Interaction of the GTP-binding and GTPase-activating Domains of ARD1 Involves the Effector Region of the ADP-ribosylation Factor Domain*

(Received for publication, September 20, 1996, and in revised form, November 6, 1996)

Nicolas Vitale Dagger , Joel Moss and Martha Vaughan

From the Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

ADP-ribosylation factors (ARFs) are a family of ~20-kDa guanine nucleotide-binding proteins and members of the Ras superfamily, originally identified and purified by their ability to enhance the ADP-ribosyltransferase activity of cholera toxin and more recently recognized as critical participants in vesicular trafficking pathways and phospholipase D activation. ARD1 is a 64-kDa protein with an 18-kDa carboxyl-terminal ARF domain (p3) and a 46-kDa amino-terminal extension (p5) that is widely expressed in mammalian tissues. Using recombinant proteins, we showed that p5, the amino-terminal domain of ARD1, stimulates the GTPase activity of p3, the ARF domain, and appears to be the GTPase-activating protein (GAP) component of this bifunctional protein, whereas in other members of the Ras superfamily a separate GAP molecule interacts with the effector region of the GTP-binding protein. p5 stimulated the GTPase activity of p3 but not of ARF1, which differs from p3 in several amino acids in the effector domain. After substitution of 7 amino acids from p3 in the appropriate position in ARF1, the chimeric protein ARF1(39-45p3) bound to p5, which increased its GTPase activity. Specifically, after Gly40 and Thr45 in the putative effector domain of ARF1 were replaced with the equivalent Asp and Pro, respectively, from p3, functional interaction of the chimeric ARF1 with p5 was increased. Thus, Asp25 and Pro30 of the ARF domain (p3) of ARD1 are involved in its functional and physical interaction with the GTPase-activating (p5) domain of ARD1. After deletion of the amino-terminal 15 amino acids from ARF1(39-45p3), its interaction with p5 was essentially equivalent to that of p3, suggesting that the amino terminus of ARF1(39-45p3) may interfere with binding to p5. These results are consistent with the conclusion that the GAP domain of ARD1 interacts with the effector region of the ARF domain and thereby stimulates GTP hydrolysis.


INTRODUCTION

ARD1,1 a 64-kDa protein, contains a 46-kDa amino-terminal domain and an 18-kDa carboxyl-terminal ADP-ribosylation factor or ARF domain (1). ARFs are 20-kDa guanine nucleotide-binding proteins initially described as GTP-dependent activators of cholera toxin-catalyzed ADP-ribosylation of the alpha  subunit of the adenylyl cyclase-stimulatory G protein (Gsalpha ) (reviewed in Ref. 2). In eucaryotic cells, ARFs are critical components of vesicular trafficking pathways (3-7) (reviewed in Ref. 8) and activators of phospholipase D (9, 10). Based on size, amino acid sequences, phylogenetic analysis, and gene structure, mammalian ARFs have been divided into three classes with ARF1, ARF2, and ARF3 in class I, ARF4 and ARF5 in class II, and ARF6 in class III (11). ARFs from these classes may function in different parts of the exocytotic and endocytotic pathways.

Like alpha  subunits of the heterotrimeric G proteins, the smaller monomeric guanine nucleotide-binding proteins are active in their GTP-bound forms and inactive when GDP is bound (12). ARFs exhibit no detectable GTPase activity (13), and the ratio of GTP/GDP bound appears to be governed by guanine nucleotide-exchange proteins (GEPs) and GTPase-activating proteins (GAPs) (2). Both GEPs (14-18) and GAPs (19-22) have been identified. Inhibition of cytosolic and Golgi-associated GEPs by brefeldin A has been reported (14-16, 18), and a cytosolic brefeldin A-insensitive GEP has been isolated (17).

Recently, we reported that ARD1 exhibited significant GTPase activity, whereas the ARF domain did not (23). Addition of the 46-kDa amino-terminal domain (p5) of ARD1 (expressed as a recombinant protein in Escherichia coli) to the carboxyl-terminal ARF domain (p3) enhanced GTPase activity (23). The stimulatory effect of the p5 domain on the GTPase activity of p3 was specific, as GTP hydrolysis by other members of the ARF family was not increased (22). ARD1, therefore, is a novel member of the Ras superfamily of the guanine nucleotide-binding protein family, which, like the alpha  subunits of the trimeric G proteins, appears to consist of two domains that contribute to its biological activity.

We report here identification of a region involved in the interaction between the two domains of the protein. Using chimeric proteins and site-specific mutagenesis, we demonstrate that amino acids Asp25 and Pro30 in the ARF domain of ARD1, corresponding to amino acids 40 and 45 in the ARF proteins, appear to be crucial for both the functional and physical interaction between the two domains of ARD1. Thus, as it is in other members of the Ras superfamily, the effector domain of ARF is a site of its interaction with GAP.


EXPERIMENTAL PROCEDURES

Materials

Bovine thrombin, sodium cholate, dimyristoyl PC, GSH, phosphatidic acid, PE, phosphatidylinositol, and PIP2 were purchased from Sigma, and brain PC was from Avanti Polar Lipids, Inc. TLC plates were purchased from VWR Scientific, and GSH-Sepharose beads were from Pharmacia Biotech Inc. PCR reagents and restriction enzymes, unless otherwise indicated, were from Boehringer Mannheim. Sources of other materials have been published (16, 23, 24).

Preparation of Recombinant Fusion Proteins (p3, p5, and p8)

Fusion proteins, synthesized using a ligation-independent cloning method (25) and purified on glutathione-Sepharose, were ~90% pure as estimated by silver staining after SDS-PAGE (23). After cleavage by bovine thrombin, glutathione S-transferase was removed with glutathione-Sepharose beads and thrombin with benzamidine-Sepharose 6B (26). The proteins were further purified by gel filtration through Ultrogel AcA 54 and Ultrogel AcA 34. Purity estimated by silver staining after SDS-PAGE was >98%. Recombinant ARF1 was expressed and purified as described (27). Amounts of purified proteins were estimated by a dyebinding assay (28) and by SDS-PAGE using bovine serum albumin as standard.

Construction and Expression of Chimeric Forms of p3 and ARF1

The 5' end of the ARF domain of ARD1 (p3), from pGEX-5G/LIC (1) containing the p3 cDNA (EMBL Data Bank 1993, accession number L04510[GenBank]), was amplified by PCR using the forward primer 5'-GTCGACCATCCTCCAGGAGG-3' (italicized sequence indicates SalI restriction site) and the reverse primer 5'-CTTAAGA<UNL>C</UNL>CC<UNL>A</UNL>GTTTTAACTTAAACAAGA-3' (italicized sequence is an EcoRI restriction site; differences from the original clone are underlined). The reverse primer introduced two replacement bases and the EcoRI restriction site. The 3' end of p3 from pGEX-5G/LIC, containing the p3 cDNA, was amplified by PCR using the forward primer 5'-GAATTC<UNL>G</UNL>TG<UNL>ACCA</UNL>CCATTCCAACAATTGGTT-3' (italicized sequence is an EcoRI restriction site; differences from the original clone are underlined) and the reverse primer 5'-GGATCCAACTGCGCCTCGCT-3' (italicized sequence is a BamHI restriction site). The forward primer introduced five replacement bases and an EcoRI restriction site. The PCR fragments were extracted from LM-agarose gel, purified by phenol/chloroform precipitation, and subcloned into pCRTMII vector using the TA cloning kit (Invitrogen) according to the manufacturer's instructions. The mutated 5' fragment was excised with SalI and EcoRI. The mutated 3' fragment was excised with EcoRI and BamHI. The two fragments were ligated in-frame through their EcoRI restriction sites. The resulting fragment was then ligated in-frame to the SalI- and BamHI-digested pGEX-5G/LIC expression vector. Ultracompetent cells (Stratagene) were transformed with the plasmid pGEX-5G/LIC/p3(24-30ARF1). The sequence of the mutated p3 was confirmed by automatic sequencing (Applied Biosystems 373 DNA sequencer) using the primer 5'-CTCGACCATCCTCCAGGA-3'. p3(24-30ARF1) fusion protein was expressed and purified as described for the non-mutant p3.

The 5' end of ARF1 (representing 43 amino acids) from the pBluescript (pT7/Nde) expression vector containing the ARF1 cDNA (GenBankTM 1992, accession number M84326[GenBank]) was amplified by PCR using the forward primer 5'-GGCGAACATATGGGGAACATCTTCGC-3' (italicized sequence is an NdeI restriction site) and the reverse primer 5'-GA<UNL>AT</UNL>TCA<UNL>T</UNL>CC<UNL>T</UNL>GCTTAAGCTTGTAGAGG-3' (italicized sequence is an EcoRI restriction site; differences from the original clone are underlined). The reverse primer introduced four replacement bases and an EcoRI restriction site. The 3' end of ARF1, from pT7/Nde containing the ARF1 cDNA, was amplified by PCR using the forward primer 5'-GA<UNL>AT</UNL>TC<UNL>A</UNL>TG<UNL>CAGC</UNL>CCATTCCCACCATAGGCTTCAACG-3' (italicized sequence is an EcoRI restriction site; differences from the original clone are underlined) and the reverse primer 5'-CTCGCTCCGGCGAAGGATCCCGTTCACTTCTGG-3' (italicized sequence is a BamHI restriction site). The forward primer introduced seven replacement bases and an EcoRI restriction site. The PCR fragments were extracted from LM-agarose gel, purified by phenol/chloroform precipitation, and subcloned into pCRTMII vector using the TA cloning kit (Invitrogen). The mutated 5' fragment was excised with NdeI and EcoRI. The mutated 3' fragment was excised with EcoRI and BamHI. The two fragments were ligated in-frame through their EcoRI restriction sites. The resulting fragment was then ligated in-frame to the NdeI- and BamHI-digested pT7/Nde expression vector. Ultracompetent cells (Stratagene) were transformed with the plasmid pT7/Nde/ARF1(39-45p3). The sequence of the mutated ARF1 was confirmed by automatic sequencing (Applied Biosystems 373 DNA sequencer) using the primer 5'-AATAATACGACTCACTAT-3'.

For large scale production of chimeric ARF1(39-45p3), 10 ml of overnight culture of the transformed bacteria were added to a flask with 500 ml of LB broth and 50 µg/ml ampicillin followed by incubation at 37 °C with shaking. When the culture reached an A600 of 0.6, 250 µl of 1 M isopropyl-beta -D-thiogalactopyranoside were added (0.5 mM final concentration). After incubation for an additional 3 h, bacteria were collected by centrifugation (Sorvall GSA, 6000 rpm, 4 °C, 10 min) and stored at -20 °C. Bacterial pellets were dispersed in 5 ml of cold phosphate-buffered saline, pH 7.4, with 20 µg/ml trypsin inhibitor, 5 µg/ml each leupeptin and aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride. Lysozyme (10 mg in 5 ml) was added. After 30 min at 4 °C, cells were disrupted by sonication and centrifuged (Sorvall SS34, 16,000 rpm, 4 °C, 20 min). The supernatant was applied to a column (5 × 100 cm) of Ultrogel AcA 54, equilibrated, and eluted with TENDS buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl, 2 mM DTT, 250 mM sucrose, 5 mM MgCl2, 1 mM NaN3). Fractions that had both high ARF activity and high purity were pooled and further purified by DEAE chromatography on a column (1.5 × 40 cm) of DEAE, eluted with a linear gradient of 50 to 500 mM NaCl (0.6-ml fractions), and gel filtered on Ultrogel AcA 34 (1.5 × 30 cm) before storage in small portions at -20 °C.

Construction and Expression of Mutated Form of ARF1

For site-directed mutagenesis of ARF1, a modification of the unique site-elimination mutagenesis procedure described by Deng and Nickoloff (29) was used. 25 pmol of a 5'-phosphorylated selection primer and 25 pmol of a 5'-phosphorylated mutagenic primer were simultaneously annealed to 600 ng of pARF1T7Nde in 20 µl of 10 mM Tris acetate (pH 7.5), 10 mM magnesium acetate, 50 mM potassium acetate by heating for 5 min at 100 °C and cooling for 5 min on ice followed by incubation at room temperature for 30 min. The selection primer 5'-CTGTGACTGGTG<UNL>ACGCGT</UNL>CAACCAAGTC-3' changed a ScaI restriction site in the ampr gene of pT7 into an MluI restriction site (underlined). The mutagenic primer 5'-TACAAGCTTAAGCTGG<UNL>A</UNL>TGAGATCGTGACCACC-3' changed Gly40 of ARF1 to Asp. The mutagenic primer 5'-GGTGAGATCGTGACC<UNL>C</UNL>CCATTCCCACCATAGGC-3' changed Thr45 of ARF1 to Pro. The mutagenic primer 5'-TACAAGCTTAAGCTGG<UNL>A</UNL>TGAGATCGTGACC<UNL>C</UNL>CCATTCCCACCATAGGC-3' changed Gly40 and Thr45 of ARF1 to Asp and Pro, respectively. Primers were extended with T7 DNA polymerase and the new strands were ligated with T4 DNA ligase for 1 h at 37 °C (final volume, 30 µl). Plasmids were then digested for 2 h at 37 °C with 20 units of ScaI (final volume, 60 µl). 4-µl samples were used to transform 90 µl of Epicurian Coli XL1-Blue competent cells (Stratagene). Plasmids ARF1 (G40D), ARF1 (T45P), and ARF1 (G40D, T45P) were purified with Miniprep Wizard (Promega) from bacteria grown overnight in 2 ml of 2× YT broth with 100 µg/ml ampicillin. Samples (500 ng) of the plasmids were digested with 20 units of ScaI for 3 h at 37 °C. 4-µl samples were used to transform 40 µl of XLmutS competent cells (Stratagene). Colonies were selectively screened by digestion with MluI, and the presence of the mutations was confirmed by automatic sequencing (Applied Biosystems 373 DNA sequencer) using the primer 5'-AATAATACGACTCACTAT-3'. Large scale production of mutated ARF1 (G40D), ARF1 (T45P), or ARF1 (G40G, T45P) was carried out as described for the chimeric ARF1 (39-45p3).

Construction and Expression of Delta 15ARF1(39-45p3)

The plasmid pT7/Nde/ARF1(39-45p3) described above was amplified by PCR in the presence of Pfu DNA polymerase (Stratagene) with the forward primer 5'-ATGGAAATGCGCATCCTCATGGTG-3' and the reverse primer 5'-CTCGCTCCGGCGAAGGATCCCGTTCACTTCTGG-3' (italicized sequence is a BamHI restriction site). The forward primer produced the deletion of 45 bases from the beginning of the coding region of ARF1(39-45p3). The PCR fragment was extracted from LM-agarose gel, purified, and subcloned into the blunt end SrFI restriction site of pCR-ScriptTM SK(+) according to the manufacturer's instructions (Stratagene) for transfection into XL1-Blue MRF'kan supercompetent cells. Because the orientation of the PCR product in 20 positive colonies (checked by PCR and restriction enzyme digestion) did not allow expression under the T7 promoter, the Delta 15ARF1(39-45p3) fragment was excised from the pCR-ScriptTM SK(+) vector with SacII and BamHI and was ligated in-frame to the SacII- and BamHI-digested pGEX-5G/LIC expression vector. Ultracompetent cells (Stratagene) were transformed with the plasmid pGEX-5G/LIC/Delta 15ARF1(39-45p3). The sequence of the ARF1 deletion mutant was confirmed by automatic sequencing (Applied Biosystems 373 DNA sequencer) using the primer 5'-GTCGACCATCCTCCAGGA-3'. GST-Delta 15ARF1(39-45p3) fusion protein was expressed and purified as described above. Glutathione S-transferase was removed (23) and the protein was further purified by gel filtration through columns of Ultrogel AcA 54 (1.5 × 45 cm) and Ultrogel AcA 34 (1.5 × 30 cm).

GTP-binding Overlay Assay with Recombinant ARD1

Samples (22 pmol) of p3 (0.4 µg), p5 (1 µg), and p8 (1.4 µg) were subjected to electrophoresis in 4-20% polyacrylamide gels with SDS and transferred to nitrocellulose. The membrane was incubated in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM DTT, 2.5 mM EDTA, 10 µg/ml soybean trypsin inhibitor, and 0.5 mM phenylmethylsulfonyl fluoride with 0.3 mg/ml BSA and 1 mg/ml cardiolipin (binding buffer) at room temperature for 3 h, transferred to fresh buffer containing 10 mM MgCl2 and 2 µCi/ml (3000 Ci/mmol) [alpha -32P]GTP for 2 h, washed 10 times with binding buffer for 5 min, briefly dried, and exposed to Kodak XAR film at -80 °C overnight with intensifying screen.

GTPgamma S Binding Assay

GTPgamma S binding to purified recombinant ARD proteins was assessed using a rapid filtration technique. Samples were incubated for 30 min at 30 °C in 20 mM Tris, pH 8.0, 10 mM DTT, 2.5 mM EDTA with 0.3 mg/ml BSA and 1 mg/ml cardiolipin, and then for 40 min at 30 °C in the same medium plus 10 mM MgCl2 (or as indicated) and 3 µM [35S]GTPgamma S (~106 cpm; total volume, 150 µl). Where indicated, cardiolipin was replaced by another lipid or detergent in the binding buffer. Samples (70 µl) were then transferred to nitrocellulose filters in a manifold (Millipore) for rapid filtration followed by washing five times each with 1 ml of ice-cold buffer (25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 5 mM MgCl2). Dried filters were dissolved in scintillation fluid for radioassay.

Assay of GTPase Activity

Samples were incubated for 30 min at 30 °C in 20 mM Tris, pH 8.0, 10 mM DTT, 2.5 mM EDTA with 0.3 mg/ml BSA and 1 mg/ml cardiolipin, and then for 40 min at 30 °C in the same medium with 0.5 µM [alpha -32P]GTP (3000 Ci/mmol) and 10 mM MgCl2 (total volume, 120 µl). After addition of p5 or vehicle (40 µl), incubation at room temperature was continued as indicated for 10 min to 2 h (final volume, 160 µl) before proteins with bound nucleotides were collected on nitrocellulose (24). Bound nucleotides were eluted in 250 µl of 2 M formic acid, of which 3-4-µl samples were analyzed by TLC on polyethyleneimine-cellulose plates (19) and 240 µl was used for radioassay to quantify total nucleotide. TLC plates were subjected to autoradiography at -80 °C for 18-28 h. Total amounts of labeled nucleotides (GTP + GDP) bound to p3, p8, or GST-p3 after incubation with GST-p5, whether quantified by radioassay of the formic acid solution, by counting total radioactivity on the filter, or by phosphorimaging (Molecular Dynamics) after TLC, were not significantly different under any conditions.

Assay of CTA-catalyzed ADP-ribosylagmatine Formation

p3 or p8 was incubated for 30 min at 30 °C in 40 µl of 20 mM Tris, pH 8.0, 10 mM DTT, 2.5 mM EDTA with 0.3 mg/ml BSA and 1 mg/ml cardiolipin before addition of 20 µl of solution to yield final concentrations of 100 µM GTPgamma S or GTP and 10 mM MgCl2. Where indicated, p5 was then added for 30 min. Components needed to quantify ARF stimulation of cholera toxin-catalyzed ADP-ribosylagmatine formation were then added in 70 µl to yield final concentrations of 50 mM potassium phosphate (pH 7.5), 6 mM MgCl2, 20 mM DTT, 0.3 mg/ml ovalbumin, 0.2 mM [adenine-14C]NAD (0.05 µCi), 20 mM agmatine, 1 mg/ml cardiolipin, and 100 µM GTPgamma S or GTP with 0.5 µg of cholera toxin (30). Where indicated, cardiolipin was replaced by another lipid or detergent. After incubation at 30 °C for 1 h, samples (70 µl) were transferred to columns of AG1-X2 equilibrated with water and eluted with five 1-ml volumes of water. The eluate, containing [14C]ADP-ribosylagmatine, was collected for radioassay.

Release of [35S]GDPbeta S from Recombinant Proteins

Samples (450 µl) were incubated for 30 min at 30 °C in 20 mM Tris (pH 8.0), 10 mM DTT, 2.5 mM EDTA with 0.3 mg/ml BSA and 1 mg/ml cardiolipin and then for 40 min in the same medium plus 10 mM MgCl2 and 3 µM [35S]GDPbeta S (2 × 107 cpm/500 µl) before addition of p5 (or water) in 0.2 reaction volume. After incubation for 15 min at 30 °C, samples (60 µl) were transferred to nitrocellulose filters that were washed five times with 1 ml of 25 mM Tris, pH 8.0, 5 mM MgCl2, 100 mM NaCl before radioassay in a liquid scintillation counter (24). To calculate zero-time values for dissociation curves, radioactivity bound to filters in the absence of protein was subtracted from the total with proteins. The remainders of the mixtures were immediately diluted with an equal volume of reaction buffer containing 2 mM GDPbeta S. Samples (120 µl) were taken after 5, 15, 30, 45, 60, 75, 90, 105, and 120 min at 30 °C for quantification of bound radioactivity as described for the zero-time samples.


RESULTS

Specificity of Guanine Nucleotide Binding to ARD1

Recombinant proteins representing the ARF domain (p3), the non-ARF domain (p5), and the entire ARD1 protein (p8) were subjected to SDS-PAGE and transferred to nitrocellulose for assessment of their ability to bind GTP. Fig. 1, inset, provides visual evidence that p3 and p8 bound [alpha -32P]GTP, whereas the non-ARF domain, p5, did not. Binding of GTPgamma S to p3 and p8 was equivalent and was dependent on magnesium concentration (Fig. 1). Maximal binding to both p3 and p8 was observed with 5 and 10 mM MgCl2 (in the presence of 2.5 mM EDTA) and was less at 20 mM (Fig. 1). Maximal binding of GTPgamma S to ARF1 and ARF3 (in the presence of 1 mM EDTA) occurred in the very narrow range of 0.5-1 mM MgCl2 (17).


Fig. 1. Binding of GTP to recombinant ARD1 and ARD1 domains. Binding of 3 µM [35S]GTPgamma S to 30 pmol of p3 (open circle , 0.54 µg), p5 (bullet , 1.38 µg), or p8 (square , 1.92 µg) was assessed using a rapid filtration technique. The medium contained 1 mM EDTA and the indicated concentration of MgCl2. Each point is the mean of three determinations with S.E. smaller than the symbol for each value. These results were repeated twice. Inset, p3 (0.4 µg), p5 (1 µg), and p8 (1.4 µg) were subjected to SDS-PAGE and transferred to nitrocellulose membrane. [alpha -32P]GTP binding in an overlay assay was carried out in the presence of 1 mg/ml cardiolipin. Identical results were obtained with three different protein preparations.
[View Larger Version of this Image (38K GIF file)]


The specificity of guanine nucleotide binding was assessed by adding unlabeled nucleotide together with 4 µM [35S]GTPgamma S in the binding buffer. ATP, TTP, and CTP (up to 100 µM) failed completely to compete with GTPgamma S for binding to p3 and p8, whereas 100 µM GTP or GDP decreased [35S]GTPgamma S binding to p3 or p8 almost 80% (Fig. 2). The fact that GDP and GTP competed similarly for binding is in agreement with the previous observation (31) that at high Mg2+ concentrations ARFs have similar affinities for GDP and GTP.


Fig. 2. Specificity of nucleotide binding to ARD1. GTPgamma S binding to 45 pmol of p3 (0.81 µg) or p8 (2.88 µg) was carried out with 10 mM MgCl2, 1 mg/ml cardiolipin, 4 µM [35S]GTPgamma S, and the indicated concentration of unlabeled GTP, GDP, ATP, CTP, or TTP. Binding in absence of protein was subtracted. Each point is the mean of three determinations ± S.E. Error bars smaller than symbols are not shown. The same result has been obtained with two different protein preparations.
[View Larger Version of this Image (26K GIF file)]


Phospholipid Requirement for GTPgamma S and CTA Activity of ARD1

It has been reported that certain phospholipids or detergents, in the presence of Mg2+, can promote guanine nucleotide exchange and activate ARF (31, 32). In the absence of GEP, phospholipids differed markedly in their effects on GTPgamma S binding to ARF (17). As shown in Table I, in the presence of several detergents GTPgamma S binding to either p3 or p8 was very low (i.e. <0.2%), whereas with certain phospholipids, especially cardiolipin, binding was considerably higher and was similar for p3 and p8. On the other hand, with the mixture of brain PC (saturated and unsaturated PC), PIP2, and PE, GTPgamma S binding by p3 was five times that by p8 (Table I).

Table I.

Effects of detergents and phospholipids on GTPgamma S binding by p3 and p8

[35S]GTPgamma S binding to 80 pmol of p3 (1.44 µg) or p8 (5.12 µg) was assessed by the rapid filtration technique in the presence of the indicated detergent/phospholipid. In the absence of detergent/phospholipid 0.035 ± 0.009 and 0.021 ± 0.012 pmol of GTPgamma S bound to p3 and p8, respectively. Concentrations not presented in the table were: 560 nM brain PC, 780 nM PIP2, 9 µM PE, 1 mg/ml cardiolipin, 3 mM dimyristoyl PC, 0.1% cholate. GTPgamma S binding in the absence of protein was subtracted. Data are means of triplicate assays ± S.E. The experiments were repeated twice with different protein preparations.
Detergent/lipid GTPgamma S bound
80 pmol of p3 80 pmol of p8

pmol
Brain PC + PIP2 + PE 0.506  ± 0.021 0.105  ± 0.021
Cardiolipin 0.904  ± 0.011 0.995  ± 0.029
Phosphatidylserine (200 µM) 0.292  ± 0.032 0.095  ± 0.019
Phosphatidylinositol (200 µM) 0.195  ± 0.024 0.124  ± 0.011
Phosphatidic acid (200 µM) 0.168  ± 0.031 0.099  ± 0.021
PIP2 (100 µM) 0.098  ± 0.011 0.086  ± 0.009
PC (200 µM) 0.085  ± 0.021 0.078  ± 0.015
PE (200 µM) 0.135  ± 0.020 0.086  ± 0.023
Dimyristoyl PC/cholate 0.155  ± 0.018 0.098  ± 0.031
Triton X-100 (0.1%) 0.062  ± 0.009 0.053  ± 0.005
Lubrol PX (0.1%) 0.045  ± 0.007 0.061  ± 0.011
Tween 20 (0.3%) 0.164  ± 0.011 0.152  ± 0.023

Like other members of the ARF family, ARD1 activates CTA ADP-ribosyltransferase activity (1, 23). As it was for GTPgamma S binding (Table I), cardiolipin was the most effective phospholipid for activation of CTA (Table II). Despite supporting relatively low GTPgamma S binding, however, the detergent Tween 20 enhanced CTA activity induced by p3 or p8 to levels comparable to those with cardiolipin (Table II).

Table II.

Effect of different detergents and phospholipids on CTA activation by p3 and p8

p3 (900 pmol) and p8 (250 pmol) were incubated with GTPgamma S in the presence of the indicated detergent/phospholipid (as described in Table I) before components needed to quantify ARF stimulation of cholera toxin-catalyzed ADP-ribosylagmatine formation were added, and incubation was continued for 60 min at 30 °C. ARF activity is the difference between cholera toxin-catalyzed formation of [14C]ADP-ribosylagmatine without and with p3 or p8 (nmol/h). In the absence of detergent/phospholipid ARF activity was 0.014 ± 0.003 and 0.013 ± 0.006 for p3 and p8, respectively. Data are means of values from quadruplicate assays ± S.E. and were repeated twice.
Detergent/lipid ARF activity
900 pmol of p3 250 pmol of p8

Brain PC + PIP2 + PE 0.54  ± 0.14 1.55  ± 0.11
Cardiolipin 4.32  ± 0.15 4.51  ± 0.14
Phosphatidylserine 2.61  ± 0.11 1.21  ± 0.08
Phosphatidylinositol 1.02  ± 0.11 0.85  ± 0.15
Phosphatidic acid 0.98  ± 0.06 0.75  ± 0.09
PIP2 0.62  ± 0.23 0.54  ± 0.05
PC 0.54  ± 0.05 0.42  ± 0.11
PE 0.82  ± 0.03 1.21  ± 0.08
Dimyristoyl PC/cholate 1.46  ± 0.09 1.01  ± 0.05
Triton X-100 0.30  ± 0.02 0.40  ± 0.08
Lubrol PX 0.23  ± 0.06 0.31  ± 0.03
Tween 20 4.31  ± 0.15 4.69  ± 0.09

Activation by p5 of GTP Hydrolysis by p3

We had reported that the 64-kDa ARD1 (p8) exhibited significant GTPase activity, whereas the ARF domain (p3), synthesized as a recombinant protein in E. coli, did not (23). Addition of p5 (the ARD amino-terminal domain) increased hydrolysis of GTP bound to p3 in a concentration-dependent manner (Fig. 3A). The maximal effect was observed with a ratio of 2 mol of p5/mol of p3 and half-maximal hydrolysis with equimolar concentrations of p3 and p5. The reaction proceeded at an essentially constant rate until it stopped at 60 min (Fig. 3B). It appears that hydrolysis of GTP bound to p3 may have stopped when p5 became limiting (with equimolar p3 and p5), because p5 apparently interacts physically with p3-GDP as well as with p3-GTP (Ref. 23 and Fig. 3) and only a small fraction of p3 (~2%) had bound GTP. Indeed, addition of p5 at 60 min maintained GTP hydrolysis (see legend to Fig. 3).


Fig. 3. Stimulation of the GTPase activity of p3 by p5. A, 55 pmol of p3 (1 µg) with [alpha -32P]GTP bound were incubated for 60 min at room temperature with 0, 5.5, 13.75, 27.5, 55, 110, or 275 pmol of p5 to give the indicated ratio of p5/p3 before analysis of bound nucleotide by TLC. Amounts of GTP and GDP were quantified by phosphorimaging. Each point is the mean of two determinations ± one-half the range. The same results were obtained with at least five different protein preparations. Inset shows TLC from a typical experiment with the indicated ratio of p5/p3. GTP is the lower spot and GDP the upper spot. B, 55 pmol of p3 with [alpha -32P]GTP bound were incubated with 55 pmol of p5 for the indicated time. Each point is the mean of two determinations ± one-half the range. Similar results were obtained with three different protein preparations. Inset shows GTP hydrolysis after 0, 20, 30, 45, and 60 min. GTP is the lower spot and GDP the upper spot. In other experiments, addition of 55 pmol of p5 in the medium (total 110 pmol of p5 and 55 pmol of p3) at 60 min increased GTP hydrolysis.
[View Larger Version of this Image (24K GIF file)]


Identification of the Specific p5-binding Site on p3

Although p5 acted as a GAP for p3, it was not effective with other ARF proteins (22). To identify the specific interaction domain in p3, we compared the amino acid sequences of p3 and other ARFs. Residues 24-30 in p3 are very different from the equivalent region (residues 39-45), which is highly conserved among the ARFs. This region is located in the so-called effector domain of other Ras superfamily members, in which it has been shown to interact with GAP. Therefore, we prepared two chimeric proteins by exchanging these sequences in ARF1 and p3 (Fig. 4A). There was no significant difference between these and the nonchimeric proteins in GTPgamma S binding (data not shown) or in GTP-dependent CTA activation (Table III), consistent with the conclusion that GTP-binding properties and CTA activation were not impaired by the mutations. The chimeric p3 protein with ARF1 sequence, termed p3(24-30ARF1), was not a substrate for p5 (Fig. 4B) and did not interact with immobilized GST-p5 (Fig. 5B). On the other hand, p5 effectively hydrolyzed GTP bound to the chimeric ARF1(39-45p3) protein (Fig. 4, A and B).


Fig. 4. Effect of p5 on GTPase activity of chimeric ARF1 and p3 proteins. A, sequences of indicated amino acids from ARF1, p3, ARF1(39-45p3), and p3(24-30ARF1). The native LGEIVTT sequence of ARF1 was replaced with QDEFMQP from p3 to create ARF1(39-45p3), and the QDEFMQP sequence of p3 was replaced with LGEIVTT from ARF1 to create p3(24-30ARF1). B, effect of p5 concentration on GTP hydrolysis by p3, ARF1, and chimeric proteins. 2 µg of p3 or p3(24-30ARF1) or 1 µg of ARF1 or ARF1(39-45p3) with [alpha -32P]GTP bound were incubated with the indicated amount of p5 for 60 min before bound nucleotides were separated by TLC. GAP activity, i.e. the decrease in bound GTP due to incubation with p5, is presented as GTP/GTPo × 100, where GTP and GTPo are the amounts of GTP bound after incubation with and without p5, respectively. Data are means of duplicates ± one-half the range. Error bars smaller than symbols are not shown. Each experiment was performed at least four times.
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Table III.

Effect of p5 on the activation of cholera toxin by p3, p3(24-30ARF1), ARF1, and ARF1(39-45p3)

900 pmol of p3 or p3 (24-30ARF1) were incubated with GTPgamma S or GTP for 30 min at 30 °C and then with p5 (1800 pmol) or water (30 µl) for 20 min at 30 °C. 50 pmol of ARF1 or ARF1(39-45p3) were incubated with GTPgamma S or GTP for 30 min at 30 °C and then with p5 (100 pmol) or water (30 µl) for 20 min at 30 °C. Components needed to quantify ARF stimulation of cholera toxin-catalyzed ADP-ribosylagmatine formation were then added and incubation was continued for 60 min at 30 °C. Cholera toxin activity is expressed as nmol of [14C]ADP-ribosylagmatine/h. Toxin activity without added protein in each condition was subtracted. Data are means of values from quadruplicate assays ± S.E. in one experiment representative of three.
Recombinant protein added
ARF activity
p3 or ARF p5 GTP GTPgamma S

nmol/h
p3 0 4.01  ± 0.06 3.93  ± 0.02
p3 + 2.38  ± 0.05a 3.85  ± 0.03
p3(24-30ARF1) 0 3.93  ± 0.09 3.88  ± 0.01
p3(24-30ARF1) + 3.78  ± 0.07 3.85  ± 0.08
ARF1 0 4.76  ± 0.23 4.48  ± 0.11
ARF1 + 4.57  ± 0.08 4.33  ± 0.09
ARF1(39-45p3) 0 4.67  ± 0.08 4.59  ± 0.04
ARF1(39-45p3) + 3.24  ± 0.08a 4.51  ± 0.10

a  With unpaired t tests, significantly different (p < 0.005) from activity in the presence of GTPgamma S.


Fig. 5. Interaction of p5 and ARF1(39-45p3). A, gel filtration of ARF1(39-45p3) and p5. Upper, ARF1(39-45p3) (100 µg) and p5 (200 µg) were incubated for 60 min at 30 °C in 500 µl of 20 mM Tris, pH 8.0, 2.5 mM EDTA before transfer to a column (1.5 × 30 cm) of Ultrogel AcA 34 equilibrated and eluted (0.2 ml/min) with 20 mM Tris, pH 8.0, 1 mM EDTA, 1 mM NaN3, 1 mM DTT, 250 mM sucrose, 50 mM NaCl. Samples (50 µl) of fractions (0.6 ml) were used for protein assay (28). Lower, proteins in the remainder (550 µl) of the indicated fractions were precipitated with 10% trichloroacetic acid (final concentration), washed twice with ether, and dissolved in 300 µl of Laemmli buffer (45). Samples (25 µl) of each were subjected to SDS-PAGE in 4-20% gradient gels followed by silver staining. Positions of standards (kDa) are on the left. The same results were obtained in two independent experiments. B, binding of ARF1(39-45p3) to immobilized GST-p5. GSH-Sepharose beads (200 µl) with bound GST-p5 (200 µg) were incubated for 1 h at room temperature with 100 µg of p3 (lane 1), p3(24-30ARF1) (lane 2), ARF1 (lane 3), or ARF1(39-45p3) (lane 4) in 1 ml of 20 mM Tris, pH 8.0, 2.5 mM EDTA. Beads were then washed four times with 20 volumes of phosphate-buffered saline before elution of bound proteins in 0.5 ml of 10 mM GSH, 50 mM Tris, pH 8.0. Samples (1.5-2.5 µg) of eluted proteins were separated by SDS-PAGE in 4-20% gels and stained with Coomassie Blue. Positions of protein standards (kDa) are on the left. These findings have been replicated three times with independent preparations of proteins.
[View Larger Version of this Image (25K GIF file)]


Stimulation of CTA activity by p3 was similar with GTP and GTPgamma S, although it was much lower than the stimulation induced by ARF1 (Table III). As reported (23), addition of p5 to p3 reduced CTA activation with GTP but not with GTPgamma S (Table III). In the presence of GTP, p5 had no effect on the stimulation of the CTA activity by the chimeric p3(24-30ARF1), whereas it reduced stimulation by ARF1(39-45p3) (Table III). These results are consistent with the conclusion that p5 decreased the activity of the chimeric ARF1(39-45p3) by accelerating the hydrolysis of bound GTP.

Physical interaction between ARFs and the non-ARF p5 domain was assessed by gel filtration under the same conditions that p3 and p5 after incubation together, were eluted together (23). Similarly treated p5 and p3(24-30ARF1) did not coelute (data not shown), whereas ARF1(39-45p3) and p5 did (Fig. 5A, peak I); noninteracting ARF1(39-45p3) and p5 were also detected (Fig. 5A, peaks II and III).

Interactions between p5 and ARF1, p3, ARF1(39-45p3), or p3(24-30ARF1) were additionally evaluated using recombinant fusion GST-p5 bound to GSH-Sepharose beads that were then incubated with the indicated ARF protein. Proteins attached to the beads or interacting with them were eluted with GSH and separated by SDS-PAGE. The ARF domain p3 clearly interacted with GST-p5 (Fig. 5B, lane 1), whereas ARF1 did not (Fig. 5B, lane 2). GST-p5 also interacted with ARF1(39-45p3) but not with p3(24-30ARF1) (Fig. 5B, lanes 3 and 4). These results confirm that amino acids 24-30 (QDEFMQP) in p3 are involved in both functional and physical interactions between the ARF and the non-ARF domains of ARD1.

To identify more precisely the amino acids involved, two single amino acid replacement mutants of ARF1 were made with the amino acid of p3 placed in the equivalent ARF position to generate ARF1(G40D) and ARF1(T45P). Although ARF1(G40D), like ARF1, was not a substrate for p5, ARF1(T45P) was (Fig. 6). Moreover, the effect of p5 was significantly greater (p < 0.01) with the double mutant ARF1(G40D,T45P), suggesting a synergistic effect of the double mutation (Fig. 6). ARF1(G40D,T45P) also interacted with recombinant fusion GST-p5 immobilized on GSH-Sepharose beads, whereas ARF1(G40D) and ARF1(T45P) did not (Fig. 7, lanes 1-3). The lower band (~19 kDa) in Fig. 7, lane 3, is apparently a contaminant, variable amounts of which copurified with the double mutant ARF1(G40D,T45P).


Fig. 6. Effect of p5 on GTP hydrolysis by ARF1, ARF1(G40D), ARF1(T45P), and ARF1(G40D,T45P). 1 µg (~50 pmol) of recombinant ARF protein with [alpha -32P]GTP bound was incubated with the indicated amount of p5 for 60 min before bound nucleotides were separated by TLC. GAP activity is expressed as GTP/GTPo × 100. Data are means of duplicates ± one-half the range. Error bars smaller than symbols are not shown. Identical results were obtained with at least three different protein preparations. Differences with unpaired t tests between p5-stimulated GTP hydrolysis by ARF1(T45P) and ARF1(G40D,T45P) were significant (*, p < 0.007; **, p < 0.01).
[View Larger Version of this Image (21K GIF file)]



Fig. 7. Binding of ARF1 mutant to immobilized GST-p5. The indicated ARF1 (100 µg) and GSH-Sepharose beads (200 µl) with bound GST-p5 (200 µg) were incubated for 1 h at room temperature in 1 ml of 20 mM Tris, pH 8.0, 2.5 mM EDTA. Beads were then washed four times with 20 volumes of phosphate-buffered saline before elution of bound proteins in 0.5 ml of 10 mM GSH, 50 mM Tris, pH 8.0. Samples (2-3 µg) of the eluted proteins were separated by SDS-PAGE in 4-20% gels and stained with Coomassie Blue. Positions of protein standards (kDa) are on the left. Lane 1, ARF1(G40D); lane 2, ARF1(T45P); lane 3, ARF1(G40D,T45P); lane 4, Delta 15ARF1(39-45p3). These findings have been replicated twice with independent preparations of proteins.
[View Larger Version of this Image (96K GIF file)]


Effect of the Amino-terminal Region of ARFs

We reported that p5 stabilized the ARF domain in the inactive GDP-bound form by inhibiting GDP release as well as by promoting GTP hydrolysis (23). Indeed, when p5 was added to p3, dissociation of GDP bound to p3 was effectively decreased (Fig. 8A). GDP dissociation from ARF1(39-45p3) as well as from ARF1, however, was completely unaffected by p5 (Fig. 8B), indicating that even when physically interacting with ARF1(39-45p3) (Fig. 5B, lane 4) p5 did not induce all of the effects that it had on p3.


Fig. 8. Effect of p5 on dissociation of GDP from p3, ARF1, and their mutants. 300 pmol of p3 or p3(24-30ARF1), 150 pmol of ARF1 or ARF1(39-45p3), or 200 pmol of Delta 15ARF1(39-45p3) with [35S]GDPbeta S bound were incubated with p5 (ratio p5/ARF = 1.5) (closed symbols) or water (open symbols). Release of [35S]GDPbeta S from p3 (circles), p3(24-30ARF1) (squares), ARF1 (inverted triangles), ARF1(39-45p3) (diamonds), and Delta 15ARF1(39-45p3) (triangles) was monitored for 120 min. Each point is the mean of three determinations. Error bars smaller than symbols are not shown. Data are representative of those from at least three independent experiments.
[View Larger Version of this Image (23K GIF file)]


To investigate a possible effect of the amino terminus of the mutant ARF1(39-45p3), the first 15 amino acids were deleted to create Delta 15ARF1(39-45p3). Hydrolysis of the GTP bound to Delta 15ARF1(39-45p3) was stimulated by p5 more effectively than was that of GTP bound to ARF1(39-45p3), suggesting that the amino-terminal part in ARF1(39-45p3) might to some extent interfere with its interaction with p5 (Fig. 9). Indeed, the effect of p5 on GTP hydrolysis by Delta 15ARF1(39-45p3) approximated that on p3 (Fig. 9). In addition, Delta 15ARF1(39-45p3) binding to GST-p5 immobilized on GSH-Sepharose beads exceeded binding of ARF1(39-45p3) (Fig. 7, lane 4).


Fig. 9. Effect of p5 on GTPase activity of p3, ARF1, and ARF1 mutants. 1 µg (~50 pmol) of recombinant p3 or ARF proteins with [alpha -32P]GTP bound was incubated with the indicated amount of p5 for 60 min before bound nucleotides were separated by TLC. GAP activity is expressed as GTP/GTPo × 100. Data are means of duplicates ± one-half range. Error bars smaller than symbols are not shown. Similar results were obtained from at least three different protein preparations.
[View Larger Version of this Image (22K GIF file)]


GDP dissociation from Delta 15ARF1(39-45p3) was faster than from ARF1 or ARF1(39-45p3) (Fig. 8, B and C). When p5 was added, release of GDP was slowed to a rate comparable to that from ARF1(39-45p3) (Fig. 8C). These results are consistent with other evidence that the amino-terminal part of ARFs may be important in regulating nucleotide dissociation and that p5 may also play this role in ARD1.


DISCUSSION

The unusual GTPase activity of ARD1 has made possible the identification of a region specifically involved in both functional and physical interaction between the guanine nucleotide-binding domain (p3) and the GAP domain (p5) of ARD1. A major difference between the heterotrimeric and monomeric guanine nucleotide-binding proteins of the Ras superfamily is the much lower intrinsic GTPase activity of the latter. The Galpha subunits of trimeric G proteins, as described by Bourne and colleagues (33), contain a GTP-binding core ("Ralph") common to the monomeric G protein family but differ by the presence of an insertion of 110-140 amino acids ("Gail") at a position corresponding to loop 2 in p21ras. Studies of chimeric recombinant proteins (33) and crystal structure (34) established that this inserted domain is responsible for the GTPase activity of the Galpha subunit. Numerous proteins that enhance the GTPase activity of members of the monomeric G proteins have now been identified (for review, see Ref. 35). ARF proteins lack detectable intrinsic GTPase activity (13), and several groups have recently reported the purification or cloning of ARF GAPs (19-22).

ARD1 exhibits significantly greater GTPase activity than other members of the Ras superfamily (23). Although its GTP-binding domain (p3) appeared not to possess GTPase activity, addition of the amino-terminal domain (p5) expressed as a recombinant protein promoted hydrolysis of GTP bound to p3. Amino acids Asp25 and Pro30 of the ARF domain of ARD1 (p3) were crucial for both the functional and physical interaction between the two domains of ARD1. This region is equivalent to the extended effector loop in Ras, which is known to interact with Ras-GAP and neurofibromin (36). Moreover, based on the three-dimensional structure of ARF (37) and computer analysis,2 amino acids 24-30 in p3 are part of a beta  sheet that is believed to be involved in dimerization in ARF crystals (37). Without excluding the existence of other interaction sites, it is reasonable to conclude that this region is involved in the interaction between the two domains of ARD1.

Based on crystal structure and studies showing that GAPs interact with the effector domain of members of the Ras superfamily (36, 37), the corresponding sequences of ARD1 and ARF1 were exchanged. Our data clearly show that replacing the LGEIVTT sequence in ARF1 with QDEFMQP (found in ARD1) conferred on it the ability to hydrolyze bound GTP in response to p5. The single replacement of Thr45 by Pro in ARF1 transformed ARF1 into a substrate for p5. We postulate that Pro creates a curve in the beta  sheet structure that might place the conserved Glu (positions 41 in ARF1 and 26 in p3) in correct position for interaction with p5. The double mutant ARF1(G40D,T45P) provided evidence that Asp influences the interaction with p5, as physical and functional interactions of the double mutant were greater than those of ARF1(T45P). p5, however, hydrolyzed GTP bound to ARF1(39-45p3) better than GTP bound to ARF1(G40D,T45P), suggesting that glutamine(s) and hydrophobic residues in this region could also be involved in the interaction with p5.

The low stoichiometry of GTP binding by the ARF domain of ARD1 and by ARD1 itself is typical of recombinant ARFs (32, 38, 39) and may be due in part to the absence of N-myristoylation, which is a predominant determinant of the phospholipid-induced transition of ARF to the active ARF-GTP conformation (40). It may also be related to the fact that among the nucleotide-binding motifs that are otherwise identical, the CAT sequence of ARFs is replaced by DAR in ARD (1). The physiological significance of the effect of magnesium concentration on nucleotide affinities may be questioned as the intracellular concentration is thought to be in the low millimolar range (0.1-1.0 mM), although some microenvironments with higher free metal concentrations may exist in cells. ARD guanine nucleotide exchange, like that of the ARFs (14-18), may well be accelerated by a GEP.

Greater GTP binding by p3 than by p8, observed with certain phospholipids (i.e. brain PC + PIP2 + PE or phosphatidylserine), may reflect differences in interactions with phospholipid vesicles due to the presence of p5. Indeed, a two-stage loading reaction (incubation first with EDTA followed by high concentrations of Mg2+) was strictly necessary for nucleotide binding to ARD1, whereas significant GTP binding to p3 could be observed without prior incubation with EDTA (data not shown). Thus, the amino-terminal p5 domain may modulate GTP binding by affecting phospholipid interactions as well as by influencing affinity for GDP. In favor of the latter possibility is the finding that deletion of 15 amino acids from the amino terminus of ARF1(39-45p3) increased GDP dissociation to a level comparable to that of p3, and the addition of p5 then reduced GDP dissociation to a level comparable to that observed with ARF1(39-45p3). These results provide evidence to add to earlier observations (27, 41, 42) that the amino terminus of ARF proteins can be important in regulating nucleotide dissociation and suggest that p5 may also play this role in ARD1.

The covalent attachment of a GAP-like domain to the GTPase core of an ARF protein may represent an example of exon shuffling in evolution (43) and seems related to the function of ARD1, which remains to be defined. Heterotrimeric G proteins couple activation of receptors with seven membrane-spanning helices to the regulation of ion channels and intracellular enzymes (44). Like the Galpha proteins, ARD1 appears to possess an intrinsic regulator of GTPase activity. However, p5 differs from the Galpha Gail domain inserts by acting as a guanine nucleotide-dissociation inhibitor rather than a guanine nucleotide-dissociation stimulator in addition to serving as a GAP (33). Therefore, p5 acts as a negative regulator of the activity of ARD1, whereas Gail is centrally involved in both turning the Galpha switch on (GDP release followed by GTP binding) and turning it off (GTP hydrolysis). These aspects seem likely to be important in the as yet unidentified biological function of the ARF-related protein ARD1.


FOOTNOTES

*   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    To whom correspondence should be addressed: Rm. 5N-307, Bldg. 10, 10 Center Dr., National Institutes of Health, Bethesda, MD 20892-1434. Tel.: 301-402-1157; Fax: 301-402-1610; E-mail: vitalen{at}fido.nhlbi.nih.gov.
1    The abbreviations used are: ARD, ARF domain protein; ARF, ADP-ribosylation factor; BSA, bovine serum albumin; CTA, cholera toxin A subunit; DTT, dithiothreitol; GAP, GTPase-activating protein; GEP, guanine nucleotide-exchange protein; GSH, glutathione; GST, glutathione S-transferase; GDPbeta S, guanosine 5'-O-2-(thio)diphosphate; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; LM, low-melting; PC, phosphatidylcholine; PCR, polymerase chain reaction; PE, phosphatidylethanolamine; PIP2, phosphatidylinositol 4,5-bisphosphate; PAGE, polyacrylamide gel electrophoresis.
2    Computer analysis using RasMol software.

Acknowledgments

We thank Dr. Walter Patton for help in obtaining and viewing the crystal coordinates using RasMol software for Macintosh, Carol Kosh for expert secretarial assistance, and Dr. V. C. Manganiello for critical review of the manuscript.


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