Figure 7.

(A) Steady-state distribution of TfR on the cell surface of PITPα +/+ and −/− cells. Steady-state distribution of TfR was performed as described previously at 37°C (Odorizzi et al., 1994; see MATERIALS AND METHODS) with the modification that PITPα +/+ and −/− ES cells were plated in triplicate in standard media containing either 10 or 3% serum. The percentage of total ¹²⁵I-Tf (and therefore TfR) located at the cell surface (solid bars) and within the cell (hatched bars) is given for each ES cell PITPα genotype (+/+ or −/−; bottom) and for each serum concentration tested (3 and 10%; top). (B) Kinetics of TfR internalization and recycling in PITPα +/+ and −/− cells. For internalization assays, PITPα +/+ (solid bars) and −/− cells (hatched bars) were preincubated in serum-free media at 37°C and prewarmed ¹²⁵I-Tf was added for various times up to 10 min. At each time point, the ratio of internalized and surface ¹²⁵I-Tf was distinguished by an acid strip of the cell surface, and Tf(internal)/Tf(surface) ratio was plotted as a function of time. The slope of each curve yields the TfR internalization constant k_in, and the data represent the mean and SDs derived from at least three independent determinations. To measure the recycling constant k_ext, cells were incubated with ¹²⁵I-Tf for 1 h at 37°C and chilled. Surface-bound ¹²⁵I-labeled Tf was removed by chelation with deferosamine at 4°C, cells were warmed to 37°C to initiate chase of ¹²⁵I-Tf from endocytic compartments to the cell surface, and measurements of cell-associated and -released radioactivity were taken at various times of chase. The Tf(secreted)/Tf(internal) ratio was plotted against time, and the slope of each curve yields k_ext. The data represent the mean and SE derived from at least three independent determinations.