Studies on the inhibition of endosome fusion by GTP gamma S-bound ARF

Using a cell free assay, we have previously shown that ARF is not required for endosome fusion but that inhibition of fusion by GTP gamma S is depende nt on a cytosolic pool of ARFs, Since ARF is proposed to function in intrac ellular membrane traffic by promoting vesicle biogenesis, and components of clathrin- and COP-coated vesicles have been localized on endosomal structu res, we investigated whether ARF-mediated inhibition of early endosome fusi on involves the recruitment or irreversible association of these proteins o nto endosomal membranes. We now report that depletion of components of clat hrin coated vesicles (clathrin, AP-1 and AP-2) or COPI vesicles (beta COP) does not affect the capacity of GTP gamma S-activated ARF to inhibit endoso me fusion, Inhibition of fusion by activated ARF is also independent of end osomal acidification since assays performed in the presence of the vacuolar ATPase inhibitor bafilomycin A1 are equally sensitive to GTP gamma S-bound ARF. Finally, in contrast to reported effects on lysosomes, we demonstrate that ARF-GTP gamma S does not induce endosomal lysis. These combined data argue that sequestration of known coat proteins to membranes by activated A RF is not involved in the inhibition of early endosome fusion and that its capacity to inhibit fusion involves other specific interactions with the en dosome surface. These results contrast with the mechanistic action of ARF o n intra-Golgi transport and nuclear envelope assembly.