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 erythrocytes 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 microM DIDS. The 70-kDa protein that is cross-linked to MCT1 was purified and shown to contain N-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.
Poole
R C
RC
Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom.
Halestrap
A P
AP
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000097:blood* / Q000187:drug effects; D003432:Cross-Linking Reagents; D019281:Dimerization; D004910:Erythrocyte Membrane / Q000378:metabolism*; D007136:Immunoglobulins / Q000737:chemistry; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D027501:Monocarboxylic Acid Transporters; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013724:Teratoma; D014357:Trypsin
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D007136:Immunoglobulins; D008562:Membrane Glycoproteins; D027501:Monocarboxylic Acid Transporters; D010446:Peptide Fragments; D014357:Trypsin; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid