Figure 4.

Inhibition of Tf recycling by dyn^ts is not due to defective endosome acidification. (A) dyn^wt (circles) and dyn^ts (squares)-expressing cells were loaded with ¹²⁵I-Tf for 1 h at 38°C. Cell surface-associated ¹²⁵I-Tf was removed at 0°C by the acidic-neutral wash procedure using either MES buffer (open symbols) or acetate buffer (closed symbols). The cells were then reincubated at 38°C, and the release of ¹²⁵I-Tf was measured. Intracellular ¹²⁵I-Tf is plotted as a percentage of total endocytosed ¹²⁵I-Tf (n = 2, mean ± SD). The experiments shown are representative of three independent experiments, each with duplicate samples. The acetate treatment assured the release of Fe³⁺ from intracellular ¹²⁵I-Tf. dyn^ts inhibited the release of ¹²⁵I-Tf irrespective of acetate or MES treatment. Lack of endosome acidification thus can not explain the results. (B) As a positive control for this procedure, dyn^wt-expressing cells were loaded for 1 h at 38°C with ¹²⁵I-Tf in the absence (○) or presence (▵ and □) of 100 nM of concanamycin A, a vacuolar proton pump inhibitor. Cell surface-associated ¹²⁵I-Tf was removed at 0°C by the acid-neutral wash procedure using either MES buffer (□) or acetate buffer (○ and▵). Cells were then reincubated at 38°C in the absence (○) or presence (▵ and □) of 100 nM concanamycin A and release of ¹²⁵I-Tf was measured. Intracellular ¹²⁵I-Tf is plotted as a percentage of total internalized ¹²⁵I-Tf. The results shown (n = 2, mean ± SD) are representative of five independent experiments. The acetate treatment restored ¹²⁵I-Tf recycling by concanamycin A-treated cells, illustrating the effectiveness of this procedure.