title: LAT1 is the transport competent unit of the LAT1/CD98 heterodimeric amino acid transporter.
abstract: 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 120kDa major band. Upon DTE treatment separated bands, corresponding to LAT1(35kDa) or CD98(80kDa), were detected. LAT1 function was evaluated in intact cells as BCH sensitive [(3)H]His transport inhibited by hydrophobic amino acids. Antiport of [(3)H]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 [(3)H]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 [(3)H]Leu and [(3)H]Gln with respect to [(3)H]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.
authors.affiliation: Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via Bucci 4C, 87036 Arcavacata di Rende, Italy.
authors.forename: Lara
authors.initials: L
authors.lastname: Napolitano
authors.affiliation: Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via Bucci 4C, 87036 Arcavacata di Rende, Italy.
authors.forename: Mariafrancesca
authors.initials: M
authors.lastname: Scalise
authors.affiliation: Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via Bucci 4C, 87036 Arcavacata di Rende, Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Galluccio
authors.affiliation: Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via Bucci 4C, 87036 Arcavacata di Rende, Italy.
authors.forename: Lorena
authors.initials: L
authors.lastname: Pochini
authors.affiliation: Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via Bucci 4C, 87036 Arcavacata di Rende, Italy.
authors.forename: Leticia Maria
authors.initials: LM
authors.lastname: Albanese
authors.affiliation: Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via Bucci 4C, 87036 Arcavacata di Rende, Italy. Electronic address: cesare.indiveri@unical.it.
authors.forename: Cesare
authors.initials: C
authors.lastname: Indiveri
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D001692:Biological Transport; D045744:Cell Line, Tumor; D004530:Egg Yolk / Q000737:chemistry; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D027283:Large Neutral Amino Acid-Transporter 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D010743:Phospholipids / Q000737:chemistry; D055503:Protein Multimerization; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein
chemical_list: D000596:Amino Acids; D027283:Large Neutral Amino Acid-Transporter 1; D010743:Phospholipids; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes
keywords: CD98; Heterodimer; Histidine; LAT1; Proteoliposomes; Transport