©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cytosolic ADP-ribosylation Factors Are Not Required for Endosome-Endosome Fusion but Are Necessary for GTPS Inhibition of Fusion(*)

David J. Spiro (1), Timothy C. Taylor (3)(§), Paul Melanon (3), Marianne Wessling-Resnick (1) (2)(¶)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

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.


INTRODUCTION

ADP-ribosylation factors (ARFs)() 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) . ARF activity appears to be regulated by guanine nucleotide exchange and hydrolysis since cytosolic ARF is in a GDP-bound state while GTP-bound ARF associates with cellular membranes (5) . Because recruitment and dissociation of the coat proteins -COP (6, 7) and AP-1 (8) are dependent on the membrane binding of ARF, this factor's guanine nucleotide binding state may play a critical role in cellular membrane traffic. Based on these and other observations, it was proposed that ARF functions in coatomer-coated vesicle formation (9) , although the GTP-binding protein's exact activity in the secretory pathway remains unclear (10-12).

In addition to its proposed function in the secretory pathway, a specific role for ARF in endosome-endosome fusion was proposed based on the effects of GTPS in several cell-free assay systems (13, 14, 15) . Stahl and co-workers (16) have demonstrated that GTPS has a dual effect on endosomal fusion, stimulating fusion at low cytosolic concentrations (<0.5 mg/ml) and inhibiting fusion at concentrations of cytosol that support maximal fusion activity (0.5-2.0 mg/ml). 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 GTPS. Treatment of cytosol with GTPS causes the recruitment of cytosolic factor(s) to vesicle membranes in both of these systems (14, 17) . Further work by the Stahl laboratory (18) demonstrated that an N-terminal ARF peptide prevents the effects of GTPS on endocytic vesicle fusion at low or high cytosol concentrations and that recombinant, myristoylated ARF1 inhibits this reaction in the presence of GTPS. Based on this evidence, Lenhard et al.(18) proposed that ARF activity is required for endosome-endosome fusion although stringent functional criteria to support this idea are lacking. For example, the N-terminal peptide's actions may be rather indirect since it alone does not affect in vitro fusion (18) . In fact, nonspecific effects associated with this peptide have been reported (19) , and a thorough investigation of the peptide's activity has revealed that this cationic amphipathic helix can cause irreversible membrane damage (20) . Moreover, recent in vivo experiments fail to support a direct role for ARF in the endocytic pathway. Brefeldin A (BFA), an agent that blocks the catalyzed exchange of guanine nucleotides necessary to activate ARF, was found to interfere with transferrin receptor membrane traffic by slowing the rate of exocytosis and not endocytosis(21) . In addition, BFA does not prevent endocytosis of the low density lipoprotein receptor but causes its missorting from endosomes and the trans-Golgi network (22) . The failure of BFA to interfere with early events of the endocytic pathway suggests that endosome fusion continues under conditions that would inactivate ARF function.

These concerns prompted us to re-evaluate the role of ARF in endocytic vesicle fusion by investigating the mechanics of this reaction in the absence of the GTP-binding protein. Our results rigorously demonstrate that cell-free endocytic vesicle fusion does not require cytosolic ARFs, although in agreement with the initial observations made by Lenhard et al.(18) , we find that the presence of ARFs is necessary for GTPS-mediated inhibition. Moreover, endocytic vesicle fusion and GTPS 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 GTPS-mediated inhibition of intra-Golgi transport (26) .


EXPERIMENTAL PROCEDURES

Preparation of Cell Extracts

K562 cells and Chinese hamster ovary (CHO) cells were maintained in -minimum Eagle's medium containing 7.5% fetal calf serum as described (14) . Cells (K562 or CHO) were collected by centrifugation and washed three times in phosphate-buffered saline (PBS) on ice. Cell pellets were resuspended in 3 volumes of uptake buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 1 mg/ml glucose, 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. Endocytic uptake was quenched by diluting the cell suspension with 5 volumes of ice-cold PBS, followed by five washes in the same buffer at 4 °C. Cells were mechanically disrupted with a stainless steel ball homogenizer with 2-7 10 cells/ml in ice-cold breaking buffer (20 mM HEPES, pH 7.4, 0.1 M KCl, 85 mM sucrose, 20 mM EGTA) until 80-90% of the cells were broken. Postnuclear supernatants (PNS) were prepared by centrifugation at 800 g for 5 min at 4 °C. Vesicle fractions were separated from cytosolic proteins over a step gradient (14) . Briefly, PNS was layered on 0.25 M sucrose, 10 mM acetic acid, 10 mM triethanolamine, 1 mM EDTA, pH 7.4, and membranes were pelleted on a cushion of isotonic Nycodenz (Accurate Chemical Co.) upon centrifugation at 380,000 g for 5 min at 4 °C. Cytosol was prepared by centrifugation of PNS at 380,000 g for 15 min at 4 °C. The supernatant was removed and stored at -85 °C until use.

Rat liver Golgi fractions were isolated according to Tabas and Kornfeld (27). Livers from five male rats were homogenized in 0.5 M sucrose in homogenization buffer (0.05 M Tris maleate, pH 6.5, 5 mM MgCl, containing 5 mM phenylmethanesulfonyl fluoride, 1 mg/ml soybean and lima bean trypsin inhibitors, 1 mg/ml leupeptin, and 1 mg/ml aprotinin). The homogenate was centrifuged at 600 g for 10 min to remove nuclei and intact cells. The PNS was layered on top of 1.3 M sucrose in homogenization buffer and centrifuged in an SW 28 rotor at 63,000 g for 2 h at 4 °C. The crude smooth membrane fraction, collected above the 1.3 M sucrose layer, was adjusted to 1.1 M sucrose and layered on a sucrose step gradient as described (27) . Golgi membranes were collected at the interface between 0.5 and 1.0 M sucrose and pelleted by centrifugation in a Ti-70 rotor at 165,000 rpm for 30 min at 4 °C. The Golgi membrane pellet was resuspended in breaking buffer and frozen at -85 °C until use.

Preparation and characterization of ARF-depleted cytosol and bovine ARF1 have been described (2, 11) . Briefly, a soluble CHO cell extract was centrifuged at 100,00 g for 1 h at 4 °C, and the resulting supernatant was fractionated over a Superdex-75 size-exclusion column. Column fractions were analyzed for the presence of 20-21-kDa GTPases using [P]GTP-ligand blot analysis. An ARF(-) pool consisted of fractions of apparent molecular mass greater than 35 kDa and smaller than 15 kDa, which were concentrated to the original volume by ultrafiltration.

Assay Conditions for in Vitro Endocytic Vesicle Fusion

Vesicle fusion was performed by incubating 2.5-µl samples of avidin -galactosidase and biotin-transferrin vesicle fractions together for 30 min at 37 °C in 25 µl of breaking buffer supplemented with 1 mM MgATP, 50 mg/ml creatine kinase, 8 mM phosphocreatine, 10 mg/ml biotin-cytochrome c, and 1 mM dithiothreitol (14) . The fusion reaction was terminated by addition of 5 µl of lysis buffer (10% Triton X-100, 1% sodium dodecyl sulfate, 50 mg/ml biotin-cytochrome c) and 200 µl of dilution buffer (0.05% Triton X-100, 50 mM NaCl, 10 mM Tris, pH 7.4, 1 mg/ml heparin); lysates were clarified by centrifugation.

The complex between avidin -galactosidase and biotin-transferrin resulting from vesicle fusion was measured by a modified enzyme-linked immunosorbent assay (23) . Microstrip wells (Labsystems) were precoated with rabbit anti-human transferrin antibodies (1:500 in 50 mM NaCO, pH 9.6). Antibody-coated wells were incubated with clarified lysates for 3 h at 37 °C (or overnight at 4 °C) and then rinsed free of unbound material by three washes with PBS, followed by four washes with 10 mM Tris, pH 7.5, 1% Triton X-100, 0.1% SDS, 10 mM NaCl, 1 mM EDTA. The wells were incubated in the final wash at 37 °C for 30 min and were rinsed once with PBS. Avidin -galactosidase activity was then measured by incubating the wells for 3 h at 37 °C with 250 µl of substrate solution (0.3 mM 4-methylumbelliferyl -galactoside in 0.1 M NaCl, 25 mM Tris, pH 7.4, 1 mM MgCl, 12 mM -mercaptoethanol). Samples were diluted into 15 volumes of 133 mM glycine, 83 mM NaC0, pH 10.7, and the fluorescence of the hydrolysis product (365 nm excitation, 450 nm emission) was measured with an Hitachi F-2000 spectrophotometer. The resulting signal, in fluorescence units, is directly proportional to the extent of vesicle fusion in vitro(14, 28) .

Measurement of GTPS-dependent Membrane Association of ARF

Membranes were isolated from PNS by centrifugation at 380,000 g for 15 min, washed once, and then resuspended in breaking buffer and stored at -85 °C. ARF binding reactions were carried out with PNS membranes (1.25-2 mg/ml) or rat liver Golgi (2 mg/ml) and 2-3 mg/ml K562 cell cytosol, with or without 100 µM GTPS; these components were incubated for 30 min at 37 °C in 100 µl of breaking buffer supplemented with 1 mM MgATP, 50 mg/ml creatine kinase, 8 mM phosphocreatine, 10 mg/ml biotin-cytochrome c, and 1 mM dithiothreitol. MgCl was added to 3 mM, and the binding reaction was terminated by centrifugation at 14,000 rpm for 30 min at 4 °C. Membrane pellets were washed once in breaking buffer plus 3 mM MgCl and then repelleted at 14,000 rpm for 5 min at 4 °C. The ability of membrane-associated ARF to stimulate the [C]NAD:agmatine ADP-ribosyltransferase activity of cholera toxin A subunit (CTA) was then assayed (29, 30) . Briefly, membranes (100 µg of protein) were incubated in the presence of 50 mM potassium phosphate, pH 7.5, 0.3 mg/ml ovalbumin, 20 mM dithiothreitol, 4 mM MgCl, 1 mM GTP, 2 mg/ml cardiolipin, 60 mM Cibachrome blue (Fluka), 20 mM agmatine, [C]NAD (3 10 cpm/reaction) with or without 0.5 mg CTA (List Laboratories) in a total volume of 100 µl. After a 60-min incubation at 30 °C, reactions were terminated by the addition of 1 ml of 1% sodium dodecyl sulfate, and the entire sample was transferred to a column (0.5 2 cm) of Dowex AG 1-X2. Eluates were collected by washing columns with 5 ml of distilled, deionized H0, and [C]ADP-ribosylagmatine was measured using a Beckman LS 5000-TD liquid scintillation counter. GTPS-dependent ARF binding was defined as the difference in [C]ADP-ribosylagmatine produced in assays with membranes that had been incubated with cytosol in the presence and absence of GTPS. Activation of CTA ADP-ribosyltransferase activity was the same in reactions with membranes incubated with or without GTPS but in the absence of cytosol, i.e. the effect was dependent on cytosolic ARFs.


RESULTS

The effects of BFA on cell-free endocytic vesicle fusion were examined to further investigate the proposed activity of ARFs in this reaction. As shown by the results of Fig. 1, treatment of cytosol and membranes with BFA did not inhibit endocytic vesicle fusion. In fact, the extent of vesicle fusion was the same with or without BFA treatment. This observation is in agreement with results demonstrating that BFA interferes with exocytic and not endocytic processes (21) . However, it should be noted that in vitro intra-Golgi transport also takes place in the presence of the drug (26), despite the fact that BFA inhibits secretion in vivo (31).


Figure 1: Brefeldin A does not prevent cell-free endocytic vesicle fusion or GTPS inhibition of fusion. K562 cell membrane vesicles and cytosol were separately incubated in the presence (openbars) or absence (filledbars) of 200 µM BFA for 10 min at 37 °C. Cytosol was further incubated for 30 min with or without 100 µM GTPS as indicated. The membrane and cytosol fractions were then combined at final concentrations of 1 mg/ml (leftpanel) or 3 mg/ml (rightpanel) cytosol in order to assay for endocytic vesicle fusion. Reaction conditions for the fusion assay were as described under ``Experimental Procedures.'' The results shown are the average of duplicate samples (±S.E.); data are representative of three independent experiments.



Although BFA does not affect cell-free intra-Golgi transport, it does block the GTPS-mediated inhibition of this reaction (26) . To investigate BFA effects on inhibition of endosome fusion, cytosol was also incubated with GTPS before addition to fusion reactions (Fig. 1). At a final concentration in the assay of 1 mg/ml, GTPS-treated cytosol inhibited fusion by 40% while at 3 mg/ml, 80% of this activity was blocked; these observations are compatible with the idea that a cytosolic factor promotes inhibition of endocytic vesicle fusion (14) . However, the presence of BFA did not block GTPS-mediated inhibition at either cytosol concentration of cytosol. Finally, although the concentration of BFA used in these experiments (200 µM) was previously documented to disrupt ARF membrane association in vitro(23, 24) , we also examined the effects of BFA over a wide concentration range (Fig. 2). The dose-response curve reveals that treatment of membranes and cytosol with up to 500 µM BFA did not diminish that extent of endocytic vesicle fusion or prevent the inhibition of vesicle fusion by GTPS. The results of Fig. 1 and Fig. 2indicate that if ARFs play a role in endosome fusion or the GTPS-mediated inhibition of fusion, as has been previously suggested (18), they most likely function by a BFA-resistant mechanism.


Figure 2: Dose-response curve for brefeldin A effects on cell-free endocytic vesicle fusion and GTPS inhibition of fusion. Endocytic vesicle fusion measurements were performed exactly as detailed under Fig. l, except that membrane and cytosol fractions were incubated for 10 min at 37 °C in the presence of BFA at the concentrations indicated. In vitro fusion was supported by 3 mg/ml cytosol incubated with (opensymbols) or without (closedsymbols) 100 µM GTPS. Data presented are the average of duplicate reactions (±S.E.) and are representative of three independent experiments.



The lack of effect on cell-free endocytic vesicle fusion might suggest that in the presence of BFA, membrane fusion proceeds via an alternate pathway independent of ARF function. For example, a mechanism of ``uncoupled'' fusion was proposed to account for the significant level of in vitro Golgi transport observed in the absence of ARF (10) . To test this possibility, the kinetic parameters of the in vitro reaction were examined, and it was found that BFA treatment did not significantly alter the rate or extent of endocytic vesicle fusion (Fig. 3). Rates of fusion of BFA-treated samples, 27.0 ± 7.1 units/min (n = 4), were comparable with those measured in control reactions, 24.1 ± 4.4 units/min (n = 4). The fact that the time course of endosome fusion is unaffected by BFA argues that the mechanism of endocytic vesicle fusion is not substantially altered by the presence of the drug.


Figure 3: Time course of endocytic vesicle fusion in the absence or presence of BFA. K562 cell cytosol and PNS fractions were separately incubated with (opencircles) or without (closedcircles) 200 µM BFA for 30 min at 20 °C. The samples were then combined (final concentration, 3 mg/ml protein), and fusion assays were carried out as described for Fig. 1. At the times of incubation indicated, samples were placed on ice, and fusion was rapidly quenched with lysis buffer. Data presented are the average of duplicate samples (±S.E.) and are representative of results obtained in four independent experiments.



As a positive control for BFA effects related to ARF activity, we examined the membrane association of the GTP-binding protein in our in vitro system based on the well characterized activity of ARF as a stimulator of cholera toxin ADP-ribosyltransferase activity (29). As shown by the results of Fig. 4, BFA reduced GTPS-dependent ARF binding to rat liver Golgi membranes by approximately 60%, similar to previous reports (23, 24, 25, 30) . In contrast, GTPS-mediated ARF binding to the K562 cell membranes present in our assay was relatively unaffected by BFA. Since the PNS membrane fraction includes Golgi, endosomes, endoplasmic reticulum, plasma membrane, and other intracellular membrane components, it is unclear from these experiments whether ARFs specifically associated with endocytic vesicle membranes persist in binding in the presence of BFA. The combined results of Fig. 1-3 do suggest, however, that BFA-insensitive membrane binding of cytosolic ARFs could account for the GTPS-mediated inhibition of endocytic vesicle fusion seen under our assay conditions.


Figure 4: Brefeldin A reduces GTPS-dependent membrane association of ARF. K562 cell PNS membranes or rat liver Golgi and cytosol were incubated in the presence (filled bars) or absence (open bars) of GTPS and BFA exactly as described in Fig. 1. This binding reaction was terminated by centrifugation; membranes were then washed and assayed for the ability to activate CTA ADP-ribosyltransferase activity as detailed under ``Experimental Procedures.'' Data are calculated as the percent GTPS-dependent binding activity (±S.D.) from results obtained in four independent experiments.



To critically examine whether cytosolic ARFs are responsible for GTPS-mediated inhibition of cell-free endocytic vesicle fusion, we measured the ability of ARF-depleted CHO cytosol (11) to support in vitro endocytic vesicle fusion. As shown in Fig. 5, ARF(-) cytosol stimulated endocytic vesicle fusion to the same extent as wild-type CHO cytosol. However, while GTPS-treated CHO cytosol inhibited the fusion reaction by 40%, ARF(-) cytosol incubated with GTPS failed to block vesicle fusion. If the ARF(-) cytosol was first supplemented with 135 ng of purified bovine ARF1 and then incubated with GTPS, inhibition of endocytic vesicle fusion was recovered to levels comparable with the GTPS-mediated inhibition observed for wild-type CHO cytosol. Under these assay conditions, the amount of ARF1 added is roughly equivalent to the native concentration of ARF in wild-type cytosol (40 ng/µl); we estimate that 200 ng of ARF is present in the wild-type CHO cytosol supporting fusion reactions in this experiment. Control assays were also performed in the presence of ARF1 but in the absence of GTPS. Despite the fact that ARF1 alone causes slight inhibition of in vitro fusion activity, this effect is clearly potentiated by preincubation with the nucleotide. Thus, factors contained in ARF-depleted cytosol are both necessary and sufficient to support cell-free endocytic vesicle fusion, although an ARF or ARF-dependent function must be responsible for the inhibition of cell-free endocytic vesicle fusion in the presence of GTPS. These results are consistent with data obtained from investigations of cell-free intra-Golgi transport in which ARF(-) cytosol was shown to support Golgi transport yet failed to inhibit transport in the presence of GTPS (11) .


Figure 5: ARF-depleted cytosol supports fusion but not GTPS inhibition of fusion. CHO or ARF(-) cytosol was incubated with (filledbars) or without (openbars) 100 µM GTPS and then assayed for the ability to support endocytic vesicle fusion (final concentration, 1.5 mg/ml); purified bovine ARF1 was also added to some samples (135 ng final in assay). Fusion activity is expressed relative to the maximal fusion signal (ARF(-) cytosol + GTPS). Data shown are representative of results obtained in four independent experiments. Conditions were: A, CHO cytosol alone; B, CHO cytosol with ARF1 added; C, ARF-depleted cytosol; and D, ARF-depleted cytosol with ARF1 added.



The reaction parameters in the absence of ARFs were also evaluated. As shown in the time course experiments of Fig. 6, the rates of fusion reactions performed in the presence of CHO cytosol or ARF(-) cytosol were found to be identical. Moreover, the extent of vesicle fusion in the presence and absence of ARFs is the same, confirming the results shown in Fig. 5 . Finally, not only is our cell-free endocytic vesicle fusion reaction dependent on cytosolic factors, but this activity also requires ATP and is inhibited by ATPS (14, 32) . Therefore, to verify that fusion in the absence of cytosolic ARFs is an ATP-dependent process, CHO and ARF(-) cytosols were added to fusion assays with or without the nucleotide. The results of Fig. 7 demonstrate that in vitro fusion activity supported by either CHO or ARF(-) cytosols requires ATP. This is an important observation because other cell-free assay systems display an alternate pathway supporting endosome fusion that can be detected in the absence of ATP and cytosol (33) . Based on the identical kinetics and ATP dependence of vesicle fusion supported by either wild-type CHO or ARF(-) cytosol, we conclude that endosome-endosome fusion in the presence or absence of cytosolic ARFs is mediated by the exact same mechanism.


Figure 6: Depletion of cytosolic ARFs does not change the time course of endocytic vesicle fusion. CHO (opensquares) or ARF(-) (closedcircles) cytosol was added to vesicle fractions at a final concentration of 1.5 mg/ml, and fusion activity was measured exactly as described for Fig. 3. Data shown are representative of results obtained on three separate occasions.




Figure 7: Endocytic vesicle fusion in the absence or presence of cytosolic ARFs requires ATP. K562 cell membrane fractions were combined with CHO or ARF(-) cytosol and incubated in the presence of ATP and an ATP-regeneration system (1 mM ATP, 100 µg/ml creatine kinase, 8 mM phosphocreatine) or in the absence of ATP plus an ATP-depleting system (10 units/ml hexokinase, 5 mM 2-deoxyglucose). Fusion reactions were carried out as described for Fig. 1. The data presented are the average of duplicate points (±S.E.) and are representative of results obtained in three independent experiments.




DISCUSSION

Our results establish that inhibition of cell-free endocytic vesicle fusion in the presence of GTPS is absolutely dependent on cytosolic ARFs, in agreement with previous observations (18) . However, the specific role originally proposed for ARFs in endosome fusion fails to be supported by our demonstration that neither the rate nor the extent of fusion are altered in the absence of cytosolic ARFs. Instead, our data suggest a rather promiscuous membrane association of cytosolic ARFs in the presence of GTPS, which disrupts vesicle-vesicle interactions and which may or may not reflect a true physiologic function. For example, ARF is known to associate with both PC12 cell membranes and liposomes in a manner that is entirely dependent on guanine nucleotides rather than specific membrane factors (5) . Furthermore, Helms et al.(34) also demonstrated the existence of a significant pool of ARFs that are loosely associated with Golgi membranes and readily extracted with liposomes. Such nonspecific interactions would not only account for our in vitro results with GTPS but would also readily explain the pleiotropic effects observed in vivo with a GTPase-defective mutant of ARF1, which disrupts membrane traffic, including endocytosis (35, 36).

Nonetheless, one could argue that a membrane-bound form of ARF that is resistant to BFA mediates endosome-endosome fusion. Such a proposal would be consistent with our observation that the effects of GTPS on endosome fusion are not reversed by BFA, unlike the results obtained for intra-Golgi transport assays (26) , and that less than 10% of GTPS-dependent ARF binding is blocked by BFA in the crude PNS fractions that provide the endocytic vesicles under study. In fact, recent studies on the overexpression of ARF6 demonstrate a plasma membrane/endosomal distribution of this isoform (37, 38) . However, the fact that GTPS does not inhibit the endosome fusion assay in the absence of cytosolic ARFs clearly argues against this idea. A model in which membrane-associated BFA-insensitive ARFs play a role in endosome fusion would require that GTP hydrolysis is not essential for ARF function or that ARFs remain stably associated with membranes in the GTP-bound state and unable to bind GTPS. Neither of these caveats is compatible with known properties of ARFs, since GDP-bound ARF does not bind membranes (5) and GTP hydrolysis is rapidly catalyzed upon ARF association with the Golgi (23, 24) . Curiously, a defective GTP-binding mutant of ARF6 does appear to remain associated with endosomal structures (presumably in the GDP-bound state); however, this mutant disrupts recycling of transferrin receptors (37) and is localized on endosomal domains that appear to be coated (38) . Thus, it has been postulated ARF6 may promote budding of transport vesicles from the endosome (37) , making a role for this isoform in endosome fusion unlikely.

It still remains to be determined if GTPS-mediated inhibition of endosome fusion by ARF involves other factors. Since specific binding of ARFs to Golgi membranes and their function in coatomer complex recruitment have been well documented (6, 7, 8, 9, 10) , it is possible that these events contribute to the pattern of inhibition observed in our assay system. One can envision that during coatomer assembly, universal factors necessary for membrane traffic may be sequestered. This recruitment by ARF of important elements of the fusion machinery onto the surface of other membranes may consequently result in the inhibition of our assay. If this possibility is correct, then inhibition of endosome-endosome fusion by ARF in the presence of GTPS must involve other cytosolic factors, a prediction that we are currently evaluating. These ongoing experiments may elucidate novel features of ARF's function in the regulation of intracellular membrane traffic.


FOOTNOTES

*
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.

§
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.

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.

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.


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