Characterization of the rabbit renal Na+-dicarboxylate cotransporter using antifusion protein antibodies

Polyclonal antibodies were prepared against the rabbit renal Na+-dicarboxylate cotransporter, NaDC-1. The antibodies were raised in chickens against a fusion protein consisting of a 60-amino acid peptide from NaDC-1 and glutathione S-transferase. These antibodies specifically recognized the fusion protein in Western blots and could immunoprecipitate the full-length NaDC-1 after in vitro translation. The antifusion protein antibodies specifically recognized a protein of 63 kDa in rabbit renal brush-border membrane vesicles (BBMV), similar to the predicted mass of 66 kDa. Two proteins of 57 and 115 kDa were recognized in rabbit intestinal brush-border membranes. A protein of 66 kDa was recognized in Xenopus oocytes injected with NaDC-1 cRNA. Enzymatic deglycosylation of rabbit renal BBMV resulted in a decrease in mass by 11 kDa, consistent with N-glycosylation at a single site. Site-directed mutagenesis of the two consensus sequences for N-glycosylation in the NaDC-1 cDNA showed that Asn-576, located near the COOH-terminal, is glycosylated. The nonglycosylated mutant of NaDC-1 exhibited 50% of wild-type succinate transport activity when expressed in Xenopus oocytes, suggesting that glycosylation is not essential for function. The revised secondary structure model of NaDC-1 contains 11 putative transmembrane domains and an extracellular glycosylated COOH-terminal.