Centro de Ciências do Ambiente, Departamento de Biologia, Universidade do Minho, 4710-057 Braga, Portugal1
Author for correspondence: Fernanda Cássio. Tel: +351 253604310. Fax: +351 253678980. e-mail: fcassio{at}bio.uminho.pt
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ABSTRACT |
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Keywords: affinity labelling, lactate transporter, Candida utilis
Abbreviations: DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate; MCT, monocarboxylate transporter
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INTRODUCTION |
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The transport of monocarboxylates into animal cells has been better characterized than transport into other systems and several approaches have been used to identify the carrier proteins. In this respect, labelling experiments using a variety of compounds, such as the stilbene disulfonate DIDS and L[U14C]lactic acid, proved to be useful (reviewed by Poole & Halestrap, 1993 ; Juel, 1997
). Additionally, studies involving molecular biological methods have led to the proposal of a family of monocarboxylate transporters (MCTs) in membranes from animal cells which comprises about 13 proteins (see Price et al., 1998
; Pao et al., 1998
; Paulsen et al., 1998
). In Saccharomyces cerevisiae, four potential MCT homologues have been identified. Furthermore, in this yeast species, it was found that the gene JEN1, included by Paulsen et al. (1998)
in the sialate proton symporter family, encodes a lactate permease (Casal et al., 1999
).
To our knowledge, the present work is the first report of an attempt to identify a plasma membrane protein responsible for lactate transport in yeast cells. We have selected the yeast C. utilis IGC 3092 since the lactate proton symport of this yeast displays a high capacity to transport lactate, suggesting high expression of the protein. Labelling experiments performed with L-[U-14C]lactic acid led to the identification in SDS-PAGE of a 43 kDa polypeptide, which is likely to be the lactate transporter protein.
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METHODS |
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Preparation of plasma membranes from C. utilis.
C. utilis cells grown in either lactic acid or glucose medium were harvested in the mid-exponential growth phase (OD640 0·50·6), and washed twice with ice-cold distilled water and once with buffer A (0·3 mM KCl, 0·1 M glycine, pH 7·0). Cells (15 g) were suspended in buffer A (15 ml) containing 0·1 mM PMSF and plasma membranes were prepared as described by Van Leeuwen et al. (1991) . Briefly, cells were homogenized (Braun Cell Homogenizer) with 35 g glass beads (0·250·32 mm) for 2 min, and the suspension was separated from the glass beads by filtration (glass filter) and centrifuged at 2100 g for 10 min. The supernatant was filtered through a glass-fibre filter (Sartorius; 13400-47-S) and centrifuged at 6200 g for 20 min. The supernatant was re-centrifuged (6200 g, 20 min) and the two pellets were combined and washed once with buffer A. For the aggregation of mitochondrial membranes the pellet was resuspended in 15 ml buffer A with 0·1 mM PMSF and titrated to pH 4·9 with 40 mM HCl in buffer A. Aggregated mitochondrial membranes were removed by centrifugation at 2100 g for 10 min and the supernatant was brought to pH 7·0; the pellet was resuspended in buffer A, acidified to pH 4·9 and re-centrifuged (2100 g for 10 min). The supernatants were combined, acidified to pH 5·0 and centrifuged at 7700 g for 3 min. The resulting supernatant was adjusted once more to pH 7·0, frozen in liquid N2 and thawed at room temperature. This suspension was then centrifuged for 25 min at 100000 g, and the pellet was washed with 50 mM potassium phosphate, 1 mM MgCl2, pH 6·2, and finally resuspended in this buffer to a final protein concentration of about 5 mg ml-1. Aliquots were stored in liquid N2.
Protein was assayed by the Lowry method, using BSA as a standard.
L[U14C]Lactate binding to plasma membranes, and SDS-PAGE analysis.
Binding assays were performed according to the method of Welch et al. (1984) . Portions of plasma membrane suspensions (1020 µl), containing approximately 100 µg protein, were incubated with 0·01 mM L-[U-14C]lactic acid (2220 Bq), pH 7·4, for 2 h at 30 °C, in a final volume of 40 µl. Prior to loading onto a polyacrylamide gel, samples were mixed with the same volume of 125 mM Tris/HCl buffer, 20% (v/v) glycerol, 4% (w/v) SDS, 0·01% (w/v) bromophenol blue and 1% (v/v) 2-mercaptoethanol, pH 6·8, and then heated to 100 °C for 5 min.
Slab polyacrylamide gels (10%, 1 mm thickness) with a 2·5% stacking gel were prepared as described by Laemmli (1970) in a Sturdier SE 400 (Hoefer) system. The run was performed at 100 V for 1 h and 250 V thereafter. Calibration markers of known molecular mass (14·494 kDa) were included in each gel. Electrophoresis was carried out until the dye was about 0·5 cm from the bottom of the gel. At the end of the run, the lanes containing the radioactive samples were sliced at 2·5 mm intervals, unless otherwise stated, and the slices were placed in vials containing 5 ml scintillation fluid. The vials were left for 24 h prior to counting (Packard Tri-Carb 2200 CA liquid scintillation spectrophotometer). In addition, non-radioactive membrane protein samples were also separated by SDS-PAGE and gels were stained for protein with either silver nitrate (Merril et al., 1994
) or Coomassie brilliant blue R.
Inhibition assays of lactate transport in intact cells by DIDS.
Cells of the yeast grown on lactic acid were harvested in the mid-exponential growth phase, washed twice with cold distilled water, and suspended in distilled water at a final concentration of about 40 mg (dry weight) ml-1. The initial uptake rates of labelled lactic acid were estimated as described previously (Leão & Van Uden, 1986 ). Briefly, 10 µl yeast suspension was mixed with 30 µl 0·1 M potassium phosphate buffer, pH 5·0, and, after 2 min incubation at 26 °C, the reaction was started by addition of 10 µl 0·0250·4 mM labelled lactic acid (67 Bq nmol-1). Samples were taken after 0, 5 and 10 s, time periods over which the uptake of labelled lactic acid was linear. The reaction was stopped by dilution with 4 ml ice-cold water and the mixtures were immediately filtered through GF/C filters (Whatman). The filters were washed with 8 ml ice-cold water, introduced into vials containing scintillation fluid and radioactivity was measured. For the non-specific 14C adsorption, at zero time, labelled lactic acid was added after cold water. To evaluate the inhibitory effect of DIDS on lactic acid uptake, the compound was included in the reaction mixture 30 s before adding labelled lactic acid, as indicated in Results.
Chemicals.
L[U14C]Lactic acid, sodium salt (5·62 GBq mmol-1), was purchased from the Radiochemical Centre (Amersham). Scintillation fluid OptiPhase HiSafe II and DIDS were from LKB FSA Laboratory Supplies and Sigma, respectively.
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RESULTS |
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L[U14C]Lactate binding to yeast plasma membranes
Labelling experiments were performed with L-[U-14C]lactic acid since this substrate, after prolonged incubation (above 30 min), binds firmly to the MCT of animal cells (Welch et al., 1984 ; McCullagh & Bonen, 1995
). Plasma membranes isolated from C. utilis grown with either lactic acid (presence of lactate proton symport) or glucose (absence of lactate proton symport) were then incubated with L-[U-14C]lactic acid and separated by SDS-PAGE, as indicated in Methods. A sample without membrane protein was included as a control. A typical pattern of labelled lactate binding to plasma membrane proteins is shown in Fig. 1
. The results showed a well-defined peak of radioactivity corresponding to the sample containing membrane proteins from lactic-acid-grown cells. Maximum binding was associated with a protein(s) with a molecular mass of approximately 43 kDa. In contrast, no defined peak of radioactivity was obtained with the sample of plasma membranes from glucose-grown cells and no radioactivity above background was detected when L-[U-14C]lactic acid was run on the gels in the absence of protein. Furthermore, when plasma membrane proteins were subjected to the SDS-PAGE denaturing treatment (see Methods), before incubation with labelled lactic acid, no peak of radioactivity was detected (not shown). To rule out the possibility that the radioactive peak was an artefact due to a contaminant bacterial protein, L-[U-14C]lactate-binding assays were performed in the presence of streptomycin (100 µg ml-1) and penicillin (100 U ml-1). Under these conditions, a radioactive peak similar to that presented in Fig. 1
was obtained (data not shown). Overall, the results suggested that the peak of radioactivity observed corresponded to a binding of labelled lactate to a plasma membrane protein of C. utilis, possibly the lactate transporter.
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The stilbene disulfonate DIDS is one of the most effective inhibitors of lactate transport in animal cells (Poole & Halestrap, 1993 ) and its inhibitory action has been used as an index for the presence of a specific monocarboxylate carrier. To substantiate the presence of a transporter protein of the MCT type, the effect of this inhibitor on the transport of lactic acid in whole cells of C. utilis was investigated. Estimates of the initial uptake rates using 0·0250·5 mM labelled lactic acid, in the presence and absence of 0·110 mM DIDS, showed that this compound behaved as a competitive inhibitor of lactate transport (not shown) and an inhibition constant (Ki) of 0·26 mM was obtained. Based on these results, and to confirm that the radioactive peak was associated with the protein of the lactate proton symport of C. utilis, lactate binding to plasma membranes was studied in the presence of 0·0210 mM DIDS. As shown in Fig. 2
, lactic acid binding was inhibited by DIDS and this effect was dependent on its concentration.
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DISCUSSION |
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In C. utilis, it appears that L-[U-14C]lactate displays the capacity to covalently bind its own carrier, since this binding was stable to the procedures involved in SDS-PAGE. It has also been reported that labelled lactate binds to plasma membranes from rat hepatocytes (Welch et al., 1984 ) and rat skeletal muscle (McCullagh & Bonen, 1995
) in a similar way. As discussed by these authors, this type of binding is unlikely to be physiological since it would make for an energetically unfavourable transport mechanism. Similar behaviour was described for the MCT inhibitor DIDS, which initially binds reversibly to the lactate carrier in mammalian erythrocytes, but upon prolonged incubation becomes irreversibly bound (Poole & Halestrap, 1991
). Indeed, our results indicated that DIDS binds reversibly to the lactate carrier during short incubations since it behaved as a competitive inhibitor of lactate transport in whole cells of C. utilis. However, the chemical nature of the binding of these compounds to plasma membranes upon prolonged incubation is not completely understood.
A possibility that has arisen from the present work is that the metabolism of L[U14C]lactate by contaminating bacteria during prolonged incubation could cause labelling of a bacterial protein, which could be responsible for the peak of radioactivity observed. However, this does not appear to be the case since no significant L[U14C]lactate binding to plasma membranes from glucose-grown cells (absence of lactate proton symport) was detected, and the inclusion of antibiotics in the labelling assays did not prevent the appearance of the peak of radioactivity.
The data presented here showed that the characteristics of L[U14C]lactate binding to plasma membrane proteins of C. utilis were similar to those of the lactate proton symport exhibited by the yeast. First, the presence of the radioactive peak in labelling experiments performed with plasma membranes from lactic-acid-grown cells and absence of the peak from glucose-grown cells was in accordance with the inducibility of this carrier. Second, both lactate and pyruvate were able to inhibit binding of labelled lactate while succinate and citrate did not show significant inhibitory effect. This inhibition pattern of lactate binding by carboxylates was identical to that of the lactate proton symport system of C. utilis, which is able to accept monocarboxylates but not di- or tricarboxylates (Leão & Van Uden, 1986 ; Gerós et al., 1996
). In addition, the MCT inhibitor DIDS inhibited, in a dose-dependent manner, lactate binding to plasma membranes and lactate uptake in intact cells.
The polypeptide, identified in SDS-PAGE, associated with the peak of radioactivity had an apparent molecular mass of 43 kDa, which was similar to the values reported for the MCTs of animal cells (for a review see Poole & Halestrap, 1993 ; Juel, 1997
). Altogether, the present study strongly supports the idea that we have identified a protein involved in the symport of lactate with protons in the plasma membrane of C. utilis, which can probably be included in the MCT family. Nevertheless, conclusive proof that this protein is the lactate proton symporter will require its purification and functional reconstitution in membrane vesicles. These approaches are in progress in our laboratory. Amino acid sequence analysis of the protein would also be important, enabling its comparison with sequences of the MCT family.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Cássio, F. & Leão, C. (1991). Low and high-affinity transport systems for citric acid in the yeast Candida utilis.Appl Environ Microbiol 57, 3623-3628.[Medline]
Cássio, F. & Leão, C. (1993). A comparative study on the transport of L(-)malic acid and other short-chain carboxylic acids in the yeast Candida utilis: evidence for a general organic acid permease.Yeast 9, 743-752.[Medline]
Cássio, F., Leão, C. & Van Uden, N. (1987). Transport of lactate and other short-chain monocarboxylates in the yeast Saccharomyces cerevisiae.Appl Environ Microbiol 53, 509-513.[Medline]
Cássio, F., Cõrte-Real, M. & Leão, C. (1993). Quantitative analysis of proton movements associated with the uptake of weak-carboxylic acids. The yeast Candida utilis as a model.Biochim Biophys Acta 1153, 59-66.[Medline]
Fleet, G. H. (1990). Food spoilage yeasts. In Yeast Technology, pp. 124-166. Edited by J. F. T. Spencer & D. M. Spencer. Berlin: Springer.
Gerós, H., Cássio, F. & Leão, C. (1996). Reconstitution of lactate proton symport activity in plasma membrane vesicles from the yeast Candida utilis.Yeast 12, 1263-1272.[Medline]
Juel, C. (1997). Lactate-proton cotransport in skeletal muscle.Physiol Rev 77, 321-358.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680-685.[Medline]
Leão, C. & Van Uden, N. (1986). Transport of lactate and other short-chain monocarboxylates in the yeast Candida utilis.Appl Microbiol Biotechnol 23, 389-393.
McCullagh, K. J. A. & Bonen, A. (1995). L(+)-Lactate binding to a protein in rat skeletal muscle plasma membranes. Can J Appl Physiol 20, 112-124.
Merril, C. R., Joy, J. E. & Creed, G. J. (1994). Ultrasensitive silver-based stains for protein detection. In Cell Biology a Laboratory Handbook, pp. 281-287. Edited by J. E. Celis. San Diego: Academic Press.
Pao, S. S., Paulsen, I. T. & Saier, M. H.Jr (1998). Major facilitator superfamily.Microbiol Mol Biol Rev 62, 134.
Paulsen, I. T., Sliwinski, M. K., Nelissen, B., Goffeau, A. & Saier, M. H.Jr (1998). Unified inventory of established and putative transporters encoded within the complete genome of Saccharomyces cerevisiae.FEBS Lett 430, 116-125.[Medline]
Poole, R. C. & Halestrap, A. P. (1991). Reversible and irreversible inhibition, by stilbene disulfonates, of lactate transport into rat erythrocytes: identification of some new high-affinity inhibitors.Biochem J 275, 855-862.
Poole, R. C. & Halestrap, A. P. (1993). Transport of lactate and other monocarboxylates across mammalian plasma membranes.Am J Physiol 264, C761-C782.
Price, N. T., Jackson, V. N. & Halestrap, A. P. (1998). Cloning and sequencing of four new mammalian monocarboxylate transporter (MCT) homologues confirms the existence of a transporter family with an ancient past.Biochem J 329, 321-328.[Medline]
Sinskey, A. J. & Batt, C. A. (1987). Fungi as a source of protein. In Food and Beverage Mycology, pp. 435-471. Edited by L. R. Beuchat. New York: Van Nostrand Reinhold.
Van Leeuwen, C. C. M., Postma, E., Van der Broek, P. J. A. & Van Steveninck, J. (1991). Proton-motive force-driven D-galactose transport in plasma membrane vesicles from the yeast Kluyveromyces marxianus.J Biol Chem 266, 12146-12151.
Welch, S. G., Metcalfe, H. K., Monson, J. P., Cohen, R. D., Henderson, R. M. & Iles, R. A. (1984). L(+)-Lactate binding to preparations of rat hepatocyte plasma membranes.J Biol Chem 259, 15264-15271.
Received 17 November 1999;
accepted 3 December 1999.
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