Fig. 4.

Functional characterization of PurT_Cp. (A) SPA-based binding of 0.5 µM ³H-xanthine (red) or ³H-guanine (blue) binding by PurT_Cp-WT or -D276A or D276N measured at pH 5.5 and pH 8.0. (B) pH dependence of 0.5 µM ³H-xanthine or ³H-guanine binding by PurT_Cp-WT. (C) The uptake of 1 µM ³H-xanthine by PurT_Cp-containing proteoliposomes requires an inwardly directed H⁺ gradient. Proteoliposomes containing PurT_Cp or control liposomes devoid of protein were prepared in either 20 mM HEPES-KOH, pH 8.5 or 20 mM MES-KOH, pH 5.5 and 100 mM KCl, 2 mM β-mercaptoethanol (pH_in) and transport was measured for 30 s in 20 mM HEPES-KOH, pH 8.5 or 20 mM MES-KOH, pH 5.5 and 100 mM NaCl, 2 mM β-mercaptoethanol (pH_out) in the presence or absence of 1 µM of the K⁺ ionophore valinomycin (Val) as indicated. (D) pH dependence of 1 µM ³H-xanthine transport by proteoliposomes containing PurT_Cp or control liposomes (pH_in = 8.5) measured for 1 min in assay buffer with pH_out between 5.0 and 9.0. Data were normalized to the maximum transport activity observed for PurT_Cp-containing proteoliposomes at pH_out = 5.0. (E) Time course of 1 µM ³H-xanthine in proteoliposomes containing PurT_Cp or control liposomes (pH_in = 8.5; pH_out = 5.5). (F) Transport kinetics of PurT_Cp incorporated into proteoliposomes (pH_in = 8.5; pH_out = 5.5) revealed a Michaelis–Menten constant (K_m) of 1.78 ± 0.23 µM and a maximum velocity of transport (V_max) of 102.0 ± 3.3 nmol × mg⁻¹ × min⁻¹. (G) Substrate uptake is dependent on the proton motive force (pmf). The uptake of 1 µM ³H-xanthine in proteoliposomes containing PurT_Cp or control liposomes was measured for 5 min in the presence or absence of 5 µM of the protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Data in panels A–G are means ± SEM of three independent experiments performed as technical triplicates. (H) Transport of xanthine is accompanied by H⁺ flux. Representative recording (n = 8) of a SSM electrophysiological measurement performed with proteoliposomes containing PurT_Cp (red) or with control liposomes (gray) using the SURFE²R N1 (Nanion Technologies, Inc.). Ten micromolar xanthine was added to the assay buffer as indicated by the bar. The Inset shows the net difference between currents (ΔI) elicited by 10 µM of the nontransported pyrimidine uracil (Ura, black) or glucose (Glc, green) in PurT_Cp-containing proteoliposomes and control liposomes. Data of a representative experiment (n = 3 to 6) are shown. All proteoliposome preparations were tested for H⁺ leakiness using an assay by Tsai and Miller (26) (SI Appendix, Fig. S6c).