Proc. Natl. Acad. Sci. USAVol. 93, pp. 1941-1944, March 1996Biochemistry

ARDi, a 64-kDa bifunctional protein containing an 18-kDaGTP-binding ADP-ribosylation factor domain and a 46-kDaGTPase-activating domainNICOLAS VITALE, JOEL MOSS, AND MARTHA VAUGHANPulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892

Contributed by Martha Vaughan, November 16, 1995

ABSTRACT The a subunits of the heterotrimeric guaninenucleotide-binding proteins (G proteins) hydrolyze GTP at arate significantly higher than do most members of the Rasfamily of '20-kDa GTP-binding proteins, which depend on aGTPase-activating protein (GAP) for acceleration of GTPhydrolysis. It has been demonstrated that an inserted domainin the G-protein a subunit, not present in the much smallerRas-like proteins, is responsible for this difference [Markby,D. W., Onrust, R. & Bourne, H. R. (1993) Science 262, 1895-1900]. We report here that ARD1, a 64-kDa protein with an18-kDa carboxyl-terminal ADP-ribosylation factor (ARF) do-main, exhibited significant GTPase activity, whereas the ARFdomain, expressed as a recombinant protein in Escherichiacoli, did not. Addition of the 46-kDa amino-terminal extension(similarly synthesized in E. coli) to the GTP-binding ARF-domain of ARD1 enhanced GTPase activity and inhibitedGDP dissociation. The kinetic properties of mixtures of theARF and non-ARF domains were similar to those of an intactrecombinant ARD1. Physical association of the two proteinswas demonstrated directly by gel filtration and by using theimmobilized non-ARF domain. Thus, like the ai subunits ofheterotrimeric G proteins, ARD1 appears to consist of twodomains that interact to regulate the biological activity of theprotein.

The GTPase superfamily comprises a diverse array of signal-transducing and regulatory proteins, which couple hydrolysisof GTP to the regulation of cellular processes ranging fromsecretion and endocytosis to vision and olfaction (1). Like thea subunits of the heterotrimeric guanine nucleotide-bindingproteins (G proteins), Ga, the smaller monomeric G proteinsare active in their GTP-bound form and inactive when GDP isbound. Thus, the cyclic alternation between these two confor-mations represents a critical mechanism to control the bio-logical activities of the proteins. Cells regulate the ratio ofactive and inactive monomeric GTPases by modulating therates of GDP release and GTP hydrolysis. A GTPase-activatingprotein (GAP) determines the rate of conversion of theGTP-bound form to an inactive GDP-bound form, whereas aguanine nucleotide-exchange protein (GEP) catalyzes releaseof bound GDP, allowing GTP binding and subsequent GTPhydrolysis (for a review, see ref. 2).ARDI, a 64-kDa protein identified by cDNA cloning,

contains an 18-kDa carboxyl-terminal ADP-ribosylation factor(ARF) domain and a 46-kDa amino-terminal domain. TheARFs are 20-kDa GTP-binding proteins initially described asGTP-dependent activators of cholera toxin A subunit (CTA)-catalyzed ADP-ribosylation of the a subunit of the adenylylcyclase-stimulatory G protein (Gsa) (for a review, see ref. 3).More recently, ARFs have been identified as essential com-ponents of vesicular trafficking pathways. They exhibit no

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detectable GTPase activity, and the ratio of GTP/GDP boundto ARF appears to be governed by GTP hydrolysis-activatingprotein (GAP) and GEP activities (3). We report here evi-dence that the additional amino-terminal domain of ARD1serves as a GAP for the ARF-domain and, therefore, mayparticipate in the regulation of its biological activity. Thephysiological function of ARD1 remains to be defined.

MATERIALS AND METHODSPreparation of Recombinant Fusion Proteins. Fusion pro-

teins synthesized by using a ligation-independent cloningmethod (4) and purified on glutathione (GSH)-conjugatedSepharose were "90% pure as estimated by silver stainingafter SDS/PAGE. After cleavage by bovine thrombin, GSHS-transferase (GST) was removed with GSH-Sepharose beads,and thrombin was removed with benzamidine-Sepharose 6B(5). The proteins were further purified by gel filtration throughUltrogel AcA54 and then Ultrogel AcA34. Purity estimated bysilver staining after SDS/PAGE was >98%. Amounts ofpurified proteins were estimated by a dye-binding assay (6) andby SDS/PAGE with bovine serum albumin (BSA) as standard.Assay of GTPase Activity. Samples were incubated for 30

min at 30°C in 20 mM Tris, pH 8/10 mM dithithreitol(DTT)/2.5 mM EDTA/0.3 mg of BSA per ml/1 mg ofcardiolipin per ml and then for 40 min at 30°C in the samemedium containing 0.5 ,tM [a-32P]GTP (3000 Ci/mmol; 1 Ci= 37 GBq) and 10 mM MgCl2. After addition of recombinantprotein or vehicle (40 ,tl), incubation was continued for 1 hr atroom temperature (final volume, 160 ,ul) before proteins withbound nucleotide were collected on nitrocellulose (7). Boundnucleotides were eluted in 250 ,ul of 2 M formic acid, of which3- to 4-,ul samples were analyzed by TLC on polyethylenei-mine-cellulose plates (8), and 240 ,ul was used for radioassayto quantify total nucleotide. TLC plates were subjected toautoradiography at -80°C for 14-24 hr. Total amounts ofnucleotides (GTP + GDP) bound to proteins, whether quan-tified by radioassay of the formic acid solution, by assay of thetotal radioactivity in the filter, or by assay with a Phosphor-Imager (Molecular Dynamics) after TLC, were not signifi-cantly different under any conditions.

Evaluation of the GTP and GDP Release from RecombinantProteins. Samples were incubated for 30 min at 30°C in 20 mMTris, pH 8/10 mM DTT/2.5 mM EDTA/0.3 mg of BSA perml/1 mg of cardiolipin per ml and then for 40 min in the samemedium containing 10 mM MgCl2 and 3 ,uM guanosine5'-[?y-(35S)thio]triphosphate (GTP[y-35S]; 2 x 107 cpm/500 p.1)or 3 p.M guanosine 5'-[,B-(5S)thio]diphosphate (GDP[f-35S];

Abbreviations: ARF, ADP-ribosylation factor; BSA, bovine serumalbumin; CTA, A subunit of cholera toxin; DTT, dithiothreitol; GEP,guanine nucleotide-exchange protein; GAP, GTP hydrolysis-activating protein; GDP[,B-S], guanosine 5'-[,B-thio]diphosphate;GTP[-y-S], guanosine 5'-[y-thio]triphosphate; GSH, glutathione; GST,GSH S-transferase; G protein, guanine nucleotide-binding protein;Ga, a subunit of G protein; Gs, stimulatory G protein.

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2 x 107 cpm/500 ,ul) (NEN). After further additions as indi-cated in 0.2 reaction volume and incubation for 15 min at 30°C,samples (60 ,ul) were transferred to nitrocellulose filters, whichwere washed five times with 1 ml of 25 mM Tris, pH 8/5 mMMgCl2/100 mM NaCl before radioassay (7). Total nucleotidebound to the proteins was determined by counting the radio-activity bound to filters and subtracting the amount bound inthe absence of protein. These are the zero-time values for thedissociation curve. The remaining mixtures were immediatelydiluted by the addition of an equal volume of reaction buffercontaining 2 mM GTP[-y-S] or GDP[P3-S]. Samples (120 ,ul)were taken after 5, 15, 30, 45, 60, 75, 90, 105, and 120 min at30°C, and bound radioactivity was quantified as described forthe zero-time samples.Assay of CTA-Catalyzed ADP-Ribosylagmatine Formation.

p3 or p8 was incubated for 30 min at 30°C in 40 [l of 20 mMTris, pH 8/10 mM DTT/2.5 mM EDTA/0.3 mg of BSA perml/1 mg of cardiolipin per ml before addition of 20 ,ul ofsolution to yield final concentrations of 100 ,uM GTP[,y-S] orGTP and 10 mM MgCl2. Further additions as indicated weremade 40 min later, and incubation was continued for 20 minat 30°C. Components needed to quantify ARF stimulation ofCTA-catalyzed ADP-ribosylagmatine formation were thenadded in 70 Al to yield final concentrations of 50 mM potas-sium phosphate (pH 7.5), 6 mM MgCl2, 20 mM DTT, 0.3 mgof ovalbumin per ml, 0.2 mM [adenine-14C]NAD (0.05 AOCi), 20mM agmatine, 1 mg of cardiolipin per ml, 100 ,uM GTP[,y-S]or GTP, and 0.5 ,ug of CTA (4). After incubation at 30°C for1 hr, samples (70 ,ul) were transferred to columns of AG1-X2equilibrated with water and were eluted with five 1-ml volumesof water. The eluate, containing [14C]ADP-ribosylagmatine,was collected for radioassay.

RESULTS AND DISCUSSIONARDI, a 64-kDa protein identified by cDNA cloning, containsan 18-kDa carboxyl-terminal ARF-domain and a 46-kDaamino-terminal domain (4). Like the ARFs, recombinantARDI bound GTP (Fig. 1, lane 1) as did the separatelyexpressed 18-kDa p3 ARF domain (4). Recombinant ARDIexhibited significant GTPase activity, whereas the recombi-nant ARF domain did not (Fig. 1, lanes p8 and p3). Additionof the amino-terminal domain pS, synthesized as a GST fusionprotein in E. coli, or the intact fusion protein GST-p5, resultedin hydrolysis of GTP bound to p3, the ARF domain (Fig. 1,

GDP 9

lanes p3 + p5 and p3 + Tp5). Addition of GST had no effect(data not shown). p5 and GST-p5 themselves did not bind(data not shown) or hydrolyze GTP (Fig. 1, lanes p5 and Tp5).Trypsin treatment or boiling of p5 completely abolished itsability to enhance the GTPase activity of p3 (data not shown).These findings are consistent with the conclusion that thenon-ARF domain is responsible for the stimulation of hydro-lysis of GTP bound to the ARF domain. Since GST-p5stimulated the GTPase activity of the p3 domain, the GSTmoiety did not interfere with the functional interaction be-tween these two domains. However, the presence of the GSTmoiety at the amino terminus of the ARF domain in GST-p3completely prevented stimulation of GTP hydrolysis by p5 orGST-p5 (Fig. 1, lanes Tp3 + p5 and Tp3 + Tp5); GTP bindingby GST-p3 was not affected. Thus, the presence of GST at theamino terminus of p3 apparently interferes with functionalinteraction between it and p5.To characterize further the effects of the p5 amino-terminal

non-ARF domain of ARDI, dissociation of bound guaninenucleotides from p3 and p8 was compared. Although there wasno significant difference between the two proteins in theamount of GTP[,y-35S] or GDP[_3-35S] bound at zero time (Fig.2), GDP[_3-35S] release (in the presence of large excess ofGDP[,3-S]) was much slower from p8 than from p3 (Fig. 2Lower). Indeed, the p3 domain released bound GDP fasterthan did recombinant ARF1 (data not shown), which isconsistent with the recent report that an amino-terminaldeletion mutant of ARF1 released GDP faster than did thewild-type protein (9). Release of GTP[y-35S] from p3 was,however, significantly slower than from p8 (Fig. 2 Upper).Prompted by these results, we assessed the ability of the

non-ARF p5 domain to influence GDP and GTP dissociationfrom p3. When p5 was added to p3, release of both nucleotidesapproximated that from p8 (Fig. 2). These data are consistentwith the hypothesis that the non-ARF domain of ARD1stabilizes the ARF-domain in the inactive GDP form byinhibiting GDP release and thereby GTP binding and bypromoting the hydrolysis of GTP via its GAP-like activity.

Physical interaction between the ARF and non-ARF do-mains was assessed by gel filtration. When chromatographedtogether, some p3 and p5 were eluted together at the sameposition as p8; noninteracting p3 and p5 were also detected(Fig. 3). Interactions between the two domains were addition-ally evaluated by using recombinant proteins GST-p5, GST-p3,or GST bound to GSH-Sepharose beads, which were then

GTP

p8 p3 p3 p3 Tp3 Tp3 Tp3 p5 Tp5+ + + + med medp5 Tp5 p5 Tp5

FIG. 1. Effect of the non-ARF domain (p5) of ARDM on GTPase activity of the ARF domain (p3). Of the recombinant GST fusion proteins,GST-p8 (Tp8) contains the entire ARDM sequence, GST-p5 (Tp5) contains the amino-terminal non-ARF domain, and GST-p3 (Tp3) contains thecarboxyl-terminal ARF domain. Respective proteins without the GST moiety are p8, p5, and p3. Tp3, p3, and p8 (45 pmol) with [a-32P]GTP boundwere incubated without or with p5 or Tp5 (100 pmol) as indicated before analysis of bound nucleotide by TLC. In lanes p5 and Tp5 are 0.25-,ulsamples of assay mixtures that were applied directly to TLC plates (i.e., not filtered, as p5 does not bind GTP), showing that p5 and Tp5 did notthemselves hydrolyze GTP. Positions of standard GTP and GDP are indicated on the left. Data are representative of those from at least threeindependent experiments.

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Time (min)

FIG. 2. Effect of the non-ARF domain (p5) ofARDl on GTP andGDP dissociation from the ARF domain (p3). p3 or p8 (300 pmol) withGTP[y-35S] or GDP[,3-35S] bound were incubated with 300 pmol of p5or water. Release of GTP[y-35S] (Upper) or GDP[p3-35S] (Lower) fromp8 (0), p3 (o), and p3 plus p5 (-) was monitored for 120 min. Eachpoint is the mean of three determinations. The SEM is smaller thanthe symbol for each value. Data are representative of those from fiveindependent experiments.

incubated with p3 or p5. Proteins attached to the beads orinteracting with them were eluted with free GSH and sepa-rated by SDS/PAGE. The ARF domain p3 clearly interactedwith recombinant fusion GST-p5 (Fig. 4, lane 1) but not withbound GST (Fig. 4, lane 2). The non-ARF domain p5 did notbind to recombinant fusion GST-p3 (Fig. 4, lane 3). Theseresults confirm the functional interactions of the two ARD1domains observed in solution and are consistent with thefailure of p5 to stimulate the GTPase activity of GST-p3.

Finally, we examined the effects of the amino-terminaldomain p5 on the biological activity of ARDL. Like all othermembers of the ARF family (11, 12) in the presence of GTPor a non-hydrolyzable analogue, ARDI serves as an allostericactivator of CTA ADP-ribosyltransferase activity (4). Stimu-lation of CTA by p3 was similar with GTP and GTP['y-S],whereas p8 was less effective with GTP than with GTP[-y-S](Table 1). Thus, the intrinsic GTPase activity of ARD1reported here may contribute to regulation of the biologicalactivity of the protein. The non-ARF domain ofARD1 had noARF activity by itself (data not shown). As expected, theaddition of the non-ARF p5 domain to the ARF domain alsoreduced stimulation with GTP but not with GTP[y-S] (Table1), confirming that the non-ARF domain influences thebiological activity of the ARF domain of ARD1.The heterotrimeric G proteins couple activation of trans-

membrane receptors with seven membrane-spanning helicesto the regulation of ion channels and intracellular enzymes (1,13). The Gas of these G proteins, as described by Bourne andcolleagues, contain a GTP-binding core ("Ralph") common tothe Ras GTPase family but are distinguished by an insertion of110-140 amino acids ("Gail") at a site corresponding to loop2 of p21 Ras. Studies of chimeric recombinant proteins estab-lished that this inserted domain is responsible for the GTPaseactivity of the Gas of trimeric G proteins (14). The covalent

FIG. 3. Analysis of the interaction of the ARF and the non-ARFdomains of ARD1 by gel filtration. (Upper) p3 (100 ,ug) and p5 (200,ug) were incubated for 1 hr at 30°C in 500 jl of 20 mM Tris, pH 8/2.5mM EDTA and then applied to a column (1.5 x 30 cm) of UltrogelAcA34 equilibrated and eluted (0.2 ml/min) with 20 mM Tris, pH 8/1mM EDTA/1 mM NaN3/1 mM DTT/250 mM sucrose/50 mM NaCl.Samples (50 ,ul) of fractions (0.6 ml) were used for protein assay (6).(Lower) Proteins in the remainder (550 ,ul) of the indicated fractionswere precipitated with 10% trichloroacetic acid (final concentration),washed twice with ether, and dissolved in 300 ,ll of Laemmli buffer(10). Samples (25 ,ul) of each were subjected to SDS/PAGE in 4-20%gradient gels followed by silver staining. The first peak fractionscontained both p3 and p5 presumably in a complex. The two followingpeaks contained noninteracting p5 or p3. Positions of protein stan-dards (kDa) are on the left. The same elution profile was obtained intwo independent experiments.

attachment of a GAP-like domain to the GTPase core of Ga,or as described here to the GTPase core of an ARF protein,may represent examples of exon shuffling in evolution (15).

97- _

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21 - am14 -

Std 1 2 3FIG. 4. Binding of the ARF domain (p3) ofARD1 to immobilized

GST-p5 (non-ARF domain). GSH-Sepharose beads (200 ,lI) withbound GST-p5 (200 jug, lane 1), GST (400 ,ug, lane 2), or GST-p3 (200,ug, lane 3) were incubated for 1 hr at room temperature with 100 jigof p3 (lanes 1 and 2) or 200 ,ug of p5 (lane 3) in 1 ml of 20 mM Tris,pH 8/2.5 mM EDTA. Beads were then washed four times with 20volumes of phosphate-buffered saline before elution of bound proteinsin 0.5 ml of 10 mM GSH/50 mM Tris, pH 8. Samples (2-4 jig) of theeluted proteins were separated by SDS/PAGE in 4-20% gels andstained with Coomassie blue. p3 was bound specifically to immobilizedGST-p5 (lane 1) but not to GST (lane 2), whereas p5 did not bind toimmobilized GST-p3 (lane 3). Positions of protein standards (kDa) are

on the left. These findings have been replicated twice with twoindependent preparations of proteins.

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Table 1. Effect of the non-ARF domain (p5) of ARD1 on theability of the ARF domain (p3) to stimulate the ADP-ribosyltransferase activity of CTA

ARF activity, nmol/hr

Protein added GTP GTP[y-S]p3 4.22 ± 0.02 4.21 ± 0.01p8 2.36 ± 0.01 4.40 ± 0.01p3 + p5 2.71 ± 0.02 3.96 ± 0.04

p3 (900 pmol) or p8 (200 pmol) was loaded with GTP[(y-S] or GTPand then incubated with p5 (900 pmol) or water (20 ,l) for 20 min at30°C. Components needed to quantify ARF stimulation of CTA-catalyzed ADP-ribosylagmatine formation were then added, and in-cubation was continued for 1 hr at 30°C. Basal activity (in the presenceof p3 or p8) in each condition has been subtracted. Data are means ofvalues from quadruplicate assays ± SEM in one experiment repre-sentative of four.

Crystallographic evidence that the two domains of Ga havefew contacts was consistent with the view that guanine nucle-otide exchange is mediated by en bloc movement of the rigidGail domain (16, 17). Therefore, this additional Gail domain,which is missing in Ras, is centrally involved in both turning theGa switch on (GDP release followed by GTP binding) and inturning it off (GTP hydrolysis). Although p5 from ARD1 andthe Gail domain of GSa both exhibit GAP activity, they havequite different effects on nucleotide binding, as GDP releasefrom p3 was inhibited by p5 GDP-dissociation inhibitor (GDI)-like activity], whereas Gail accelerated GDP release and GTPbinding, a GEP activity (14). It could be useful, nevertheless,to compare the structure of p5 with those of Gail domains fromGa proteins and ARF-GAP (17). Are there, for example,similarities among these sequences that may offer clues tofunctional structures, related to GAP or to GDI- or GEP-likeeffects? Is there an arginine that corresponds to Arg-201 inGsa, which is the site modified by CTA and is required for theGTPase activity of Ga-i.e., the GAP activity of Gail? Thepossible functional role of this Arg-201 and a perhaps cognate

arginine in Ras-GAP was discussed in detail by Markby et al.(14). Structural information about ARD1 in GDP- and GTP-bound forms will surely be helpful in trying to understand theinteractions between the two domains as well as the molecularalterations associated with the GDP-GTP switch.

We thank Carol Kosh for expert secretarial assistance and Dr. V. C.Manganiello for critical review of the manuscript. N.V. was supportedby a grant from the Institut National de la Sante et de la RechercheMedicale.

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