Figure 6.

Arf6-Q67L affects trafficking of clathrin-independent cargo but not clathrin-derived cargo. (A) Cos cells were transfected with Arf6 Q67L and Tac (first column), Tac-LL (second column), or Arf6 Q67L alone (third column). Cells were allowed to internalize mouse anti-Tac (first and second column) or DiI-LDL (10 μg/ml; third column) for 1 h at 37°C. (B) Cos cells were cotransfected with Arf6 Q67L and PH-GFP or Q67L alone, and EEA1 was detected in fixed cells using monoclonal anti-EEA1. Cells in A and B were labeled with polyclonal anti-ARF6 followed by the appropriate secondary antibodies. (C) HeLa cells coexpressing Arf6 Q67L and Tac or Tac-LL were incubated with polyclonal anti-Tac antibody for 30 min at 4°C and then incubated for 11 h at 37°C (in the presence of 15 mM NH₄Cl) to allow delivery of PM-bound antibody to lysosomes. After acid wash, cells were fixed, and lysosomes were visualized with monoclonal anti-Lamp1 followed by secondary antibodies. Bar, 10 μm. (D) HeLa cells coexpressing Arf6 wt or Q67L and Tac or Tac-LL were subjected to biotinylation pulse-chase as described in MATERIALS AND METHODS. Lysates were immunoprecipitated with anti-Tac mAb and analyzed as described in Figure 5C.