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

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.

Inhibition of in Vitro Endosomal Vesicle Fusion Activity by Aminoglycoside Antibiotics

“…This group of investigators demonstrated that addition of exogenous PLD had similar effects; hence, the activation of endogenous PIP 2 -dependent PLD by ARF is likely to be responsible for AP-2 recruitment. Although these results point to a functional role for ARF-dependent recruitment of AP-2 to support endosomal membrane trafficking, in vitro studies from our laboratory indicate that soluble ARF activity is not required for endosome-endosome fusion despite the fact that cytosolic ARFs inhibit this activity when bound to GTP␥S (18). Nonetheless, PLD has been implicated to function in endosome fusion; an inhibitor of PA production by PLD, 1-butanol, suppresses in vitro fusion activity, whereas exogenously added PLD can stimulate this reaction (19).…”

“…Because of the relative specificity of neomycin for PIP 2 binding (39), and the fact that this lipid cofactor is required for ARF-stimulated PLD activity (3-7), we next explored the relationship between aminoglycoside-and GTP␥S-sensitive steps of endosome fusion. We (18,28) and others (40) have demonstrated that when activated by GTP␥S, cytosolic ARFs will inhibit endosome fusion. To examine the temporal steps of endosome fusion affected by neomycin and GTP␥S, cell-free assays were carried out at 37°C in the absence of added inhibitor until the times indicated in Fig.…”

“…Shown is the amount of fusion activity measured as percentage of control (untreated) membranes treated in the same way except in the absence of drug. (19) would ordinarily function in a manner regulated by membrane-associated factors as depletion of cytosolic ARFs does not affect fusion activity but eliminates the inhibitory effects of GTP␥S (18). If ARF or an ARF-like activity associated with endosomes is involved in PLD activation, then the binding of PIP 2 by neomycin might interfere with its GDP/GTP exchange (41,42), the activity of the phospholipase itself, which requires PIP 2 as a cofactor (3), or the hydrolysis of GTP stimulated by ARF-GTPase-activating factor, which is enhanced by acidic phospholipids (43).…”

“…Post-nuclear supernatant (PNS) fractions were prepared as described previously (18). Briefly, K562 cells were washed in phosphate-buffered saline (PBS), resuspended in 25 mM Hepes, pH 7.5, 150 mM NaCl, 1 mg/ml dextrose, 1 mg/ml bovine serum albumin, and incubated with either 0.5 mg/ml avidin ␤-galactosidase or 100 nM biotin-transferrin for 60 min at 20°C.…”

In Vitro Reconstituted Dictyostelium discoideum Early Endosome Fusion Is Regulated by Rab7 but Proceeds in the Absence of ATP-Mg2+ from the Bulk Solution

“…The presence of an activated G protein, but not its deactivation, is therefore required for the fusion process. This stimulatory G protein probably does not belong to the Arf subfamily, because impairment of GTP hydrolysis on Arf proteins is known to block homotypic or heterotypic fusions (31)(32)(33). In contrast, impairment of GTP hydrolysis on Rab5 protein activates early endosome fusion in mammalian cells (29,33,34).…”