©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Myristoylation of ADP-ribosylation Factor 1 Facilitates Nucleotide Exchange at Physiological Mg Levels (*)

(Received for publication, September 13, 1994; and in revised form, November 14, 1994)

Michel Franco (§) Pierre Chardin (¶) Marc Chabre Sonia Paris (**)

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
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Recombinant N-myristoylated bovine ADP-ribosylation factor 1 (myr-rARF1) has been expressed in bacteria and purified to near homogeneity with a high (85%) myristoylation efficiency. Myr-rARF1 and nonmyristoylated rARF1 have been compared with respect to their kinetics of guanine nucleotide exchange and their interactions with phospholipids. Myristoylation is shown to allow the release of bound GDP at physiological (mM) concentrations of Mg. GDP dissociation is slow in the absence of phospholipids but is accelerated 2-fold in the presence of phospholipid vesicles. On the contrary, myristoylation decreases 10-fold the rate of dissociation of GTP or guanosine 5`-O-(thiotriphosphate) (GTPS) in the presence of phospholipids. As a result, myr-ARF1 can be spontaneously activated by GTP or GTPS (t 30 min at 37 °C) at 1 mM Mg, in the sole presence of phospholipid membranes without the need for a nucleotide exchange factor.

In contrast to the nonacylated protein, the GDP-bound form of myr-ARF1 interacts with phospholipids, as demonstrated by its cosedimentation with phospholipid vesicles and its comigration with phospholipid/cholate micelles on gel filtration. The interaction is, however, weaker than for the GTP-bound form, suggesting that only the myristate in myr-ARF1 interacts with phospholipids, whereas both the myristate and the amino-terminal hydrophobic residues in myr-ARF1 bind to phospholipids.


INTRODUCTION

ADP-ribosylation factors (ARFs) (^1)are a family of 20-kDa guanine nucleotide-binding proteins. ARFs were originally discovered as cofactors for cholera toxin-catalyzed ADP-ribosylation of G(s)(1) , but, as a result of cDNA cloning, they now appear to constitute a large family that includes both the ARF proteins, acting as cofactors for cholera toxin activity, and the ARF-like proteins, which lack this activity(2) . All known members of the ARF family have a glycine residue at position 2, which is the site of N-myristoylation, a cotranslational modification.

ARFs have been recognized as regulators of intracellular vesicular transport and more recently as activators of phospholipase D (for review, see (3) ). Both GTP binding and myristoylation appear to be required for ARF activities(4, 5, 6, 7) , but the precise role of myristoylation is not clear. Myristoylation was found to be necessary for GTP-dependent binding of ARF to Golgi membranes (8, 9) and to phospholipid vesicles(10) , and some authors favor the view that binding of GTP to ARF induces a conformational switch that exposes the N-myristoyl group, enabling its binding to the lipid bilayer (9, 11, 12) according to the model of the Ca-myristoyl switch proposed for recoverin(13) . However, our previous report (14) that nonmyristoylated rARF1 strongly binds to phospholipids once it is activated by GTPS does not support this hypothesis. Instead, we believe that myr-ARF interacts with lipid bilayers mostly through the amphiphatic alpha helix found at the amino terminus (15) and that the GTP-induced conformational change primarily affects that alpha helix rather than the fatty acid.

In an attempt, therefore, to understand at which step of ARF activation myristoylation plays a role, we have compared in this study the myristoylated and nonmyristoylated forms of rARF1 for their interactions with phospholipids and their nucleotide exchange properties. We demonstrate that myristoylation of ARF allows GDP dissociation at physiological Mg concentrations, especially in the presence of phospholipids, because myr-rARF1 interacts with lipid membranes in marked contrast to rARF1. This interaction appears to further open the nucleotide binding site and facilitate nucleotide exchange. Moreover, myristoylation enhances the phospholipid-dependent stabilization of the GTP- or GTPS-bound form.


EXPERIMENTAL PROCEDURES

Materials

Nucleotides were purchased from Boehringer Mannheim; myristic acid, fatty acid-free bovine serum albumin, azolectin, sodium cholate, and L-alpha-dimyristoylphosphatidylcholine (DMPC) were from Sigma. [S]GTPS was from DuPont NEN; [^3H]GDP and [^3H]dipalmitoylphosphatidylcholine (DPPC) were from Amersham Corp. [alpha-P]GTP and [-P]GTP were from ICN Biomedicals Inc.

Expression Vectors

The wild-type bovine ARF1 cDNA, with a glycine at position 2, was generated by polymerase chain reaction amplification from our first pET3c/Ile-2 ARF1 vector (14) with two mutagenic oligonucleotides.

Oligonucleotide 1 (5`-TGCACCATGGGGAATATCTTTGCAAACCTCTTC-3`) introduced a NcoI site at the 5` end without changing the coding sequence.

Oligonucleotide 2 (5`-ATGAGGATCCTCATTTCTGGTTCCGGAGCT-3` introduced a BamHI site right after the stop codon. The NcoI-BamHI fragment was inserted in the pET11d expression vector (Novagen Inc.). Saccharomyces cerevisiaeN-myristoyltransferase expression vector (pBB131) was kindly provided by J. I. Gordon(16) .

Production and Purification of Myristoylated and Nonmyristoylated ARF1

Nonmyristoylated Gly-2 ARF1 (rARF1) was expressed and purified by a single QAE-Sepharose (Pharmacia Biotech Inc.) chromatography as described for Ile-2 ARF1(14) .

For production of myristoylated ARF1 (myr-rARF1), BL21(DE3) bacteria were cotransformed with the pET11d/Gly-2 ARF1 and pBB131 (yeast N-myristoyltransferase) plasmids and selected for both ampicillin and kanamycin resistance. Transformed cells were grown at 37 °C to A = 0.6. Myristate (50 µM) bound to bovine serum albumin (6 µM) was added as a 100-fold concentrated solution prepared by slowly adding warm sodium myristate 100 mM, pH 9, to fatty acid-free bovine serum albumin. After 10 min, the coexpression of ARF1 and N-myristoyltransferase was induced with 0.3 mM isopropyl-1-thio-beta-D-galactopyranoside, and the temperature was reduced to 27 °C to increase the yield of myristoylation (see ``Results''). After 3 h, the cells were harvested and lysed(17) . The supernatant was precipitated at 35% saturation of ammonium sulfate and centrifuged at 8,000 times g for 30 min. The pellet was dissolved in 50 mM Tris/HCl, pH 8, 1 mM MgCl(2), 1 mM DTT, 5 µM GDP, dialyzed against the same buffer, and applied to a DEAE-Sepharose column equilibrated with 50 mM Tris/HCl, pH 8, 1 mM MgCl(2), 1 mM DTT. The flow-through fractions containing ARF1 were pooled, concentrated on a Centricon-10 (Amicon) in 10 mM MES, pH 5.7, 1 mM MgCl(2), 1 mM DTT, and applied to a Mono S column (Pharmacia) equilibrated in the same buffer. The bound proteins were eluted with a gradient of NaCl (0-500 mM). Fractions containing myr-rARF1 (eluted between 130 and 170 mM NaCl) were pooled, concentrated on a Centricon-10 (Amicon) in 50 mM Tris/HCl, pH 8, 1 mM MgCl(2), 1 mM DTT, 2 µM GDP, to a protein concentration of 0.8 mg/ml and stored at -70 °C.

Preparation of Phospholipid Vesicles

Large unilamellar vesicles of azolectin were prepared as described(14) .

Nucleotide Binding Assay

rARF1 or myr-rARF1 (1 µM) was incubated at 37 °C with [S]GTPS, [^3H]GDP, [-P]GTP or [alpha-P]GTP (10 µM, 1000 cpm/pmol, unless otherwise stated) in 50 mM Hepes-NaOH, pH 7.5, 1 mM DTT, and EDTA and MgCl(2) as indicated, with or without 1.5 mg/ml phospholipid vesicles. Samples of 50 µl were diluted into 2 ml of ice-cold 20 mM Hepes, pH 7.5, 100 mM NaCl, 10 mM MgCl(2) and filtered on 25-mm BA 85 nitrocellulose filters (Schleicher & Schuell). Filters were washed twice with 2 ml of the same buffer, dried, and counted.

Sedimentation Analysis of the Binding of ARF to Phospholipid Vesicles

rARF1 or myr-rARF1 (3 µM) was incubated with 1.5 mg/ml phospholipid vesicles and 50 µM GDP in 25 mM Tris/HCl, pH 7.5, 80 mM NaCl, 1 mM MgCl(2), for 30 min at 30 °C, in 100 µl. After centrifugation for 6 min at 200,000 times g, the supernatant and the pellet (resuspended in 100 µl) were subjected to 15% SDS-PAGE, and the gel was analyzed by densitometry after Coomassie Blue staining.

Gel Filtration Analysis of the Binding of ARF to Phospholipid-Cholate Micelles

rARF1 or myr-rARF1 (3 µM) was incubated with 10 µM GDP in 20 mM Tris/HCl, pH 7.5, 120 mM NaCl, 1 mM MgCl(2), 1 g/liter DMPC and 4 g/liter sodium cholate, for 30 min at 30 °C. A 200-µl sample was loaded on a Superose 12 HR 10/30 column (Pharmacia), equilibrated and eluted at room temperature with the same DMPC/cholate-containing buffer, which was supplemented with 5 mM 2-mercaptoethanol and 0.1 mM phenylmethylsulfonyl fluoride. The flow rate was 30 ml/h, and 300-µl fractions were collected. When [^3H]DPPC was included, radioactivity was determined by counting 20 µl of each fraction. For SDS-PAGE analysis, the fractions were concentrated by precipitation of the proteins with methanol/chloroform(18) .


RESULTS

Preparation of Highly Myristoylated ARF1

Recombinant bovine ARF1 can be myristoylated in bacteria by coexpression of yeast N-myristoyltransferase(16) . As reported for rARF5(8) , the mobility of myr-rARF1 on SDS-PAGE was greater than that of rARF1. As shown in Fig. 1B (lane2), it was even possible to detect three bands around 20 kDa in a cytosolic extract of bacteria induced at 37 °C for coexpression of rARF1 and N-myristoyltransferase. Amino-terminal sequencing and labeling with [^3H]myristate indicated that the three bands corresponded, with increasing mobility, to Met-1 rARF1, Gly-1 rARF1 formed by cleavage of the amino-terminal Met, and myr-Gly1 ARF1, respectively. Interestingly, decreasing the induction temperature from 37 to 27 °C notably increased the efficiency of N-myristoylation with a marked fading of the Gly-1 rARF1 band (Fig. 1B, lanes2 and 3), which suggests that cleavage by methionine aminopeptidase becomes rate-limiting at this lower temperature. Nonmyristoylated rARF1 could be easily purified to near homogeneity by a single QAE-Sepharose chromatography (14) and appeared as a mixture of Met-1 and Gly-1 rARF1 (Fig. 1B, lane1). In contrast, myr-ARF1 was more strongly adsorbed on the QAE-Sepharose column and could not be efficiently purified by this method. Another purification procedure was therefore applied to the myristoylated form; myr-rARF1 was precipitated at 35% saturation of ammonium sulfate, which eliminated the majority of bacterial proteins and most of the nonmyristoylated ARF in the soluble fraction (Fig. 1A, lane3). Sequential chromatography on DEAE-Sepharose and Mono S columns removed the remaining contaminants (Fig. 1A, lanes4 and 5). myr-rARF1 was thus obtained with a purity > 95%, and it was estimated to be at least 85% myristoylated by densitometry of Coomassie-stained gels (Fig. 1A, lane5, and Fig. 1B, lane4).


Figure 1: Expression and purification of myr-rARF1. A, coexpression of rARF1 and yeast N-myristoyltransferase was induced at 27 °C, and myr-rARF1 was purified from the cytosolic fraction as described under ``Experimental Procedures.'' Samples were subjected to SDS-PAGE in a 15% gel and stained with Coomassie Blue. Lane 1, molecular mass markers; lane 2, total cytosol; lane 3, proteins precipitated at 35% saturation of ammonium sulfate; lane 4, pooled DEAE-Sepharose fractions; lane 5, pooled Mono S fractions. Arrows on the right indicate the positions of nonmyristoylated rARF1 (upper) and myristoylated rARF1 (lower). B, compared electrophoretic mobilities of the three forms of rARF1: Met-1 ARF (M), nonmyristoylated Gly-1 ARF (G), and myristoylated Gly-1 ARF (myrG). Lane 1, nonmyristoylated rARF1 obtained without expression of yeast N-myristoyltransferase and purified on QAE-Sepharose; lane 2, cytosolic extract after coexpression of rARF1 and N-myristoyltransferase at 37 °C; lane 3, cytosolic extract after coexpression of rARF1 and N-myristoyltransferase at 27 °C; lane 4, myr-rARF1 purified on Mono S column. Arrows on the right indicate the positions of Met-1 ARF1 (upper) and myr-rARF1 (lower).



Myristoylation of rARF1 Affects Nucleotide Exchange

As previously reported(14) , nonmyristoylated rARF1 can be optimally loaded with [S]GTPS in the presence of phospholipids at 1 µM free Mg (Fig. 2A), whereas at physiological (mM) concentrations of Mg, the exchange is extremely slow (Fig. 2B). myr-rARF1 behaved quite differently since, first, it was loaded with [S]GTPS much more rapidly at 1 µM Mg, with a tof 1 min instead of 21 min for rARF1 (Fig. 2A) and, most importantly, it could be efficiently loaded with GTPS at 1 mM Mg, with a tof 35 min (Fig. 2B). Because [S]GTPS binding is very likely to be rate-limited by the dissociation of GDP, we directly investigated [^3H]GDP dissociation from rARF1 and myr-rARF1 (Fig. 3, A and B). The ARFs were first loaded with [^3H]GDP in the presence or absence of phospholipids. Optimal GDP-[^3H]GDP exchange was obtained at 0.1 µM Mg in all cases except for myr-rARF1 in the presence of lipids; in this case, denaturation of nucleotide-free protein occurred at low Mg levels and the loading with [^3H]GDP was therefore performed at 100 µM Mg. When the plateau was attained in each condition, the concentration of free Mg was adjusted to either 1 µM (Fig. 3A) or 1 mM (Fig. 3B), and an excess of unlabeled GDP was added to initiate the release assay. As expected, very similar rates were found for [S]GTPS association and [^3H]GDP dissociation under identical conditions. At 1 µM Mg in the presence of lipids, [^3H]GDP dissociated much more rapidly from myr-rARF1 (t= 0.8 min) than from rARF1 (t= 13 min). The fact that the binding of GTPS was slightly slower than the dissociation of GDP may be explained by the dissociation rate of GTPS (see Fig. 3C). At 1 mM Mg, [^3H]GDP dissociation from rARF1 was undetectable, both in the presence and absence of phospholipids. Thus, the low level of [S]GTPS binding observed in Fig. 2B might be due to the presence in the rARF1 preparation of a small fraction of nucleotide-free ARF that renaturated upon binding of GTPS. By contrast, GDP significantly dissociated from myr-rARF1 at 1 mM Mg, and the rate of dissociation was doubled in the presence of phospholipids (Fig. 3B), reaching a value very similar to the rate of association measured for GTPS (Fig. 2B).


Figure 2: Time course of [S]GTPS binding to rARF1 and myr-rARF1 in the presence of phospholipid vesicles at 1 µM and 1 mM Mg. Binding of [S]GTPS to 1 µM rARF1 (circle) or myr-rARF1 (bullet) was determined as described under ``Experimental Procedures'' in the presence of phospholipid vesicles. A,1 µM free Mg (2 mM EDTA, 1 mM MgCl(2)); B, 1 mM MgCl(2). Values are means of two or three independent experiments and were normalized relative to the maximum binding observed for each protein at 1 µM Mg measured after 30 min for myr-rARF1 and 150 min for rARF1. The maximum binding corresponded to 50% of the concentration of rARF1, and to 60-90% of the concentration of myr-rARF1, depending on the preparation used. The curves (except for circle in B) were obtained by fitting the data to the model y = 100bullet(1 - e) with k = 0.68 min for myr-rARF1 and 0.03 min for rARF1 at 1 µM Mg, and k = 0.02 min for myr-rARF1 at 1 mM Mg.




Figure 3: Dissociation of GDP and GTPS from rARF1 and myr-rARF1. A and B, rARF1 (1 µM) was loaded for 60 min at 37 °C with 10 µM [^3H]GDP in the presence of 0.1 µM Mg (1 mM EDTA, 0.1 mM MgCl(2)), with (circle) or without (box) phospholipid vesicles; myr-rARF1 (1 µM) was loaded with 10 µM [^3H]GDP for 60 min in the presence of 100 µM MgCl(2) and phospholipid vesicles (bullet) or for 30 min at 0.1 µM Mg without phospholipids (). [^3H]GDP dissociation was initiated by adding 1 mM unlabeled GDP with MgCl(2) ± EDTA to give a final concentration of 1 µM (A) or 1 mM (B) free Mg. The concentration of bound [^3H]GDP just prior to the initiation of the exchange reaction (taken as 100%) was 0.60 and 0.50 µM for rARF1, and 0.46 and 0.40 µM for myr-rARF1 with and without phospholipids, respectively. The curves are best fits assuming first-order kinetics for nucleotide dissociation. At 1 µM Mg in the presence of phospholipids k = 0.05 min for rARF1 and 0.91 min for myr-rARF1; at 1 mM Mg, there was no detectable dissociation from rARF1, with or without phospholipids, whereas k values were 15 times 10 and 8 times 10 min for myr-rARF1 with and without phospholipids, respectively. C and D, in the presence of phospholipid vesicles, rARF1 (circle) and myr-rARF1 (bullet) were loaded for 90 and 30 min, respectively, with 10 µM [S]GTPS (900 cpm/pmol) at 1 µM free Mg. In the absence of phospholipids, loading was performed with 2 µM [S]GTPS (6000 cpm/pmol) for 1 h at 1 µM Mg for rARF1 (box) and for 3 h at 1 mM Mg for myr-rARF1 (). [S]GTPS exchange was initiated by the addition of 1 mM unlabeled GTPS either alone (C) or with 1 mM Mg, final concentration (D). 100% corresponds to 27 and 31 pmol (per 50-µl aliquot) in the presence of lipids, and to 6 and 1 pmol in the absence of lipids, for rARF1 and myr-rARF1, respectively. Dissociation rates are: 0.074 min (circle) and 0.027 min with a maximal exchange of 77% (bullet) in C and 5 times 10 min (bullet), 5 times 10 min (circle), 0.043 min (box), and 0.54 min () in D.



We next compared the rates of dissociation of [S]GTPS from rARF1 and myr-rARF1 at low and high Mg (Fig. 3, C and D). We previously reported that phospholipids stabilize the GTPS-bound form of rARF1 by decreasing the k rate of GTPS(14) . Myristoylation of rARF1 accentuates this effect, since in the presence of phospholipids the dissociation of [S]GTPS was notably slower from myr-rARF1 than from rARF1, both at low and at high Mg concentrations. In contrast, in the absence of lipids, GTPS dissociated very rapidly from myr-rARF1, even at 1 mM Mg (Fig. 3D). For that reason, it was difficult to load myr-rARF1 with [S]GTPS in the absence of phospholipids. In the experiment described in Fig. 3D, only 2% of total ARF had bound [S]GTPS at time 0 of the dissociation, but we are confident that this fraction represented only myr-rARF1 because GTPS loading was performed at 1 mM Mg, which precluded nucleotide exchange on the contaminating rARF1.

Thus altogether, the effect of phospholipids on the dissociation of GTPS is much more dramatic for myr-rARF1 than for rARF1, since for the myristoylated protein the k rate was 1000-fold lower in the presence of lipids.

myr-rARF1 can also be loaded with GTP at 1 mM Mg in the presence of phospholipids but less efficiently than with GTPS (Fig. 4A). This difference is not due to the hydrolysis of GTP by myr-rARF1 but to a faster dissociation of GTP than of GTPS (Fig. 4B). Two observations demonstrate that myr-rARF1 lacks any GTPase activity: 1) similar kinetics were obtained with [alpha-P]GTP and [-P]GTP both for GTP binding and dissociation (Fig. 4); 2) no liberation of P(i) from [-P]GTP could be detected with either myr-rARF1 or rARF1 (not shown) provided that 3 mM phosphate was included in the assay to inhibit a nucleotidase (or phosphatase) activity that frequently contaminated the ARF preparations to a varying degree. This contaminant, which appeared only slightly smaller than ARF by gel filtration, was very difficult to separate from rARF1 (myristoylated or not). It hydrolyzed ATP as well as GTP and could be completely inhibited by a few mM P(i) or by AlF(x) (5 mM F and 10 µM AlCl(3)). We cannot exclude that it might be a degradation product of ARF.


Figure 4: Comparison of rARF1 and myr-rARF1 for the binding and the dissociation of GTP at 1 mM Mg in the presence of phospholipids. A, 1 µM of rARF1 (circle) or myr-rARF1 (bullet, down triangle, ) was incubated in the presence of phospholipid vesicles and 1 mM Mg with 10 µM [-P]GTP (circle, bullet), [alpha-P]GTP (down triangle) or [S]GTPS (), and 3 mM phosphate (see ``Results''). Values are expressed as pmol of bound nucleotide/50 pmol of ARF. Fitting the data to the model y = A(max)bullet(1 -e) gave the following parameters: A(max) = 45 pmol, k = 0.021 min (), and A(max) = 26 pmol, k = 0.022 min (bullet, down triangle). B, 1 µM of rARF1 (circle) or myr-rARF1 (bullet, down triangle) was loaded for 30 min or 5 min, respectively, with 10 µM [-P]GTP (circle, bullet) or [alpha-P]GTP (down triangle) in the presence of phospholipids and 1 µM free Mg. Dissociation was initiated by the addition of 1 mM unlabeled GTP and 1 mM Mg (final concentration). Nucleotide bound at the initiation of the exchange (100%) was 32 pmol for myr-rARF1 and 15 pmol for rARF1/50 pmol ARF. Dissociation rates are 5 times 10 min (bullet, down triangle) and 0.042 min (circle).



The rate of dissociation of GTP from myr-rARF1 at 1 mM Mg in the presence of lipids (Fig. 4B) was intermediate between the rates of dissociation of GTPS measured at 1 µM and 1 mM Mg (Fig. 3, C and D). This suggests that the interaction of Mg is stronger with GTPS than with GTP on myr-rARF1. The same is true for rARF1, since GTP dissociates 8-fold more rapidly than GTPS at 1 mM Mg (Fig. 3D and Fig. 4B).

Myr-rARF Binds to Phospholipid Vesicles

The observation that phospholipids accelerate the dissociation of GDP from myr-rARF1 (Fig. 3B) suggested an interaction between lipids and myr-rARF1. This was therefore directly investigated in a sedimentation experiment described in Fig. 5. Indeed, 40% of myr-rARF1 sedimented with the phospholipid vesicles, whereas rARF1 was almost entirely recovered in the supernatant, as previously reported(14) . It can be noted also that the small fraction of nonmyristoylated ARF contaminating the myr-rARF1 preparation remained totally in the supernatant.


Figure 5: Binding of myr-rARF1 to phospholipid vesicles. rARF or myr-rARF was incubated with phospholipid vesicles as described under ``Experimental Procedures.'' After centrifugation, pellet (P) and supernatant (S) were analyzed by SDS-PAGE and Coomassie Blue staining. Arrows on the right indicate the positions of Met-1 rARF1 (openarrow) and myr-rARF1 (closedarrow). The figures below indicate the distribution (in percent) of ARF between pellet and supernatant.



Myr-rARF1 Binds to DMPC/Cholate Micelles

We previously reported that rARF1 binds totally to phospholipid/cholate micelles and migrates with them on a gel filtration column(14) . In contrast, rARF1 does not associate to micelles and elutes at a volume corresponding to a 20-kDa protein, as illustrated in Fig. 6B. A quite different result was obtained with myr-rARF1 (Fig. 6A). Two peaks of ARF were obtained, a small one eluting at the same volume as rARF1, and corresponding to the contaminating nonmyristoylated ARF, as indicated by the SDS-PAGE analysis, and a major peak eluting more rapidly and containing only myr-rARF1. Labeling of the micelles with [^3H]DPPC indicated that micelles eluted at a volume slightly smaller than the elution volume of myr-rARF1. This suggests that myr-rARF1 binds to DMPC/cholate micelles but not as strongly as ARF, which exactly coeluted with the micelles, whether myristoylated or not(14) . Thus, it is likely that myr-rARF1 associates to and dissociates from micelles all along the column, whereas myr-rARF1 and rARF1 remain firmly bound to the micelles. This rather weak interaction between myr-rARF1 and phospholipids can explain why only 40% of the myr-rARF1 was found in the pellet in the sedimentation experiment of Fig. 5.


Figure 6: Binding of myr-rARF1 to phospholipid-cholate micelles. rARF1 or myr-rARF1 was incubated with DMPC/cholate micelles, and the mixture was analyzed by gel filtration as described under ``Experimental Procedures.'' The solid curves indicate protein absorbance at 280 nm, and the fractions were analyzed by SDS-PAGE, as shown below. A, myr-rARF1 and DMPC/cholate micelles. [^3H] DPPC (0.2 µM, 40,000 dpm/µl) was included to indicate the elution volume of the micelles (bullet-bullet). Arrows indicate the positions on the gel of myr-rARF1 (closedarrow) and nonmyristoylated rARF1 (openarrow). B, nonmyristoylated rARF1 and DMPC/cholate micelles.




DISCUSSION

Recombinant bovine ARF1 has been expressed in bacteria as unmodified ARF1 or N-myristoylated ARF1. In contrast to Randazzo et al.(10) who could not separate the two forms, we found that myr-rARF1 and rARF1 migrate differently on SDS-polyacrylamide gels and can be efficiently separated in a few steps. Obtention of myr-ARF1 that is at least 85% myristoylated allowed us to compare the properties of the two forms with regard to their interactions with phospholipids and the Mg dependence of nucleotide exchange. Two major differences between myristoylated and nonmyristoylated ARF1 are described.

The first one concerns the interaction of the GDP-bound form with phospholipids. Nonmyristoylated ARF1 binds to phospholipids exclusively when it is in the GTP-bound form. We confirm here with wild-type Gly-2 ARF1 what we previously reported for Ile-2 ARF1(14) . In fact, we have not detected any difference between nonmyristoylated Gly-2 and Ile-2 ARF1 proteins in their interactions with phospholipids or in the kinetics of nucleotide binding, despite the fact that the amino-terminal Met was partially cleaved in Gly-2 ARF1 and totally conserved in Ile-2 ARF1. In contrast with the nonmyristoylated protein, the GDP-bound form of myr-rARF1 also interacts with phospholipids, either as phospholipid vesicles or as phospholipid/cholate micelles. However, the interaction is weaker than for the GTP-bound form, consistent with the idea that in myr-rARF1 only myristate interacts with lipids with a rather low affinity(19) , while in myr-rARF1 both the myristate chain and the amino-terminal hydrophobic residues of the protein moiety interact with phospholipids. Thus, myr-ARF should not be considered as totally cytosolic as it is commonly described (20, 21, 22) but more likely as predominantly membrane-bound in intact cells due to the high cellular ARF concentration. Cell fractionation might cause its release into the soluble fraction as a consequence of the resulting dilution.

Another salient difference between rARF1 and myr-rARF1 concerns the Mg dependence of nucleotide exchange. GDP can be released from nonmyristoylated ARF1 only at low (µM) concentrations of free Mg(14) . No significant nucleotide exchange occurs at physiological (mM) Mg concentrations, which led us to hypothesize that a guanine nucleotide-exchange factor is required for ARF activation in vivo(14) . However, we show in the present study that, in marked contrast to the nonmyristoylated protein, myr-rARF1 can release its GDP at high Mg concentrations. GDP dissociation is slow in the absence of phospholipids (t= 90 min at 37 °C) but is accelerated by about 2-fold in the presence of lipid vesicles. This suggests that hydrophobic interaction of the myristate chain with either the protein (in the absence of lipids) or, more efficiently, with the phospholipid bilayer somehow results in the opening of the nucleotide binding site. A possible mechanism is the displacement of the amino-terminal peptide, since its removal was shown to greatly increase the release of bound GDP(15) .

If GDP can dissociate from myr-rARF1 with or without phospholipids, it should be noted that its replacement by GTP or GTPS will efficiently occur only in the presence of lipids, because in their absence GTP or GTPS dissociate much faster than GDP. However, if as discussed above myr-ARF remains essentially membrane-bound in cells, the GTP-bound form should be immediately stabilized by interaction with the bilayer. Myristoylation appears to be crucial for the lipid-dependent stabilization of the GTP-bound form (Fig. 4B). It should be noted that with GTPS, the importance of myristoylation is less obvious, as Mg seems to stabilize GTPS more strongly than GTP in the nucleotide binding site and therefore GTPS dissociates notably more slowly than GTP from both rARF1 and myr-rARF1 at 1 mM Mg.

Thus, in summary, myr-ARF1 can be spontaneously activated by GTP or GTPS at physiological concentrations of Mg in the sole presence of phospholipids without any nucleotide exchange factor. However, the activation remains rather slow (t 30 min), which does not preclude the existence in cells of such factors, which would accelerate the exchange and might also account for the specific localization of the various ARFs. Indeed, several groups have reported evidence of an ARF-specific guanine nucleotide-exchange activity in Golgi membranes (10, 22, 23) , and a partial purification of this activity from bovine brain cytosol has been recently described(24) . Brefeldin A inhibits the exchange activity in the crude preparation but no longer in the partially purified fraction(24) . It is noteworthy that brefeldin A has no effect on the spontaneous lipid-dependent activation of myr-ARF1 described here (not shown).

Nevertheless, in most cell-free systems in which myristoylation has been reported to be essential for ARF functions, including its role in intra-Golgi (4, 6) or endoplasmic reticulum to Golgi (5) transports, in endosome-endosome fusion(25) , and in phospholipase D activation(7, 26) , it is not necessary to postulate the involvement of a specific nucleotide exchange factor to account for the higher activity of myr-ARF. Our present finding that at mM Mg concentrations only the myristoylated protein can be spontaneously activated provides a reasonable explanation for the observed requirement for myristoylation.


FOOTNOTES

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§
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To whom correspondence should be addressed. Tel.: 33-93-95-77-71; Fax: 33-93-95-77-10.

(^1)
The abbreviations used are: ARF, ADP-ribosylation factors; GTPS, guanosine 5`-O-(thiotriphosphate); myr, N-myristoylated; DMPC, L-alpha-dimyristoylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine; DTT, dithiothreitol; MES, 4-morpholineethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank J. I. Gordon for the gift of the pBB131 yeast N-myristoyltransferase expression vector, M. Frech for helpful discussions during myristoylated ARF purification, and G. Imbs for expert secretarial assistance.


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