(Received for publication, September 14, 1994; and in revised form, December 23, 1994)
From the
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.
ADP-ribosylation factors (ARFs) ()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 GTP
S 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.
GAP activity was assayed
in a final volume of 10 µl in the presence of 5 mM MgCl, 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 [
-
-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 [
-
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.
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.
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).
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 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
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), 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.
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 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
ARF
GAP 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(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. (
)
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.