Activated ADP-ribosylation factor assembles distinct pools of actin on Golgi membranes

The small GTP-binding protein ADP-ribosylation factor (ARF) has been shown to regulate the interaction of actin and actin-binding proteins with the Ge ls apparatus. Here we report that ARF activation stimulates the assembly of distinct pools of actin on Golgi membranes. One pool of actin cofractionat es with coatomer (COPI)-coated vesicles and is sensitive to salt extraction and the plus end actin-binding toxin cytochalasin D. A second ARF-dependen t actin pool remains on the Golgi membranes following vesicle extraction an d is insensitive to cytochalasin D, Isolation of the salt-extractable ARF-d ependent actin from the Golgi reveals that it is bound to a distinct repert oire of actin-binding proteins, The two abundant actin-binding proteins of the ARF-dependent actin complex are identified as spectrin and drebrin. We show that drebrin is a specific component of the cytochalasin D-sensitive, ARF-dependent actin pool on the Golgi. Finally, we show that depolymerizati on of this actin pool with cytochalasin D increases the extent of the salt- dependent release of COPI-coated vesicles from the Golgi following cell-fre e budding reactions. Together these data suggest that regulation of the act in-based cytoskeleton may play an important role during ARF-mediated transp ort vesicle assembly or release on the Golgi.