©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ADP-ribosylation Factor-directed GTPase-activating Protein
PURIFICATION AND PARTIAL CHARACTERIZATION (*)

(Received for publication, September 14, 1994; and in revised form, December 23, 1994)

Vardit Makler Edna Cukierman Miriam Rotman Arie Admon Dan Cassel (§)

From the Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The small GTP-binding protein ARF plays an established role in the control of vesicular traffic and in the regulation of phospholipase D activity. Like other GTP binding proteins, ARF becomes activated upon the binding of GTP, whereas GTP hydrolysis acts as a turn-off signal. The fact that purified ARF proteins have negligible GTPase activity has suggested that GTP hydrolysis by ARFs is dependent on a GTPase-activating protein (GAP). Here we report the complete purification of an ARF GAP from rat liver cytosol. Advanced stages in the purification were carried out in the presence of denaturing agents, making use of an unusual conformational stability, or refolding capacity, of the GAP. The GAP was purified about 15,000-fold and was identified as a protein of 49 kDa. Partial amino acid sequence analysis showed that the GAP is a previously uncharacterized protein.

Both crude and purified GAP migrated on a Superdex 200 column as a 200-kDa complex, suggesting a tetrameric structure. The purified ARF GAP was stimulated by phosphoinositides and was inhibited by phosphatidylcholine, similar to the results previously reported for a preparation from brain (Randazzo, P. A., and Kahn, R. A.(1994) J. Biol. Chem. 269, 10758).

The availability of the ARF GAP molecule will advance the understanding of the regulation of the cellular processes in which ARF proteins participate.


INTRODUCTION

ADP-ribosylation factors (ARFs) (^1)are a distinct subfamily of 21-kDa GTP binding proteins(1, 2) . The proteins derive their name from the ability of ARFs to stimulate the ADP-ribosyl transferase activity of cholera toxin(3, 4) . Although a specific role has not yet been assigned to each ARF, the proteins are apparently involved in the regulation of vesicular traffic among different cellular compartments (for review see (5) ). Best characterized is ARF1, an abundant cellular protein that has been purified both from tissues (3, 6, 7) and from recombinant sources(8) . ARF1 is required for the assembly of coat proteins on Golgi stacks (9, 10) and of AP-1 adaptor particles on the trans-Golgi network(11, 12) . Coat protein binding is triggered by the association of ARF1 with the Golgi membrane following a GDP-to-GTP exchange reaction that is stimulated by a membrane-bound guanine nucleotide exchange factor(13, 14, 15) . The subsequent hydrolysis of ARF-bound GTP is required for the release of coat proteins from Golgi membranes and vesicles. Thus, the binding of coat proteins to the Golgi in the presence of the hydrolysis-resistant derivative GTPS is irreversible(16) . Furthermore, a stabilization of coat protein association with the Golgi is observed in the presence of ARF mutants displaying a decreased rate of GTP hydrolysis(17, 18) .

The fact that pure ARFs have undetectable intrinsic GTPase activity (4) has suggested that GTP hydrolysis by ARF requires an interaction with a GTPase-activating protein. Structurally diverse GAPs are associated with all families of small GTP binding proteins, acting as signal terminators and possibly also in some cases as effectors downstream of the GTP binding protein(19) . The molecular characterization of an ARF-directed GAP has been particularly challenging, in view of its likely role in triggering the uncoating of Golgi-derived membranes and vesicles(17, 18) . Recently, Randazzo and Kahn (20) demonstrated the presence of ARF GAP activity in a salt extract from bovine brain membranes and showed that this activity depends on acid phospholipids. We now report the purification and characterization of an ARF-directed GAP from rat liver cytosol. The availability of this molecule will allow the study of new aspects of ARF-regulated systems.


EXPERIMENTAL PROCEDURES

Materials

[alpha-P]GTP (800 Ci/mmol) was from DuPont NEN. Recombinant, myristoylated ARF1 was prepared from bacteria co-expressing ARF1 and N-myristoyltransferase, kindly provided by Dr. Richard D. Kahn, as described in (8) . A monoclonal antibody to hnRNP F/H was kindly provided by Dr. Gideon Dreyfuss.

Preparation of Rat Liver Cytosol

2-month-old rats were killed with either ether or CO(2), and livers were removed and rinsed with cold phosphate-buffered saline. Livers were minced and homogenized in 28 ml of homogenization buffer (300 mM sucrose, 25 mM NaCl, 20 mM Tris-Cl pH 8.0, 4 mM EGTA, 1 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM 1:10 phenanthroline, 2 µM pepstatin A, 2 µg/ml aprotinin, and 2 µg/ml leupeptin) per 10 g of tissue by two 30-s bursts of the Polytron homogenizer at speed 6. The homogenate was centrifuged at 14,000 rpm for 30 min in a Sorvall SS34 rotor, and the supernatant was collected and centrifuged at 43,000 rpm for 1 h in a Beckman 50Ti rotor. Supernatants were frozen in liquid nitrogen and stored at -80 °C.

Assay of ARF-directed GAP Activity

The assay measures a single round of a GTPase reaction(21) . ARF1 was first loaded with [alpha-P]GTP. In order to achieve high loading efficiency, loading was carried out in the presence of dimyristoyl phosphatidylcholine (DMPC) and cholate (4) and using a relatively high ARF concentration. The loading reaction mixture contained ARF1 (0.5 mg/ml), [alpha-P]GTP (0.2 mCi/ml, 0.25 µM), 5 mM MgCl(2), 1 mM DTT, 1 mM ATP plus ATP/GTP regeneration system (5 mM phosphocreatine and 50 µg/ml creatine phosphokinase), 25 mM MOPS buffer, pH 7.5, 150 mM KCl, and a mixture of DMPC and sodium cholate, added last from a 10 times stock to give 3 mM and 1 mg/ml, respectively. Loading was carried out for 90 min at 30 °C, and the preparation was divided into small aliquots and stored at -80 °C. Filter binding assays (4) showed that between 30 and 60% of the [alpha-P]GTP becomes associated with different preparations of recombinant ARF.

GAP activity was assayed in a final volume of 10 µl in the presence of 5 mM MgCl(2), 1 mM DTT, 1 mM ATP plus the above mentioned ATP/GTP regeneration system, 25 mM MOPS buffer, pH 7.5, 0.5 mM unlabeled GTP, 0.1 unit/ml guanylate kinase, and 1 µl of [alpha--P]GTP-loaded ARF. Following incubation for 15 min at 30 °C, reactions were boiled for 1 min to release the nucleotides from ARF, and the nucleotides were separated by thin layer chromatography on PEI-cellulose sheets, developed with 1.2 M Tris-Cl, pH 7.4. [P]GDP formation was determined by autoradiography or by cutting the GDP and GTP areas, and determination of radioactivity was made using Cerenkov radiation. Data are presented as the percentage of ARF-bound [alpha-P]GTP that is converted to [-P]GDP. Background values were 4-5%, and these values were not increased during incubations in the absence of a GAP. Where appropriate, GAP activity was assessed by carrying out serial dilutions of the sample, and specific activity was calculated at the protein concentration that resulted in 50% hydrolysis of ARF-bound GTP.

Purification of ARF GAP from Rat Liver Cytosol-The following solutions were used during the purification: solution A, 25 mM Tris-Cl, pH 7.4 (25 °C), 1 mM DTT; solution AU, solution A plus 5 mM ammonium chloride and 5 M urea; solution AG, solution A plus 20% glycerol. The first two steps in the purification (I and II) were carried out in the cold, and the last ones were at room temperature.

Step I: Ammonium Sulfate Precipitation

100 ml of cytosol containing 3-3.5 g of protein were thawed and diluted with 2 volumes of 25 mM Tris-Cl, pH 7.5, 1 mM EDTA, 1 mM DTT. A saturated ammonium sulfate solution was added to give a final concentration of 38.5%, and the mixture was allowed to stand for 20 min then centrifuged at 8,000 rpm for 10 min in a Sorvall GSA rotor. Supernatants were completely removed, and pellets were suspended in a total of 40 ml of solution A containing 2.5 µg/ml leupeptin. The suspension was cleared by centrifugation at 15,000 rpm for 15 min in a SS34 rotor, removing floating fat if present.

Step II: DEAE Chromatography

The cleared suspension from step I was diluted with 60 ml of solution A and applied at 8 ml/min to a 4.4-cm diameter column containing 100 ml of DEAE-Toyopearl (Fractogel TSK DEAE-650 S, from Merck) pre-equilibrated with solution A. The column was washed with 100 ml of solution A containing 1 µg/ml leupeptin and 100 mM NaCl and then with 350 ml of the same solution with NaCl adjusted to 155 mM. GAP activity was eluted by the stepwise increase of the NaCl concentration to 255 mM, collecting the entire peak of UV absorption (70-80 ml).

Step III: DEAE/Urea Chromatography

Step II eluate was diluted with 1.5 volumes of solution A containing 1 µg/ml leupeptin and applied in the cold at 4 ml/min to a 3.2-cm diameter column containing 35 ml of DEAE-Toyopearl. The column was transferred to room temperature and washed with 35 ml of solution A and then with 50 ml of solution AU containing 20 mM NaCl. Elution was carried out at a flow rate of 4 ml/min by linear NaCl gradient in solution AU (20-120 mM NaCl over 50 min, then to 500 mM NaCl in 10 min). 4-ml fractions were collected, and aliquots representing 0.1-0.2 µl of each fraction were assayed for GAP activity. An activity peak containing 7-9 fractions was pooled.

Step IV: Resource Q/Glycerol Chromatography

The pooled fractions from step III were directly loaded at 3 ml/min onto a 1-ml Resource Q column (Pharmacia Biotech Inc.) equilibrated with solution AU containing 60 mM NaCl, then washed with 10 ml of the same solution. The column was then equilibrated with solution AG containing 60 mM NaCl, and proteins were eluted at 1 ml/min by a 20-min gradient, 60-500 mM NaCl in solution AG. 0.25-ml fractions were collected, and 25-50-nl aliquots were assayed for GAP activity.

Renaturation of ARF GAP Activity Following SDS-PAGE

Renaturation was carried out by a modification of the method described in (22) . Following SDS-PAGE (10 cm of 10% separating gels), a gel lane was sliced into 2-3-mm portions. The slices were briefly rinsed with water and then homogenized in microfuge tubes using a conical Teflon pestle in 0.3 ml of 50 mM Tris-Cl, pH 8.0, 0.15 M NaCl, 5 mM DTT, 0.1 mM EDTA, 0.1% SDS, and 50 µg/ml lactoglobulin. The suspension was subjected to rotary inversion for 60 min at room temperature and spun in the microfuge, and 0.2 ml of the supernatants were transferred to another microfuge tube. Proteins were precipitated by the addition of 0.8 ml of acetone (prechilled to -20 °C) and incubation for 30 min at -80 °C. The tubes were centrifuged in the microfuge for 15 min at 4 °C, and pellets were carefully rinsed with 50 µl of 80% acetone and then left open in the hood for 10 min. The pellets were extracted for 30 min at 37 °C with 50 µl of 5 M urea containing 25 mM Tris-Cl, pH 7.4, 0.2 M NaCl, and 1 mM DTT, and 0.5 µl aliquots were directly taken for assay of GAP activity.

Partial Amino Acid Sequence Determination

The ARF GAP was first purified to homogeneity by reverse phase HPLC. One-third of the activity pool from stage IV was separated on a C8 RP300 column (2.1 times 30 mm, Applied Biosystems), and proteins were eluted by a linear acetonitrile gradient in the presence of 0.1% trifluoroacetic acid (15-70% acetonitrile in 25 min). Peaks of UV absorption were manually collected, and the GAP peak was identified by performing activity assays on 0.1-0.5-µl aliquots from each peak. The protein was digested overnight with trypsin or with Lys-C, and peptides were resolved on a microbore C8 HPLC column (RP300, 1 times 100 mm). Peptides were subjected to microsequencing using the Applied Biosystems model 476A sequencer and standard chemistry.

Miscellaneous

Protein was determined by the method of Lowry et al.(23) using bovine serum albumin as standard. SDS-PAGE (10% gels) was according to Laemmli(24) .


RESULTS

Purification of an ARF-directed GAP from Rat Liver Cytosol

We chose the rat liver as the starting material for the purification of the ARF GAP since cytosol from this tissue was found to be a relatively rich and reproducible source of activity. GAP activity from the high speed supernatant was quantitatively precipitated with 38.5% ammonium sulfate. The redissolved pellet was applied to a DEAE-Toyopearl column, which was washed with 155 mM NaCl, and GAP activity was subsequently eluted with 255 mM NaCl.

Further attempts to achieve a high degree of purification using a variety of chromatography techniques were initially unsuccessful because of either broad elution profiles or loss of GAP activity. This problem was overcome by carrying out a chromatography of the DEAE activity pool on a second DEAE column in the presence of 5 M urea. Under these conditions, ARF GAP activity eluted from the DEAE column at much lower salt concentrations (75-85 mM), thereby separating it from most other proteins (Fig. 1). Such a shift in the elution profile may reflect a dissociation of subunits brought about by the presence of urea. GAP activity was detected in column fractions following a direct dilution into the assay. Experiments in which crude preparations were treated with 5 M urea and were subsequently added to the assay systems to give final urea concentrations of 0.5-1 M showed that most GAP activity (50-60%) is retained in urea-treated samples.


Figure 1: Purification of ARF GAP by DEAE chromatography in the presence of urea. Protein eluted from a DEAE-Toyopearl column by 255 mM NaCl was chromatographed on a second DEAE column in the presence of 5 M urea as described under ``Experimental Procedures,'' step III.



Chromatography of the DEAE/urea activity pool on a Resource Q column in the presence of 20% glycerol (Fig. 2) resulted in further enrichment of ARF GAP activity. Analysis of column fractions by SDS-PAGE (Fig. 3) revealed a major 49-kDa band in Resource Q fractions in which GAP activity was observed (fractions 34-41). It can also be noticed that the peak of the 49-kDa band (Fig. 3, fractions 34-36) appears slightly earlier than the peak of GAP activity (Fig. 2, fractions 36-38). This is because of the presence of an additional 49-kDa protein in the earlier fractions, the nature of which will be described later.


Figure 2: Resource Q chromatography. The peak of GAP activity from Fig. 1(fraction 35-42) was chromatographed on a Resource Q column in the presence of 20% glycerol (see ``Experimental Procedures,'' step IV).




Figure 3: SDS-PAGE analysis of stages in the purification of the ARF GAP. CY, crude cytosol; AS, ammonium sulfate precipitate; DE, the eluate from the first DEAE column (see ``Experimental Procedures,'' 30 µg of each fraction); DU, pooled GAP activity fractions from DEAE/urea chromatography (5 µg, see Fig. 1). Resource Q, analysis of 25-µl aliquots of the fractions from the experiment presented in Fig. 2.



In order to determine whether ARF GAP activity is indeed contained within the major 49-kDa band observed on SDS gels, a gel lane containing proteins from a Resource Q-purified material was divided into small slices, and SDS was removed from eluted proteins by acetone precipitation. This procedure resulted in the renaturation of ARF GAP activity and showed that activity co-migrates with the 49-kDa band (Fig. 4, A and B, lane 6).


Figure 4: ARF GAP activity recovered from slices of an SDS gel. 150 µl of a Resource Q-purified GAP (Fig. 2) was separated by SDS-PAGE. The gel was sliced into 2-3-mm portions, and proteins were recovered from the slices as described under ``Experimental Procedures.'' Panel A presents the thin layer chromatography results of the GAP assay of the gel slice extracts, and panel B shows SDS-PAGE and Coomassie Blue staining of 20-µl aliquots of the slice extracts. The slices are numbered according to a decreasing molecular weight. LG, lactoglobulin.



The purification results obtained in the experiments shown in Fig. 1Fig. 2Fig. 3are summarized in Table 1. GAP activity was enriched by about 4,000-fold with a 25% yield. Similar results were obtained in most experiments, although in some preparations the degree of purification varied between 2,500- and 9,000-fold. The increase in total activity in the Resource Q step probably reflects some renaturation of the GAP following the previous step in which urea was employed.



Partial Sequence Analysis of the ARF GAP

In preparation of the GAP for amino acid sequence analysis, the Resource Q-purified material was subjected to reverse phase HPLC (Fig. 5). GAP activity could be detected in column fractions when the protein contained in the acetonitrile/trifluoroacetic acid eluant was directly added to the assay, indicating that activity is either resistant to this organic solvent or is regained upon dilution. GAP activity eluted from the reverse phase column as a sharp peak (marked *), resulting in a highly pure 49-kDa protein (Fig. 5, inset). The profile shown in Fig. 5was obtained from a particularly active Resource Q preparation, yet a homogenous 49-kDa protein was also obtained from HPLC of other, less active preparations in which the GAP peak only comprised 20-25% of the total protein (data not shown). Based on the increase in specific activity of GAP preparations up to the Resource Q step and the further resolution of the Resource Q preparation during reverse phase chromatography, we estimate that the GAP comprises approximately 0.005-0.01% of cytosolic protein.


Figure 5: Reverse phase HPLC purification of the ARF GAP. The activity peak from a Resource Q column was chromatographed on a C8 column as described under ``Experimental Procedures.'' Inset shows a silver-stained SDS gel of 1.5 µg of the peak containing GAP activity (marked *).



Analysis of peptides obtained from the HPLC-purified GAP following digestion with trypsin or Lys-C gave the following sequences: L(S)V(S)DSXDIXGX(S)GXAXNNK, IFDDVSSGVSQLASK, KFREFLEAQDDYEP, KAEDTSDRPL, KTLQFTAHRPAGQPQNVTTXG, YVGFGNTV(P), and AL(S)E(G)L(V)G(H)H?SLNENVLKPA.

Analysis using the Blast program showed that none of the above peptides is part of a protein whose sequence is present in the SwissProt databank, demonstrating that the ARF GAP that we have isolated is a previously unidentified protein. It should be cautioned that even though the ARF GAP was purified to an apparent homogeneity, we cannot exclude the possibility that some of the peptides that we have sequenced represent contaminant proteins that are nevertheless present in the preparation.

As already mentioned, fractions of the Resource Q column immediately preceding the peak of GAP activity possessed a protein identical in size to the GAP (see Fig. 3). The protein in these fractions was identified by microsequencing as the hnRNP F/H(25) . This result was confirmed using a monoclonal antibody directed against the RNP(25) . The hnRNP was present as a minor contamination in the Resource Q pool of GAP activity. The two proteins separated from each other during the reverse phase HPLC and could also be resolved by affinity chromatography on polyguanylic acid(25) , which retained the RNP but not the GAP (data not shown).

Biochemical Characterization of the ARF GAP

The specificity of the purified GAP for ARF is indicated by the finding that it had no effect on H-Ras (data not shown). Similar results were reported for a partially purified ARF GAP from bovine brain(20) .

The molecular size of the ARF GAP was determined by gel filtration chromatography. ARF GAP activity in crude, ammonium sulfate-precipitated preparation and in the highly purified preparation obtained after Resource Q chromatography both showed a similar migration on a Superdex 200 FPLC column, corresponding to a molecular mass of approximately 200 kDa (Fig. 6). This result suggests that the GAP may exist as a homotetramer. A slight difference in migration between the crude and purified preparations, corresponding to a size difference of approximately 20 kDa, was observed in the experiment shown in Fig. 6. A similar difference in size was also observed in two other experiments, whereas in two additional ones the migration of GAP activity was identical in crude and purified preparations. This variability in the results may reflect a limitation of the resolution of the methods employed.


Figure 6: Analysis of GAP preparations by gel filtration. 200-µl aliquots of the resuspended ammonium sulfate pellet (A.S.) or the pooled Resource Q fraction (Q) were brought to 0.5% of hydrogenated Triton X-100 and were chromatographed on a 24-ml Superdex 200 column (Pharmacia Biotech Inc.). Elution buffer contained 0.5 M NaCl, 25 mM Tris-Cl, pH 7.4, 1 mM DTT, and 0.1% hydrogenated Triton X-100. Flow rate was 0.5 ml/min. 0.25-ml fractions were collected, and 2-µl aliquots were assayed for GAP activity. The position of molecular weight markers is indicated by arrows. 100% GDP amounts to 75 fmol/assay system.



Randazzo and Kahn (20) have recently reported that ARF GAP from bovine brain membranes is strongly stimulated by phosphoinositides and is inhibited by phosphatidylcholine. Qualitatively similar results were obtained with our GAP preparations (Fig. 7). In both crude (ammonium sulfate-precipitated cytosol) and Resource Q-purified preparations, PIP(2) caused a reproducible 2-2.5-fold increase in GAP activity. A similar 2-fold stimulation was observed upon addition of crude brain phosphoinositides to the purified GAP (Fig. 7B). The brain phosphoinositides were somewhat more effective with the ammonium sulfate-precipitated material (Fig. 7A), apparently reflecting stimulation by components other than PIP(2) that are present in the crude brain preparation. The above effects of phosphoinositides were all significantly lower than those reported by Randazzo and Kahn(20) . Phosphatidylcholine from egg strongly inhibited GAP activity in both crude and purified GAP preparations. However, in contrast to the results of Randazzo and Kahn (20) , DMPC had no effect. Since DMPC is employed in our standard assays for loading ARF with GTP, resulting in a final concentration of 0.3 mM, the finding that 1.3 mM DMPC does not affect GAP activity under our assay conditions (Fig. 7) indicates that DMPC does not interfere with the assay of ARF GAP preparations from rat liver.


Figure 7: Effect of lipids on ARF GAP activity. Activity was assayed on the ammonium sulfate (A) or Resource Q (B) fractions in the presence of 0.02% hydrogenated Triton X-100 and in the presence or absence of 100 µM phosphatidylinositol 4`,5`-bisphosphate (PIP(2)), 100 µg/ml of a phophoinositide mixture from brain (PI's, Sigma P-6023), 1.3 mM of phosphatidylcholine from egg yolk (PC), and 1.3 mM dimyristoyl phosphatidylcholine (DMPC). 100% GDP amounts to 88 fmol per assay system.




DISCUSSION

This work describes for the first time the isolation of an ARF-directed GAP, a previously unidentified 49-kDa protein. A key finding that facilitated the purification was that the presence of urea during anion exchange chromatography causes a shift in the elution profile of the GAP to much lower salt concentrations, allowing the separation of the GAP from the majority of the cytosolic proteins. A most likely explanation for this phenomenon is a dissociation of subunits as a result of the denaturing effect of urea and a consequent decrease in total surface charge. Evidence for a multisubunit structure of the GAP is provided by the finding that GAP activity in both crude and purified preparations migrates as a 200-kDa entity in molecular sieve chromatography, a size approximately 4 times that of the purified GAP on SDS gels. This may imply that the GAP is a homotetramer, although we cannot exclude the possible existence of additional subunits that replace part of the 49-kDa subunits in the native complex. The fact that GAP activity was relatively resistant to treatment with 5 M urea indicated that the protein is capable of readily refolding to its native structure. Furthermore, ARF GAP activity could also be recovered following reverse phase chromatography in the presence of an organic solvent as well as after SDS-PAGE. Renaturation following SDS-PAGE was also observed with multiple forms of GAPs acting on members of the p21rho subfamily of GTP binding proteins(26) .

The high purification factor that was required to reach a homogeneous 49-kDa protein (about 10,000-20,000-fold) indicates that the ARF GAP has low abundance in liver cytosol. This is somewhat surprising considering the fact that ARF proteins are quite abundant in different cell types(4, 27) . A small amount of GAP may suffice if only a low proportion of the ARF pool is in the GTP-bound state or if the GAP has a high turnover rate. It is also possible that the cytosolic GAP represents only part of the cellular pool of this protein. Randazzo and Kahn (20) recently reported the detection and enrichment of ARF GAP activity associated with bovine brain membranes. In unpublished experiments we also observed ARF GAP activity in a crude membrane fraction from rat liver, but preliminary attempts to purify this activity were unsuccessful. The relationship between the cytosolic and membrane pools of ARF GAP remains to be explored.

The fact that ARF has undetectable intrinsic GTPase activity suggests that the ARF GAP is an essential terminator of ARF-regulated processes. ARF proteins in their GTP-bound form are required for coatomer binding to Golgi stacks (9, 10) and for the binding of clathrin adaptor particles to the trans-Golgi network(11, 12) . GTP hydrolysis is required for the dissociation of these proteins from Golgi-derived membranes and vesicles(17, 18) , a process in which an ARF GAP is most likely involved, thereby acting as an uncoating factor.

Randazzo and Kahn (20) have recently reported that ARF GAP activity in a partially purified preparation from bovine brain membranes is modulated by phospholipids. GAP activity was strongly stimulated by PIP(2) and was inhibited by phosphatidylcholine. We obtained qualitatively similar results using both crude and Resource Q-purified GAP preparations, indicating that phospholipid sensitivity is an intrinsic characteristic of the ARFbulletGAP complex. The stimulatory effects of phosphoinositides that we observed are significantly lower than those reported by Randazzo and Kahn, possibly reflecting the different source of ARF GAPs employed in the two studies (liver cytosol versus brain membranes) or differences in the GAP assay conditions. The former possibility is further suggested by the qualitative difference between the brain and liver preparations in their sensitivity to the synthetic phospholipid DMPC. Whereas DMPC was found to strongly inhibit brain ARF GAP(20) , the GAP from liver cytosol was not affected by DMPC. In fact, DMPC was routinely used in our assays to facilitate the loading of ARF with GTP, and unlike the preparation from brain(20) , GAP activity could be readily detected in crude liver cytosol in the presence of DMPC.

The effects of phospholipids on the ARF GAP may be related to a recently discovered role of ARF in the regulation of phospholipid metabolism(28, 29, 30) . ARF was identified as the cytosolic GTP binding protein that activates phospholipase D. Activated phospholipase D cleaves phosphatidylcholine to produce phosphatidic acid and choline. A feedback loop mechanism has been proposed (20) where following the activation of phospholipase D by GTP-bound ARF, an increase in local phosphatidic acid concentration (and possibly also a decrease in phosphatidylcholine concentration) brings about an increase in the activity of the ARF GAP, resulting in the hydrolysis of ARF-bound GTP and the cessation of phospholipase D activity. The fact that, like the ARF GAP, phospholipase D activity is also stimulated by PIP(2)(28) might suggest a direct relationship between the two proteins, but in preliminary experiments the GAP that we have purified was found to be devoid of phospholipase D activity. (^2)

Partial sequence analysis of the 49-kDa protein clearly demonstrates that the ARF GAP is a so far uncharacterized protein. The availability of the purified protein opens the way to a molecular characterization of the ARF GAP and to studying its role in the regulation of different ARF-dependent cellular pathways.

Addendum-We have recently isolated from a rat liver library cDNA clones with a 1245-base pair open reading frame encoding a polypeptide that includes all seven peptides that we have found in the 49-kDa protein. Coupled in vitro transcription/translation in reticulocyte lysate resulted in a protein that co-migrated on SDS-PAGE with the protein that we have purified, and the translated protein possessed ARF GAP activity.


FOOTNOTES

*
Work on the purification of the ARF GAP was initiated while D. C. was on a Sabbatical year with Dr. Rick Klausner at the Cell Biology and Metabolism Branch, National Institutes of Health. This work was supported by grants from the Israel Academy of Sciences and from the Fund for the Promotion of Research at the Technion. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 972-4-293408; Fax: 972-4-225153.

(^1)
The abbreviations used are: ARF, ADP ribosylation factor; GAP, GTPase-activating protein; PAGE, polyacrylamide gel electrophoresis; DMPC, dimyristoyl phosphatidylcholine; PIP(2), phosphatidylinositol 4`,5`-bisphosphate; DTT, dithiothreitol; MOPS, 4-morpholinepropanesulfonic acid; HPLC, high performance liquid chromatography; RNP, ribonucleoprotein; hnRNP, heterogeneous RNP; GTPS, guanosine 5`3-O-(thio)triphosphate.

(^2)
V. Chalifa, M. Liscovitch, and D. Cassel, unpublished observations.


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