citrate synthase 0.23 malate dehydrogenase 0.27

Indicative of soluble fraction glucose-6-phosphate dehydrogenase 0.02

(a) ratio of isolated vacuoles/specific activity in spheroplast lysate. Specific activities of enzymes (nmol. min-~.mg pro- tein -j) in the spheroplast lysate were: y-glutamyltranspepti- dase, 2.3; ¢t-mannosidase, 0.26; alkaline phosphatase, l.l; leucine aminopeptidase, 4.3 : protease B, 0.04; citrate synthase, 0.08; malate dehydrogenase, 0.46; glucose-6ophosphate dehy- drogenase, 0.34.

BIOCHIMIE, 1984, 66, n ° I.

Location of y-glutamyhranspeptidase in yeast 73

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, - -

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2

15 o t~ .

o

5 ,-e

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1.5

I

T

/ / / / / /

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AP

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cs

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_ , ' / / . , , ' " . J / J J

50 100 0 50 100 Retotive protein(%}

FIG. I. - - Subcelhdar distributions for marker enzymes and y-glutamyitranspeptidase hi Saccharomyces cerevisiae.

Cell-free extracts were prepared, the fractions separated and enzyme activities est imated as described in Materials and Methods. The data are plotted as histograms of relative specific activity versus relative iJrotein [16] with the SEM for specific activity indicated by the lines on the b.~fs. The data for the particulate fraction is indicated as , - . . . . and as r : , for the 40.000 x g soluble fraction. The activities of the enzymes in the spheroplast lysate are reported in table I. The extent of yield of activities in the two fractions obtained by differential centrifugation were : alkaline phosphatase (AP), 95 ___ 12 per cent; citrate synthase (CS), 102 +_ 6 p e r cent: glucose-6-phosphate dehydrogenase ( G D P H ) 92 ___ 7 per cent: y-glutamyltranspeptidase (y-GT). 101 + 7 per cent. In u,,-" cases the results given are ,ho...~ means . . . . . . . . . . . . . . . . . + ql:M af threa ~xnerimenlS_r . . . . . .

3.1. Association of y-glutamyitranspeptidase with the 'particulate fraction"

A first approach to the definition of the subcellular location of y-glutamyltranspeptidase in S. cerevisiae was provided by fractionation of the cell-free extract by differential centrifugation, as described in Materials and Methods. This procedure resulted in the fractionation of the extract into a 40,000 x g "'soluble fraction" and in a "particulate fraction". Analysis of the enzy- mic activities and protein contents of these frac- tions gave the results shown in figure ! when plotted according to de Duve [16]. The y-gluta- myltranspeptidase appeared as a particulate en- zyme that followed the distribution of alkaline phosphatase, a typical vacuolar marker [7]. This result prompted us to prepare purified vacuoles.

BiOCHIMIE, 1984, 66, n ° I .

3.2. Location of S. cerevisiae y-glutamyitrans- peptidase in vacuoles

Enzyme activities in isolated vacuoles as com- pared with the enzyme activities in the total spheroplast lysate are shown in table I. Activity of glucose-6-phosphate dehydrogenase, which is the soluble fraction indicator, is barely detectable in the isolated vacuoles. Small amounts of citrate synthase and malate dehydrogenase, both mito- chondrial markers, are present :m the preparation, but high activities of the vacuolar indicators and of y-glutamyltranspeptidase are found in the same preparation. This result led us to conclude that y-glutamyltranspeptidase is most probably associated with the vacuoles.

74 C.J. Jaspers and M.J. Penninck.x

Discussion

Histochemical studies of animal tissues indi- cate substantial y-glutamyitranspeptidase activity in the membranes of cells exhibiting secretory or absorptive functions [!]. From those observations and others it was suggested that transpeptidase could be plausibly integrated in a translocation system for amino acids [171. More than 80 per cent of the total soluble amino acid pool in a yeast cell is located in the vact, oles [18] and it has been shown that exchanges between the cyto- plasmic and vacuolar contents are regulated by specific carders II9]. Transport of amino acids in erythrocytes containing artificia!!y implanted ~,-glutamyltranspeptidase was shown to be media- ted by this enzyme [20]. Giutathione, the natural substrate of ~,-glutamyltranspeptidase, is present at a concentration of about 5-10mM in the vacuole sap (Jaspers & Pennincks, unpublished). This seriously opens the possibility that y-gluta- myltranspeptidase could be involved in the va- cuolar transport. This hypothesis is currently under investigation in our laboratory.

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BIOCHIMIE, 1984, 66, n ° !.