Interaction of the Erythrocyte Lactate Transporter (Monocarboxylate Transporter 1) with an Integral 70-kDa Membrane Glycoprotein of the Immunoglobulin Superfamily

Treatment of intact erythrocytes with 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS) causes irreversible inhibition and chemical labeling of the lactate transporter, monocarboxylate transporter 1 (MCT1) (Poole, R. C., and Halestrap, A. P. (1992) Biochem. J. 283, 855–862). In rat eythrocytes DIDS also causes cross-linking of MCT1 to another protein in the membrane to give a product of 130 kDa on SDS-polyacrylamide gel electrophoresis. Cross-linking is markedly reduced by those compounds that protect against irreversible inhibition of lactate transport by DIDS and enhanced by imposition of a pH gradient across the plasma membrane to recruit the substrate binding site of MCT1 to an exofacial conformation. These data indicate that DIDS cross-linking is via the same site on MCT1 as is responsible for inhibition of transport. Antibodies raised against the cross-linked conjugate react with proteins of approximately 40 kDa (MCT1) and 70 kDa on Western blots of erythrocyte membranes and an additional band of 130 kDa after treatment of erythrocytes with 100 μm DIDS. The 70-kDa protein that is cross-linked to MCT1 was purified and shown to containN-linked carbohydrate; the apparent core molecular mass is 40 kDa. Amino acid sequencing showed that the protein is the rat equivalent of the membrane-spanning mouse teratocarcinoma glycoprotein GP-70, a member of the immunoglobulin superfamily related to basigin (Ozawa, M., Huang, R. P., Furukawa, T., and Muramatsu, T. (1988)J. Biol. Chem. 263, 3059–3062). Possible implications of the specific interaction between MCT1 and this protein are discussed. Treatment of intact erythrocytes with 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS) causes irreversible inhibition and chemical labeling of the lactate transporter, monocarboxylate transporter 1 (MCT1) (Poole, R. C., and Halestrap, A. P. (1992) Biochem. J. 283, 855–862). In rat eythrocytes DIDS also causes cross-linking of MCT1 to another protein in the membrane to give a product of 130 kDa on SDS-polyacrylamide gel electrophoresis. Cross-linking is markedly reduced by those compounds that protect against irreversible inhibition of lactate transport by DIDS and enhanced by imposition of a pH gradient across the plasma membrane to recruit the substrate binding site of MCT1 to an exofacial conformation. These data indicate that DIDS cross-linking is via the same site on MCT1 as is responsible for inhibition of transport. Antibodies raised against the cross-linked conjugate react with proteins of approximately 40 kDa (MCT1) and 70 kDa on Western blots of erythrocyte membranes and an additional band of 130 kDa after treatment of erythrocytes with 100 μm DIDS. The 70-kDa protein that is cross-linked to MCT1 was purified and shown to containN-linked carbohydrate; the apparent core molecular mass is 40 kDa. Amino acid sequencing showed that the protein is the rat equivalent of the membrane-spanning mouse teratocarcinoma glycoprotein GP-70, a member of the immunoglobulin superfamily related to basigin (Ozawa, M., Huang, R. P., Furukawa, T., and Muramatsu, T. (1988)J. Biol. Chem. 263, 3059–3062). Possible implications of the specific interaction between MCT1 and this protein are discussed. Proton-monocarboxylate transporters (MCTs) 1The abbreviations used are:MCTmonocarboxylate transporter;CHCα-cyano-4-hydroxycinnamate;C12E8octaethylene glycol monododecyl ether;DBDS4,4′-dibenzamidostilbene-2,2′-disulfonate;DIDS4,4′-diisothiocyanostilbene-2,2′-disulfonate;DNDS4,4′-dinitrostilbene-2,2′-disulfonate;DTSSP3,3′-dithiobis[sulfosuccinimidyl]-propionate;TMtransmembrane;PAGEpolyacrylamide gel electrophoresis;TricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;MOPS4-morpholinepropanesulfonic acid. are essential for the well being of almost all mammalian cells and play a central role in the transport of lactate between tissues (1Poole R.C. Halestrap A.P. Am. J. Physiol. 1993; 264: C761-C782Google Scholar). Recently two distinct MCTs (MCT1 and MCT2) have been cloned from mammalian cells (2Kim Garcia C. Goldstein J.L. Pathak R.K. Anderson R.G.W. Brown M.S. Cell. 1994; 76: 865-873Google Scholar, 3Garcia C.K. Brown M.S. Pathak R.K. Goldstein J.L. J. Biol. Chem. 1995; 270: 1843-1849Google Scholar, 4Jackson V.N. Price N.T. Halestrap A.P. Biochim. Biophys. Acta. 1995; 1238: 193-196Google Scholar, 5Takanaga H. Tamai I. Inaba S. Sai Y. Higashida H. Yamamoto H. Tsuji A. Biochem. Biophys. Res. Commun. 1995; 217: 370-377Google Scholar, 6Carpenter L. Poole R.C. Halestrap A.P. Biochim. Biophys. Acta. 1996; 1279: 157-163Google Scholar). MCT1 is widely distributed in mammalian cells, and has been well characterized at the functional (1Poole R.C. Halestrap A.P. Am. J. Physiol. 1993; 264: C761-C782Google Scholar, 7Carpenter L. Halestrap A.P. Biochem. J. 1994; 304: 751-760Google Scholar) and structural level (8Poole R.C. Sansom C.E. Halestrap A.P. Biochem. J. 1996; 320: 817-824Google Scholar), especially in erythrocytes (1Poole R.C. Halestrap A.P. Am. J. Physiol. 1993; 264: C761-C782Google Scholar, 8Poole R.C. Sansom C.E. Halestrap A.P. Biochem. J. 1996; 320: 817-824Google Scholar). It is inhibited by a variety of stilbene disulfonates, such as 4,4′-dibenzamidostilbene-2,2′-disulfonate (DBDS) and 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS), which bind in competition with substrates and some other inhibitors of transport (9Poole R.C. Halestrap A.P. Biochem. J. 1991; 275: 307-312Google Scholar). DIDS has two isothiocyanate groups that are reactive toward susceptible amino groups, and with prolonged incubation these cause irreversible inhibition and chemical labeling of the transporter (9Poole R.C. Halestrap A.P. Biochem. J. 1991; 275: 307-312Google Scholar, 10Poole R.C. Halestrap A.P. Biochem. J. 1992; 283: 855-862Google Scholar). The two isothiocyanate groups of DIDS give it the potential to cross-link MCT1 with closely associated membrane proteins. Indeed, SDS-PAGE of rat erythrocyte ghosts prepared from cells preincubated with DIDS revealed a protein band of approximately 130 kDa that might be such a cross-linked product (10Poole R.C. Halestrap A.P. Biochem. J. 1992; 283: 855-862Google Scholar). Here we show that this 130-kDa band is the result of a highly specific DIDS-mediated cross-linking of MCT1 to a glycoprotein of approximately 70 kDa. Internal sequencing of the protein shows that is is the rat equivalent of the mouse teratocarcinoma glycoprotein, GP-70, which is a member of the immunoglobulin gene superfamily related to basigin (11Ozawa M. Huang R.-P. Furukawa T. Muramatsu T. J. Biol. Chem. 1988; 263: 3059-3062Google Scholar). Cross-linking appears to occur via a site at or in communication with the substrate binding site of MCT1 and might play a role in regulating MCT1 activity. monocarboxylate transporter; α-cyano-4-hydroxycinnamate; octaethylene glycol monododecyl ether; 4,4′-dibenzamidostilbene-2,2′-disulfonate; 4,4′-diisothiocyanostilbene-2,2′-disulfonate; 4,4′-dinitrostilbene-2,2′-disulfonate; 3,3′-dithiobis[sulfosuccinimidyl]-propionate; transmembrane; polyacrylamide gel electrophoresis; N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; 4-morpholinepropanesulfonic acid. Chemicals and biochemicals were obtained from the sources given previously (8Poole R.C. Sansom C.E. Halestrap A.P. Biochem. J. 1996; 320: 817-824Google Scholar, 9Poole R.C. Halestrap A.P. Biochem. J. 1991; 275: 307-312Google Scholar, 12Poole R.C. Halestrap A.P. Biochem. J. 1994; 303: 755-759Google Scholar). The protease Lys-C was from Sigma, Poole, Dorset, UK. Anti-peptide antibodies to various regions of MCT1 were raised and purified as described elsewhere (8Poole R.C. Sansom C.E. Halestrap A.P. Biochem. J. 1996; 320: 817-824Google Scholar). Erythrocytes were collected in a citrate buffer (84 mm sodium citrate, 1 mm EGTA, pH 7.4). Cells were then usually washed once in a bicarbonate-buffered saline buffer (121 mm NaCl, 25 mm NaHCO3, equilibrated with 95% O2, 5% CO2), and then at least twice more in citrate buffer before resuspending in the same buffer to 10% hematocrit and adjusting the pH to 7.4. Incubations with DIDS, removal of nonbound inhibitor, and preparation of ghost membranes were performed as described previously (10Poole R.C. Halestrap A.P. Biochem. J. 1992; 283: 855-862Google Scholar). Proteolytic digestion of red cell ghosts and separation of membrane proteins by SDS-PAGE were performed as described elsewhere (8Poole R.C. Sansom C.E. Halestrap A.P. Biochem. J. 1996; 320: 817-824Google Scholar). Rat erythrocyte membranes, prepared from cells treated with 100 μm DIDS, were subjected to chromatography on aminoethyl-Sepharose, essentially as described previously (12Poole R.C. Halestrap A.P. Biochem. J. 1994; 303: 755-759Google Scholar). Elution of the cross-linked product of 130 kDa was monitored both by Western blotting with anti-MCT1 antibodies, and by silver staining of protein. The peak fractions containing the cross-linked product (which co-eluted with free MCT1) were concentrated by centrifugal filtration (Amicon, Centriprep 10) and separated on a 6% (w/v) SDS-PAGE gel, and the protein was located with copper staining (13Lee C. Levin A. Branton D. Anal. Biochem. 1987; 166: 308-312Google Scholar). The band of 130 kDa was excised, destained, and then electroeluted as described below. This preparation was used to immunize a New Zealand White rabbit, as described previously (8Poole R.C. Sansom C.E. Halestrap A.P. Biochem. J. 1996; 320: 817-824Google Scholar). The membrane protein of approximately 70 kDa that is cross-linked to MCT1 upon incubation of rat erythrocytes with DIDS was shown to co-elute with MCT1 on ion-exchange fractionation (Q-Sepharose). Thus membranes (from either control or 5 μm DIDS-treated erythrocytes) were stripped of peripheral membrane proteins and solubilized with 1% (w/v) C12E8 at a protein concentration of 1 mg/ml, prior to batch purification using Q-Sepharose (10Poole R.C. Halestrap A.P. Biochem. J. 1992; 283: 855-862Google Scholar). Nonbound protein was removed by washing with buffer containing 0.5% C12E8, and a 0.2 m NaCl eluate was prepared by mixing the washed matrix with an equal volume of buffer containing 0.5% C12E8 and 0.4 mNaCl. The eluate was concentrated by centrifugal filtration to 2 ml, and proteins were separated by SDS-PAGE. A broad band at approximately 70 kDa (identified by staining with Coomassie Blue) was electroeluted overnight in buffer containing 20 mm Tris, 2 mmEDTA, 0.1% (v/v) 2-mercaptoethanol, and 0.1% SDS (w/v), pH 8.0, before concentrating to 1.5 ml by centrifugal filtration. C12E8 was added to 1% (v/v), and the protein was incubated with N-glycanase F (400 Oxford Glycosystems units/ml) for at least 2 h, and usually overnight. The sample was then subjected to SDS-PAGE, where the deglycosylated binding protein migrated at approximately 40 kDa. Direct N-terminal microsequencing was performed after transfer onto a ProBlot membrane and staining with Serva Blue G (12Poole R.C. Halestrap A.P. Biochem. J. 1994; 303: 755-759Google Scholar). Alternatively, the polyacrylamide gel was stained with Coomassie Blue, and the 40-kDa band was excised, electroeluted overnight, and concentrated by centrifugal filtration. After addition of C12E8 (1% (v/v) in a final volume of 200 μl), a 50-μl aliquot was incubated overnight at 37 °C with 0.2 μg of Lys-C, and the products were separated by SDS-PAGE using a Tricine buffer system able to separate small peptides (14Schagger H. von Jaggow G. Anal. Biochem. 1987; 166: 368-379Google Scholar). Peptides were transferred onto ProBlot membrane and stained with Serva Blue G to reveal discrete bands of about 11 and 3.5 kDa. These were cut out for N-terminal microsequencing. The data of Fig. 1 show that the 130-kDa band formed following incubation of rat erythrocytes with 100 μm DIDS is recognized by specific anti-MCT1 antibodies. It is of note that this cross-linking is highly specific; normally no other additional bands were detected upon treatment with DIDS. These observations indicate that the cross-linking is probably a reflection of a close association of the two proteins in the erythrocyte membrane, which may in turn be of functional relevance. Since DIDS is membrane impermeant, the binding protein must be either a membrane-spanning protein or an exofacial peripheral protein. It could be argued that the new MCT-containing band is a result of cross-linking MCT1 to itself, forming oligomers. This seems unlikely for several reasons. First, 130 kDa is not a simple multiple of 40 kDa (especially as trimer formation seems unlikely), and second, the products of self-aggregation of MCT1 do not seem to co-migrate with the cross-linked product as is also shown in Fig. 1. Aggregation was induced by solubilizing rat erythrocyte membranes with the detergent C12E8 (1% w/v) and leaving on ice for varying periods of time before separating on SDS-PAGE and probing Western blots with anti-MCT1 antibodies. There was a time-dependent aggregation of MCT1 to produce a product of approximately 80 kDa, which corresponds to a dimer of MCT1. Such aggregation phenomena are common with membrane proteins (15Furthmayr H. Marchesi V.T. Biochemistry. 1976; 15: 1137-1144Google Scholar, 16Jhun B.H. Berenski C.J. Craik J.D. Paterson A.R. Jung C.Y. Biochim. Biophys. Acta. 1991; 1061: 149-155Google Scholar). To investigate more fully the cross-linking reaction, we performed the labeling reaction in the absence and presence of various inhibitors of MCT1 activity. DBDS and CHC, two potent inhibitors of lactate transport that also inhibit labeling of MCT1 by DIDS (9Poole R.C. Halestrap A.P. Biochem. J. 1991; 275: 307-312Google Scholar, 10Poole R.C. Halestrap A.P. Biochem. J. 1992; 283: 855-862Google Scholar), reduced markedly the DIDS-induced cross-linking of MCT1, whereas the poor inhibitor 4,4′-dinitrostilbene-2,2′-disulfonate (DNDS) (9Poole R.C. Halestrap A.P. Biochem. J. 1991; 275: 307-312Google Scholar) had little effect (Fig. 2). These results demonstrate that the cross-linking of MCT1 is via a site on the transporter that is either at the substrate binding site, consistent with competitive inhibition of lactate transport by DIDS, or affected by conformational changes induced by substrate or inhibitor binding. In Fig. 3 we show that the cross-linking reaction is dependent upon the nature of the buffer used for the reaction. The reaction was performed in either a citrate buffer, pH 7.4, which induces a large pH gradient (0.6–0.8 pH unit, alkaline inside) or a saline buffer, also at pH 7.4, in which there is very little gradient (approximately 0.2 pH unit, acid inside) (17Edlund G.L. Halestrap A.P. Biochem. J. 1988; 249: 117-126Google Scholar). It is clear that in citrate buffer the rate of cross-linking of MCT1 is more rapid and the extent is greater. This result might be expected since an alkaline-inside pH gradient would be predicted to cause recruitment of an outward-facing binding site of the transporter, according to the accepted kinetic model for the carrier in which proton binding precedes monocarboxylate binding (1Poole R.C. Halestrap A.P. Am. J. Physiol. 1993; 264: C761-C782Google Scholar). Thus the empty carrier will preferentially take up the conformation in which the substrate binding site is exposed to the face of the membrane at which the proton concentration is highest. The inhibitor then binds and traps the carrier in this conformation. This model is supported by the observation that the Ki for inhibition of lactate transport by the reversibly binding stilbene disulfonate DBDS is lower in citrate than in saline buffer (results not shown). Such data provide further evidence that the cross-linking occurs at or near the external substrate binding site.Figure 3Time course of formation of DIDS cross-linked MCT1 conjugate in citrate and saline media. The protocol was the same as for Fig. 2, but incubation with 100 μm DIDS was performed for the time shown in either citrate medium (see “Methods”) or saline medium (150 mm NaCl, 20 mm HEPES, pH 7.4) as indicated.View Large Image Figure ViewerDownload (PPT) The cross-linking reaction can also be used to locate the region of MCT1 containing the DIDS-labeled lysine residue, by investigation of the fragments produced upon proteolysis of MCT1. Using a series of anti-peptide antibodies, we have characterized the pattern of cleavage by several proteases and used this information to derive information on the topology of the protein with respect to the membrane (8Poole R.C. Sansom C.E. Halestrap A.P. Biochem. J. 1996; 320: 817-824Google Scholar). Rat MCT1 has only 5 lysine residues predicted to be extracellular in location, and these are the most likely targets for labeling by DIDS. Two of these lysine residues are within a well characterized N-terminal fragment, and the remaining three in fragments reactive with antibodies raised against the TM7/8 and TM11/12 loops. Thus, if the binding protein is linked to either of these fragments by DIDS, there should be additional immunoreactve fragments derived from proteolytic digestion. Data demonstrating this are shown in Fig. 4. Trypsin digests MCT1 quantitatively to yield a fragment of approximately 20 kDa that reacts with the TM11/12 antibody. Upon digestion of membranes from DIDS-pretreated erythrocytes, additional antibody reactive bands of approximately 100 and 35 kDa were detected. These data indicate that DIDS reacts with a lysine in the C-terminal half of the protein, which, following trypsin cleavage, remains conjugated to the binding protein (100 kDa) or a proteolytically cleaved fragment of it (35 kDa). Membranes from DIDS-treated erythrocytes were treated with N-glycanase F, which resulted in an apparent reduction in molecular mass of the binding protein on SDS-PAGE, to approximately 100 kDa (Fig. 5). Since MCT1 is not glycosylated (6Carpenter L. Poole R.C. Halestrap A.P. Biochim. Biophys. Acta. 1996; 1279: 157-163Google Scholar), this must reflect N-linked glycosylation of the binding protein. Antibodies raised against the MCT1-binding protein conjugate, as described under “Experimental Procedures,” recognized two proteins on Western blots of control erythrocyte membranes. These were a weak band at 40 kDa that reacted with antibodies against MCT1 (not shown) and a stronger band at 70 kDa, which is likely to represent the unconjugated binding protein (Fig.6). In membranes prepared from cells pretreated with 100 μm DIDS, these bands were still detected, but an additional band at 130 kDa was detected that presumably represents the DIDS cross-linked MCT1 conjugate to which the antibody was raised.Figure 6Western blots of control and DIDS-treated erythrocyte membrane proteins probed with an antibody against the DIDS cross-linked MCT1-conjugate protein. Control erythrocytes and those treated with 100 μm DIDS were prepared for SDS-PAGE as described for Fig. 2. Subsequent Western blotting was performed with an antibody against the DIDS-cross-linked MCT1-conjugate protein raised as described under “Methods.” Parallel Western blots using control serum showed no specific bands.View Large Image Figure ViewerDownload (PPT) We attempted to purify the putative 70-kDa binding protein by replacing DIDS as cross-linker with the cleavable membrane impermeant cross-linking reagent showed that erythrocytes incubated for 1 at 37 °C with 1 mm a 130-kDa cross-linked product that reacted with MCT1 antibody on Western purification of this protein was performed on and SDS-PAGE, by of the 130-kDa band as described for the DIDS-labeled conjugate under “Methods.” of the protein was by incubation with for and the cleaved products were separated by SDS-PAGE. A broad band of about 70 kDa was but N-terminal sequencing showed it to more than Thus we attempted to purify the binding protein. Western blotting with the antibody raised against the 130-kDa MCT1-binding protein conjugate showed that the 70-kDa putative binding protein from Q-Sepharose at a concentration to MCT1 m NaCl in batch The protein was further purified by SDS-PAGE, and a broad band 70 kDa, presumably of a of was electroeluted and then subjected to treatment The 70-kDa free of MCT1, was detected with the and treatment F reduced apparent molecular mass to kDa. staining of these fractions revealed the band to be a of the deglycosylated as shown in The core protein prepared in this was subjected to N-terminal but no was Thus it was with the protease Lys-C, and the peptides were separated by SDS-PAGE before to ProBlot membrane for N-terminal microsequencing. Two peptides were of approximately and 3.5 kDa, but only the a of amino This was used in a of the protein data via the for and was to have a to an of mouse teratocarcinoma glycoprotein as shown in This 70-kDa glycoprotein is a cell member of the immunoglobulin superfamily that has a (11Ozawa M. Huang R.-P. Furukawa T. Muramatsu T. J. Biol. Chem. 1988; 263: 3059-3062Google Scholar). It to yield a core protein with a on SDS-PAGE to about 40 the molecular being Thus it is that MCT1-binding protein is the rat equivalent of mouse GP-70, or a closely related protein. The is further by the presence of a lysine on the N-terminal of the of the since was derived by Lys-C of some amino acid of the MCT1-binding protein with the mouse and rat The represents amino of the mouse membrane teratocarcinoma glycoprotein The to that obtained from N-terminal microsequencing a derived from Lys-C digestion of the purified and deglycosylated MCT1-binding protein as described in the The at the is in of the of the is derived from of a rat that was by the data with the as described in the Large Image Figure ViewerDownload (PPT) that the MCT1-binding protein is the rat equivalent of the mouse was obtained by the data with the Two closely related were and of which only the to the region of mouse this fragment the region of the protein to the we had The the is shown in Fig. and with in all but The was a which we as an This is a common sequencing since the two amino with very but it may also be the result of a sequencing in the The data demonstrate that DIDS can cross-link MCT1 to a 70-kDa glycoprotein in rat erythrocyte membranes via an amino acid This protein is likely to be the rat equivalent of mouse teratocarcinoma glycoprotein GP-70, a member of the immunoglobulin Since DIDS is an that binds in competition with substrates and inhibitors of MCT1, it is likely that cross-linking occurs at the substrate binding it is that binding occurs at a site conformation and for DIDS is affected by substrate binding. of to the substrate binding site of MCT1 the that this interaction may have an effect on the of MCT1. In two distinct monocarboxylate transporters have been detected Poole R.C. Halestrap A.P. Biochem. J. 1993; Scholar, Halestrap A.P. Am. J. Physiol. 1994; Scholar, Halestrap A.P. Am. J. Physiol. 1996; 270: Scholar), and of have that to those of MCT1. there is a level of of MCT1 in the region of the (2Kim Garcia C. Goldstein J.L. Pathak R.K. Anderson R.G.W. Brown M.S. Cell. 1994; 76: 865-873Google Scholar). and A. P. Halestrap, Such observations may be if the absence or presence of a protein in cells could the functional of MCT1.