Coatomer vesicles are not required for inhibition of Golgi transport by G-protein activators

The G-protein activators guanosine 5'-O-(3-thiodiphosphate) (GTP gamma S) a nd aluminum fluoride (AIF) are thought to inhibit transport between Golgi c isternae by causing the accumulation of nonfunctional coatomer-coated trans port vesicles on the Golgi. Although GTP gamma S and AIF inhibit transport in cell-free intra-Golgi transport systems, blocking coatomer vesicle forma tion does not. We therefore determined whether inhibition of in vitro Golgi transport by these agents requires coatomer vesicle formation. Depletion o f coatomer was found to completely block coated vesicle formation on Golgi cisternae without affecting inhibition of in vitro transport by either GTP gamma S or AIF. Depletion of ADP-ribosylation factor (ARF) prevented inhibi tion of transport by GTP gamma S. but not by Air, suggesting that the AIF-s ensitive component in transport may not be a GTP-binding protein. Surprisin gly, depletion of cytosolic ARF did not prevent the GTP gamma S-induced for mation of Golgi-coated vesicles. whereas ARF was required for AIF-induced v esicle formation. Although ARF or coatomer depletion caused an increase in the fenestration of cisternae, no other utrastructural changes were observe d that might explain the inhibition of transport by GTP gamma S or Air. The se findings suggest that ARF-GTP gamma S and AIF act by distinct and coatom er-independent mechanisms to inhibit membrane fusion in cell-free intra-Gol gi transport.