LAT1 is the transport competent unit of the LAT1/CD98 heterodimeric amino acid transporter

摘要: LAT1 (SLC7A5) and CD98 (SLC3A2) constitute a heterodimeric transmembrane protein complex that catalyzes amino acid transport. Whether one or both subunits are competent for transport is still unclear. The present work aims to solve this question using different experimental strategies. Firstly, were immuno-detected in extracts from SiHa cells. Under oxidizing conditions, i.e., without addition of SH (thiol) reducing agent DTE, proteins revealed as 120kDa major band. Upon DTE treatment separated bands, corresponding LAT1(35kDa) CD98(80kDa), detected. function was evaluated intact cells BCH sensitive [(3)H]His inhibited by hydrophobic acids. Antiport measured proteoliposomes reconstituted with cell extract presence internal His. Transport increased DTE. Hydrophobic acids best inhibitors hydrophilic Tyr, Gln, Asn Lys. Cys, Tyr included the intraliposomal space, transported antiport external [(3)H]His. Similar experiments performed recombinant purified hLAT1. Results overlapping those obtained native achieved. Lower [(3)H]Leu [(3)H]Gln respect Kinetic asymmetry found Km His lower than one. No detected hCD98. data demonstrate sole subunit heterodimer. This conclusion has important outcome following studies on functional characterization identification specific potential application human therapy.