Insights into the transport side of the human SLC38A9 transceptor |
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Authors: | Mariafrancesca Scalise Michele Galluccio Lorena Pochini Jessica Cosco Miriam Trotta Manuele Rebsamen Giulio Superti-Furga Cesare Indiveri |
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Institution: | 1. 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;2. CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria;3. Center for Physiology and Pharmacology, Medical University of Vienna, 1090 Vienna, Austria;4. CNR Institute of Biomembranes, Bioenergetics and Molecular Biotechnology, via Amendola 165/A, 70126 Bari, Italy |
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Abstract: | The lysosomal amino acid transporter SLC38A9 is referred to as transceptor, i.e. a transporter with a receptor function. The protein is responsible for coupling amino acid transport across the lysosomal membrane according to the substrate availability to mTORC1 signal transduction. This process allows cells to sense amino acid level responding to growth stimuli in physiological and pathological conditions triggering mTOR regulation. The main substrates underlying this function are glutamine and arginine. The functional and kinetic characterization of glutamine and arginine transport was performed using human SLC38A9 produced in E. coli, purified by affinity chromatography and reconstituted in liposomes. A cooperative behaviour for the wild type protein was revealed for both the substrates. A novel Na+ binding site, namely T453, was described by combined approaches of bioinformatics, site-directed mutagenesis and transport assay. Stimulation by cholesterol of glutamine and arginine transport was observed. The biological function of SLC38A9 relies on the interaction between its N-terminus and components of the mTOR complex; a deletion mutant of the N-terminus tail was produced and transport of glutamine was assayed revealing that this portion does not play any role in the intrinsic transport function of the human SLC38A9. Different features for glutamine and arginine transport were revealed: human SLC38A9 is competent for glutamine efflux, while that of arginine is negligible. In line with these results, imposed ?pH stimulated glutamine, not arginine transport. Arginine plays, on the contrary, a modulatory function and is able to stimulate glutamine efflux. Interestingly, reciprocal inhibition experiments also supported by bioinformatics, suggested that glutamine and arginine may bind to different sites in the human SLC38A9 transporter. |
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Keywords: | Corresponding author at: 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 MeAIB 2-Methylaminoisobutyric acid mTORC1 mammalian target of rapamycin complex 1 12 Octaethylene Glycol Monododecyl Ether CHS Cholesteryl HemiSuccinate NEM N-EthylMaleimide MTSEA 2-aminoethyl methanethiosulfonate hydrobromide PLP pyridoxal phosphate drSLC38A9 mTOR Proteoliposomes Cholesterol Glutamine Transceptor hSLC38A9 |
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