FIGURE 4:

Model of acute pharmacological perturbation to clathrin and AP-3 in PC12 cells stably expressing mCh-FKBP-CLC. Step 1: AP-2 vesicles bud from the plasma membrane (PM) in a clathrin-dependent manner. Acute AP20187 treatment perturbs FKBP-CLC, and thus clathrin function, in these cells. AP-2-clathrin coated vesicles (CCV) feed into endosomal organelles. Step 2: AP-3 vesicles, presumably coated with clathrin, bud from an early endosomal compartment to form SLMVs. Acute treatment of these cells with brefeldin A disrupts AP-3 vesicle formation. We tested whether acute treatment of these cells with AP20187 similarly affects SLMV formation. We hypothesized that if clathrin is required for AP-3–derived SLMV formation, then AP20187 treatment after release of a brefeldin A block in SLMV formation should further prohibit SLMV formation.