Role of Protein-Phospholipid Interactions in the Activation of ARF1 by the Guanine Nucleotide Exchange Factor Arno*

(Received for publication, April 30, 1997)

Sonia Paris Dagger , Sophie Béraud-Dufour , Sylviane Robineau , Joëlle Bigay , Bruno Antonny , Marc Chabre and Pierre Chardin §

From the CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

Arno is a 47-kDa human protein recently identified as a guanine nucleotide exchange factor for ADP ribosylation factor 1 (ARF1) with a central Sec7 domain responsible for the exchange activity and a carboxyl-terminal pleckstrin homology (PH) domain (Chardin, P., Paris, S., Antonny, B., Robineau, S., Béraud-Dufour, S., Jackson, C. L., and Chabre, M. (1996) Nature 384, 481-484). Binding of the PH domain to phosphatidylinositol 4,5-bisphosphate (PIP2) greatly enhances Arno-mediated activation of myristoylated ARF1. We show here that in the absence of phospholipids, Arno promotes nucleotide exchange on [Delta 17]ARF1, a soluble mutant of ARF1 lacking the first 17 amino acids. This reaction is unaffected by PIP2, which suggests that the PIP2-PH domain interaction does not directly regulate the catalytic activity of Arno but rather serves to recruit Arno to membranes. Arno catalyzes the release of GDP more efficiently than that of GTP from [Delta 17]ARF1, and a stable complex between Arno Sec7 domain and nucleotide-free [Delta 17]ARF1 can be isolated. In contrast to [Delta 17]ARF1, full-length unmyristoylated ARF1 is not readily activated by Arno in solution. Its activation requires the presence of phospholipids and a reduction of ionic strength and Mg2+ concentration. PIP2 is strongly stimulatory, indicating that binding of Arno to phospholipids is involved, but in addition, electrostatic interactions between phospholipids and the amino-terminal portion of unmyristoylated ARF1GDP seem to be important.

We conclude that efficient activation of full-length ARF1 by Arno requires a membrane surface and two distinct protein-phospholipid interactions: one between the PH domain of Arno and PIP2, and the other between amino-terminal cationic residues of ARF1 and anionic phospholipids. The latter interaction is normally induced by insertion of the amino-terminal myristate into the bilayer but can also be artificially facilitated by decreasing Mg2+ and salt concentrations.


INTRODUCTION

ADP-ribosylation factors (ARFs),1 which were originally identified as cofactors for cholera toxin, are small GTP-binding proteins involved in intracellular vesicular transport (1, 2). Their functions include acting as regulators of the binding of coat proteins and adaptins to intracellular membranes (3, 4) and as activators of phospholipase D (5-7). It is still unclear whether phospholipase D mediates ARF signals to initiate coated vesicle formation (8, 9).

Like other G proteins, ARFs cycle between inactive GDP-bound and active GTP-bound conformations, and they are thought to shuttle during the activation cycle between cytosol and membranes, with the possible exception of ARF6 (10). Taking ARF1, the most abundant ARF family member, as a model, we previously assessed the importance of N-myristoylation for GTP-dependent binding to phospholipid vesicles in vitro (11, 12). We concluded that the increased affinity of ARFGTP for membranes cannot be ascribed to a myristoyl switch, i.e. a nucleotide exchange-dependent exposure of the myristate, as frequently proposed in reviews (3, 13). We believe that in ARFGDP, the myristate is accessible for interacting with phospholipids and that this interaction mostly accounts for the weak binding of ARFGDP to membranes. The tight binding of ARFGTP to phospholipids can be explained by a GTP-dependent release of the amino-terminal helix from the protein core, leading to the exposure of several hydrophobic residues that insert into the lipids in addition to the myristate (14).

Conversion of ARFGDP to ARFGTP is promoted in vivo by a guanine nucleotide exchange factor. Complementation studies in yeast recently led to the discovery of a family of ARF nucleotide exchange factors that share a region of sequence homology with Sec7, a yeast protein necessary for intra-Golgi transport (15). We identified as a member of this family an ubiquitously expressed ~47-kDa human protein termed Arno (for ARF nucleotide-binding site opener) (16). Another protein of very similar sequence but mostly expressed in hematopoietic cells (17) turned out to be identical to cytohesin-1, a protein described as a regulator of beta 2 integrin in lymphocytes (18). Cytohesin-1 also promotes nucleotide exchange on ARF1 (16) as well as on ARF3 (19). Arno and cytohesin-1 contain an amino-terminal coiled-coil motif, a central Sec7 domain, and a pleckstrin homology (PH) domain followed by a cluster of positively charged residues at the carboxyl terminus. Optimal exchange activity on myrARF requires the presence of negatively charged phospholipid vesicles supplemented with PIP2. Studies with truncated mutants of Arno have demonstrated that the Sec7 domain is responsible for the exchange activity, whereas the PH domain binds to PIP2 (16).

In the present study, we have addressed two questions concerning the activation of ARF1 by Arno. (a) What is the role of the binding of the PH domain to PIP2: does it serve only to recruit Arno to membranes, or is it required to activate the exchange factor? (b) What is the role of the amino-terminal helix of ARF1 in the interaction ARF-Arno?


EXPERIMENTAL PROCEDURES

Materials

Egg phosphatidylcholine, egg phosphatidylglycerol, and bovine brain PIP2 were purchased from Sigma. [35S]GTPgamma S was from NEN Life Science Products, [3H]GDP was from Amersham Corp., and unlabeled nucleotides were from Boehringer Mannheim.

Expression and Purification of Recombinant Proteins

Bovine recombinant ARF1 was expressed in Escherichia coli and purified by a single QAE-Sepharose chromatography as described previously (11). Myristoylated ARF1 was made by co-expression with a yeast N-myristoyltransferase (20) and separated from the contaminating nonmyristoylated protein by a precipitation at 35% saturation of ammonium sulfate followed by sequential chromatography on DEAE-Sepharose and Mono-S columns (12). The mutant [Delta 17]ARF1, which lacks the first 17 amino acids, was purified by gel filtration with Ultrogel AcA44 (14). Wild-type Arno, Delta PH Arno (a deletion mutant lacking the PH domain), and Arno Sec7 domain were isolated by anion exchange on QAE-Sepharose and gel filtration on Sephacryl S-100 HR (Pharmacia Biotech Inc.) as described previously (16). As judged by Coomassie Blue staining after SDS-polyacrylamide gel electrophoresis, final purity was >95% for ARF proteins and was between 70 and 90% for Arno proteins.

Preparation of Phospholipid Vesicles

Unilamellar phospholipid vesicles were prepared by the extrusion method of Hope et al. (21). A film of phospholipids of the desired composition in PC (phosphatidylcholine), PG (phosphatidylglycerol), and PIP2 was formed in a Rotavapor and resuspended at 4 mg/ml in 50 mM Hepes (pH 7.5) with or without 100 mM KCl, as indicated. The suspension was vortexed for 20 min and freeze-thawed five times. Unilamellar vesicles were produced by extrusion through 0.1-µm polycarbonate filters (14).

Nucleotide Binding Assay

Unless otherwise stated, wild-type or truncated ARF (1 µM) was incubated at 37 °C with [35S]GTPgamma S or [3H]GDP (10 µM, ~1000 cpm/pmol) in 50 mM Hepes (pH 7.5), 1 mM dithiothreitol, 1 mM MgCl2 with or without (as indicated in the figure legends) 2 mM EDTA (1 µM or 1 mM free Mg2+), 100 mM KCl, and 1 mg/ml phospholipid vesicles. At the indicated times, samples of 25 µl (25 pmol of ARF) were removed, diluted into 2 ml of ice-cold 20 mM Hepes (pH 7.5), 100 mM NaCl, and 10 mM MgCl2, and filtered on 25-mm BA 85 nitrocellulose filters (Schleicher & Schüll). Filters were washed twice with 2 ml of the same buffer, dried, and counted.

Gel Filtration Analysis of the Interaction of [Delta 17]ARF1 with Arno Sec7 Domain

[Delta 17]ARF1 (10 µM) and Arno Sec7 domain (10 µM) were incubated, separately or together, for 10 min at 25 °C in 20 mM Tris/HCl (pH 7.5), 100 mM NaCl, 5 mM 2-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM MgCl2, with or without 2 mM EDTA (1 µM or 1 mM free Mg2+). A 200-µl sample of each incubation was applied to a Superose 12 HR 10/30 column (Pharmacia) and eluted with the same buffer at a flow rate of 0.5 ml/min. Fractions of 300 µl were collected, and 60-µl samples were concentrated and analyzed by SDS-polyacrylamide gel electrophoresis.


RESULTS

Arno Activates [Delta 17]ARF1 in the Absence of Phospholipids

As previously reported (16), optimal nucleotide exchange activity of Arno on myrARF1 requires the presence of PC/PG/PIP2 vesicles in the assay (Fig. 1A). The stimulatory effect of PIP2 is most likely due to an interaction between this phospholipid and the PH domain of Arno (16), but two possible mechanisms can be proposed. The binding of the PH domain to PIP2 could simply concentrate Arno on the surface of vesicles and thereby facilitate its interaction with the membrane-bound fraction of myrARF1GDP. Alternatively, binding to PIP2 could induce a conformational change of Arno, leading to an increased intrinsic catalytic activity, for example, by releasing a negative constraint. To discriminate between these alternatives, we examined the effect of Arno on [Delta 17]ARF1, a soluble substrate. Indeed, this deletion mutant of ARF1 lacking the 17 amino-terminal amino acids has lost the requirement for phospholipids in the nucleotide exchange reaction (22) and remains totally soluble in the GTP-bound state, as demonstrated by lipid-protein fluorescence energy transfer measurements (14) and by a sedimentation assay with phospholipid vesicles (data not shown). This allows monitoring of the GDP-to-GTPgamma S exchange in the absence of phospholipids. Fig. 1B shows that Arno is indeed active on [Delta 17]ARF1, which first indicates that the amino-terminal alpha -helix of ARF1 is not essential for the interaction ARF-Arno. Most notably, the exchange activity of Arno on [Delta 17]ARF1 was exactly the same in the absence of phospholipids and in the presence of PC/PG/PIP2 vesicles. Moreover, a similar activity was observed with Delta PH Arno, a mutant lacking the PH domain, or Arno Sec7 domain, a deletion mutant restricted to the central catalytic domain (Fig. 1C). Altogether, these results demonstrate that in full-length Arno, there is no constraint on the catalytic site that would be alleviated by the binding of the PH domain to PIP2-containing vesicles. Accordingly, the interaction of the PH domain with PIP2 does not regulate the enzymatic activity of Arno but simply mediates its membrane association.


Fig. 1. PIP2 is not required for Arno exchange activity on [Delta 17]ARF1. Binding of [35S]GTPgamma S to 1 µM myrARF1 (A) or [Delta 17]ARF1 (B and C) was determined as described under "Experimental Procedures" in the presence of 100 mM KCl and 1 mM MgCl2. A, the assay buffer was supplemented with 1 mg/ml phospholipid vesicles made of PC/PG (70:30, w/w; (triangle , open circle ) or PC/PG/PIP2 (65:30:5, w/w/w; black-down-triangle , bullet ) with (open circle , bullet ) or without (triangle , black-down-triangle ) 75 nM wild-type Arno. B, [Delta 17]ARF1 was incubated with (open circle , bullet ) or without (triangle , black-down-triangle ) 75 nM wild-type Arno in the presence (bullet , black-down-triangle ) or the absence (open circle , triangle ) of 1 mg/ml phospholipids (PC/PG/PIP2, 65:30:5). C, [Delta 17]ARF1 was incubated in the absence of phospholipids either alone (triangle ) or with 75 nM Delta PH Arno (square ) or Arno Sec7 domain (diamond ).
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Arno Sec7 Domain Forms a Stable Complex with [Delta 17]ARF1

The observation that Arno Sec7 domain catalyzes nucleotide exchange on [Delta 17]ARF1 (Fig. 1C) prompted us to examine whether the interaction between these two soluble proteins could be demonstrated by gel filtration chromatography. Fig. 2 shows that this is indeed the case. When a 1:1 mixture of Arno Sec7 domain and [Delta 17]ARF1 (10 µM each) was applied to and eluted from a Superose 12 column, the two proteins were found to be partly associated at 1 mM Mg2+ (Fig. 2B) and nearly totally associated at 1 µM Mg2+ (Fig. 2D). Addition of 10 µM GDP to the elution buffer at 1 µM Mg2+ markedly reduced the interaction between the two proteins (Fig. 2E), which indicates that Arno Sec7 domain forms a stable complex specifically with the nucleotide-free state of [Delta 17]ARF1.


Fig. 2. Binding of nucleotide-free [Delta 17]ARF1 to Arno Sec7 domain. [Delta 17]ARF1 (10 µM), Arno Sec7 domain (10 µM), or a mixture of the two proteins was applied to a gel filtration column as described under "Experimental Procedures." Protein absorbance was monitored at 280 nM, and a sample of each fraction was analyzed by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining. The concentration of free Mg2+ was 1 mM (A and B) or 1 µM (C, D, and E). In E, the elution buffer was supplemented with 10 µM GDP. A and C, elution profiles obtained when [Delta 17]ARF1 and Arno Sec7 domain are loaded separately on the Superose 12 column. B, D, and E, elution profile obtained with the mixture of the two proteins. The dotted line represents the calculated sum of the two absorbance profiles obtained for the separate proteins under the same Mg2+ condition, shown in the panel above. The upper band on the gels corresponds to Arno Sec7 domain, and the lower band corresponds to [Delta 17]ARF1.
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Arno Catalyzes GDP and GTPgamma S Release from ARF1 but Prefers ARFGDP as a Substrate

We previously reported that a soluble nucleotide exchange factor present in a retinal extract can promote GTPgamma S release as well as GDP release from ARF1 (23). We investigated whether purified Arno is also able to catalyze the exchange reaction in both directions. This was first examined in the absence of phospholipids with [Delta 17]ARF1 as a substrate. As shown in Fig. 3, Arno stimulates both GDP and GTPgamma S dissociations from [Delta 17]ARF1, but the effect is much more pronounced on GDP release. Addition of 0.1 µM Arno to 1 µM [Delta 17]ARF1 increased the rate of GDP dissociation 40-fold and increased the rate of GTPgamma S dissociation only 3-fold, and this was not affected by the nature of the displacing nucleotide (Fig. 3). Thus, Arno seems to have in solution a better affinity for ARFGDP than it does for ARFGTPgamma S. This preference is less obvious when the nucleotide exchange is measured with myrARF1 in the presence of PC/PG/PIP2 vesicles (Fig. 4). Under these conditions, GDP and GTPgamma S releases seem to be stimulated to a similar extent by 20 nM Arno, but this is likely due to the fact that the reaction then occurs on the membrane surface, with both substrates. Indeed, if phospholipids are omitted from the assay medium, the stimulation of [3H]GDP release from myrARF1 by Arno becomes detectable only above 0.1 µM of the exchange factor (data not shown). This indicates that only the membrane-bound fraction of myrARF1GDP is a good substrate for Arno, and because myrARF1GDP is mostly soluble, this fraction must be very low as compared with myrARF1GTPgamma S, which is totally bound to phospholipids. Thus, differential binding of the proteins to membranes greatly complicates the kinetic analysis. This is also evidenced by the observation that in these conditions, GDP and GTPgamma S are no longer equivalent as displacing nucleotide. Both [3H]GDP and [35S]GTPgamma S dissociations from myrARF1 were more accelerated by a catalytic amount of Arno when GDP was used as the displacing nucleotide (Fig. 4). This suggests that after the exchange, Arno dissociates more rapidly from myrARFGDP than it does from myrARFGTPgamma S, which could be linked to the low affinity of myrARFGDP for membranes. But on the other hand, Arno-stimulated [35S]GTPgamma S release was less complete in the presence of GDP (Fig. 4, right panel). It did not fit a first-order kinetics, as if with time, less and less Arno was acting on [35S]GTPgamma S-bound myrARF1, because of a progressive trapping by the accumulating GDP-bound form of myrARF1. Altogether, these results suggest that Arno has indeed a higher affinity for ARFGDP, even though membrane-bound Arno dissociates more rapidly from myrARFGDP than it does from myrARFGTP, because the former species is less retained on phospholipids.


Fig. 3. Effect of Arno on the dissociation of [3H]GDP and [35S]GTPgamma S from [Delta 17]ARF1 in the absence of phospholipids. 1 µM [Delta 17]ARF1 was first incubated with 10 µM [3H]GDP (left panel) or [35S]GTPgamma S (right panel) in a buffer containing 100 mM KCl and 1 mM free Mg2+ for 20 min at 37 °C. Then sufficient MgCl2 was added to provide 1 mM free Mg2+, with 1 mM unlabeled GDP (open circle , bullet ) or GTPgamma S (triangle , black-triangle). When present (bullet , black-triangle), wild-type Arno (0.1 µM) was added at zero time. The amount of labeled [Delta 17]ARF1 at that time was taken as 100% (~15 pmol with [3H]GDP and ~20 pmol with [35S]GTPgamma S in a 25-µl sample). Spontaneous dissociation of [3H]GDP or [35S]GTPgamma S was independent of the unlabeled nucleotide (GDP or GTPgamma S) added in excess (data not shown). Dissociation rates are 0.04 min-1 without Arno and 1.6 min-1 with 0.1 µM Arno for [3H]GDP and 10-4 min-1 without Arno and ~3 × 10-4 min-1 with Arno for [35S]GTPgamma S.
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Fig. 4. Effect of Arno on the dissociation of [3H]GDP and [35S]GTPgamma S from myrARF1 in the presence of PC/PG/PIP2 vesicles. 1 µM myrARF1 was first incubated with 10 µM [3S]GTPgamma S in the presence of 0.1 mM MgCl2 for 1 h at 37 °C (left panel) or with 10 µM [35S]GTPgamma S in the presence of 1 mM MgCl2 and 2 mM EDTA (1 µM free Mg2+) for 10 min at 37 °C (right panel). In each case, the assay buffer also contained 100 mM KCl and 1 mg/ml PC/PG/PIP2 (65:30:5, w/w/w) vesicles. Thereafter, free Mg2+ was raised to 1 mM, and 1 mM unlabeled GDP (open circle , bullet ) or GTPgamma S (Delta , black-triangle) was added, with (bullet , black-triangle) or without (open circle , Delta ) 20 nM Arno at zero time. 100% corresponds to 16-18 pmol of bound [3H]GDP and ~20 pmol of bound [35S]GTPgamma S/25 pmol of myrARF1.
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Activation of Nonmyristoylated ARF1 by Arno Requires Low Mg2+ and Low Salt Concentrations

We previously reported that Arno is inactive on unmyrARF1 when tested at 1 mM Mg2+ in the presence of 100 mM KCl and azolectin vesicles (16). Replacement of azolectin, whose composition in PIP2 is not known, by defined phospholipids of optimal composition (PC/PG/PIP2, 65:30:5) allows the detection of a small activation of GTPgamma S binding to unmyrARF1 on the addition of a 2-fold molar excess of Arno (Fig. 5, left panel), but this effect remains marginal as compared with that on myrARF1 or [Delta 17]ARF1. However, this effect could be greatly amplified by changing the composition of the assay buffer: (a) decreasing the ionic strength by omission of KCl led to the rapid accumulation of a substantial fraction of activated unmyrARF1 at 1 mM Mg2+ (Fig. 5, left panel), and (b) lowering the concentration of free Mg2+ to 1 µM further enhanced the activated fraction (Fig. 5, right panel). We have shown that 1 µM free Mg2+ allows optimal spontaneous GDP-to-GTPgamma S exchange on unmyrARF1 (11). Thus, Mg2+ conditions that favor spontaneous nucleotide exchange seem to also favor the activation by Arno, whereas decreasing the salt concentration presumably facilitates ionic interactions between the two proteins and phospholipids.


Fig. 5. Effects of Mg2+ and KCl concentrations on Arno-stimulated [35S]GTPgamma S binding to unmyrARF1 in the presence of PC/PG/PIP2 vesicles. Unmyristoylated ARF1 (1 µM) was incubated with 10 µM [35S]GTPgamma S in the presence of 1 mg/ml PC/PG/PIP2 (65:30:5, w/w/w) vesicles, 1 mM (A) or 1 µM (B) free Mg2+, and various concentrations of KCl: 0 (open circle , bullet ), 25 (black-down-triangle ), or 100 mM (Delta , black-triangle) with (closed symbols) or without (open symbols) 2 µM Arno.
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Lipid Dependence of the Activation of unmyrARF1 by Arno

With the optimal Mg2+ and salt concentrations defined above (1 µM free Mg2+, no KCl), we reexamined the effect of phospholipids on the stimulation of GTPgamma S binding to unmyrARF1 by wild-type Arno and Arno Sec7 domain. In the absence of phospholipids (Fig. 6A), [35S]GTPgamma S binding to 1 µM unmyrARF1 was accelerated poorly by 0.1 µM Arno and accelerated more significantly by 2 µM Sec7 domain. In fact, the initial rate of exchange was a linear function of the exchange factor concentration and was identical for full-length Arno and Arno Sec7 domain (Fig. 6A, inset). Thus, in the absence of phospholipids, Arno and the Sec7 domain are equivalent toward unmyrARF but are both very poor activators. In the presence of PC/PG (70:30) vesicles, the stimulation by 2 µM Sec7 domain was only slightly increased, but wild-type Arno became much more active because 0.1 µM Arno was roughly equivalent to 2 µM Sec7 domain under these conditions (Fig. 6B). Inclusion of 5% PIP2 in the vesicles did not change the stimulation by the Sec7 domain but further markedly increased the stimulatory effect of Arno (Fig. 6C).


Fig. 6. Effects of phospholipids on the stimulation of GTPgamma S binding to unmyrARF1 by wild-type Arno and Arno Sec7 domain. Main panels, [35S]GTPgamma S binding to 1 µM unmyrARF1 was assayed in a buffer containing 1 µM free Mg2+ and no KCl in the absence of lipids (A) or in the presence of 1 mg/ml phospholipid vesicles composed of PC/PG (70:30, w/w; B) or PC/PG/PIP2 (65:30:5, w/w/w; C). Nucleotide exchange was assayed without exchange factor (open circle ) or in the presence of 0.1 µM Arno (bullet ) or 2 µM Sec7 domain (black-triangle). Inset, the initial rate of [35S]GTPgamma S binding was measured in the absence of lipids as a function of the concentration of wild-type Arno (bullet ) or Sec7 domain (black-triangle).
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Thus, although unmyrARF1GDP is essentially soluble (12), its activation by Arno is greatly facilitated by the binding of Arno to PIP2-containing vesicles, which is in marked contrast with [Delta 17]ARF1 (Fig. 1B). The lack of PIP2 effect on the Sec7 domain was expected because the PH domain is missing in this mutant protein. In fact, the Sec7 domain is completely soluble; hence, the small difference in its activity observed without lipids (Fig. 6A) or with lipids (Fig. 6, B and C) cannot be due to an interaction between the Sec7 domain and phospholipids. It must be therefore ascribed to unmyrARF1. We previously reported that phospholipids stabilize the active form of unmyrARF1 and thereby increase the binding of [35S]GTPgamma S (11). To determine whether this is the explanation for the effect observed here, we repeated the same experiment by monitoring [3H]GDP dissociation. Interestingly, the activation by the Sec7 domain remained higher in the presence of phospholipids than it was in their absence (Fig. 7). Sec7-catalyzed exchange (after subtraction of the spontaneous exchange) was accelerated 2-fold in the presence of lipids. This result demonstrates that interaction of unmyrARF1GDP with phospholipids facilitates its activation by the soluble truncated exchange factor, whereas it does not affect the spontaneous nucleotide dissociation at all. It can be concluded, therefore, that both Arno-phospholipid and unmyrARF1GDP-phospholipid interactions play a role in the activation of unmyrARF1 by Arno.


Fig. 7. Effect of PC/PG vesicles on the stimulation of [3H]GDP dissociation from unmyrARF1 by Arno Sec7 domain. 2 µM unmyrARF1 was preloaded with 10 µM [3H]GDP in a low Mg2+ buffer (0.1 mM MgCl2 and 2 mM EDTA) containing no KCl and no phospholipid. After 30 min at 37 °C, MgCl2 was added to provide 1 µM free Mg2+. For each dissociation assay, 100 µl of this mixture was combined with 100 µl of a similar buffer containing 1 µM free Mg2+, 2 mM unlabeled GDP, 0 (open circle ) or 4 µM (black-triangle) Sec7 domain, and 0 (left panel) or 2 mg/ml (right panel) PC/PG (70:30, w/w) vesicles. Initial binding of [3H]GDP (100%) was ~15 pmol/25 pmol of unmyrARF1. Dissociation rates are 0.06 min-1 in the absence of Sec7 domain with or without lipids, 0.11 min-1 with 2 µM Sec7 in the absence of lipids (left panel), and 0.16 min-1 with 2 µM Sec7 in the presence of PC/PG vesicles (right panel).
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DISCUSSION

The present study demonstrates that wild-type Arno is able to promote guanine nucleotide exchange on [Delta 17]ARF1, a soluble truncated mutant of ARF1, in the absence of phospholipids. The exchange activity is unaffected by the addition of phospholipid vesicles composed of 65% PC, 30% PG, and 5% PIP2, vesicles that have been shown to bind >60% Arno (16). Moreover, a comparable activity is observed with an equivalent concentration of Arno Delta PH, a mutant of Arno lacking the PH domain, or of Arno Sec7 domain, a deletion mutant restricted to the central catalytic domain. These results indicate that in wild-type Arno the catalytic site is likewise accessible to the soluble substrate, whether Arno is membrane-bound or in solution. Therefore, the binding of the PH domain to PIP2-containing vesicles does not affect the catalytic activity of Arno but simply promotes membrane recruitment of the exchange factor.

The role of PIP2 binding has been examined for a number of other PH domain-containing enzymes. In two cases, namely the dynamin GTPase (24) and the Akt/PKB kinase (25), the binding of phosphoinositides has been proposed to directly regulate the catalytic activity, even though in the latter case the physiological relevance of this stimulation has been recently questioned by the characterization of a 3-phosphoinositide-dependent protein kinase that phosphorylates and activates Akt/PKB (26). Most frequently however, the PH domain is thought to have a simple recruiting function, by facilitating anchoring of the enzyme in PIP2-enriched regions of the membrane. This has been clearly demonstrated for G protein-coupled receptor kinases (27) and both delta  and beta  isoforms of phosphoinositide-specific phospholipase C (28, 29). Also, for other small G protein-specific nucleotide exchange factors of the Dbl family acting on Rho-like G proteins (30), the PH domain is believed to serve primarily to target the exchanger to specific cellular locations. Because ARF activates PLD1 (5, 6), and PLD1 activity is dependent on PIP2 even after purification of the enzyme (31), it is possible that the recruitment of Arno by PIP2 serves to concentrate ARF in the vicinity of its PLD effector. It should be stressed that PIP2-mediated membrane association of Arno greatly enhances the activity of the exchange factor toward its membrane-bound substrate myrARF1, even though the catalytic site is not directly affected. This is likely to result from an increased local concentration of the two proteins on the membrane surface that facilitates their interaction.

The fact that Arno efficiently catalyzes GDP dissociation from [Delta 17]ARF1 demonstrates that the amino-terminal helix of ARF1 is not the site of interaction with the exchange factor, which contradicts the prediction of Amor et al. (32) based on the crystallographic structure of unmyrARFGDP. The formation of a stable complex between Arno Sec7 domain and nucleotide-free [Delta 17]ARF1 (Fig. 2) should allow preparation of crystals for x-ray diffraction analysis and identification of the true contacts between the two proteins.

It is noteworthy that in contrast to [Delta 17]ARF1, the equally water-soluble full-length unmyrARF1GDP is not readily activated by Arno in solution. This suggests that the amino-terminal helix somehow hinders the catalyzed exchange reaction, just as it hinders the spontaneous exchange. Indeed, we previously reported that at physiological Mg2+ levels, GDP does not spontaneously dissociate from unmyrARF, whereas it is significantly released from myrARF in the presence of phospholipid vesicles (12). Because the amino-terminal helix in unmyrARFGDP is held in a hydrophobic cleft (32, 33), we proposed that insertion of the myristate into the lipid bilayer might slightly displace the helix away from the protein core and thereby facilitate the opening of the nucleotide binding site (23). Accordingly, when the myristate is lacking, the amino-terminal helix would stick to the protein core and maintain the nucleotide binding site in a locked conformation. In this case the only way to obtain GDP dissociation is to reduce the affinity of ARF for GDP by decreasing the Mg2+ concentration (11). The present study shows that to accelerate Arno-catalyzed GDP release from unmyrARF1, it is necessary to add phospholipids, to decrease the ionic strength of the medium and to lower the concentration of free Mg2+. Moreover, we show that the effect of phospholipids is not entirely due to Arno binding to the vesicles, because a lipid-dependent enhancement of the catalyzed exchange is still observed when Arno is replaced by the sole Sec7 domain. Previous studies measuring the co-sedimentation of ARF with vesicles of defined lipid composition have shown that myrARFGDP weakly interacts with phospholipids through both hydrophobic interaction of the myristate with the bilayer and electrostatic interactions of cationic residues with anionic lipids (14). The ionic interactions, presumably due to the positive patch formed on the surface of the protein by residues K10, K15, K16, K59, and R178 (32, 33), were not detected between unmyrARFGDP and phospholipids at a physiological ionic strength (14), but they should be stronger at a lower salt concentration. Accordingly, the requirement for a low ionic strength observed here could reflect the need to reinforce these electrostatic interactions to compensate for the absence of the myristate. Whether decreasing the Mg2+ concentration only affects the affinity for GDP or also facilitates the movement of the amino-terminal helix is not known, but altogether our observations suggest that efficient activation of full-length ARF1 by Arno requires a membrane surface and involves an interaction of the bilayer with the amino-terminal portion of ARFGDP.

An unresolved issue, however, is the exact sequence of events during the exchange reaction. Does the helix interact with the bilayer before or after the binding of Arno to ARF? In the first alternative, the binding site of Arno would be unmasked consecutively to the movement of the helix. In the case of the bacterial elongation factor EF-Tu and its nucleotide exchange factor EF-Ts, it has been proposed that the exchanger breaks open the nucleotide binding site by disrupting the coordination pattern of the magnesium ion (34). If the same mechanism applies to Arno, it is possible that the nucleotide binding site of ARF has to be unlocked before the interaction with Arno. Accordingly, the nucleotide binding site of ARFGDP would take two possible conformations: (a) a closed conformation, corresponding to the crystal structure of unmyrARFGDP (32, 33), and (b) a looser conformation induced by a slight release of the amino-terminal helix or/and by chelating the magnesium. Arno would recognize only the latter conformation.

Alternatively, Arno could bind normally to ARFGDP with the amino-terminal helix packing against the protein core but would not be able to open the nucleotide binding site on its own, without the help of phospholipids to pull away the amino-terminal helix. We cannot presently discriminate between the two possibilities.

Whatever the exact mechanism, the present findings point to the importance of the amino-terminal helix of ARF1 and of the protein-phospholipid interactions in the activation of ARF1 by Arno. We propose that efficient catalysis of GDP release by Arno can only occur on a membrane surface with the two proteins interacting with phospholipids: Arno interacting with PIP2 by its PH domain, and ARF interacting with anionic phospholipids by its amino-terminal portion. Thus, even though Arno and ARFGDP can potentially interact in solution, the presence of the amino-terminal helix precludes any futile nucleotide exchange and warrants the membrane localization of the exchange, whereas the PIP2 requirement for Arno binding may serve to target activated ARF to specific locations.


FOOTNOTES

*   This work was supported in part by grants from the Ministère de l'Enseignement Supérieur et de la Recherche (ACC SV5 9505025 Interface chimie-physique-biologie) and from the Centre National de la Recherche Scientifique (Biologie Cellulaire 96 121).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Supported by INSERM.
Dagger    To whom correspondence should be addressed. Tel.: 33-4-93-95-77-71; Fax: 33-4-93-95-77-10.
1   The abbreviations used are: ARF, ADP ribosylation factor; PH, pleckstrin homology; myrARF1, myristoylated ARF1; unmyrARF1, unmyristoylated ARF1; PIP2, phosphatidylinositol 4,5-bisphosphate; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; PC, phosphatidylcholine; PG, phosphatidylglycerol.

ACKNOWLEDGEMENT

We thank Gabrielle Imbs for skillful secretarial assistance.


REFERENCES

  1. Donaldson, J. G., and Klausner, R. D. (1994) Curr. Opin. Cell Biol. 6, 527-532 [Medline] [Order article via Infotrieve]
  2. Moss, J., and Vaughan, M. (1995) J. Biol. Chem. 270, 12327-12330 [Free Full Text]
  3. Rothman, J. E. (1994) Nature 372, 55-62 [CrossRef][Medline] [Order article via Infotrieve]
  4. Robinson, M. S. (1997) Trends Cell Biol. 7, 99-102 [CrossRef]
  5. Brown, H. A., Gutowski, S., Moomaw, C. R., Slaughter, C., and Sternweis, P. C. (1993) Cell 75, 1137-1144 [Medline] [Order article via Infotrieve]
  6. Cockroft, S., Thomas, G. M. H., Fensome, A., Geny, B., Cunningham, E., Gout, I., Hiles, I., Totty, N. F., Truong, O., and Hsuan, J. J. (1994) Science 263, 523-526 [Medline] [Order article via Infotrieve]
  7. Frohman, M. A., and Morris, A. J. (1996) Curr. Biol. 6, 945-947 [Medline] [Order article via Infotrieve]
  8. Ktistakis, N. T., Brown, H. A., Waters, M. G., Sternweis, P. C., and Roth, M. G. (1996) J. Cell Biol. 134, 295-306 [Abstract]
  9. Zhao, L., Helms, J. B., Brügger, B., Harter, C., Martoglio, B., Graf, R., Brunner, J., and Wieland, F. T. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4418-4423 [Abstract/Free Full Text]
  10. Cavenagh, M. M., Whitney, J. A., Carroll, K., Zhang, C., Boman, A. L., Rosenwald, A. G., Mellman, I., and Kahn, R. A. (1996) J. Biol. Chem. 271, 21767-21774 [Abstract/Free Full Text]
  11. Franco, M., Chardin, P., Chabre, M., and Paris, S. (1993) J. Biol. Chem. 268, 24531-24534 [Abstract/Free Full Text]
  12. Franco, M., Chardin, P., Chabre, M., and Paris, S. (1995) J. Biol. Chem. 270, 1337-1341 [Abstract/Free Full Text]
  13. Harter, C., and Wieland, F. (1996) Biochim. Biophys. Acta 1286, 75-93 [Medline] [Order article via Infotrieve]
  14. Antonny, B., Béraud-Dufour, S., Chardin, P., and Chabre, M. (1997) Biochemistry 36, 4675-4685 [CrossRef][Medline] [Order article via Infotrieve]
  15. Peyroche, A., Paris, S., and Jackson, C. L. (1996) Nature 384, 479-481 [CrossRef][Medline] [Order article via Infotrieve]
  16. Chardin, P., Paris, S., Antonny, B., Robineau, S., Béraud-Dufour, S., Jackson, C. L., and Chabre, M. (1996) Nature 384, 481-484 [CrossRef][Medline] [Order article via Infotrieve]
  17. Liu, L., and Pohajdak, B. (1992) Biochim. Biophys. Acta 1132, 75-78 [Medline] [Order article via Infotrieve]
  18. Kolanus, W., Nagel, W., Schiller, B., Zeitlmann, L., Godar, S., Stockinger, H., and Seed, B. (1996) Cell 86, 233-242 [Medline] [Order article via Infotrieve]
  19. Meacci, E., Tsai, S.-C., Adamik, R., Moss, J., and Vaughan, M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1745-1748 [Abstract/Free Full Text]
  20. Duronio, R. J., Jackson-Machelski, E., Heuckeroth, R. O., Olins, P. O., Devine, C. S., Yonemoto, W., Slice, L. W., Taylor, S. S., and Gordon, J. I. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1506-1510 [Abstract]
  21. Hope, M. J., Bally, M. B., Webb, G., and Cullis, P. R. (1985) Biochim. Biophys. Acta 812, 55-65
  22. Kahn, R. A., Randazzo, P., Serafini, T., Weiss, O., Rulka, C., Clark, J., Amherdt, M., Roller, P., Orci, L., and Rothman, J. E. (1992) J. Biol. Chem. 267, 13039-13046 [Abstract/Free Full Text]
  23. Franco, M., Chardin, P., Chabre, M., and Paris, S. (1996) J. Biol. Chem. 271, 1573-1578 [Abstract/Free Full Text]
  24. Salim, K., Bottomley, M. J., Querfurth, E., Zvelebil, M. J., Gout, I., Scaife, R., Margolis, R. L., Gigg, R., Smith, C. I. E., Driscoll, P. C., Waterfield, M. D., and Panayotou, G. (1996) EMBO J. 15, 6241-6250 [Abstract]
  25. Franke, T. F., Kaplan, D. R., and Cantley, L. C. (1997) Cell 88, 435-437 [Medline] [Order article via Infotrieve]
  26. Alessi, D. R., James, S. R., Downes, C. P., Holmes, A. B., Gaffney, P. R. J., Reese, C. B., and Cohen, P. (1997) Curr. Biol. 7, 261-269 [Medline] [Order article via Infotrieve]
  27. Pitcher, J. A., Fredericks, Z. L., Stone, W. C., Premont, R. T., Stoffel, R. H., Koch, W. J., and Lefkowitz, R. J. (1996) J. Biol. Chem. 271, 24907-24913 [Abstract/Free Full Text]
  28. Garcia, P., Gupta, R., Shah, S., Morris, A. J., Rudge, S. A., Scarlata, S., Petrova, V., McLaughlin, S., and Rebecchi, M. J. (1995) Biochemistry 34, 16228-16234 [Medline] [Order article via Infotrieve]
  29. James, S. R., Paterson, A., Harden, T. K., and Downes, C. P. (1995) J. Biol. Chem. 270, 11872-11881 [Abstract/Free Full Text]
  30. Zheng, Y., Zangrilli, D., Cerione, R. A., and Eva, A. (1996) J. Biol. Chem. 271, 19017-19020 [Abstract/Free Full Text]
  31. Hammond, S. M., Jenco, J. M., Nakashima, S., Cadwallader, K., Gu, Q., Cook, S., Nozawa, Y., Prestwich, G. D., Frohman, M. A., and Morris, A. J. (1997) J. Biol. Chem. 272, 3860-3868 [Abstract/Free Full Text]
  32. Amor, J. C., Harrison, D. H., Kahn, R. A., and Ringe, D. (1994) Nature 372, 704-708 [CrossRef][Medline] [Order article via Infotrieve]
  33. Greasley, S. E., Jhoti, H., Teahan, C., Solari, R., Fensome, A., Thomas, G. M. H., Cockroft, S., and Bax, B. (1995) Nat. Struct. Biol. 2, 797-806 [Medline] [Order article via Infotrieve]
  34. Kawashima, T., Berthet-Colominas, C., Wulff, M., Cusak, S., and Leberman, R. (1996) Nature 379, 511-518 [CrossRef][Medline] [Order article via Infotrieve]

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