反转运蛋白
氨基酸
生物化学
蛋白质亚单位
氨基酸转运体
化学
跨膜蛋白
运输机
重组DNA
基因
膜
受体
作者
Lara Napolitano,Mariafrancesca Scalise,Michele Galluccio,Lorena Pochini,Leticia Maria Albanese,Cesare Indiveri
标识
DOI:10.1016/j.biocel.2015.08.004
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, LAT1 and CD98 were immuno-detected in protein extracts from SiHa cells. Under oxidizing conditions, i.e., without addition of SH (thiol) reducing agent DTE, both proteins were revealed as a 120 kDa major band. Upon DTE treatment separated bands, corresponding to LAT1(35 kDa) or CD98(80 kDa), were detected. LAT1 function was evaluated in intact cells as BCH sensitive [3H]His transport inhibited by hydrophobic amino acids. Antiport of [3H]His was measured in proteoliposomes reconstituted with SiHa cell extract in presence of internal His. Transport was increased by DTE. Hydrophobic amino acids were best inhibitors in addition to hydrophilic Tyr, Gln, Asn and Lys. Cys, Tyr and Gln, included in the intraliposomal space, were transported in antiport with external [3H]His. Similar experiments were performed in proteoliposomes reconstituted with the recombinant purified hLAT1. Results overlapping those obtained with native protein were achieved. Lower transport of [3H]Leu and [3H]Gln with respect to [3H]His was detected. Kinetic asymmetry was found with external Km for His lower than internal one. No transport was detected in proteoliposomes reconstituted with recombinant hCD98. The experimental data demonstrate that LAT1 is the sole transport competent subunit of the heterodimer. This conclusion has important outcome for following studies on functional characterization and identification of specific inhibitors with potential application in human therapy.
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