Specificities for the Small G Proteins ARF1 and ARF6 of the Guanine Nucleotide Exchange Factors ARNO and EFA6*

Eric MaciaDagger, Marc Chabre, and Michel Franco§

From the Institut de Pharmacologie Moléculaire et Cellulaire, CNRS-UMR 6097, 660 Route des Lucioles, 06560 Valbonne Sophia-Antipolis, France

Received for publication, April 12, 2001, and in revised form, May 3, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ARF1 and ARF6 are distant members of the ADP-ribosylation factor (ARF) small G-protein subfamily. Their distinct cellular functions must result from specificity of interaction with different effectors and regulators, including guanine nucleotide exchange factors (GEFs). ARF nucleotide-binding site opener (ARNO), and EFA6 are analogous ARF-GEFs, both comprising a catalytic "Sec7" domain and a pleckstrin homology domain. In vivo ARNO, like ARF1, is mostly cytosolic, with minor localizations at the Golgi and plasma membrane; EFA6, like ARF6, is restricted to the plasma membrane. However, depending on conditions, ARNO appears active on ARF6 as well as on ARF1. Here we analyze the origin of these ARF-GEF selectivities. In vitro, in the presence of phospholipid membranes, ARNO activates ARF1 preferentially and ARF6 slightly, whereas EFA6 activates ARF6 exclusively; the stimulation efficiency of EFA6 on ARF6 is comparable with that of ARNO on ARF1. These selectivities are determined by the GEFs Sec7 domains alone, without the pleckstrin homology and N-terminal domains, and by the ARF core domains, without the myristoylated N-terminal helix; they are not modified upon permutation between ARF1 and ARF6 of the few amino acids that differ within the switch regions. Thus selectivity for ARF1 or ARF6 must depend on subtle folding differences between the ARFs switch regions that interact with the Sec7 domains.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The small GTP-binding protein ADP-ribosylation factors (ARFs),1 originally identified as cofactors for the ADP-ribosyltransferase activity of cholera toxin, form a distinct six-member subgroup within the small G proteins (1). Like all G proteins, the ARFs cycle between inactive GDP-bound and active GTP-bound states, which differ mainly by the conformation of two regions called switch I and switch II. These two switches constitute a major site of interaction with regulator and effector proteins, including the guanine nucleotide exchange factors. But the ARFs differ from all other small G proteins by a myristoylated N-terminal amphipathic helix, which also changes conformation between the ARF-GDP and ARF-GTP states and is responsible for the GTP-dependent binding of ARF to the phospholipid membrane (2-4). The ARFs are key regulators of vesicular trafficking in eukaryotic cells. The best characterized members of the family, ARF1 and ARF6, are the most distant and appear to have very distinct functions. ARF1, which is mostly cytosolic, is clearly involved in intra-Golgi transport by regulating coat protein recruitment onto the Golgi membranes (5, 6); an additional possible role of ARF1 in focal adhesion assembly in regulating paxillin recruitment onto the plasma membrane has recently been suggested (7). ARF6, which localizes mostly at the cell periphery, has been implicated in regulating endocytosis and the organization of the cortical actin cytoskeleton (8-10).

Guanine nucleotide exchange factors (GEFs) mediate the activation of G-proteins by stimulating the dissociation of the bound GDP that rate-limits the binding of GTP. About 10 ARF-GEFs have been identified. They all share a conserved domain of about 200 amino acids, homologous to a portion of the Sec7 protein (11), which bears the nucleotide exchange catalytic activity (12, 13). The ARF GEFs can be divided into two major classes based on their size and primary sequence; one is a high molecular weight class composed of the GEA/GBF and Sec7/BIG subgroups, and the other is a low molecular weight class. This latter class shares a common structure organization in which the catalytic Sec7 domain is always linked to a PH domain specifically interacting with membrane-embedded polyphosphoinositides. This small ARF-GEF class can be further divided in the ARNO/cytohesin and EFA6 subclasses. The human ARNO/cytohesin subclass includes four members that are more than 77% identical: ARNO, cytohesin-1, ARNO3, and cytohesin-4; their N-terminal region contains a coiled-coil domain. The EFA6 subclass includes three members: EFA6 and two close analogs, Tic and the coding sequence KIAA0942, which have been deposited in data bank but not yet characterized. The N-terminal domain of EFA6 contains a proline-rich motif, and the long C-terminal extension to the PH domain contains a coiled-coil and two proline-rich motifs.

Within one cell several ARF proteins coexist with several GEFs, raising the question of the different functions of the various ARFs and of the specificities of their interactions with the various GEFs. We and others (14, 15) observe that overexpression of ARNO leads to fragmentation and loss of function of the Golgi complex, which suggests a role for ARNO in ARF1 activation in the Golgi. This suggestion was reinforced by the observation of a Golgi localization for the N-terminal coiled-coil domain of ARNO fused to green fluorescent protein (16). But another experiment indicated a recruitment to the plasma membrane of adipocytes of full-length ARNO fused to green fluorescent protein and of a green fluorescent protein-PH domain of ARNO upon insulin stimulation of phosphatidylinositol 3-kinase (17); moreover, a small fraction of overexpressed ARNO and ARF6 appeared in epifluorescence microscopy to partially colocalize at the plasma membrane (18). Overall these results suggest that the ARNO/cytohesins are able to localize at and probably act on both Golgi and plasma membranes, but their ARF substrates at these two localizations are not clearly identified. On the other hand EFA6 localizes at the plasma membrane, where it couples actin cytoskeleton remodeling with endocytic trafficking (19). This is compatible with a role of EFA6 in ARF6 activation. Indeed, exogenous expression of EFA6 or of ARF6-Q67L, a constitutively active ARF6 mutant, induces large plasma membrane invaginations enriched in polymerized actin where both proteins localize (9, 19). Likewise, both EFA6 and ARF6-Q67L decrease the rate of transferrin uptake. This phenotype is dependent on the nucleotide exchange activity of EFA6, as a mutant devoid of exchange activity has no effect on transferrin internalization (19).

In the present study, we analyze in vitro, but in the presence of lipid membrane, the specificities for ARF1 versus ARF6 of ARNO and EFA6 constructs. We also study the influence of the PH domain of ARNO on the substrate specificity. Finally, we try to determine by site-directed mutagenesis within the switch regions of the ARFs the molecular basis of their specificity of interaction with their GEF.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- Egg phosphatidylcholine, egg phosphatidylethanolamine, brain phosphatidylserine, and azolectin were purchased from Sigma. Brain phosphatidyl-4,5-bisphosphate ((4,5)PIP2) was from Avanti Polar Lipids (Birmingham, AL). [3H]GDP was from Amersham Pharmacia Biotech, and unlabeled nucleotides were from Sigma.

Expression and Purification of Recombinant Proteins-- Myristoylated ARF1 and ARF6 were expressed in Escherichia coli by co-expression with a yeast N-myristoyltransferase (20) and purified as previously described (3, 21). The myrARF1 protein is essentially purified as a GDP-bound form, whereas myrARF6 is totally in the GTP-bound form. Before use, the concentrated myrARF6 was loaded with GDP by a 45-min incubation at 37 °C in a buffer containing 2 mM GDP and 1 µM free Mg2+.

N-terminal-truncated [Delta 17]ARF1 and [Delta 13]ARF6 mutants, which lack, respectively, the first 17 and 13 amino acids, were expressed in E. coli, purified on an ACA44 (Ultrogel) gel filtration and GDP-loaded before storage at -70 °C (4). Full-length ARNO and ARNO-Sec7 domain were expressed in E. coli and isolated by anion exchange on Q-Sepharose and gel filtration on Sephacryl S-100 HR (Amersham Pharmacia Biotech) as previously described (12).

For production of full-length EFA6, BL21(DE3) bacteria were transformed with the pET3a-EFA6 plasmid. Transformed cells were grown at 37 °C to A600 = 0.6. The expression was induced with 0.1 mM isopropyl-1-thio-beta -D-galactopyranoside, and the temperature was reduced to 27 °C to increase the yield of proteins in the supernatant. After 3 h, the cells were harvested and lysed using a French press in 50 mM Tris/HCl, pH 8.0, 1 mM EDTA, 0.15% sodium cholate, 1 mM dithiothreitol and a tablet of protease mixture inhibitors (Roche Molecular Biochemicals). After sequential centrifugation at 8,000 × g and at 150,000 × g, the supernatant was applied to a Q-Sepharose column equilibrated with 20 mM Tris/HCl, pH 8.0, 1 mM MgCl2, 0.15% sodium cholate, and 0.5 mM dithiothreitol. The flow-through fractions containing EFA6 were pooled and applied directly to a Mono S column (Amersham Pharmacia Biotech) equilibrated with 10 mM MES, pH 6.0, 1 mM MgCl2, and 0.5 mM dithiothreitol (Mono S buffer). The bound protein was eluted with a gradient of NaCl (0-1 M). Fractions containing EFA6 (eluted between 350 and 450 mM NaCl) were pooled, concentrated on a Centricon 30 (Amicon Corp., Beverly MA), diluted 3 times in Mono S buffer, and stored at -70 °C. Several EFA6-Sec7 constructs were tested before obtaining a stable and active protein. EFA6-Sec7 domain (from His-125 to Asp-347) was generated by high fidelity polymerase chain reaction and inserted into pET11a expression vector. The resulting construct was sequenced and used to transformed BL21(DE3) bacteria. After protein expression and lysis of the bacteria, purification was achieved by separation on Q-Sepharose, concentration on Centricon 10 (Amicon Corp.), gel filtration on Sephacryl S-200 (Amersham Pharmacia Biotech), concentration, and purification by Mono S column. As judged by Coomassie Blue staining after SDS-polyacrylamide gel electrophoresis, final purity was >90% for ARF proteins, 50-60% for full-length ARNO and EFA6, and 90% for the two isolated Sec7 domains.

Preparation of Phospholipid Vesicles-- Large unilamellar vesicles of azolectin were prepared as described in Szoka and Papahadjopoulos (22) and extruded through a 0.1 µM pore polycarbonate filter (Isopore, Millipore). Vesicles of defined composition were prepared as described (23). Concentration of each phospholipid is indicated for each experiment in the legend.

Fluorescence Measurements-- The large increase in intrinsic fluorescence of ARF-GTP over ARF-GDP (24) was used to monitor in real time ARF activation upon GDP to GTP exchange. All measurements were performed at 37 °C in HKM buffer (50 mM Hepes, pH 7.5, 100 mM KCl, 1 mM MgCl2, 1 mM dithiothreitol) supplemented with azolectin vesicles (final lipid concentration 0.4 g/liter). The protein was diluted into the cuvette (0.5 µM myrARF with 50 µM GDP or 1 µM Delta -ARF with 1 µM GDP) to perform fluorescence measurements. Then, GDP/GTP exchange was allowed by the addition of GTP as indicated.

GDP Dissociation Assay-- MyrARF protein (approx 20 µM) was first loaded with ([3H]GDP) by incubating 45 min at 37 °C in a buffer containing [3H]GDP (500 µM, 500 dpm/pmol) with 1 µM free Mg2+ (1 mM MgCl2 and 2 mM EDTA). When maximal loading was reached, the concentration of free Mg2+ was raised to 1 mM to stop the reaction. This solution was diluted 10 times (2 µM final) in HKM buffer supplemented with lipid vesicles, and nucleotide dissociation was monitored as a loss of protein-bound radiolabel after the addition of 2 mM GTP.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ARF6-GDP Is Fully Soluble in Vitro, like ARF1-GDP, and Has a Higher Spontaneous GDP Exchange Rate-- Upon production in bacteria and purification as described under "Experimental Procedures," the full-length myristoylated ARF1 protein was obtained as a GDP-bound form, whereas myrARF6 was totally in the GTP-bound form. Before use the concentrated myrARF6 was fully loaded with GDP by incubation at 37 °C for 45 min in a buffer containing 2 mM GDP and 1 µM free Mg2+ (1 mM total Mg2+ and 2 mM EDTA). Returned to a medium with a physiological magnesium concentration, myrARF6-GDP remained fully soluble in vitro in the presence of phospholipid vesicles. This contrasts with the in vivo situation where ARF6 appears mainly membrane bound.

The kinetics of GDP/GTP exchange in myrARF1 and myrARF6 were followed in real time by fluorescence (see "Experimental Procedures"); the rates of nucleotide exchange are monitored as the rates of tryptophan fluorescence increase in ARF upon its trans-conformation from ARF-GDP to ARF-GTP. In the absence of GEF in vitro at millimolar magnesium concentration and in the presence of azolectin vesicles, myrARF1-GDP spontaneously exchanged its bound nucleotide very slowly (Fig. 1, A and B). Under the same conditions the spontaneous GDP/GTP exchange in ARF6 was about 15 times faster than in ARF1 (Fig. 1, C and D). Altogether these results might indicate that in cells ARF6 is primarily in its GTP-bound form associated to the membrane or that there exists an ARF6-GDI (GDP dissociation inhibitor) that would retain ARF6-GDP at the membrane.


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Fig. 1.   ARNO preferentially activates myrARF1, and EFA6 exclusively activates myrARF6. Kinetics of GDP to GTP exchange in the ARFs were monitored by the correlated variation in tryptophan fluorescence (Fluo.) as detailed under "Experimental Procedures." The purified proteins myrARF1 (A and B) and myrARF6 (C and D) were preloaded with GDP and injected at 0.5 µM in a fluorescence cuvette containing azolectin vesicles (0.4 g/liter) in HKM buffer. As indicated by the arrows, the purified GEF protein (50 nM) was injected (black line) or not (gray line) followed by the addition of GTP (250 µM). GTP activation kinetics were fitted to single exponentials. The value of the GDP/GTP exchange rate (kexch) for each experiment is shown in the inset. Fold stimulation is the ratio of the GEF-catalyzed over the spontaneous exchange rates. A.U., units.

ARNO Preferentially Activates myrARF1 but Also myrARF6, whereas EFA6 Exclusively Activates myrARF6-- We then compared the activity of the purified full-length exchange factors, ARNO and EFA6, also produced in bacteria (see "Experimental Procedures"), on full-length myristoylated ARF1 and ARF6. The catalytic efficiency of a GEF is best expressed by the fold stimulation of the spontaneous nucleotide exchange rate of the substrate protein. 50 nM ARNO increased about 20-fold the nucleotide exchange rate in ARF1 but only 5-fold the nucleotide exchange rate in ARF6 (Fig. 1, A and C). Conversely, 50 nM EFA6 increased the rate of nucleotide exchange in ARF6 about 17-fold, whereas it had no detectable effect on the rate of nucleotide exchange in ARF1 (Fig. 1, B and D). Thus ARNO is rather selective for ARF1, and EFA6 is strictly specific for ARF6. Furthermore, in terms of fold-stimulation of the spontaneous exchange rate, the efficiency of ARNO on ARF1 is about the same as that of EFA6 on ARF6.

ARNO PH Domain Interaction with (4,5)PIP2 Increases the Activity of ARNO on both ARF1 and ARF6 and Does Not Change Its Selectivity-- We have previously shown that the nucleotide exchange activity of ARNO on myrARF1 is increased in the presence of (4,5)PIP2-containing vesicles, and that this stimulation is strictly dependent on the PH domain of ARNO (12). In addition, we have observed that (4,5)PIP2 had no effect on the nucleotide exchange activity of ARNO when tested on a soluble mutant of ARF1 lacking the first 17 amino acids, [Delta 17]ARF1 (25). These results suggest that (4,5)PIP2, by recruiting ARNO onto the vesicles, increases its probability of interaction with the vesicle-bound myrARF1 but has no effect on the intrinsic catalytic activity of the Sec7 domain. Now we address the role of (4,5)PIP2 on the specificity of ARNO for ARF1 versus ARF6. The composition in phospholipids, and particularly in phosphoinositides of azolectin vesicles being unknown, we tested the effect of (4,5)PIP2 on ARNO activity on purified myrARF1 and myrARF6 using pure phospholipid vesicles (phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine: 35/35/30%) supplemented or not with 2% (4,5)PIP2 (Fig. 2). This (4,5)PIP2 addition did not modify the spontaneous nucleotide exchange rate of either myrARF, but it increased the nucleotide exchange rate on both myrARF1 and myrARF6 in the presence of ARNO. The PIP2-induced increase of ARNO activity appears larger on ARF1 than on ARF6, suggesting that in the presence of (4,5)PIP2, the specificity of ARNO for ARF1 is conserved or even increased. We conclude that (4,5)PIP2 does not affect either the intrinsic catalytic activity nor the selectivity of ARNO.


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Fig. 2.   Dependence on (4,5)PIP2 of the activity of ARNO on ARF1 and ARF6. As described under "Experimental Procedures," the kinetics of GDP/GTP exchange in ARF1 and ARF6 were measured by following the dissociation of [3H]GDP from [3H]GDP-ARF (2 µM) upon the addition of an excess nonradioactive GTP (2 mM). The measurements were performed in the presence or not of ARNO (20 nM) and in the presence of 0.4 g/liter phospholipid vesicles of defined composition, 35% phosphatidylcholine, 35% phosphatidylethanolamine, 30% phosphatidylserine, supplemented or not with 2% (4,5)PIP2 (A). The dissociation kinetics were fitted to single exponentials. Values of the GDP/GTP exchange rate under the various conditions are represented in B. PIP2 is seen to increase the activity of ARNO on both ARF proteins, not changing significantly its specificity for ARF1.

The Specificities of ARNO and EFA6 for Distinct ARFs Lie within the Sec7 Domain of the GEFs and the Core Domain of the ARFs-- We had previously shown that the isolated Sec7 domain of ARNO was sufficient to catalyze the guanine nucleotide exchange reaction on myristoylated ARF1 in the presence of azolectin vesicles (12). Moreover, in the absence of any phospholipids, the Sec7 domain of ARNO could stimulate nucleotide exchange in a soluble [Delta 17]ARF1 mutant deleted of its amphipathic N-terminal helix. In the absence of GTP, these two proteins formed a stoichiometric nucleotide-free [Delta 17]ARF1· Sec7 domain complex (25, 26). This minimal complex between ARNO-Sec7 and the ARF1 core, without its N-terminal helix, includes all the structural components required to catalyze the nucleotide exchange. We now ask whether the specificity of a given GEF for a particular ARF is maintained within this minimal complex? We produced and purified isolated Sec7 domains of ARNO and EFA6 (that are less that 30% identical) and ARF proteins deleted from their N-terminal myristoylated amphipathic helix, [Delta 17]ARF1 and [Delta 13]ARF6, that remain soluble in their GTP-bound state. Using the tryptophan fluorescence method, we analyzed the effect of increasing concentrations of ARNO-Sec7 (Fig. 3A) and of EFA6-Sec7 (Fig. 3B) on the rate of GDP/GTP exchange by [Delta 17]ARF1 and [Delta 13]ARF6 and in the absence of lipid vesicles. We observed that the selectivity of the isolated Sec7 domains was conserved or even increased; ARNO-Sec7 (50 nM) stimulated the GDP dissociation rate on [Delta 17]ARF1 50-fold and [Delta 13]ARF6 only 2.7-fold (Fig. 3) as compared with 21- and 5-fold, respectively, for the full-length ARNO (Fig. 1). EFA6-Sec7 (800 nM) stimulated the rate of GDP dissociation on [Delta 13]ARF6 8-fold, and as full-length EFA6, did not display any activity on [Delta 17]ARF1. Thus the Sec7 domains of ARNO and EFA6 contain all the determinants of specificity for distinct ARFs.


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Fig. 3.   The specificity of ARNO and EFA6 for distinct ARF proteins lies within their Sec7 domain. Kinetics of GDP to GTP exchange were measured by fluorescence as in Fig. 1. The N-terminal-deleted ARFs, [Delta 17]ARF1 and [Delta 13]ARF6, were injected (1 µM) in HKM buffer in the absence of lipid vesicles. The rates of exchange were measured as a function of the added concentrations of ARNO-Sec7 domain (panel A) or EFA6-Sec7 domain (panel B). Fold stimulation indicates the ratio of the Sec7 catalyzed over the spontaneous exchange rate for [Delta 17]ARF1 (open circle ) or [Delta 13]ARF6 (). The upper insets show the exchange rate (kexch) on the N-terminal-deleted ARFs in the presence (black bar) or not (gray bar) of ARNO-Sec7 (50 nM) or EFA6-Sec7 (400 nM).

Residues That Differ between ARF1 and ARF6 within the Switch I and II Regions Do Not Determine the Specificities of the ARF-Sec7 Domain Interaction-- The ARFs switch I and II regions are the principal sites of interaction with the GEFs. The first step of an ARF interaction with its GEF is the docking of ARF-GDP onto the Sec7 domain, as modeled in Fig. 4 (26). Upon conformational change of the switch I and II of ARF, which interact with the hydrophobic groove region of Sec7, the Glu-156 "glutamic finger" of Sec7 dislodges the GDP from ARF, resulting in a high affinity nucleotide-free complex (25-27). Within the switch I and II regions, ARF1 and ARF6 have identical primary sequences except for seven residues exposed on the surface of the molecule. To test if these seven residues account for the selectivity of the interaction between ARF1-ARNO and ARF6-EFA6, we produced mutants of [Delta 17]ARF1 in which each one of these seven residues was substituted by their equivalent found in ARF6. We constructed 3 point mutants in the switch I region ([Delta 17]ARF1-E41Q, [Delta 17]ARF1-I42S, [Delta 17]ARF1-E57T), one point mutant in the interswitch region ([Delta 17]ARF1-S62K), and a triple mutant in the switch II region ([Delta 17]ARF1-F82Y-Q83T-N84G). Using the tryptophan fluorescence method we tested their sensitivity to the Sec7 domain of ARNO and EFA6. We observed that ARNO-Sec7 activated the mutants about as much as the [Delta 17]ARF1 wild type, whereas EFA6-Sec7 was not much more active on the mutants than on the [Delta 17]ARF1 wild type (Fig. 5). Hence, none of the ARF1/ARF6 point mutations in the switch I and II domains affected the specificity of the ARNO and EFA6 Sec7 domains.


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Fig. 4.   Schematic representation for the docking complex between ARF1GDP and ARNO-Sec7 domain. ARF1 interacts with the Sec7 domain via its switch I and II regions. In this Sec7-interacting surface, the seven residues represented in the figure are different between ARF1 and ARF6. We produced mutants of [Delta ]17ARF1 in which each one of these residues was replaced by the equivalent found in ARF6. The catalytic glutamic residue (Glu-156) of ARNO-Sec7 is also shown.


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Fig. 5.   Permutation of the distinct amino acids between ARF6 and ARF1 in the switch regions of ARF1 does not change the specificity of the ARF1-Sec7 domain interaction. Kinetics of the GDP to GTP exchange were monitored by the correlated variation in tryptophan fluorescence. Measurements were performed on [Delta 17]ARF1-WT (1 µM) and the various [Delta 17]ARF1 mutants with the indicated ARF1->ARF6 mutations in the presence of 25 nM ARNO-Sec7 (panel A) and 400 nM EFA6-Sec7 (panel B). The GEF activities, deduced from the activation rates, are normalized to that observed for WT [Delta 17]ARF1. Relative activities of ARNO-Sec7 and of EFA6-Sec7 on wild-type (WT) [Delta 13]ARF6 are also shown for comparison. TM corresponds to the [Delta 17]ARF1-F82Y-Q83T-N84G triple mutant.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Determining the specificities of the various ARF-GEFs for their substrates is fundamental for deciphering the cellular functions of the different ARF proteins. The very different cellular functions of ARF1 and ARF6 must be related to their distinct cellular localizations and to specificities of interactions with different GEFs. In contrast with ARF1 and all the other ARFs, ARF6 appears mainly membrane-bound in the cells (28-30). The cellular localizations of the ARF-GEFs of the small ARNO/cytohesin and EFA6 types seemed to coincide, respectively, with that of ARF1 and ARF6. This strongly suggested that ARNO would be specific to ARF1 and EFA6 specific to ARF6, as was proposed in the initial studies characterizing ARNO and EFA6 (14, 19). However, depending on the cellular context ARNO has been found to also partially localize with and activate ARF6 (17, 18, 31). In this study we have further analyzed in vitro the ARF1 versus ARF6 specificities of ARNO and EFA6. We first noticed that, at physiological magnesium concentration and in the presence of phospholipid vesicles, myristoylated ARF6-GDP is soluble and that its spontaneous GDP/GTP exchange rate is 15 times higher than that of ARF1-GDP. We then demonstrated that the full-length ARNO catalyzes the GDP-GTP exchange reaction preferentially for ARF1, and the full-length EFA6 catalyzes the GDP-GTP exchange reaction exclusively for ARF6. It is also worth noting that the two GEFs have very comparable activities on their specific substrate.

Next, we attempted to determine whether the PH domains of ARNO and EFA6 are implicated in the GEF specificities for the ARFs. The PH domain of ARNO has been shown to mediate membrane association via specific interaction with polyphosphoinositides (12, 23, 32). Here we observed that (4,5)PIP2 incorporated into lipid vesicles does not modify the selectivity of ARNO towards ARF proteins. These data demonstrate that the PH domain participates in the membrane localization of ARNO but is not involved in the specificity of the interaction of ARNO with ARF1. Recently, Knorr et al. proposed that the interaction of cytohesin-1 with phosphoinositides could regulate its GEF activity and control its ARF specificity (33). They observed that the addition of PIP2 or phosphatidyl inositol 1,4,5-trisphosphate (PIP3) into liposomes strongly suppressed the activity of cytohesin-1 on ARF6, whereas it increased its activity on ARF1. Since ARNO and cytohesin-1 are 82% identical and 90% in their PH domain, it is surprising that they behave differently in the presence of PIPs. The differences may arise from the methods of purification and the preparation of the ARF proteins used in the assay. We further confirmed that the catalytic Sec7 domains of ARNO and EFA6 alone, without their adjacent PH domain and N- and C-terminal extensions, contain all the determinants of their specificities for ARF1 and ARF6.

The coiled-coil motif of ARNO seems to be a Golgi-targeting signal (16). Indeed, a green fluorescent protein fusion of the coiled-coil domain predominantly localizes to the Golgi apparatus. A single substitution of one of the hydrophobic residues is sufficient to alter this localization. These results suggest that the localization at the Golgi apparatus of ARNO is regulated by the coiled-coil domain, whereas its localization at the plasma membrane is controlled by the PH domain via phosphatidyl inositol 1,4,5-trisphosphate (PIP3) binding. EFA6 also contains both a PH and a coiled-coil domain. It has been clearly demonstrated that the PH domain is responsible for the strong association of EFA6 to the plasma membrane (19). However, no information is available regarding the precise function of the coiled-coil domain. In addition, EFA6 contains some other adaptor modules such as a few proline-rich regions. All these domains may be involved in the formation of a multi-protein complex serving as a central platform to coordinate the plasma membrane dynamics with the actin cytoskeleton remodeling.

As for the ARF1 and ARF6 substrates, their differential recognition by the GEF Sec7 domains is not related to their very different N-terminal amphipathic helix motifs. Thus the specificity determinants must be contained within the "core" of the ARFs, most probably within the effector region, including the switch I and switch II domains that are known to strongly interact with the GEF (27). The primary sequence of these two switch regions is strictly identical in ARF1 and ARF6, except for seven residues. We investigated whether these few differences were sufficient to account for the different substrate specificities we observed. Individual substitutions of each of these different residues between ARF1 and ARF6 do not affect the specificity of the ARF1-Sec7 interaction. Thus these seven distinct residues do not define the different selectivities of interaction of the ARFs with the Sec7 domains. The recently resolved crystallographic structure of ARF6 in the GDP-bound form (34) appears similar to that of ARF1-GDP, except in the switch regions; the two switch I regions have comparable conformations but are markedly displaced by several Angströms, and the switch II of ARF6-GDP is structurally ordered, in contrast to the switch II of ARF1-GDP, which is flexible. Thus the specificity of the ARF-GEF interaction does not result from primary sequence within the switch regions but rather from a folding difference dictated to the switch regions from elsewhere in the protein.

In situ ARF6 appears mainly membrane-bound, in contrast with the other ARFs, which appear mainly cytosolic (28-30). But in our hands, in vitro myristoylated ARF6 remained fully soluble when fully converted to the GDP form, even in the presence of lipid membrane. This could suggest that in situ the membrane-associated ARF6 is primarily in its GTP-bound form. However, it seems unlikely that ARF6 could be constitutively activated in vivo. Indeed, we have previously shown that the effects of overexpression of EFA6 are mediated through the activation of ARF6 and can be mimicked by the expression of ARF6-Q67L, a constitutively activated form of ARF6 (19). Therefore, we rather propose that in vivo a large pool of ARF6-GDI (GDP dissociation inhibitor) is responsible for retaining ARF6-GDP at the membrane and slowing down the GDP dissociation rate.

Our in vitro studies provide information as to how the GEFs could control in vivo the specific functions mediated by their cognate ARF. Our previous studies demonstrated that the formation of the complex between ARF1 and ARNO, which precedes the activation of ARF1 by GDP/GTP exchange, occurs at the membrane (35, 36). In vitro and in vivo ARF proteins appear soluble in their inactive GDP-bound state, whereas the GTP form is strongly associated with the phospholipid membranes, suggesting that the specific localization of the GEF determines the membrane site where the ARF protein will be activated and consequently anchored to the membrane.

Regarding ARNO, different laboratories using various cellular models and assays led to controversial conclusions as to its role of GEF for ARF6 (14, 18, 19, 31, 37, 38). Although it is well accepted that in vitro ARNO works better on ARF1, two observations on the effects of the fungus toxin brefeldin A (BFA) have been used to support its role on ARF6; it was observed long ago that the Golgi localization of ARF1 is BFA-sensitive, whereas ARF6 localization at the cell periphery is not affected by BFA (9). Then, in vitro studies demonstrated that the GEF activity of ARNO was not sensitive to BFA (12, 39, 40). The conjunction of these two observations suggested that ARNO is the BFA-insensitive ARF6 GEF. However, a high molecular weight BFA-insensitive ARF-GEF, GBF1, which does not localize at the periphery but to the Golgi apparatus, has recently been found (41). Thus BFA-insensitive GEFs are probably not restricted to the activation of ARF6.

Several groups have detected by subcellular fractionation or immunofluorescence a fraction of ARNO at the plasma membrane together with ARF6 (18, 31, 37, 42). However, although ARF1 is enriched in the Golgi area, it is predominantly a soluble protein. Therefore, one cannot exclude that the fraction of ARNO detected at the plasma membrane could recruit ARF1 onto it as well. In agreement with this hypothesis, it has been recently described a role for ARF1 in the recruitment of paxillin to the focal adhesion complexes (7). A thorough localization analysis of ARF1 at the cell periphery is needed.

In conclusion, our results suggest that EFA6 and/or its two close analogs Tic and the coding sequence KIAA0942 are the major GEFs for ARF6 and that ARNO is mostly involved with ARF1 activation. The first indication is that in vitro but in the presence of phospholipid membranes at a physiological magnesium concentration, full-length ARNO is less efficient on ARF6 than on ARF1. This specificity of ARNO for ARF1 is not affected by the interaction of its PH domain with (4,5)PIP2, which implies that in vivo it would not depend on the phosphoinositide composition of the membrane. The specificity of ARNO for ARF1 is entirely contained within the Sec7 domain of ARNO, and the Sec7 domain of EFA6, which is very different from that of ARNO (only 30% identity), achieves a markedly better activity on ARF6. Moreover, the analysis of this in vitro study appears consistent with our earlier results on the effects of the overexpression of these two GEFs in vivo (14, 19). Exogenous expression of EFA6, but not of ARNO, mimics the effects of expression of constitutively activated ARF6, i.e. cortical actin reorganization and inhibition of transferrin uptake. Conversely, ARNO, but not EFA6, affects the morphology and the function of the Golgi apparatus where ARF1 plays a major role.

    ACKNOWLEDGEMENTS

We thank Mariagrazia Partisani for skillful technical assistance. We are grateful to Drs. F. Luton, K. Singer, and C. Favard for very helpful comments on this manuscript.

    FOOTNOTES

* This study was supported by CNRS institutional funding and a specific grant from the Groupement des Entreprises Françaises dans la Lutte contre le Cancer (to M. F.).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.

Dagger Supported by a doctoral fellowship from the Association pour la Recherche sur le Cancer.

§ To whom correspondence should be addressed. Tel.: 33 4 93 95 77 70; Fax: 33 4 93 95 77 10; E-mail: franco@ipmc.cnrs.fr.

Published, JBC Papers in Press, May 7, 2001, DOI 10.1074/jbc.M103284200

    ABBREVIATIONS

The abbreviations used are: ARF, ADP-ribosylation factor; myrARF, myristoylated ARF; GEF, guanine nucleotide exchange factor; PH, pleckstrin homology; (4, 5)PIP2, phosphatidyl-4,5-bisphosphate; MES, 4-morpholineethanesulfonic acid; BFA, brefeldin A.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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