Cytosolic ADP-ribosylation Factors Are Not Required for Endosome-Endosome Fusion but Are Necessary for GTPS Inhibition of Fusion

doi:10.1074/jbc.270.23.13693

Cytosolic ADP-ribosylation Factors Are Not Required for Endosome-Endosome Fusion but Are Necessary for GTPS Inhibition of Fusion(*)

From the (1) Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts 02115, the
(2) Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115, and the
(3) Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309

^¶ Recipient of a Junior Faculty Award from the American Cancer Society. To whom correspondence should be addressed:
Dept. of Nutrition, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115.
Tel.: 617-432-3267; Fax: 617-432-2435.

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 (GTPS). Addition of purified ARF1 restored the ability of the ARF-depleted cytosol to inhibit endosome fusion when incubated with GTPS. Both endocytic vesicle fusion and the GTPS-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 GTPS and cytosolic factors in a brefeldin A-resistant manner.

Footnotes

↵^§ Supported by a National Institutes of Health predoctoral fellowship. Present address: Dept. of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520.
↵* This work was supported by Grant CB-15 from the American Cancer Society (to M. W.-R.) and Grant GM43378 from the National Institutes of Health (to P. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ARF

ADP-ribosylation factor

GTPS

guanosine 5′-3-O-(thio)triphosphate

BFA

brefeldin A

CHO

Chinese hamster ovary

PBS

phosphate-buffered saline

PNS

postnuclear supernatants

CTA

cholera toxin A subunit.