(Received for publication, September 20, 1996, and in revised form, November 6, 1996)
From the Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892
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.
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 subunit of the adenylyl cyclase-stimulatory
G protein (Gs
) (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 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 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.
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 ARF1The 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
CC
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
TG
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
TCA
CC
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
TC
TG
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--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.
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
CAACCAAGTC-3
changed a
ScaI restriction site in the ampr
gene of pT7 into an MluI restriction site (underlined). The
mutagenic primer 5
-TACAAGCTTAAGCTGG
TGAGATCGTGACCACC-3
changed Gly40 of ARF1 to Asp. The mutagenic primer
5
-GGTGAGATCGTGACC
CCATTCCCACCATAGGC-3
changed
Thr45 of ARF1 to Pro. The mutagenic primer
5
-TACAAGCTTAAGCTGG
TGAGATCGTGACC
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).
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
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/
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-
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).
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)
[-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.
GTPS 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]GTP
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.
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 [-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.
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 GTPS
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
GTP
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.
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]GDPS (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 GDP
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.
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 [-32P]GTP, whereas the non-ARF domain,
p5, did not. Binding of GTP
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 GTP
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).
The specificity of guanine nucleotide binding was assessed by adding
unlabeled nucleotide together with 4 µM
[35S]GTPS in the binding buffer. ATP, TTP, and CTP (up
to 100 µM) failed completely to compete with GTP
S for
binding to p3 and p8, whereas 100 µM GTP or GDP decreased
[35S]GTP
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.
Phospholipid Requirement for GTP
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 GTPS binding to
ARF (17). As shown in Table I, in the presence of
several detergents GTP
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, GTP
S
binding by p3 was five times that by p8 (Table I).
|
Like other members of the ARF family, ARD1 activates CTA
ADP-ribosyltransferase activity (1, 23). As it was for GTPS binding
(Table I), cardiolipin was the most effective phospholipid for
activation of CTA (Table II). Despite supporting
relatively low GTP
S binding, however, the detergent Tween 20 enhanced CTA activity induced by p3 or p8 to levels comparable to those
with cardiolipin (Table II).
|
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).
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 GTPS 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).
|
Stimulation of CTA activity by p3 was similar with GTP and GTPS,
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 GTP
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).
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.
To investigate a possible effect of the amino terminus of the mutant
ARF1(39-45p3), the first 15 amino acids were deleted to create
15ARF1(39-45p3). Hydrolysis of the GTP bound to
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
15ARF1(39-45p3) approximated that on p3 (Fig. 9). In addition,
15ARF1(39-45p3) binding to GST-p5 immobilized on GSH-Sepharose beads
exceeded binding of ARF1(39-45p3) (Fig. 7, lane 4).
GDP dissociation from 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.
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 G 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 G
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 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 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 G proteins, ARD1 appears to
possess an intrinsic regulator of GTPase activity. However, p5 differs
from the G
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 G
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.
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.