Extended Data 7. Characterization of SLC38A9-mediated amino acid transport in proteoliposomes.

a, Purification of SLC38A9. Lanes represent empty vector control and SLC38A9 expressed in E. coli and purified on Ni-chelating chromatography. Immunoblot of the same fractions using anti-His antibody or anti-SLC38A9 are shown. b, Orientation of SLC38A9 in proteoliposomes. Purified His-SLC38A9 protein or proteoliposomes reconstituted with SLC38A9 were incubated overnight at 37°C in presence or in absence of 1 U thrombin. Proteoliposomes were then solubilized with SDS and analysed by immunoblot. Results are representative of two independent experiments (n=2) c, Time course of glutamine uptake by SLC38A9 in proteoliposomes reconstituted with the purified protein fraction. The uptake of 10 μM [³H]-glutamine was measured at different time intervals in the presence of the indicated intraliposomal sodium concentrations. Transport was calculated by subtracting the radioactivity associated to proteoliposomes reconstituted with the empty vector fraction. Values represent means of specific transport ± s.d. from three independent experiments (n=3) d, Time course of glutamine uptake in proteoliposomes reconstituted with purified SLC38A9 wild-type or N128A mutant protein. Values represent means of specific transport ± s.d. from 3 independent experiments (n=3). Significance was estimated by Student’s t test (* P < 0.01). Immunoblot analysis of purified protein reconstituted in the proteoliposomes e. Effect of pH on the reconstituted SLC38A9. Reconstitution and transport assay were performed at the indicated pH. Results are means of specific transport rate ± s.d. from three different experiments (n=3). f, Inhibition of the [³H]-glutamine uptake in proteoliposomes. 1 mM MeAIB (α-(methylamino)isobutyric acid) was added together with 10 μM [³H]-glutamine. Transport was measured at 60 min. Values represent means of percent residual activity with respect to control (without added inhibitor) ± s.d. from three independent experiments (n=3).