Figure 8: PbNPT1 is a cationic amino acid transporter.

(a) Uptake of [¹⁴C]Arg and [¹⁴C]Lys into X. laevis oocytes expressing PbNPT1-HA (black), or into uninjected controls (grey). The data are derived from the 30?min time points of Supplementary Fig. 5e,f. Uptake was measured in the presence of 289?nM [¹⁴C]Arg, 100?μM unlabelled arginine and 125?μM unlabelled methionine or 307?nM [¹⁴C]Lys, 100?μM unlabelled lysine and 500?μM unlabelled methionine. The data are averaged from three independent experiments and are shown±s.e.m. (*P<0.05; Student’s t test). (b) Uptake of [¹⁴C]Arg into oocytes expressing PbNPT1-HA (black) or uninjected oocytes (grey), incubated in the presence of 289?nM [¹⁴C]Arg and in either the absence (control) or presence of a 1?mM concentration of a range of unlabelled amino acids, and 125?μM unlabelled methionine. The data are averaged from three experiments, each conducted on oocytes from a different frog, and are shown±s.e.m. (*P<0.05; ***P<0.001; ****P<0.0001; n.s.=not significant; ANOVA. Where P is not indicated for comparisons between uptake in the presence and absence of unlabelled amino acids in PbNPT1-HA expressing oocytes, the differences are not significant). (c,d) Concentration-dependence of PbNPT1-mediated arginine and lysine transport in X. laevis oocytes expressing PbNPT1-HA. The uptake of [¹⁴C]Arg (c) and [¹⁴C]Lys (d) was measured over 30?min, in the presence of 125?μM (c) or 500?μM (d) unlabelled methionine. At each of the concentrations tested, uptake measured in control (uninjected) oocytes was subtracted from that in oocytes expressing PbNPT1 to yield PbNPT1-mediated transport. The data were averaged from three experiments, each conducted on oocytes from a different frog, and are shown±s.e.m. The Michaelis–Menten equation was fitted to the data by non-linear regression. For arginine, K_m=41±9?μM, mean±s.e.m.; V_max=2.6±0.1?pmol oocyte⁻¹?min⁻¹, mean±s.e.m. For lysine, K_m=130±26?μM, mean±s.e.m.; V_max=4.4±0.2?pmol oocyte⁻¹?min⁻¹, mean±s.e.m.