Journal Article

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

Indiveri, Cesare, Pochini, Lorena, Pingitore, Piero, Hedfalk, Kristina, Scalise, Mariafrancesca, Pappacoda, Gilda, Console, Lara

2018

Overview

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.

• Publisher: MDPI AG,MDPI
• Subject: amino acid / Amino Acid Transport System ASC - chemistry / Amino Acid Transport System ASC - genetics / Amino Acid Transport System ASC - metabolism / amino-acid transporter / anticancer drugs / Biochemistry & Molecular Biology / Bioinformatics / Biological Transport - drug effects / Chemical Sciences / Chemistry / cysteine / Cysteine - genetics / Disulfides - chemistry / Energy Metabolism / glutamine / Glutamine - metabolism / Glutamine - pharmacology / glutathionylation / Humans / inhibition / Kemi / Kinetics / liposome / liposomes / membrane / Minor Histocompatibility Antigens - chemistry / Minor Histocompatibility Antigens - genetics / Minor Histocompatibility Antigens - metabolism / mitochondrial carnitine/acylcarnitine carrier / Models, Molecular / Molecular Biology / Molekylärbiologi / Mutagenesis / Mutagenesis, Site-Directed / over-expression / Oxidation-Reduction / pichia-pastoris / Protein Conformation / reconstitution / site-directed mutagenesis / Structure-Activity Relationship / Substrate Specificity / transport