BiochimicaelBu)ph'sicaActa l(17~;q1991187 ~95
c Ig91 ElsevierScience Puhlishcr,~B,V. Ol¢~7-4838/ql/g0351~ ADONIS 01~7483~,q(~12583
Purification and functional charactcrisation of the pyruvatc (monocarboxylate) carrier from baker's yeast mitochondria
( Saccharomyces ceret'isiae) M a c i e j J. N a l ~ c z *, K a t a r r ~ ' n a A . ~-ra~cz * a n d A n g e l o Az~'i ! slit te of Biochemi.gtt3 a~ I Molec da.." Ri ;fogy. Unitcr.~ityo[ Berilr'. Berm" (Switzerland
Key ~ord,,: P~jra~zltc(monocarbcixylatcc~.,ier Y¢;I',I; Mil~ch~mddt~n:'l~,n,p,~rt:Purillc~dion;RcconMitutitln
Isolated yeast mitochondria were subjected ~o solubilization by Triton X-l14 and the detergent e~tract was subsequently chromatographed cn dry hydrox3apatite. Purification of the yeast monocarboxylate (pyruvate) carrier was achieved by affinity chromatography on immobilized 2.cyano-,l-hydroxyeinnamate, as described previously for bovine heart mitochondria (Bolli, R., Nalgcz K.A. and Azzi, A. (1989) J. Biol. Chem. 264, 18024-18030L The final preparation contained two polypeptides of apparent molecular mass 26 and 50 Id)a. The yeast ca,~er appeared to be less abundant, but more active, than the analogot,.s protein from higber eukaryotes. The carrier was able to catalyse the pyruvate/pyruvate and pyh'uvate/acetoacetate exchange reactions, both reactions being sensitive t~ cyanocinnamate and its derivatives, to phenylpyruvate and to mcrsalyl and p~chlorGmercuribenzoate. In the pyruvate/acetoacetate exchange reaction (200 mM internal acetoacetate, enzymatic assay), the Km value for external pyrovate was found to be 0.8 mM and the Vma~ 135/zmol/min per mg protein. Among other substrates of the yeast carrier, all transported with .,imilar affinity and identical maximal velocity against acetoacetate, we identified 2-oxoisacaproate, 2-oxoisovalerate and 2-oxo-3-methylvalerate. Lactate was not translocated by this carrier with a measurable rate, neither ~ere di- or tricarboxylates.
Introduction The monocarboxy~ate (pyruvat¢) transporting system in mit(~hondria from higher eukaryotes catalyses an exchange reaction and a net, Jpl-~-dependcnt, uptake of pyruvate [ 1 3]. In liver, both reactions were shown to be c~*mpetitively inhibited by other 2-oxoaeids, especially those originating from branched-chain amino acids su~.h as leucine, isoleucinc and valine , and the uptake of 2-oxoisoc~tproic acid was demonstrated to bc
* Permanent address: Polish Academyof Sciences. Nencki Instilatt: of Experimental Biology,02-093 Warsaw. Poland. Abbreviations: DTE, 1,4-dithioerYthritol: DTT. 1,4-dirhio-ot.-threitol: EDTA, ethylenediamineletraaceticacid; KIC. 2-oxoisocaproic aci4; KP'. 2-oxoisovalericacid; KMV, 2-oxo-3-me,'hylvalericacid; LM. lauJ3"lmaltoside; M(,ps. 4-morpholinepropanesnll3htmicacid; pCMB, p-hydrox~mercuribenzoicacid (p-ehloromereuribenzoate). Co;tvspondence: M.J. Nat~cz. Instituteof Biochenlislry and Mnlecl*lar ~iology.Universityof Berr!e. BiJHstr~,e 28, C/-I-31}12Berne. Svdtzerland.
driven by )he proton gradient . In heart, however, the existence of a separate transport system for br:mchcd-cham 2-oxoacids has been postulated , which may indicate possible differences in the monocarboxylate carriers present in mitochondria from different tissues. The pyruvate carrier has been exlracted and partially purificd from bovim, heart  ~md rat liver mitochondria . Its final purification to homog~meity from the inner membrane of b,Jvine heart mitochondria was achieved by affinity chromatography and yielded a single pt)lypcptide with tin apparent molecular mass of 34 kDa [8,9]. It has been sho,,~,n that mitochondria from S a c c h a romyce~ ceret'isiue are abte to catalyse a ApH-dcpenden t uptake of pyruvate, sensitive to 2-cyano-3-hyd r ~ , c i n n a m a t e , and it has t',:,.'n postuJ;,ted that the same cartier may traJ,;!oeate other monocarhox3'lic oxoacids as well as lact~te . The carrier protein. however, has not been purified. Here wc present the purification proccdnre and the functiondl ct]:~racteristics of the pyruvatc transporting protein from baker's yeast mitochondria.
8~ Materials and Methods
Materials [l-t~C]Py;uvic acid, sodium salt, was obtained from Amcrsham lnlcrnatio~al. Triton X-114, 3-sn-phosphati,:y~cholinc from egg yolk (catalogue No. 61755), 1,4dithioeJylhfitol (aTE), 1,4-dithio-DL-threitol (DTT) and Ambedite XAD-2 were from Fluka. Sodium deox3,cholate, Mops, mannitol, sorbitol, p-hydroxymercuribenzoic acid (pCMB) and EDTA were from Merck. Yeast extract, peptone powder from meat, a DL-lactic acid 80% aqueous solution, agarose, pyruvic acid, L-lactic acid, succinic acid, 2-cyano-4-hydroxycinnamie acid, acetoacetic acid, 2-oxoisocaproic acid (KIC), 2-oxoisovaleric acid (KIV), 2-oxo-3-methylvaleric ac!d IKMV), phenyl methylsulfonyl fluoride, leupeptin, pepstatin A, NADH and bovine serum albumin wetc from Sigma. Sephadex G-25 medium and CNBr-aetivated Sepharose 4B were obtained from Pharmacia. Dowex I-X8, CI- form, 100-.200 mesh and hydroxyapatite (Bio-Gel HTP) were from Bio-Rad. Liquid scintillation cattail Ultima Gold was from Packard. All other chemicals were of analytical grade. Affinity resin with immobilized 2-cyano-4-hydroxycinnamate was synthesized as described in Ref. 8.
Growth of ~,'eastcells and preparation of mitochondria The cells of Saccharomyces cere~'isiae wild strain HRI-2A ÷, as well as of the porin-deficient mutant st:ain HRI-2A , were obtained from Prof. G. S~hatz (Biozentrum, Basel). The cells were grown on rich medium containing (per liter): 10 g yeast extract, 20 g peptone, 30 g glycerol, 1 g KHzPO 4, I g NH4CI, 0.5 g CaCI 2, 0.5 g NaCI, 0.75 g MgSO4, 1.5 ml of 1% FeCl 3 and 23 ml of 80% at.-lactate, at a final pH of 5.5. Mutant cells had to be preadapted to glycerol on solid rich medium before incubation of liquid cultures. Cells were grown aerobically, in the Aqua-Shaker (Kiihner AG, Switzerland), at 28°C and 180 min-i, for about 24-26 h (wild strain) or 30-32 h (mutant). Growth was monitored at 650 nm and continued until the absorbance of a cell suspension, diluted 1 : 1 with H20, reached about 1 absorbance unit. The usual yield was 5 g (wet weight)/1. For the isolation of mitochondria, cells were harvested by centrifugation (15 rain at 3000 xg, MSECoolspin 6 × 1 liter swing-out rotor), washed once with distilled water and suspended to 0.5 g wet weight/ml in 0.1 M Tris-SO4 (pH 9.4), 10 mM DTI'. After incubation for 10 rain at 30~C, cells were washed twice with 1.2 M sorbitol and re-suspended in 1.2 M sorbitol, 0.1 M EDTA, 10 mM 2-mercaptoethanol (pH 7.5) to give an A~Jnm ef approx. 0.3 when diluted 1 : 20 with 1.2 M sorbitol. Yeast cells were converted to spheroplasts by the use of I Jtiease (Sigma), exactly as described in the product description material. In brief, lyticase was dis-
solved in 50 mM potassium phosphate buffer (pH 7.5, 100 units/ml) and 1 part of thc enzyme solution was added to 4 parts of yeast suspension. Production of spheroplasts was conducted at 28°C, with gentle shaking (70-90 min-l). Usually, alter 80-100 min, the majority of cells had been converted to spheroplasts, as checked by the absorbance decrease at 650 nm (down to I).03-0.05 absorbance units, measured after approx. l0 rain from 1 : 1 dilution with water). For homogenization, spheroplasts were suspended in 0.6 M mannitol, l0 mM Tris-CI (pH 7.4), 0.1% bovine serum albumin, l mM EDTA, 0.2 mM phenyl methylsulfonyl fluoride, 1 #M teupeptin, and l #M pepstatin A to a concentration of 0.15 g of spheroplasts (wet weight)/ml. After cooling on ice, spheroplasts were homogenized by 15 strokes in a tight-fitting teflon-glass homogenizer, diluted with 1 volume of the homogenization buffer and centrifuged for 5 min at 4000 × g (SorvaU SS-34 rotor, 4°C). This step was repeated twice in order to assure disruption of all spheroplasts present in the preparation. Mitochondria were isolated from pooled supernatants exactly as described in Ref. I I. They were resuspended in 0.6 M mannitol, 10 mM Tris-CI (pH 7.4) to give an approx, final concentration of 15 mg protein/ml and stored at -70°C.
Isolation and reconstitution of the pyrut~ate carrier Yeast mitochondria were solubilized in 3% Triton X-114, 20 mM Mops (pH 7.2), 50 mM NaCI, l mM EDTA. at a protein concentration of about 5 mg/ml. Unsolubilized material was sedimented by centrifugation at 100000 × g for 40 min. Membrane extract (0.6 ml) was loaded onto 0.6 g of dry hydroryapatite and eluted with 2 ml of the solubilization buffer. The eluate was supplemented with deoxycholate to a final concentration of 0.7%, and the pH was adjusted to 6.3 prior to subsequent affinity chromatography. 1 ml of wet affinity resin was placed in a small column (0.8 cm diameter) and equilibrated with 10 mM Mops (pH 6.3), 0.7% deox3,cholate and 2% Triton X-114. Hydroxyapatite eluate (0.7 ml) was loaded on the column and kept in the resin for about 20 min. The column was then washed twice with 5 ml of equilibration buffer, followed by 3 ml of the buffer supplemented with 0.2 M NaCI and, subsequently, by 3 ml of the buffer containing l M NaCI. Fractions of 1 ml each were collected throughout the experiment. Reconstitution of the carrier-containing Triton Xll4-suspended material into egg yolk phosphatidylcholine vesicles was performed by the Amberlite XAD2 method . Liposomes (45 mg lipids/ml) were prepared by sonication ill 50 mM KCI, 20 mM Mops, 1 mM EDTA (pH 7.2). Amberlite XAD-2 moist beads were packed in 0.5 g portions into small columns (Pasteur pipettes) and equilibrated by repetitive passage of liposomes diluted five times with the corre-
89 sponding medium supplemented with 50 or 2(X) mM substrate (a~ specified). Samples containing the pyruvate carrier w~,~re mLxed with the liposomes at a 4:1 (v/v) ratio and the mixture was repeatedly passed, in 0.5 ml portions, through Amberlite XAD-2 columns. It was found that the activity of the pyruvate transport could already be measured after seven passages, and that it reached a plateau between 12 and 24 passages (not shown). 20 passages were thus used routinely for equilibration of the column and 16-18 passages for the reconstitution process. The ratio of detergent to lipid and Amberlite beads was the same as in the batch procedure used previously .
As my of the carrier actit'ity Kf!er reconstitut~on, the carrier activity was assayed either by the accumulation of radioactive pyruvate (0.6 mM final conceraration in the medium) in proteoliposomes pre-loaded with 50 or 200 mM (as specified) pyruvate or other substrates (exchange reaction, for detailes see Ref. 9), or by the enzymatic assay with the use of 3-hydroxybutyrate dehydrogenase, as described in Ref. 7. In the latter case, protcoliposomes were reconstituted in the presence of 200 mM acetoacetate. Its subsequent appcarenee in the external medium upon diffusion a n d / o r carrier-driven exchange reaction was monitored spectrophotometrically (NADH oxidation catalysed by 3-hydroxybutyrate dehydrogenase reacting with acetoaeetate as substrate). Measurements were performed with the Aminco DW-2 spectrophotometer at 360-374 nm, using an extinction coefficient for NADH of 2.3 mM-~, according to Ref. 7. Modifications of the procedure described in Ref. 7 were as f'~llows: the exchange reaction was always started by addition of 0.66 mM external substrate. A 3 ml euvette, continuously stirred with a small magnetic bar, contained 2 ml of the medium (50 mM KCI, 1 mM EDTA, 20 mM MOPS, pH 7.2), 10 #1 of 10 mM NADH and 200 #1 proteoliposomes. About 1 U of 3-hydroxybutyrate dehydrogenase was added prior to the addition of counter substrate. Reconstituted samples were also passed through Dowex (removal of external acetoacetate) just before adding to the spectrophotometer cuvette. This prevented emptying of vesicles before the assay and allowed monitoring of a diffusion-driven leak of acetoacetate from proteoliposomes, occurring prior to the addition of external substrate (carrier-mediated exchange). Miscellaneous techniques Protein concentration by precipitation with acetone, SDS-PAGE, staining of gels with silver nitrate and protein determination in the presence ol SDS were standard procedures, performed exactly as described previously .
Results and Discussion
Purification of the pymtate carrwr I1 is known that, as in higher eukaryotcs, transport of pyruvate in yeast mitochondria is inhibited by cyanocinnamate and its derivatives . Thus, our technique fo: purifying the pyruvat,, carrier from bovine heart mitochondria [8,9]. ;~,amely affinity chromatography on immobtlized 4-h~ oxy-cyanoeinnamatc, was alto tried with yeast. The result of such an experiment is presented in Fig. 1. Mitoehondria from yeast strain HRI-2A + (wild type) cells were solubilized with Triton X-114, ehromatographed on dry hydroxyapatite (HTPeluate, lane A) and loaded onto the affinity chromatography column. Subsequently, the column was washed with loading medium (unbound material, lanes B and CI, followed by medium containing 0 2 M NaCI (lane D) and by medium containing 1 M NaCI (lane E). Samples collected at all steps of the purification procedure were reconstituted and their pyruvate activity (radioactive assay, pyruvate/pyruvate exchange) was
Fig. 1. Purificationof the pyruvatecarrier from mitochondriaof wild strain S. cerevisiaeHRI-2A* cells. SDS-PAGEof fractionscollected at different steps of the purification procedure, stained with silver nitrate. A, hydroxyapatitu(HTP) eluate; B-E, fr:~tions collected during affinity chromatography:B, unbound material recovered in the voidvolumeof the column;C, retarded materialwashedout with the loading buffer; D, material eluted in the presence of 200 mM NaCI; E, material eluted in the presenceof I M NaCL Aliquotsof samples were precipitated with acetone and re-dissolved in the sample buffer [13). Material presented in the fugure correspondsto 100 ,u.Iof HTP eMareand 3 ml of subsequentfractionsfrom affinity chromatography. Numbers below SDS-PAGE lanes correspond to the pyruvate/pyruvateexchangeactivity measured after reconstitution of a givenfractionby the radioactiveassay.Activityis expressed in nmol/min per ml of reconstitutedfraction.The pyrovatecarrier activity measured in the Triton extract of mitochondria(not shown) was Iound in this experiment to be 1310 nmol/min per ml of reconstitutedfraction.
9/) measured (Fig. I). The carrier extracted from mitochondria and prc-purified by hydroxyapatite chromatography was bound to the affinity chromatography column and was not cluted by extensive washing using the same medium. However. addition of salt to the washing buffer elutcd a fraction which contained carrier activity. Such a result points to the ionic nature of the protein-matrix interactions and is not consistent with covalent binding of cyanocinnamate to the pyruvate carrier SH groups + It cannot be excluded, however, that cyanocinnamate, when immobilized on the agarose column through a long spacer [8,9], still retains affinity for the protein, but can no longer react with its SH groups. The experiment presented in Fig. 1 also shows that the fractions displaying pyruvate transport activity were etuted by 1 M NaCI, a concentration higher than that used for the bovine heart carrier (200 mM NaCI [8,9]). SDS-PAGE of the active fractions revealed the presence of at least three polypeptidcs (lanes D and E). One of the common problems in the purification of mitochondrial carriers is contamination by the outer membrane porin [15-17]. Such contamination may le. d to a fast efflux of enclosed substrates from prote iliposomes [15,16] and to difficulties in the identificztion of the carrier protein in SDS-PAGE . Mor~over, porin is thought to regulate the sensitivity of mitochondrial anion carriers to inhibitors , ° suggestion which has not been unanimously accepted [17,20]. In this case, however, porin contamination could also alter the functional characteristics of "he isolated carriers. Purification of the monocarboxylate carrier was, therefore, carried out using a yeast mutant lacking mitochondrial porin, constructed in the laboratory of Prof. G. Sehatz and kindly provided to us . Fig. 2 sho~s a comparison between the carrier preparations obtained by a similar purification procedure, from bovine heart, wild-strain ~'east and mutantstrain yeast mitochondria. At the hydroxyapite eluate stage (lanes A-C), it was already clear that yeast material differed from analogous samples from bovine heart. In general, the major protein bands recovered in the yeast eluate (lanes B and C) had a lower molecular mass than the ones extracted from bovine heart (lane A). In addition, numerous polypeptides of very low molecular mass were present in the yeast material (lanes B, C and D-G), which may point to higher proteolytic activity in yeast relative to heart mitoehondria preparations. Hydro~apatite eluate of the y,'.ast mutant (lane C) differed from that of the wildstrain material (lane B), mainly because of the absence of one well staining bana at about 30 kDa, most likely rcptc~ntiag mitochondrial porin. Such a result is in agreement with the original observations of Schatz's group . The fractions collected from the affinity chromatography column (lanes D-G) contained many
Fig. 2. Comparison of pyruvate carrier preparations obtained by similar purification procedure from bovine heart, yeast wild strain and yeast porin defficient mutant bells. SDS-PAGE of different fractions, stained with silver nitrate. A, B, C, hydroxyapatite (HTP) eluates from bovine hearl, wild strain and porin-deficient mutant cells, respectively; D. E, unbound (void volume) fractions from affinity chromatography column, of material from wild strain and porin-deficlent yeast mutant cells, rcspectively; F, G. H, fractions elated from affinity chromatography by 200 mM NaCI, of wild strain yeast cells, yeast porin-dehcient mutant and bovine heart mitochondria, respectively. Samples were prepared and loaded as in Fig. 1. Arrov,' p~fints to pure bovine heart pyruvate carrier of 34 kDa [8,9]. Molecular ,nass values of polypeptides found in yeast fractions were estimated from a standard cur','e obtained by plotting positions of the bovine heart carrier and of Serva mixture 4 protein standards supplemented with cytochrome c (not shown).
more loa nole-ular mass bands in the yeast material as compared to the bovine heart samples (not shown here, sec Rcfs. 8 and 0 for comparisonl. It is difficult to establish the reason for these differences. However, the presence of some additional components (e.g., lipids, ions, free fatty acids), different in bovine and yeast material, may have influenced the final outcome of the purification procedure. Isolation of the pyruvate carrier from bovine heart mitochondria resulted in a single polypeptide of molecular mass of 34 kDa [8,9] (Fig. 2, lane H). Two additional bands visible near the top of the gel and present in all lanes (Figs. 1 and 2), represent a well described silver nitrate-staining artifact, appearing in the presence of 2-mereaptoethanol . The bovine heart 34 kDa polypeptide was shown to behave as a bona fide pyruvate carrier after reconstitution [8,9]. However, no protein of the same molecular mass was found in the fractions eluted from the cyanocinnamate column in the case of yeast (Fig. 2, lanes F and G). Instead, three major protein bands were visible in the final preparation from the wild strain cells (Fig. 1, lanes D and E; Fit 2, lane F): 50, 30 and 26 kDa, respectively. Two of these bands, of 50 and 26 kDa, also appeared in the reconstitutively active fractions purifizd from the
91 porin-deficient mutant (Fig. 2, lane G). Thus, it seems likely that the 30 kDa protein represerted mitochondrial porin, which co-purified with the pyruvate carrier from the wild strain yeast cells, and was absent in the mutant. Either both or one of the two bands must, therefore, represent the yeast pyruvate carrier. The polypeptide of 26 kDa seemed to be more specifically bound and eluted at higher ionic strength, since no trace of it was visible in fractions washed out with the loading buffer (Fig. 1, lanes B and C; Fig. 2, lanes D and E). At the same time, however, some carrier activity was found also in the washed material devoid of the 26 kDa protein (Fig. 1, B and C), which might indicate that the 50 kDa polypeptide is a better candidate for the carrier. Alternatively, the two bands may represent a monomer and dimer of the same polypeptide, of 26 and 50 kDa, respectively. In such a case, the formation of a non-covalent dimer, resistant to the presence of SDS, could be postulated. In fact, the lack of a covalent dimer, obtained by an S-S bridge, may be inferred from the unchanged SDS-PAGE pattern after reducing the protein with tri-n-butylphosphine , or after running the eleetrophoresis in the presence or absence of 2-mercaptoethanol (not shown). Thus, the final identification of the yeast pyruvate carrier requires further investigation. At present, it appears clear that the inner mitoehondrial membrane of Saccharomyces cerecisiae contains a pyruvate carrier of a different molecular mass from that of bovir~e heart mitoehondria, and that the presence of two proteins, of 26 and 50 kDa, can be associated with the yeast carrier activity. Table I shows the protocol of pyruvate carrier purification from the two types of yeast cell studied in the
present investigation. In agreemerJt with the data of Figs. 1 and 2, a smaller amount of protein with a higher specific activiD, was purified from the mutant cells relative to the yeast wild strain. However, in both cases, approximately the same total carrier activity was recovered in the final preparation. Observed differences are, therefore, most likely due to the additional contamination by a third polypeptide found in the wild strain material. Apparently, this contamination, although it increases the total protein amount and thus lowers the calculated specific activity, does not interfere with the measured rate of transport. It is also worth noting that the totat amount of protein recovered in final preparations of the yeast pyruvate carrier (18 or 14 p.g, see Table 1) is lower than in similar preparations from bovine heart mitochondria (usually about 30 p.g for the single polypeptide preparation, see Refs. 8 and 9). This suggests a lower abundance of pyruvate carrier in yeast mitochondria. At the same time, however, the specific activity of the yeast carrier (about 100 p.mol/mg protein per min, see Table I) was found to be much higher than that of the analogous protein from bovine heart (up to 10 p.mol/mg protein per rain, see Refs. 8 and 9). Functional properties of the reconstituted )'east pynwate carrier
Pyruvate, as other monocarboxylic acids, is able to diffuse through lipid membranes at a relatively high rate. The observation that this diffusion is strongly pH dependent and can be accelerated by counter-ions (exchange diffusion) has even lead to the proposal that no carrier protein is needed to catalyse pyruvate trans-
TABLE 1 Purification of pyrut'ate carrierfrom two cell lines of S. cerelisiae
The purificationprocedurewas alwaysstarted from 100 mg of mitochondrialprotein, taken as 1110%.Activitywas measured by the radioactive assay, as the pyruvate/pyruvale exchangereaclion. Only mersalyl-sensiliveaccumulationwas taken into account. Detailed conditionsare as described in Materialsand Methods. Strain
soluhilizedmaterial hydroxyapafite-eluate fraction eluted with NaCI from affinitycolumn
solubilizedmaterial hydrox3'apatite-eluate fractioneluted with NaCI from affinitycolumn
4 ,) .,,~
Activily total (nmol/min) (}229
specific (~mol/mg per rain) 0.48
Purification (fold) 1 10.8
92 greater. This was surprising attd has not been previously reported, but i~ adds to the complexity of the system discussed above. It should be noted here that a similar effect of 2-eyanoeinnamate on the rate of efflux of acetoacetatc from egg yolk phosphatodylcholinc liposomes, containing no protcin, was also observed. In this case, howevcr, the efflux of acetoacetate was found to be independent of the presence of external pyruvate, either before or after addition of 2-cyanocinnamate (not shown). This points to a simple diffusion phenomenon being present in this system. Thus, the stimulatory effect of 2-cyanocinnamate on the initial leak of acetoacetate from the vesicles (liposomes or proteoliposomes) can be interpreted as resulting from changes in the lipid structure of the membrane induced by this compound, producing an increased permeability of the membrane to monocarboxvlates. In the experiment discussed here (Fig. 3B), however, addition of pyruvatc to cyanocinnamate-treated samples still stimulated acetoacetate efflux, although to a much iesscr extent than in control samples (Fig. 3A). Since a sufficient amount of substrate was still trapped inside the proteoliposomes (Fig. 3B), the observation points to the specific inhibition of the yeast pyruvate carrier by 2-cyanocinnamate, in addition to non-specific effects induced by this compound at the membrane level. Similar experiments were also performed with other substances reported to inhibit pyruvate carrier activity in different mitochondria. Table 11 summarizes this study and shows that among the inhibitors of the yeast carrier are 3-hydroxy-2-cyano-cinnamate, 4-hydroxy-2-
port in mitochondria . Although such a view does not seem to bc correct, since in different laboratorics it was possible IO observe substratc and inhibitor specificity and estimate kinetic parameters of the monocarbox3qate transport system from different mitochondria [1-1[)]. simple pyruvatc diffusion should he taken into consideration in the quantitative analysis of the Iransloeation phenomenon. For this purpose, the enzymatic assay to monitor pyruvate or acetoacetate efflux from proteoliposomes [7,24] was found especially suitable. Fig. 3 presents charts of the experiment in which the enzymatic assay was used to study the activity of the yeast pyruvate carrier (fraction eluted from affinity chromatography with 200 mM NaC1) and its inhibition by 2-cyanocinnamate. Vesicles were loaded with 200 mM aeetoacetate and passed through the Dowex column just before the assay. After addition of NADIt and 3-hydroxybutyrate dehydrogenase, a slow leak of aeetoacetate from proteoliposomes could be observed (Fig. 3A). Subsequent addition of pyruvate to the medium stimulated the appearence of ac"~toacetate. Finally, addition of laurylmaltoside to dissolve the membrane, released the remaining acetoacetate [Fig. 3, A and B). The latter control points to the fact that, indeed, the measured rate of NADH oxidation was limited by the accessibility, of acetoaeetate, and that a substantial amount of substrate was kept inside the vesicles at the time of the assay. In the presence of 2-cyanocinnamate (Fig. 3B), the initial leak of aect:~acetate from proteoliposomes was found to be much
L ~ PYRUVATE
002 Abs unit
1 ----t 2mln
Fig. 3. Acetoacetatem/pyruvate,.~,texchangeactivityof the pyruvatecarrier purifiedfrom yeast porin-deficientmutant cells(fractioneluted from affinitychromatographywith 200 mM NaCI).Conditionsof the enzymaticassayas describedin Material and Methods.In each sample,200 u,I of reconsiilutedproteoliposomeswere present (approx. 62 ng of protein), In order to disrupt membranestructure. 100 pl of 10% laurylmaltoside was added (final concentration0.43c~). A, eonlrol sam#c: B, sample preineubatedin the presenceof l mM 2-eyanocinnamate.The following abbreviations are used in the ti.~ure: PL, proteoliposomes;3dIBDIt, 3-hydro~butyrate dehydrogenase;C-CIN, 2-cyanocinnamate;LM, laurylmaltoside.
93 TABLE II
tiposomes (here. at 200 mM final concentration). The
h~hibitor w~itil io t~f t/~e reconstiI.ted 5'cilst l~vrut age ('arri~'r
e x c h a n g e r e a c t i o n wa,~ s t a r t e d by the a d d i t i o n o f 0.(r m M r a d i o a c t i v c p ~ r u v a t e to t h e m e d i u m , a n d tr.e.'t-
Acetoacelalc m/pyru',ate,,~, t exch:.lnge acli~it)(if I hc pyruv;itc carrier purified from yeast wild strain I-IRI-2A ~ cell line mil~thondlia (fraction elutcd from affinity chromal~gr~lphy v, ith 2(1() mM Na('ll. Conditions of the assay were as m Fig. 3, except that approx. 75 ng t~f protein ~erc used per assay (wild str~lin '.'east material), lnhihitor~ were either added directly to the spcctrophotometcr cu',ette prior to the additinn of pyruvatc or (*) wore preincubated wi'~h protcolipom*mes liar 3 t i n attd subsequently, remtr,'ed by quick passage through a small Dowex column. The latter prtx:edure was used for compounds strongly absorbing al the wavelength used to monitor the reation (360-374 nrnk !n addition, it was checked that none of the compounds used had a significant effi:ct on the 3-hydroxybutyral¢ dehydrogenase activity in concentrations applied here (not shownl. Substrate + inhibitor added to the medium
Velocity of aeetoaeetalc cfflux from proteoliposomes (nmol/min per ml of reconstituted fraction)
None Pyruvale 10.6,6 mM) +2-c~,anocinnamate (1 raM) + 3-Otl-2-cyanocinnamat¢ (I raM) * + 4-OH-2-cyanocinnamate (1 mMI * + phenylpyruvale (2 mM) * +mcrsalyl ((I,1 mM) + pCMB (0.2 raM) + benzenelricarboxylate (2 raM) + phenylsuc¢inate (5 raM)
39.9 313. I 62,9 52.4 575 78 8 5q.I 4.2.1 298.2 304.7
proteoliposomes after 5 t i n of incubation. It was observed thai pyruvatein / py "uvate ,~t exchange led to the aceumultion of 220 nmol of pyruvatc per ml of proteoliposomcs in this experiment, and this value was taken as 100¢L When either KIC or aeetoacetate were inside, a larger accumulation of pyruvate was observed. amounting to 109% or 120% of that of the control, respectively. With 2-oxoglutaratc inside, however, the accumulation of only 28% of that of the control was measured. Furthermore, the pyruvate~,/pyruvate,,,, exchange activity was inhibited by externally added 6 mM KIC or 6 mM acetoacetate (by 30 or 50%, respectively), and not at all inhibited by 6 mM 2-oxoglutarate. All this confirms observations reported above (Fig. 4) on the subslrate specificity of lhe yeast pyruvate carrier. 360~374nm 3.-HBDH PYRUVATE
1 cyanocinnamate, phenylpyruvate, mersalyl and pCMB, but not benzenetricarboxylate and phenyisuccin~Ac. ;t should be added here that some of these inhibitors (4-hydroxy-2-cyanocinnam ate, 3-hydroxy-2-cyanocinnamate and pCMB) were also found to increase diflusion of acetoacetate through proteoliposomes, which had to be corrected for (as in Fig. 3B). Other inhibitors had no similar non-specific effect (not shown). The fact that a carrier-mediated reaction is responsible for the translocation of monocarboxylic acids in the experiments reported here, is supported further by studies on substrate specificity of this process. Fig. 4 shows traces from the enzymatic assay experiment in which the same concentration of different metabolitcs was added to proteoliposomes containing 200 mM acetoacetate inside. Only some of these induced a fainter, mersalyl-sensitive (not shown) efflux of acetoacetate to the external medium, indicating the carrier-driven exchange actMty. As shown in Fig. 4, among the substrates for the yeast pyruvate carrier are: acetoacetate, pyruvate, KIC, KIV and KMV, but not 2-oxoglutarate. lactate and citrate. Malate, succinate and glutamate were also found not to be transloeated by the yeast monocarboxylate carrier (not shown). The activity of the reconstituted carrier was further studied using the radioactive assay. This assay allows different substrates to be enclosed inside the proteo-
Fig. 4. Substrate specificity of the purified yeast pyruvate carrier (fraction eluted from affinity, chromatography with 200 mM NaCI; wild strain I t R I / 2 A ÷ cells material). Conditions of the assay were as in Fig, 3 and Table II. Numbers given at the recorder traces refer to the activity of the carrier in nmol/min per ml of reconstituted fraction, not corrected for the initial efflux of acetoacetate from proteoliposomes~ Abbreviations as in Fig. 3, plus: OG, 2-oxoglutarate: LAC, lactate; CIT, cilrate.
94 the environment. This is consistent with a general flexibility of yeast cells to adjust their metabolism to the environment . Since the yeast mitochondrial pyruvate carrier appears to be expressed in a lesser amount than it normally is in cells of higher eukaryores, its higher activity possibly counteracts this differ-
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Acknowledgements ° l 0 i " "'~~ -1
. . . . . . .
:/(s) Fig. 5. Double reciprocal plot of 3celoacetatein/pyruvalc (KIC. KIV or KMV),,,, exchange acti'~ity of the pyruvate carrier from I~)rin-deficient HRI-2A" yeast ceils (ti-aetion eluted from affinity chromatography with 200 mM NaCI). Conditions of the assay as in Materials and Methods. The rates of exchange reactions were corr-cted for the initial leak of aeetoacetate from proteoliposomes. Lines of best fit were obtained by the method of least-squares. ( • ) . pyru'vate; ( • ) , KIC; (,¢.). KIV; Co), KMV. Substrate concentration iS) is plotted as mM, and the reaction velocity (vl is in arbitrary units.
This work was supported by the Swiss National Science Foundation, grants Nos. 3100-27745.89 and 70PP-029614. The authors are grateful to Prof. G. Schatz for providing the yeast cells lines used in this study and wish to thank Mr. Krzysztof Drabikowski for his skillful technical assistance in growing yeast cells and in the preparation of mitochondria. Dr. Moira Glerum is kindly acknowledged for carefully reading the manuscript. References
The enzymatic assay was used to study kinetics of monocarboxylic acids transport by the reconstituted yeast pyruvate carrier. As shown in Fig. 5, pyruvate and different branched-chain monocarboxylic oxoacids were transported with approximately the same Vmx but had different affinities for the carrier. For the exchange with acetoacetate (enclosed inside proteoliposomes in the enzymatic assay), the best counter-substrate was found to be pyruvate (Km of 0.83 raM, Vm~, of 135 #mol/min per mg protein). The K m values for other substrates increase in the order of KIC-KIV-KMV (Fig. 5), and were found to be 1.25, 1.47 and 1.82 mM, respectively. Pyruvate carriers purified from bovine heart ([8,9] and above) and rat liver  were found to be less active than the one from yeast, suggesting that the differences between the yeast carrier and the analogous proteins from higher eukaryotes concern not only the structure (molecular mass), but also functional features of these proteins. This is further supported by the fact that the K m for pyruvate in the pyruvate/pyruvate exchange reaction measured in rat liver mitochondria was reported to be about 0.1 mM , significantly lower than found in the present investigation. At the same time, other monocarboxylates were reported to reveal much h i g h e r K m values, up to about 4 mM . It thur may be concluded that the carrier from rat liver mitochondria is more specific for pyruvate and can better distinguish between different monocarboxylates than the carrier from yeast. The high transport velocity revealed by the yeast carrier may also reflect the ability of a unicellular organism to more efficiently utilize different metabolites, once they become available in
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