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Int. J. Mol. Sci. 2018, 19(3), 648; doi:10.3390/ijms19030648

Cys Site-Directed Mutagenesis of the Human SLC1A5 (ASCT2) Transporter: Structure/Function Relationships and Crucial Role of Cys467 for Redox Sensing and Glutamine Transport

Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via P. Bucci 4C, 87036 Arcavacata di Rende, Italy
Department of Molecular and Clinical Medicine, Wallenberg Laboratory, Sahlgrenska Academy, University of Gothenburg, 413 45 Gothenburg, Sweden
Department of Chemistry and Molecular Biology, University of Gothenburg, P.O. Box 462, SE-405 30 Göteborg, Sweden
CNR Institute of Biomembranes, Bioenergetics and Molecular Biotechnology, via Amendola 165/A, 70126 Bari, Italy
Author to whom correspondence should be addressed.
Received: 10 January 2018 / Revised: 12 February 2018 / Accepted: 23 February 2018 / Published: 25 February 2018
(This article belongs to the Special Issue Amino Acids Transport and Metabolism)
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The human plasma membrane transporter ASCT2 is responsible for mediating Na- dependent antiport of neutral amino acids. New insights into structure/function relationships were unveiled by a combined approach of recombinant over-expression, site-directed mutagenesis, transport assays in proteoliposomes and bioinformatics. WT and Cys mutants of hASCT2 were produced in P. pastoris and purified for functional assay. The reactivity towards SH reducing and oxidizing agents of WT protein was investigated and opposite effects were revealed; transport activity increased upon treatment with the Cys reducing agent DTE, i.e., when Cys residues were in thiol (reduced) state. Methyl-Hg, which binds to SH groups, was able to inhibit WT and seven out of eight Cys to Ala mutants. On the contrary, C467A loses the sensitivity to both DTE activation and Methyl-Hg inhibition. The C467A mutant showed a Km for Gln one order of magnitude higher than that of WT. Moreover, the C467 residue is localized in the substrate binding region of the protein, as suggested by bioinformatics on the basis of the EAAT1 structure comparison. Taken together, the experimental data allowed identifying C467 residue as crucial for substrate binding and for transport activity modulation of hASCT2. View Full-Text
Keywords: amino acid; glutamine; transport; over-expression; site-directed mutagenesis; liposome amino acid; glutamine; transport; over-expression; site-directed mutagenesis; liposome

This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Scalise, M.; Pochini, L.; Console, L.; Pappacoda, G.; Pingitore, P.; Hedfalk, K.; Indiveri, C. Cys Site-Directed Mutagenesis of the Human SLC1A5 (ASCT2) Transporter: Structure/Function Relationships and Crucial Role of Cys467 for Redox Sensing and Glutamine Transport. Int. J. Mol. Sci. 2018, 19, 648.

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