Abstract
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.
Highlights
ADP-ribosylation factors (ARFs)l are members of a family of 20-21-kDa GTP-binding proteins implicated in protein transport from the endoplasmic reticulum to the cis-Golgi stacks [1], intercisternal Golgi transport [2], exocytic vesicle traffic [3], and nuclear vesicle dynamics [4]
Our results rigorously demonstrate that cell-free endocytic vesicle fusion does not require cytosolic ARFs, in agreement with the initial observations made by Lenhard et al [18], we find
Our results establish that inhibition of cell-free endocytic vesicle fusion in the presence ofGTP-yS is absolutely dependent on cytosolic ARFs, in agreement with previous observations [18]
Summary
Vol 270, No 23, Issue of June 9, pp. 13693-13697, 1995 Printed in U.S.A. Cytosolic ADP-ribosylation Factors Are Not Required for Endosome-Endosome Fusion but Are Necessary for GTPl'S Inhibition of Fusion*. Using a different cell-free assay, Wessling-Resnick and Braell [14] described the inhibition of endocytic vesicle fusion in response to the preincubation of cytosol with GTPyS. Further work by the Stahl laboratory [18] demonstrated that an N-terminal ARF peptide prevents the effects ofGTPyS on endocytic vesicle fusion at low or high cytosol concentrations and that recombinant, myristoylated ARFI inhibits this reaction in the presence of GTPyS. Based on this evidence, Lenhard et al [18] proposed that ARF activity is required for endosome-endosome fusion stringent functional criteria to support this idea are lacking. Endocytic vesicle fusion and GTPyS inhibition of vesicle fusion are not responsive to BFA under conditions where BFA inhibits the binding of ARF to Golgi membranes [23,24,25] and can antagonize GTPyS-mediated inhibition of intra-Golgi transport [26]
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