Cytosolic ADP-ribosylation factors are not required for endosome-endosome fusion but are necessary for GTP gamma S inhibition of fusion.

A specific role for ADP-ribosylation factors (ARFs) in in vitro endosome-endosome fusion has been proposed (Lenhard, J. M., Kahn, R. A., and Stahl, P. D. (1992) J. Biol. Chem. 267, 13047-13052). However, in vivo studies have failed to support a function for ARFs in the endocytic pathway, since an antagonist of ARF activities, brefeldin A, does not interfere with receptor internalization (Schonhorn, J. E., and Wessling-Resnick, M. (1994) Mol. Cell. Biochem. 135, 159-164). This controversy surrounding the exact function of ARF in endocytic vesicle traffic prompted us to critically re-examine the involvement of ARFs in cell-free endosome fusion. Cytosol depleted of ARF activity was capable of supporting in vitro endocytic vesicle fusion but failed to support inhibition of this reaction in the presence of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S). Addition of purified ARF1 restored the ability of the ARF-depleted cytosol to inhibit endosome fusion when incubated with GTP gamma S. Both endocytic vesicle fusion and the GTP gamma S-mediated inhibition of vesicle fusion were unaffected by brefeldin A. Moreover, the ATP requirement and kinetics of cell-free fusion are not altered by brefeldin A or depletion of cytosolic ARFs. These results suggest that cytosolic ARFs are not necessary for endosomal vesicle fusion in vitro but are responsible for inhibition of fusion in the presence of GTP gamma S and cytosolic factors in a brefeldin A-resistant manner.