Interactions between the thiol-group reagent N-ethylmaleimide and neutral and basic amino acid transporter-related amino acid transport.
The neutral and basic amino acid transport protein (NBAT) expressed in renal and jejunal brush-border membranes is involved in amino acid and cystine absorption. NBAT mutations result in Type 1 cystinuria. A C-terminal myc-tagged NBAT (NBATmyc) retains the amino acid transport and protein-protein interaction properties of NBAT when expressed in Xenopus oocytes. Neutral amino acid (Ala, Phe)-cationic amino acid (Arg) heteroexchanges related to NBATmyc expression in oocytes are inactivated by treatment with the thiol-group reagent N-ethylmaleimide (NEM), although significant Arg-Arg and Ala-Ala homoexchanges persist. Inactivation of heteroexchange activity by NEM is accompanied by loss of >85% of alanine and cystine uptake, with smaller (<50%) inhibition of arginine and phenylalanine uptake. NEM-sensitive cystine uptake and arginine-alanine heteroexchange (system b(0,+) activity) are not expressed by an NBAT truncation mutant (NBATmyc-Sph1) lacking the 13 C-terminal amino acid residues, but the mutant expresses NEM-resistant transport activity (system y(+)L-like) equivalent to that of full-length NBATmyc. The deleted region of NBATmyc-Sph1 contains two cysteine residues (671/683) which may be the targets of NEM action. The synthetic amino acid 2-trifluoromethylhistidine (TFMH) stimulated alanine efflux at pH 7.5 and arginine at pH 5.5, but not vice versa, establishing the existence of distinct pathways for cationic and neutral amino acid homoexchange (TFMH is zwitterionic at pH 7.5 and cationic at pH 5.5). We suggest that NBAT expresses a combination of system b(0,+) and y(+)L-like activities, possibly by interacting with different light-chain subunits endogenous to oocytes (as does the homologous 4F2hc protein). The C-terminus of NBAT may also have an additional, direct role in the mechanism of System b(0,+) transport (the major transport activity that is defective in Type 1 cystinuria).