Characterization of the lysosomal arginine transporter SLC7A14

SLC7A14 is a putative amino acid transporter whose physiological role remains poorly characterized, despite its association with retinal degeneration, auditory defects, and metabolic dysfunctions. Confocal microscopy was performed in HEK293 transiently transfected with the human SLC7A14 (hSLC7A14), demonstrating a lysosomal localization, as previously suggested. To carry on functional and kinetic characterization, the protein was overexpressed in E. coli, and purified from insoluble fraction through affinity chromatography followed by SEC with a yield of 156 mg/L of bacterial cell culture. The homogeneous hSLC7A14 showed an apparent molecular mass of 72 kDa on SDS-PAGE and was reconstituted into proteoliposomes for functional assays. hSLC7A14 showed high specificity towards arginine, not histidine or glutamine, with a measured Km of 1.2 ± 0.21 mM. The arginine transport was inhibited by cysteine and threonine, but not by other amino acids or GABA. Inhibition kinetics identified metformin as an inhibitor of the transporter showing a mixed type of inhibition with a measured Ki in the same order of that of arginine. These findings are in agreement with hSLC7A14 being a specific lysosomal arginine transporter, with potential involvement in the mTORC1 signalling. The described results represent a first step to further elucidate hSLC7A14 role in human physiology and disease. Moreover, the identification of experimental conditions for measuring specific transport activity will allow screening of ligands as inhibitors or functional modulators opening perspectives for structure/function relationship studies.