title: Residue cysteine 232 is important for substrate transport of neutral amino acid transporter, SNAT4.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center 7703 Floyd Curl Drive, San Antonio, TX.
authors.forename: Rugmani
authors.initials: R
authors.lastname: Padmanabhan
authors.forename: Sumin
authors.initials: S
authors.lastname: Gu
authors.forename: Bruce J
authors.initials: BJ
authors.lastname: Nicholson
authors.forename: Jean X
authors.initials: JX
authors.lastname: Jiang
keywords: Cys-232; Neutral amino acid transporter; SNAT4; substrate transport