RESUMEN
SNAT4 is a system A type amino acid transporter that primarily expresses in liver and mediates the transport of L-alanine. To determine the critical amino acid residue(s) involved in substrate transport function of SNAT4, we used hydrosulfate cross-linking MTS reagents - MMTS and MTSEA. These two reagents caused inhibition of L-alanine transport by wild-type SNAT4. There are 5 cysteine residues in SNAT4 and among them; residues Cys-232 and Cys-345 are located in the transmembrane domains. Mutation of Cys-232, but not Cys-345, inhibited transport function of SNAT4 and also rendered SNAT4 less sensitive to the cross-linking by MMTS and MTSEA. The results suggested that TMD located Cys-232 is an aqueous accessible residue, likely to be located close to the core of substrate binding site. Mutation of Cys-232 to serine similarly attenuated the transport of L-alanine substrate. Biotinylation analysis showed that C232A mutant of SNAT4 was equally capable as wild-type SNAT4 of expressing on the cell surface. Moreover, single site mutant, C232A was also found to be more resistant to MTS inhibition than double mutant C18A,C345A, further confirming the aqueous accessibility of Cys-232 residue. We also showed that mutation of Cys-232 to alanine reduced the maximal velocity (Vmax), but had minimal effect on binding affinity (Km). Together, these data suggest that residue Cys-232 at 4(th) transmembrane domain of SNAT4 has a major influence on substrate transport capacity, but not on substrate binding affinity.
RESUMEN
Members of system N/A amino acid transporter (SNAT) family mediate transport of neutral amino acids, including l-alanine, l-glutamine, and l-histidine, across the plasma membrane and are involved in a variety of cellular functions. By using chemical labeling, glycosylation, immunofluorescence combined with molecular modeling approaches, we resolved the membrane topological structure of SNAT4, a transporter expressed predominantly in liver. To analyze the orientation using the chemical labeling and biotinylation approach, the "Cys-null" mutant of SNAT4 was first generated by mutating all five endogenous cysteine residues. Based on predicted topological structures, a single cysteine residue was introduced individually into all possible nontransmembrane domains of the Cys-null mutant. The cells expressing these mutants were labeled with N-biotinylaminoethyl methanethiosulfonate, a membrane-impermeable cysteine-directed reagent. We mapped the orientations of N- and C-terminal domains. There are three extracellular loop domains, and among them, the second loop domain is the largest that spans from amino acid residue â¼242 to â¼335. The orientation of this domain was further confirmed by the identification of two N-glycosylated residues, Asn-260 and Asn-264. Together, we showed that SNAT4 contains 10 transmembrane domains with extracellular N and C termini and a large N-glycosylated, extracellular loop domain. This is the first report concerning membrane topological structure of mammalian SNAT transporters, which will provide important implications for our understanding of structure-function of the members in this amino acid transporter family.
