Studies of the membrane topology of the rat erythrocyte H+/lactate cotransporter (MCT1).

Abstract

1. Hydrophobicity analysis of the monocarboxylate/proton cotransporter MCT1 (lactate transporter) suggests a structure with 12 transmembrane (TM) segments, presumed to be alpha-helical. 2. A series of anti-peptide antibodies have been raised against regions of the MCT1 sequence, which each recognize a polypeptide of approx. 40 kDa in rat erythrocytes. The topology of rat MCT1 was investigated by studying the immunoreactive fragments derived from proteolytic digestion of the protein in intact rat erythrocytes and leaky membranes. 3. Reactivity with an anti-(C-terminus) antibody was prevented on treatment of leaky membranes, but not intact cells, with carboxypeptidase Y, indicating that the C-terminus of the protein is cytoplasmically disposed. 4. Treatment of intact cells in saline buffer with trypsin, chymotrypsin, bromelain and protease K (up to 1 mg/ml) resulted in no degradation of MCT1, indicating the absence of any large exposed extracellular loop. In a buffer of low ionic strength (containing sucrose), cleavage was observed with bromelain at an extracellular site, probably TM9/10.5. Treatment of leaky membranes with low (less than 100 micrograms/ml) concentrations of several proteases resulted in fragmentation of MCT1, reflecting cleavage at the cytoplasmic face of the membrane. These treatments generated N-terminal fragments of apparent molecular mass approx. 17-19 kDa that were resistant to further degradation. The epitopes for the TM6/7 and C-terminal antibodies were either lost from the membrane or destroyed under most of these conditions, indicating that these regions of the protein are located in the cytoplasm. 6. More detailed structural prediction analysis of MCT-related sequences was made assuming the constraints placed upon the possible arrangements by the experimental data outlined above. This analysis provided additional strong evidence for the 12-TM-segment model, with cytoplasmic N- and C-terminal ends and a large internal loop between TM6 and TM7. The predicted helices were assigned moments of hydrophobicity and residue substitution; for a number of TM segments this permitted the prediction of the sides of the helix that faced membrane lipid and the interior of the protein.