Figure 9.

Functional characterization of C467A. (A) Dose–response curve for the inhibition of the hASCT2 C467A in proteoliposomes by Cys. The reconstitution was performed as described in Section 4.5 and transport was measured adding 50 mM Na-gluconate and 50 µM [³H]Gln to proteoliposomes containing 10 mM Gln in the presence of indicated concentrations of Cys. Transport activity was calculated as percent of residual activity with respect to condition without any addition. (B) Efflux of Gln from proteoliposomes reconstituted with C467A hASCT2 mutant. The reconstitution was performed as described in Section 4.5 and uptake of 50 µM [³H]Gln in proteoliposomes was performed in the presence of 50 mM Na-Gluconate. After accumulation of [³H]Gln for 120 min, [³H]Gln efflux was measured in the presence of 50 mM Na-gluconate and 1 mM Gln or 1 mM Cys. After complete Gln efflux, aliquots of proteoliposomes were passed through Sephadex G75 column to remove external substrates. Percent of intraliposomal residual radioactivity is reported compared to control. Student’s two tailed unpaired t-test was performed on the WT sample; p value was symbolized as follows: ** p < 0.01. (C) The reconstitution was performed as described in Section 4.5 and transport was measured adding 50 mM Na-gluconate and 50 µM [³H]Gln to proteoliposomes containing 10 mM Gln in the presence of 5 mM GSH, 100 µM NaHS (H₂S donor) or 1 mM GSNO (NO donor). Transport was calculated as percent of residual activity with respect to condition without any addition. (A–C) Results are means ± S.D. from three experiments.