Figure 5.

AP-1 and clathrin stability to reincubation at 37°C. Golgi membranes from 1.2-ml binding assays containing either GTP or GTPγS were pelleted, and the membranes were resuspended in 600 μl of assay buffer. Three aliquots of 100 μl of the resuspended membranes were transferred to new tubes, mixed with 100 μl of assay buffer, and then incubated for 0, 5, or 15 min at 37°C. Another set of three aliquots of 100 μl was mixed with 100 μl of 10 mg/ml rat liver cytosol and incubated similarly for 0, 5, or 15 min. After each time point, samples were cooled on ice, and the membranes were pelleted and analyzed for clathrin, AP-1, and ARF content by immunoblotting. The elevated level of clathrin found in the pellet when the reincubation step is performed with cytosol (lanes 7–12) reflects some nonspecific sedimentation of clathrin from the cytosolic pool. A similar amount of clathrin to that seen in lane 9 is found in the pellet fraction of cytosol incubated without added membranes (our unpublished observations). This explains why clathrin appears to dissociate more slowly than the AP-1 adaptor in lanes 7–9.