Figure 4. LAPTM4b promotes uptake of Leu into lysosomes.

(a) ³H-Leu uptake into cells is not affected by LAPTM4b: HeLa cells stably knocked down for LAPTM4b (control and LAPTM4b KD, upper panel) or overexpressing LAPTM4b (lower panel) were incubated for 1–10 min in uptake solution containing 5 μM [³H]L-Leu. C (control) represents competition with 5 mM L-Leu. (b) ³H-Leu uptake into lysosomes is reduced upon knockdown of LAPTM4b and restored upon its re-expression: HeLa cells stably knocked down for control KD, LAPTM4b (LAPTM4b KD) or reconstituted with LAPTM4b (LAPTM4b KD+LAPTM4b) were transfected with Flag-LAMP1, serum- and nutrient-starved and stimulated for 10 min with EAA that contains 0.4 mM Leu plus [³H]-Leu. Lysosomes were isolated by anti-Flag immunoprecipitation and their radioactivity quantified by scintillation counting, as described11. Data are mean±s.e.m. (N=3 independent experiments, each performed in triplicates). P values were calculated from the Student's t-test.