CHARACTERIZATION OF THE RABBIT RENAL NA-DICARBOXYLATE COTRANSPORTER USING ANTIFUSION PROTEIN ANTIBODIES()

Polyclonal antibodies were prepared against the rabbit renal Na+-dicar boxylate cotransporter, NaDC-1. The antibodies were raised in chickens against a fusion protein consisting of a 60-amino acid peptide from N aDC-1 and glutathione S-transferase. These antibodies specifically rec ognized the fusion protein in Western blots and could immunoprecipitat e the full-length NaDC-1 after in vitro translation. The antifusion pr otein 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 rab bit intestinal brush-border membranes. A protein of 66 kDa was recogni zed in Xenopus oocytes injected with NaDC-1 cRNA. Enzymatic deglycosyl ation of rabbit renal BBMV resulted in a decrease in mass by 11 kDa, c onsistent with N-glycosylation at a single site. Site-directed mutagen esis of the two consensus sequences for N-glycosylation in the NaDC-1 cDNA showed that Asn-576, located near the COOH-terminal, is glycosyla ted. The nonglycosylated mutant of NaDC-1 exhibited 50% of wild-type s uccinate transport activity when expressed in Xenopus oocytes, suggest ing that glycosylation is not essential for function. The revised seco ndary structure model of NaDC-1 contains 11 putative transmembrane dom ains and an extracellular glycosylated COOH-terminal.