Sec7 domains catalyze the replacement of GDP by GTP on the G protein ADP-ribosylation factor 1 (myrARF1) by interacting with its switch I and II regions and by destabilizing, through a glutamic finger, the β-phosphate of the bound GDP. The myristoylated N-terminal helix that allows myrARF1 to interact with membrane lipids in a GTP-dependent manner is located some distance from the Sec7 domain-binding region. However, these two regions are connected. Measuring the binding to liposomes of functional or abortive complexes between myrARF1 and the Sec7 domain of ARNO demonstrates that myrARF1, in complex with the Sec7 domain, adopts a high affinity state for membrane lipids, similar to that of the free GTP-bound form. This tight membrane attachment does not depend on the release of GDP induced by the Sec7 domain but is partially inhibited by the uncompetitive inhibitor brefeldin A. These results suggest that the conformational switch of the N-terminal helix of myrARF1 to the membrane-bound form is an early event in the nucleotide exchange pathway and is a prerequisite for a structural rearrangement at the myrARF1-GDP/Sec7 domain interface that allows the glutamic finger to expel GDP from myrARF1. Sec7 domains catalyze the replacement of GDP by GTP on the G protein ADP-ribosylation factor 1 (myrARF1) by interacting with its switch I and II regions and by destabilizing, through a glutamic finger, the β-phosphate of the bound GDP. The myristoylated N-terminal helix that allows myrARF1 to interact with membrane lipids in a GTP-dependent manner is located some distance from the Sec7 domain-binding region. However, these two regions are connected. Measuring the binding to liposomes of functional or abortive complexes between myrARF1 and the Sec7 domain of ARNO demonstrates that myrARF1, in complex with the Sec7 domain, adopts a high affinity state for membrane lipids, similar to that of the free GTP-bound form. This tight membrane attachment does not depend on the release of GDP induced by the Sec7 domain but is partially inhibited by the uncompetitive inhibitor brefeldin A. These results suggest that the conformational switch of the N-terminal helix of myrARF1 to the membrane-bound form is an early event in the nucleotide exchange pathway and is a prerequisite for a structural rearrangement at the myrARF1-GDP/Sec7 domain interface that allows the glutamic finger to expel GDP from myrARF1. ADP-ribosylation factor Sec7 domain of ARNO brefeldin A guanine nucleotide exchange factor mutated-form of ARNO-Sec7 carrying the following mutations: F190Y,A191S,S198D,P208M polyacrylamide gel electrophoresis ADP ribosylation factors (ARFs)1 are small G proteins of the Ras superfamily involved in intracellular trafficking (1Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1993) Google Scholar, 2Chavrier P. Goud B. Curr. Opin. Cell Biol. 1999; 11: 466-475Crossref PubMed Scopus (418) Google Scholar). The best studied member of this family is ARF1. Its crystal structure has been determined in several forms (3Amor J.C. Harrison D.H. Kahn R.A. Ringe D. Nature. 1994; 372: 704-708Crossref PubMed Scopus (247) Google Scholar, 4Greasley S.E. Jhoti H. Teahan C. Solari R. Fensome A. Thomas G.M.H. Cockcroft S. Bax B. Nat. Struct. Biol. 1995; 2: 797-806Crossref PubMed Scopus (99) Google Scholar, 5Goldberg J. Cell. 1998; 95: 237-248Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar), and its function in the formation of transport vesicles has been well established by reconstitution studies (6Spang A. Matsuoka K. Hamamoto S. Schekman R. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11199-11204Crossref PubMed Scopus (160) Google Scholar, 7Bremser M. Nickel W. Schweikert M. Ravazzola M. Amherdt M. Hugues C.A. Söllner T.H. Rothman J.E. Wieland F.T. Cell. 1999; 96: 495-506Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). ARF1 controls the binding to Golgi membranes of coatomer, a large (700 kDa) cytoplasmic complex, which upon oligomerization deforms the lipid bilayer and induces the formation of "COPI"-coated vesicles (6Spang A. Matsuoka K. Hamamoto S. Schekman R. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11199-11204Crossref PubMed Scopus (160) Google Scholar, 7Bremser M. Nickel W. Schweikert M. Ravazzola M. Amherdt M. Hugues C.A. Söllner T.H. Rothman J.E. Wieland F.T. Cell. 1999; 96: 495-506Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). In addition, ARF1 activates phospholipase D (8Brown H.A. Gutowski S. Moomaw C.R. Slaughter C. Sternweis P.C. Cell. 1993; 75: 1137-1144Abstract Full Text PDF PubMed Scopus (819) Google Scholar, 9Cockcroft S. Thomas G.M.H. Fensome A. Geny B. Cunningham E. Gout I. Hiles I. Totty N.F. Truong O. Hsuan J.J. Science. 1994; 263: 523-526Crossref PubMed Scopus (584) Google Scholar). The molecular mechanism by which ARF1 recruits coatomer to the membrane is poorly understood but is based on the ability of ARF1 to interact directly, simultaneously, and in a GTP-dependent manner both with its protein target and with lipid membranes. This dual interaction can be ascribed to two regions of ARF1. The classical switch I and II regions of the "Ras-like" core domain of ARF1 are probably the main determinants of its interaction with coatomer (10Zhao L. Helms J.B. Brugger B. Harter C. Martoglio B. Graf R. Brunner J. Wieland F.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4418-4423Crossref PubMed Scopus (121) Google Scholar), whereas the interaction of ARF1 with membrane lipids involves a region specific to ARF1: the N-terminal α-helix, which is amphipathic and myristoylated (11Franco M. Chardin P. Chabre M. Paris S. J. Biol. Chem. 1993; 268: 24531-24534Abstract Full Text PDF PubMed Google Scholar, 12Franco M. Chardin P. Chabre M. Paris S. J. Biol. Chem. 1995; 270: 1337-1341Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 13Antonny B. Béraud-Dufour S. Chardin P. Chabre M. Biochemistry. 1997; 36: 4675-4684Crossref PubMed Scopus (271) Google Scholar, 14Losonczi J.A. Prestegard J.H. Biochemistry. 1998; 37: 706-716Crossref PubMed Scopus (91) Google Scholar). [Δ17]ARF1, a truncated form of ARF1 that lacks the N-terminal helix, is still able to interact with coatomer but remains soluble in the GTP-bound state and hence cannot promote the membrane recruitment of coatomer (15Jones D.H. Bax B. Fensome A. Cockcroft S. Biochem. J. 1999; 341: 185-192Crossref PubMed Scopus (50) Google Scholar, 16Goldberg J. Cell. 1999; 96: 893-902Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). The exchange of GDP for GTP on ARF proteins is catalyzed by a class of guanine nucleotide exchange factors (GEFs) that share a conserved catalytic domain of ≈200 residues, named the Sec7 domain (17Peyroche A. Paris S. Jackson C.L. Nature. 1996; 384: 479-481Crossref PubMed Scopus (231) Google Scholar, 18Chardin P. Paris S. Antonny B. Robineau S. Béraud Dufour S. Jackson C.L. Chabre M. Nature. 1996; 384: 481-484Crossref PubMed Scopus (408) Google Scholar, 19Meacci E. Tsai S.-C. Adamik R. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1745-1748Crossref PubMed Scopus (136) Google Scholar). The Sec7 domain is found in proteins of variable size and domain organization (2Chavrier P. Goud B. Curr. Opin. Cell Biol. 1999; 11: 466-475Crossref PubMed Scopus (418) Google Scholar). In the simplest case the Sec7 domain is flanked by a coiled-coil region and a PH domain. Recent structural and site-directed mutagenesis studies have led to a determination of the mechanism by which Sec7 domains interact with the core domain of ARF1 and promote the release of GDP (5Goldberg J. Cell. 1998; 95: 237-248Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 20Cherfils J. Ménétray J. Mathieu M. Le Bras G. Robineau S. Béraud Dufour S. Antonny B. Chardin P. Nature. 1998; 392: 101-105Crossref PubMed Scopus (148) Google Scholar, 21Mossessova E. Gulbis J.M. Goldberg J. Cell. 1998; 92: 415-423Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 22Betz S.F. Schnuchel A. Wang H. Olejniczak E.T. Meadows R.P. Lipsky B.P. Harris E.A.S. Staunton D.E. Fesik S.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7909-7914Crossref PubMed Scopus (33) Google Scholar, 23Béraud-Dufour S. Robineau S. Chardin P. Paris S. Chabre M. Cherfils J. Antonny B. EMBO J. 1998; 17: 3651-3659Crossref PubMed Scopus (151) Google Scholar). Sec7 domains are characterized by a groove that contains mainly exposed hydrophobic residues and that binds the switch I and II regions of ARF1 (5Goldberg J. Cell. 1998; 95: 237-248Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 20Cherfils J. Ménétray J. Mathieu M. Le Bras G. Robineau S. Béraud Dufour S. Antonny B. Chardin P. Nature. 1998; 392: 101-105Crossref PubMed Scopus (148) Google Scholar, 21Mossessova E. Gulbis J.M. Goldberg J. Cell. 1998; 92: 415-423Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 22Betz S.F. Schnuchel A. Wang H. Olejniczak E.T. Meadows R.P. Lipsky B.P. Harris E.A.S. Staunton D.E. Fesik S.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7909-7914Crossref PubMed Scopus (33) Google Scholar, 23Béraud-Dufour S. Robineau S. Chardin P. Paris S. Chabre M. Cherfils J. Antonny B. EMBO J. 1998; 17: 3651-3659Crossref PubMed Scopus (151) Google Scholar). This interaction positions an essential glutamate residue of the Sec7 domain, which lays on one edge of the groove, near the Mg2+ and β-phosphate region of the guanine nucleotide-binding site of ARF1 (5Goldberg J. Cell. 1998; 95: 237-248Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 23Béraud-Dufour S. Robineau S. Chardin P. Paris S. Chabre M. Cherfils J. Antonny B. EMBO J. 1998; 17: 3651-3659Crossref PubMed Scopus (151) Google Scholar). By its long negatively charged side chain this "glutamic finger" probably destabilizes GDP by displacing Mg2+ and the β-phosphate, allowing very rapid nucleotide exchange on ARF1 (23Béraud-Dufour S. Robineau S. Chardin P. Paris S. Chabre M. Cherfils J. Antonny B. EMBO J. 1998; 17: 3651-3659Crossref PubMed Scopus (151) Google Scholar). In the recent crystal structure of nucleotide-free [Δ17]ARF1 complexed with the Sec7 domain of Gea2p, the carboxylate group of the Glu finger occupies nearly the position of the β-phosphate of the expelled GDP and is coordinated to Lys 30 of ARF1, a residue that interacts with the β-phosphate of bound nucleotide (5Goldberg J. Cell. 1998; 95: 237-248Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). Minimalistic studies with [Δ17]ARF1 and Sec7 domains have thus given a simple picture of the mechanism by which Sec7 domains induce the dissociation of bound GDP. However, catalysis of GDP/GTP exchange on authentic ARF1 (hereafter abbreviated myrARF1) is more complex than on [Δ17]ARF1 because the reaction is correlated with a change in the interaction of the G protein with membrane lipids. The substrate (myrARF1-GDP) is mostly soluble, whereas the product (myrARF1-GTP) is tightly attached to membrane lipids. This increase in membrane affinity is due to a change in the membrane exposure of the N-terminal helix of myrARF1. In the GDP-bound state, the helix is buried in a hydrophobic pocket at the surface of the core domain of ARF1 (3Amor J.C. Harrison D.H. Kahn R.A. Ringe D. Nature. 1994; 372: 704-708Crossref PubMed Scopus (247) Google Scholar, 4Greasley S.E. Jhoti H. Teahan C. Solari R. Fensome A. Thomas G.M.H. Cockcroft S. Bax B. Nat. Struct. Biol. 1995; 2: 797-806Crossref PubMed Scopus (99) Google Scholar), whereas in the GTP-bound state the hydrophobic pocket is eliminated (5Goldberg J. Cell. 1998; 95: 237-248Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar) and the N-terminal helix is fully available for membrane interaction (13Antonny B. Béraud-Dufour S. Chardin P. Chabre M. Biochemistry. 1997; 36: 4675-4684Crossref PubMed Scopus (271) Google Scholar, 14Losonczi J.A. Prestegard J.H. Biochemistry. 1998; 37: 706-716Crossref PubMed Scopus (91) Google Scholar). In addition to the membrane insertion of the myristate, this direct protein/lipid interaction allows myrARF-GTP to be strongly attached to the membrane surface (13Antonny B. Béraud-Dufour S. Chardin P. Chabre M. Biochemistry. 1997; 36: 4675-4684Crossref PubMed Scopus (271) Google Scholar). At which step of the nucleotide exchange reaction does the switch of the N-terminal helix occur? The reaction proceeds through the formation of a series of ARF-Sec7 domain complexes in which the nucleotide-binding site is initially occupied by GDP, then vacant, and finally occupied by GTP. In these transient complexes, what is the state of the N-terminal helix? Is it held in the hydrophobic pocket on the core domain of ARF1 as in free ARF1-GDP, or does it interact with membrane lipids as in free ARF1-GTP? In this paper, we have studied the binding to lipid vesicles of full-length myristoylated ARF1 (myrARF1) in complex with the Sec7 domain of ARNO, a GEF active in vitro on ARF1 and on other members of the ARF family including ARF6 (18Chardin P. Paris S. Antonny B. Robineau S. Béraud Dufour S. Jackson C.L. Chabre M. Nature. 1996; 384: 481-484Crossref PubMed Scopus (408) Google Scholar, 24Frank S. Upender S. Hansen S.H. Casanova J.E. J. Biol. Chem. 1998; 273: 23-27Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). We have taken advantage of the fact that the ARF1-Sec7 domain complex can be "frozen" at different stages of the nucleotide exchange reaction pathway, upon removal of GDP (25Paris S. Béraud-Dufour S. Robineau S. Bigay J. Antonny B. Chabre M. Chardin P. J. Biol. Chem. 1997; 272: 22221-22226Crossref PubMed Scopus (137) Google Scholar), mutagenesis of the essential glutamic finger of the Sec7 domain (23Béraud-Dufour S. Robineau S. Chardin P. Paris S. Chabre M. Cherfils J. Antonny B. EMBO J. 1998; 17: 3651-3659Crossref PubMed Scopus (151) Google Scholar), or addition of the uncompetitive inhibitor, brefeldin A (26Peyroche A. Antonny B. Robineau S. Acker J. Cherfils J. Jackson C.L. Mol. Cell. 1999; 3: 275-285Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). We show that in the complex between nucleotide-free myrARF1 and ARNO-Sec7, the key intermediate of the nucleotide exchange pathway, myrARF1 interacts tightly with membrane lipids like free myrARF1-GTP. Analysis of the membrane interaction of abortive complexes in which GDP remains bound to myrARF1 suggests that the tight membrane interaction of myrARF1 in complex with the Sec7 domain is not a consequence of Sec7-induced GDP dissociation on myrARF1. Rather, our data support the idea that the interaction of the N-terminal helix of ARF1 with membrane lipids is an early event in the Sec7-domain catalyzed nucleotide exchange reaction that is required for all subsequent steps including destabilization of GDP by the glutamic finger of the Sec7 domain. Together with previous kinetics studies (25Paris S. Béraud-Dufour S. Robineau S. Bigay J. Antonny B. Chabre M. Chardin P. J. Biol. Chem. 1997; 272: 22221-22226Crossref PubMed Scopus (137) Google Scholar, 27Franco M. Chardin P. Chabre M. Paris S. J. Biol. Chem. 1996; 271: 1573-1578Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) and with the crystal structure of the soluble [Δ17]ARF1-Sec7 domain complex (5Goldberg J. Cell. 1998; 95: 237-248Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar), these results lead to a model for coordinated conformational changes at the myrARF1/lipid membrane interface and at the myrARF1/Sec7 domain interface during catalysis of guanine nucleotide exchange. Brefeldin A, alkaline phosphatase (from bovine intestinal mucosa), and all lipids, including azolectin (a mixture of soybean lipids) were purchased from Sigma. Unlabeled nucleotides were from Roche Molecular Biochemicals. [3H]GDP and all chromatography columns were from Amersham Pharmacia Biotech. The expression in Escherichia coli and purification of full-length myristoylated ARF1 (myrARF1), [Δ17]ARF1 (residues 18–181 of ARF1), and the Sec7 domain of ARNO (residues 52–242 of ARNO) have been described elsewhere (12Franco M. Chardin P. Chabre M. Paris S. J. Biol. Chem. 1995; 270: 1337-1341Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 13Antonny B. Béraud-Dufour S. Chardin P. Chabre M. Biochemistry. 1997; 36: 4675-4684Crossref PubMed Scopus (271) Google Scholar, 18Chardin P. Paris S. Antonny B. Robineau S. Béraud Dufour S. Jackson C.L. Chabre M. Nature. 1996; 384: 481-484Crossref PubMed Scopus (408) Google Scholar, 23Béraud-Dufour S. Robineau S. Chardin P. Paris S. Chabre M. Cherfils J. Antonny B. EMBO J. 1998; 17: 3651-3659Crossref PubMed Scopus (151) Google Scholar). Briefly, myrARF1 was purified from bacteria coexpressing bovine ARF1 and yeast N-myristoyltransferase in three steps: (i) precipitation at 35% saturation ammonium sulfate of the supernatant obtained after bacteria lysis; (ii) DEAE-Sepharose chromatography; and (iii) MonoS chromatography. [Δ17]ARF1 and ARNO-Sec7 (wild-type or mutated) were purified in two steps: anion exchange chromatography (DEAE-Sepharose or Q Sepharose, respectively) and gel filtration (Sephacryl HR 200). All proteins were 70–90% pure. Azolectin vesicles were prepared by the reverse phase method, and sucrose-loaded vesicles of defined lipid composition were prepared by the extrusion procedure as described previously (12Franco M. Chardin P. Chabre M. Paris S. J. Biol. Chem. 1995; 270: 1337-1341Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 13Antonny B. Béraud-Dufour S. Chardin P. Chabre M. Biochemistry. 1997; 36: 4675-4684Crossref PubMed Scopus (271) Google Scholar, 18Chardin P. Paris S. Antonny B. Robineau S. Béraud Dufour S. Jackson C.L. Chabre M. Nature. 1996; 384: 481-484Crossref PubMed Scopus (408) Google Scholar). All experiments were performed in 50 mm Hepes, pH 7.5, 100 mm KCl, 1 mm MgCl2, 1 mm dithiothreitol (buffer A). MyrARF1-GDP (2 μm) was preloaded with [3H]GDP (10 μm, ≈2000 dpm/pmol) by incubation in buffer A supplemented with 2 mm EDTA and 1 mg/ml bovine serum albumin at 37 °C. After 15 min, 2 mmMgCl2 was added to provide 1 mm free Mg2+. [3H]GDP dissociation was initiated by diluting the sample 2-fold in buffer A supplemented with 2 mm cold GDP and 2 mm MgCl2, and with or without azolectin vesicles and ARNO-Sec7 as indicated. Samples of 25 μl (25 pmol of myrARF1) 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 and counted directly (without drying). MyrARF1-GDP and ARNO-Sec7 (wild-type or mutant) were incubated separately or together with lipid vesicles in buffer B (20 mm Tris, pH 7.5, 120 mm NaCl, 1 mm MgCl2; total volume, 50 μl). Except where otherwise indicated the concentration of each protein was 3 μm. When indicated, alkaline phosphatase (6 μg/ml, 30 units/ml), BFA (0 - 200 μm), EDTA (2 mm), or guanine nucleotides were added. After 15 min of incubation at 25 °C, the sample was centrifuged at 400,000 ×g for 10 min at 25 °C in a TLA 100.1 rotor (Beckman Instruments, Inc.). The percentage of myrARF1 and ARNO-Sec7 in the supernatant and in the lipid pellet (which was resuspended in the same volume of buffer) was determined by densitometry of Coomassie Blue-stained SDS-polyacrylamide gels. The membrane-bound complex between nucleotide-free myrARF1 and ARNO-Sec7 was prepared by incubating 12.5 μm myrARF1-GDP with 12.5 μm ARNO-Sec7, 1 mg/ml azolectin vesicles, and 30 μg/ml alkaline phosphatase (150 units/ml) in buffer B. After 20 min at room temperature, membrane-bound proteins were recovered by centrifugation (400,000 × g, 15 min, 4 °C) and washed once with buffer A supplemented with 2 mm EDTA to remove residual alkaline phosphatase. The sample was centrifuged again, and the pellet was finally resuspended in buffer A. For fluorescence measurements, the sample was diluted in buffer A at a final concentration of 0.1 mg/ml azolectin vesicles in a 7-mm-diameter cylindrical quartz cell (13Antonny B. Béraud-Dufour S. Chardin P. Chabre M. Biochemistry. 1997; 36: 4675-4684Crossref PubMed Scopus (271) Google Scholar). The cuvette was continuously stirred with a small magnetic bar and thermostated at 25 °C. Fluorescence was recorded at 340 nm (bandwidth, 20 nm) upon excitation at 297.5 nm (bandwidth, 5 nm) and sampling rate of 1/0.3 s. The signal resulting from vesicles light scattering was subtracted. At the time indicated, GDP or GTP (final concentration, 50 μm) was added from concentrated stock solutions. The mixing time in the cuvette was about 1 s. The formation of complexes between myrARF1 and wild-type ARNO-Sec7 or [E156K]ARNO-Sec7 or [F190Y-A191S-S198d-P208M]ARNO-Sec7 in the absence of lipid vesicles was analyzed by gel filtration on a superose 12 HR 10/30 column as described for the corresponding complexes with [Δ17]ARF1 (23Béraud-Dufour S. Robineau S. Chardin P. Paris S. Chabre M. Cherfils J. Antonny B. EMBO J. 1998; 17: 3651-3659Crossref PubMed Scopus (151) Google Scholar, 25Paris S. Béraud-Dufour S. Robineau S. Bigay J. Antonny B. Chabre M. Chardin P. J. Biol. Chem. 1997; 272: 22221-22226Crossref PubMed Scopus (137) Google Scholar, 26Peyroche A. Antonny B. Robineau S. Acker J. Cherfils J. Jackson C.L. Mol. Cell. 1999; 3: 275-285Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). MyrARF1-GDP (40 μm) in a buffer containing 1 mm MgCl2 was first labeled with [3H]GDP by incubation for 15 min at room temperature with tracer [3H]GDP (final concentration, 10 μCi/ml). Labeled myrARF1-GDP (10 μm) was then incubated in buffer B for 15 min at room temperature with a stoichiometric amount of ARNO-Sec7 (either wild-type or mutated). For the interaction with wild-type ARNO-Sec7, the sample was supplemented with alkaline phosphatase (13 μg/ml, 100 units/ml). For the interaction with [E156K]ARNO-Sec7, the sample was supplemented with 2 mmEDTA (1 μm free Mg2+). For the interaction with [F190Y,A191S,S198D,P208M]ARNO-Sec7, 0 or 50 μm BFA was added. The sample (210 μl) was loaded on the column and eluted at a flow rate of 0.5 ml/min with buffer B. When indicated, the elution buffer was supplemented with 2 mm EDTA (for the interaction with [E156K]ARNO-Sec7) or with 50 μm BFA (for the interaction with [F190Y,A191S,S198D,P208M]ARNO-Sec7. Fractions (300 μl) were collected and two samples of 60 μl were used for SDS-PAGE analysis and [3H]GDP counting. Monitoring the rate of futile [3H]GDP/GDP exchange on myristoylated ARF1 catalyzed by the Sec7 domain of ARNO (ARNO-Sec7) eliminates two protein/lipid interactions that occur when full-length ARNO catalyzes GDP/GTP exchange on myrARF1: the tight binding of myrARF1-GTP to the bilayer and the binding of ARNO to phosphoinositides through its PH domain. Because ARNO-Sec7 is fully soluble and myrARF1-GDP interacts only weakly with membrane lipids, one might expect that ARNO-Sec7 would catalyze [3H]GDP/GDP exchange on myrARF1 in the absence of lipid vesicles. However, Fig. 1 A shows that ARNO-Sec7 is very inefficient in catalyzing [3H]GDP/GDP exchange on myrARF1 in solution. In contrast, a 7-fold stimulation was observed when the same experiment was carried out in the presence of 2 mg/ml azolectin vesicles (Fig. 1 B). Thus, apart from being essential for the stabilization of myrARF1 in the GTP bound state, membrane lipids are also essential for the catalysis of GDP release on myrARF1 by the Sec7 domain of ARNO. This is in agreement with a previous study on an unidentified ARF exchange factor from bovine retinas (27Franco M. Chardin P. Chabre M. Paris S. J. Biol. Chem. 1996; 271: 1573-1578Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) and suggests that productive interaction between myrARF1 and ARNO-Sec7 can occur only at the surface of lipid membranes. The effect of lipid vesicles on the association of myrARF1 with the Sec7 domain of ARNO was assessed by sedimentation experiments. MyrARF1-GDP or ARNO-Sec7 or a stoichiometric mixture of the two proteins was incubated with azolectin vesicles in an isotonic buffer containing 1 mm MgCl2. After centrifugation, the amount of protein in the pellet and in the supernatant was determined by scanning Coomassie-stained polyacrylamide gels. In the presence of 0.5 mg/ml azolectin vesicles, myrARF1-GDP displayed weak membrane binding (Fig. 2 A, lanes 1 and2), in agreement with previous studies (12Franco M. Chardin P. Chabre M. Paris S. J. Biol. Chem. 1995; 270: 1337-1341Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 13Antonny B. Béraud-Dufour S. Chardin P. Chabre M. Biochemistry. 1997; 36: 4675-4684Crossref PubMed Scopus (271) Google Scholar). Under the same conditions, ARNO-Sec7 was totally soluble (Fig. 2 A,lanes 3 and 4). When the same experiment was repeated with a stoichiometric mixture of the two proteins, a significant amount (20%) of ARNO-Sec7 was found in the lipid pellet, and the amount of membrane-bound myrARF1 doubled (Fig. 2 A,lanes 5 and 6). If ARNO-Sec7 displayed no preference for membrane-bound myrARF1 versus membrane-free myrARF1, it should not affect the membrane partitioning of myrARF1. Therefore, the synergy between the two proteins for their binding to lipid vesicles suggests that a complex between myrARF1 and ARNO-Sec7 displays a higher "affinity" for lipid membranes than isolated myrARF1-GDP and isolated ARNO-Sec7. To determine whether myrARF1 in the membrane-bound complex with ARNO-Sec7 has lost its nucleotide, as is usually the case for functional complexes between GEFs and G proteins, sedimentation experiments were repeated in the presence of excess GDP or GTP. With 100 μm GDP, no enhancement in the membrane binding of myrARF1 and ARNO-Sec7 was observed when the two proteins were incubated together (Fig. 2 A, lanes 7 and 8). With 100 μm GTP, ARNO-Sec7 was found soluble, whereas myrARF1 became totally membrane-bound, as expected because of its conversion to the GTP-bound form (Fig. 2 A, lanes 9 and 10). Thus, the synergy between myrARF1 and ARNO-Sec7 for their binding to lipid vesicles observed in the absence of added nucleotide probably reflects the formation of a membrane-bound complex between myrARF1 and ARNO-Sec7, in which the nucleotide-binding site of myrARF1 is vacant. However, even when carried out in the absence of added nucleotide, these experiments were performed under conditions that do not favor the stabilization of the nucelotide-free complex between myrARF1 and ARNO-Sec7. Indeed, the stoichiometric GDP that was initially associated with myrARF1 could antagonize the formation of the nucleotide-free complex with ARNO-Sec7. Therefore, the experiment was repeated in the presence of a catalytic amount of alkaline phosphatase which hydrolyzes GDP as it is released from myrARF1. Strikingly, under these conditions myrARF1 and ARNO-Sec7 bound almost completely to azolectin vesicles (Fig. 2 A, comparelanes 13 and 14 with lanes 5 and6). EDTA, which chelates magnesium and hence weakens the affinity of GDP for the nucleotide-binding site of myrARF1, has a similar effect (data not shown). These results suggest that the complex between nucleotide-free myrARF1 and ARNO-Sec7 displays a high affinity for membrane lipids. Like myrARF1-GTP, the membrane-bound complex between nucleotide-free myrARF1 and ARNO-Sec7 interacts with membrane lipids through the N terminus of ARF1. No cotranslocation of ARNO-Sec7 and ARF1 to azolectin vesicles could be detected when myrARF1-GDP was replaced by [Δ17]ARF1-GDP (data not shown). This was expected because the complex between nucleotide-free [Δ17]ARF1 and ARNO-Sec7 is soluble and can be isolated by gel filtration (23Béraud-Dufour S. Robineau S. Chardin P. Paris S. Chabre M. Cherfils J. Antonny B. EMBO J. 1998; 17: 3651-3659Crossref PubMed Scopus (151) Google Scholar, 25Paris S. Béraud-Dufour S. Robineau S. Bigay J. Antonny B. Chabre M. Chardin P. J. Biol. Chem. 1997; 272: 22221-22226Crossref PubMed Scopus (137) Google Scholar). Thus, both [Δ17]ARF1 and myrARF1 are able to form a nucleotide-free complex with ARNO-Sec7, but the complex with [Δ17]ARF1 is soluble like isolated [Δ17]ARF1-GDP or [Δ17]ARF-GTP, whereas the complex with myrARF1 is tightly bound to membrane lipids, like myrARF1-GTP. It should be noted that when myrARF-GDP and ARNO-Sec7 were incubated with alkaline phosphatase in the absence of lipid vesicles and then loaded on a gel filtration column, no complex between the two proteins could be detected (data not shown). Therefore, the complex between nucleotide-free myrARF and ARNO-Sec7 is so lipophilic that it can form only in the presence of lipids. To determine whether specific interactions with membrane lipids are required for the formation of the complex between nucleotide-free myrARF1 and ARNO-Sec7, sedimentation experiments were performed with artificial vesicles of defined lipid composition (Fig. 2 B). With neutral vesicles containing 50% phosphatidylcholine and 50% phosphatidylethanolamine, about 70% of ARNO-Sec7 and myrARF1 were found associated with vesicles after incubation in the presence of alkaline phosphatase. With 5% phosphatidylinositol-4,5-bisphosphate, or 20% phosphatidylserine and 5% phosphatidic acid in the vesicles, the amount of myrARF1 and ARNO-Sec7 bound to the lipid vesicles was slightly enhanced. This suggests that the complex between nucleotide-free myrARF1 and ARNO-Sec7 interacts with membrane lipids mainly through hydrophobic interactions and that the contribution of electrostatic interactions with anionic lipids is weak. By its hydrophobic nature and the essential role of the N terminus of ARF1, the membrane interaction of nucleotide-free myrARF1 in complex with ARNO-Sec7 closely resembles that of myrARF1-GTP (13Antonny B. Béraud-Dufour S. Chardin P. Chabre M. Biochemistry. 19
The small G protein Rap1 regulates diverse cellular processes such as integrin activation, cell adhesion, cell-cell junction formation and cell polarity. It is crucial to identify Rap1 effectors to better understand the signalling pathways controlling these processes. Krev interaction trapped 1 (Krit1), a protein with FERM (band four-point-one/ezrin/radixin/moesin) domain, was identified as a Rap1 partner in a yeast two-hybrid screen, but this interaction was not confirmed in subsequent studies. As the evidence suggests a role for Krit1 in Rap1-dependent pathways, we readdressed this question. In the present study, we demonstrate by biochemical assays that Krit1 interacts with Rap1A, preferentially its GTP-bound form. We show that, like other FERM proteins, Krit1 adopts two conformations: a closed conformation in which its N-terminal NPAY motif interacts with its C-terminus and an opened conformation bound to integrin cytoplasmic domain associated protein (ICAP)-1, a negative regulator of focal adhesion assembly. We show that a ternary complex can form in vitro between Krit1, Rap1 and ICAP-1 and that Rap1 binds the Krit1 FERM domain in both closed and opened conformations. Unlike ICAP-1, Rap1 does not open Krit1. Using sedimentation assays, we show that Krit1 binds in vitro to microtubules through its N- and C-termini and that Rap1 and ICAP-1 inhibit Krit1 binding to microtubules. Consistently, YFP-Krit1 localizes on cyan fluorescent protein-labelled microtubules in baby hamster kidney cells and is delocalized from microtubules upon coexpression with activated Rap1V12. Finally, we show that Krit1 binds to phosphatidylinositol 4,5-P(2)-containing liposomes and that Rap1 enhances this binding. Based on these results, we propose a model in which Krit1 would be delivered by microtubules to the plasma membrane where it would be captured by Rap1 and ICAP-1.
GDP/GTP exchange modulates the interaction of the small G-protein ADP-ribosylation factor-1 with membrane lipids: if ARFGDP is mostly soluble, ARFGTP binds tightly to lipid vesicles. Previous studies have shown that this GTP-dependent binding persists upon removal of the N-terminal myristate but is abolished following further deletion of the 17 N-terminal residues. This suggests a role for this amphipathic peptide in lipid membrane binding. In the ARFGDP crystal structure, the 2−13 peptide is helical, with its hydrophobic residues buried in the protein core. When ARF switches to the GTP state, these residues may insert into membrane lipids. We have studied the binding of ARF to model unilamellar vesicles of defined composition. ARFGDP binds weakly to vesicles through hydrophobic interaction of the myristate and electrostatic interaction of cationic residues with anionic lipids. Phosphatidylinositol 4,5-bis(phosphate) shows no specific effects other than strictly electrostatic. By using fluorescence energy transfer, the strength of the ARFGTP−lipid interaction is assessed via the dissociation rate of ARFGTPγS from labeled lipid vesicles. ARFGTPγS dissociates slowly (τoff ≈ 75 s) from neutral PC vesicles. Including 30% anionic phospholipids increases τoff by only 3-fold. Reducing the N-terminal peptide hydrophobicity by point mutations had larger effects: F9A and L8A-F9A substitutions accelerate the dissociation of ARFGTPγS from vesicles by factors of 7 and 100, respectively. This strongly suggests that, upon GDP/GTP exchange, the N-terminal helix is released from the protein core so its hydrophobic residues can interact with membrane phospholipids.
Inhibition of the potassium channels TREK-1 by spadin (SPA) is currently thought to be a promising therapeutic target for the treatment of depression. Since these channels are expressed in pancreatic β -cells, we investigated their role in the control of insulin secretion and glucose homeostasis. In this study, we confirmed the expression of TREK-1 channels in the insulin secreting MIN6-B1 β -cell line and in mouse islets. We found that their blockade by SPA potentiated insulin secretion induced by potassium chloride dependent membrane depolarization. Inhibition of TREK-1 by SPA induced a decrease of the resting membrane potential (ΔVm~12 mV) and increased the cytosolic calcium concentration. In mice, administration of SPA enhanced the plasma insulin level stimulated by glucose, confirming its secretagogue effect observed in vitro. Taken together, this work identifies SPA as a novel potential pharmacological agent able to control insulin secretion and glucose homeostasis.
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