From the Pulmonary-Critical Care Medicine Branch, NHLBI, National
Institutes of Health, Bethesda, Maryland 20892-1590 and
The Howard Hughes Medical Institute and the Department of
Molecular Physiology and Biophysics and Pharmacology, Vanderbilt
University School of Medicine, Nashville, Tennessee 37232
Arfaptin 1, a ~39-kDa protein based on the
deduced amino acid sequence, had been initially identified in a yeast
two-hybrid screen using dominant active ARF3 (Q71L) as bait with an
HL-60 cDNA library. It was suggested that arfaptin 1 may be
involved in Golgi functions, since the FLAG-tagged protein was
associated with Golgi membranes when expressed in COS-7 cells and could
be bound to Golgi in vitro in an ADP-ribosylation
factor (ARF)- and GTP
S-dependent, brefeldin A-inhibited
fashion. Arfaptin 2, found in the same two-hybrid screen as arfaptin 1, is 60% identical in amino acid sequence and may or may not have an
analogous function. We now report some effects of arfaptin 1 on ARF
activation of phospholipase D and cholera toxin ADP-ribosyltransferase.
Arfaptin 1 inhibited activation of both enzymes in a
concentration-dependent manner and was without effect in
the absence of ARF. Two ARF1 mutants that activated the toxin, one
lacking 13 N-terminal amino acids and the other, in which 73 residues
at the N terminus were replaced with the analogous sequence from ARL1,
were not inhibited by arfaptin, consistent with the conclusion that
arfaptin interaction requires the N terminus of ARF. This region has
also been implicated in phospholipase D activation, but whether the two
proteins interact with the same structural elements in ARF remains to
be determined. Arfaptin inhibition of the action of ARF5 and ARF6 was
less than that of ARF1 and ARF3; its effects were less on
nonmyristoylated than myristoylated ARFs. Arfaptin effects on guanine
nucleotide binding by ARFs were minimal whether or not a purified ARF
guanine nucleotide-exchange protein was present. These findings
indicate that arfaptin acts as an inhibitor of ARF actions in
vitro, raising the possibility that it has a similar role
in vivo.
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INTRODUCTION |
ADP-ribosylation factors
(ARF)1 (1), ubiquitous
~20-kDa GTP-binding proteins, are present in all eukaryotic cells
from Giardia to mammals and are essential for vesicle
formation at Golgi, endosomal, and probably nuclear membranes. Six
mammalian ARFs have been identified by cDNA cloning. Based on
deduced amino acid sequence, phylogenetic analysis, and gene structure,
they have been divided into three classes: class I, ARF1, -2, and -3;
class II, ARF4 and -5; class III, ARF6 (1). By definition, all ARFs
stimulate cholera toxin (CTA) ADP-ribosyltransferase activity. In
addition, all ARFs activate a specific phospholipase D (PLD) that can
serve as an effector in cellular signal transduction (2, 3). Whereas,
the domain necessary for CTA activation resides in the C-terminal
portion of the ARF molecule, activation of PLD is a function of an
N-terminal region (4). The identification and isolation of ARF
GTPase-activating proteins, termed ARF GAPs (5-7), and GEPs, or
guanine nucleotide-exchange proteins (8-10), have extended our
understanding of ARF action and its regulation. ARF GAP accelerates the
hydrolysis of GTP bound to ARF, yielding inactive ARF·GDP. ARF GEP
catalyzes the replacement of bound GDP by GTP to produce active
ARF·GTP. It has been recognized relatively recently that several
proteins initially characterized in other contexts exhibit ARF GEP
activity. These include proteins with Sec7 domains, such as p200 from
bovine brain (11), yeast Gea1 and Gea2 (12), and human ARNO (13) and
cytohesin 1 (14).
Arfaptin 1, a ~39-kDa protein based on the deduced amino acid
sequence, was identified in a yeast two-hybrid screen using dominant
active ARF3(Q71L) as bait with an HL-60 cDNA library (15). The
recombinant arfaptin2 bound
tightly to both myristoylated and nonmyristoylated ARF1 and ARF3 but
much less to ARF5 and ARF6. The native arfaptin, immunoprecipitated
from an HL-60 cell lysate, behaved as a molecule of ~44 kDa on gel
electrophoresis (15). The physiological role(s) of arfaptins remains to
be defined. It was suggested that arfaptin 1 may be involved in Golgi
functions based on observations that the FLAG-tagged protein was
associated with Golgi membranes when expressed in COS-7 cells and could
be bound to Golgi in vitro in an ARF- and
GTP
S-dependent, brefeldin A-inhibited fashion (15).
Arfaptin 2, which has 60% amino acid identity to arfaptin 1, may or
may not have an analogous function. Both of the proteins were
phosphorylated to a limited extent by protein kinase C (15). A
Rac-binding protein termed POR1, which may have a role in
Rac-dependent membrane ruffling, is identical to the
C-terminal 303 amino acids of arfaptin 2 (15).
To understand better the arfaptin-ARF interaction, we investigated some
effects of arfaptin 1 on ARF activation of CTA and PLD, and on ARF
guanine nucleotide exchange. To begin to define regions of the ARF
molecule that influence its interaction with arfaptin, native and
recombinant ARFs (with and without myristoylation) were studied as well
as chimeric molecules that include portions of the ARF amino acid
sequence along with that from human ARL1, an
ARF-like protein that is 56% identical in
sequence to human ARF1 (4) and which, unlike ARFs, does not
activate cholera toxin or rescue Saccharomyces cerevisiae
with the lethal
arf1
/arf2
double deletion (16).
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EXPERIMENTAL PROCEDURES |
Materials--
GST-arfaptin 1 (15) was added to assays as the
fusion protein (~67 kDa) in 50 mM Tris-HCl, pH 8.0, 10 mM glutathione. Amounts of vehicle were equal in all
assays. Mixed ARF (mostly ARFs 1 and 3 after DEAE and Ultrogel AcA 54 chromatography), native ARF1, ARF3, and ARF5 were purified from bovine
brain cytosol (11, 17, 18). Recombinant myristoylated mARF1, mARF3, and
mARF5 and nonmyristoylated rARF1, rARF3, and rARF5 were synthesized in Escherichia coli (19). Preparation of
13ARF1 (20) and ARF/ARL chimeric proteins (4) is published. ARF GEP was purified from
rat spleen cytosol through the heparin-agarose step (9). ARF-dependent phospholipase D was partially purified from
bovine brain membranes (21). Sources of other materials are published (8, 9).
Assay of ARF Activity--
ARF activity was assayed by three
methods, guanine nucleotide binding, CTA activation, and PLD
activation. All data reported are means of values from duplicate assays
and have been replicated twice or more except those in Table IV.
To assay nucleotide binding, ARF was incubated at 37 °C with or
without arfaptin and either [35S]GTP
S or
[3H]GDP for the indicated time. Protein-bound nucleotide
was collected on nitrocellulose for radioassay (10).
For assay of ARF activation of CTA, ARF was incubated with or without
arfaptin for 40 min at 37°C with 10 µM GTP
S in a
50-µl volume, then placed in an ice bath. After addition of CTA,
[14C]NAD and agmatine in a volume of 250 µl, samples
were incubated for 60 min at 30 °C and
[14C]ADP-ribosylagmatine was collected for radioassay
(8). The activity of partially purified ARF-dependent PLD
was assayed by a published method (4). Briefly, mixed lipids with
choline[methyl-3H]dipalmitoyl
phosphatidylcholine were added to PLD, ARF, and GTP
S with or without
arfaptin and incubated for 1 h at 37 °C before addition of
CHCl3/CH3OH/HCl, followed by centrifugation. [3H]choline in the aqueous phase was quantified by liquid
scintillation spectroscopy.
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RESULTS |
Effects of Arfaptin on Stimulation of Cholera Toxin
ADP-ribosyltransferase Activity by Native, Recombinant, and Mutant
ARFs--
In an effort to define the functions of arfaptin 1, its
in vitro effects on some known ARF activities were first
tested. Stimulation of cholera toxin ADP-ribosyltransferase activity by
native ARF3 (8) or ARF5 (18) was assayed with or without arfaptin. As shown in Fig. 1, inhibition of ARF
activity was dependent on, but not linearly proportional to, the
concentration of arfaptin. The activities of 10 pmol of native ARF1 and
ARF3 were similar and were similarly inhibited ~30% by 10 pmol and
60-70% by 40 pmol of arfaptin (Table
I). The activities of recombinant ARF proteins myristoylated or nonmyristoylated, synthesized in E. coli were variable and much lower than those of native proteins. Inhibition, which was dependent on arfaptin concentration, was apparently somewhat less for ARF5 and ARF6 than for ARF1 and ARF3 (Table I).

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Fig. 1.
Effect of arfaptin 1 on activation of CTA by
ARF3 or ARF5. Native ARF3, 0.2 µg, ~10 pmol ( ) or ARF5,
0.35 µg, ~17.5 pmol ( ) was incubated (50 µl final volume) for
40 min with the indicated amount of arfaptin 1 before assay of its
effect on CTA activity. In the absence of arfaptin 1, CTA activity was
increased 2.8 and 4.8 nmol of ADP-ribosylagmatine formed/hr by ARF3 and
ARF5, respectively. Activity of CTA alone, which was not inhibited by
arfaptin 1, was subtracted before calculation of ARF activity as
percentage of that in the absence of arfaptin 1 = 100%.
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Table I
Effect of arfaptin 1 on activation of CTA by ARFs and related proteins
The indicated amount of native or recombinant nonmyristoylated,
myristoylated ARF, or related protein was incubated without or with 10 or 40 pmol of arfaptin in a volume of 50 µl as described in Ref. 8,
except that 72 µg of cardiolipin replaced PS before assay of
activated ARF by its effect on CTA ADP-ribosyltransferase activity. CTA
activity without ARF or arfaptin (1.3 nmol/h) was subtracted from the
total to calculate the increment due to ARF = ARF activity. CTA
activities in the absence of ARF with 10 and 40 pmol of arfaptin were,
respectively, 1.4 and 1.2 nmol/h.
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CTA activity was enhanced by ARL73/ARF, which contains the first 73 amino acids of ARL1 and the last 108 of ARF1, but arfaptin did not
interfere with its activity (Table I). The considerably greater
activation by rARF1
13 (ARF1 lacking the N-terminal 13 amino acids)
likewise was not prevented by arfaptin. These data are consistent with
the possibility that arfaptin interactions require the N terminus of
ARF. ARF73ARL, a recombinant protein containing the first 73 amino
acids of human ARF1 and the last 108 of human ARL1 did not activate CTA
as previously reported (4).
Effects of Arfaptin on Stimulation of Phospholipase D by
Recombinant and Mutant ARFs--
In the absence of added ARF,
partially purified PLD from bovine brain exhibited a low level of
activity, probably due to ARF contamination (Table
II), a possibility consonant with its
inhibition by arfaptin. The same recombinant ARF preparations used for
the experiments in Table I stimulated PLD activity from 10- to 25-fold (Table II). In these assays, mARF3 and mARF5 were clearly more active
than nonmyristoylated forms. The dramatic activation by mARF3 was
virtually completely inhibited by arfaptin, which was less effective
against nonmyristoylated rARF3 and also against the ARF5 and ARF6
preparations (Table II). ARF activation of PLD required the N-terminal
domain of the ARF molecule as shown earlier (4). The chimeric ARF
73/ARL, containing the N-terminal 73 amino acids of ARF1, increased
activity 14-fold and was clearly inhibited by arfaptin (Table II). ARL
73/ARF with the C-terminal region of ARF1 and the N-terminal 73 amino
acids of ARL1 as reported (4) did not activate PLD nor did the mutant
rARF1
13, which lacks 13 amino acids at the N terminus, confirming
that the N terminus of the ARF molecule is important for PLD activation
as well as for arfaptin inhibition of CTA activation by ARF (Table I).
Whether arfaptin and PLD interact with the same structural determinants
in ARF remains to be determined.
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Table II
Effect of arfaptin on activation of phospholipase D by recombinant
and mutant ARFs
The indicated amount of recombinant ARF protein was assayed for
phospholipase D activation (4) with or without arfaptin 1 as indicated
(total volume 300 µl).
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Effect of Arfaptin on GTP
S and GDP Binding to
ARF3--
Replacement of ARF-bound GDP by GTP to produce the active
ARF·GTP (in the presence of 2 mM MgCl2) is
very slow in the absence of an ARF GEP. Although exchange can be
accelerated by decreasing MgCl2 to <1.0 mM
(9), it is known that the higher MgCl2 concentration enhances stability of ARF-nucleotide complexes and that at low MgCl2 concentrations ARF is rather unstable. Using 0.7 or
3.5 mM MgCl2, the time course of
[35S]GTP
S binding to ARF3 was determined with or
without arfaptin (Fig. 2). At the lower
MgCl2 concentration, the initial rate of binding was
10-fold that at 3.5 mM MgCl2. Arfaptin
increased the amount of [35S]GTP
S bound after longer
incubations, perhaps by stabilizing ARF, but the initial rate of
binding was not markedly accelerated. At 3.5 mM
MgCl2 [35S]GTP
S binding to ARF3 was much
slower (Fig. 2), and arfaptin had little effect. Replacement of
ARF-bound [3H]GDP with GDP from the medium was assayed
without and with arfaptin at two concentrations of MgCl2
(Fig. 3). At 0.1 mM
MgCl2 [3H]GDP binding to ARF3 was only 30%
higher than it was at 3.4 mM MgCl2. Binding was
slow, and the rate was approximately constant for 80 min. Replacement
binding of GDP was <20% greater with arfaptin than without it at both
MgCl2 concentrations (Fig. 3).

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Fig. 2.
Effect of arfaptin 1 on GTP S binding to
mARF3. mARF3 (1.2 µg, ~60 pmol) was incubated (100 µl final
volume) without ( , ) or with ( , ) 60 pmol of arfaptin, 4 µM [35S]GTP S (2.7 × 106 cpm), 1 mM EDTA, and either 0.7 ( , )
or 3.5 mM MgCl2 ( , ) at 37 °C for the
indicated time before collection of proteins on nitrocellulose for
radioassay. Arfaptin 1 alone did not bind GTP S.
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Fig. 3.
Effect of arfaptin 1 on GDP binding to
ARF3. ARF3 (0.3 µg, ~15 pmol) was incubated (100 µl final
volume) with 4 µM [3H]GDP, without ( ,
) or with 60 pmol of arfaptin 1 ( , ), 0.7 mM EDTA,
and 1.2 ( , ) or 3.4 mM MgCl2 ( , )
at 36.5 °C for the indicated time before collection of proteins on
nitrocellulose for radioassay. Arfaptin 1 alone did not bind GDP.
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Effect of MgCl2 Concentration and Arfaptin on GTP
S
Binding to mARF5--
Stable binding of GTP
S to mARF5 required
relatively high concentrations of MgCl2 (Fig.
4). Binding was maximal with 2-3
mM MgCl2 and much lower with 1 mM
MgCl2 (in the presence of 1 mM EDTA). Arfaptin
1 increased GTP
S binding 4-fold with 1 mM
MgCl2, but only ~40% with 2-3 mM
MgCl2. The enhancement by arfaptin 1 (Figs. 2 and 4) could
reflect slowing of nucleotide dissociation as a result of ARF-GTP
S
interaction with arfaptin.

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Fig. 4.
Effect of MgCl2 concentration and
arfaptin 1 on GTP S binding to mARF5. mARF5 (2 µg, ~100
pmol) was incubated (50 µl final volume) with 4 µM
[35S]GTP S, 1 mM EDTA, and the indicated
concentration of MgCl2 without ( ) or with 60 pmol of
arfaptin 1 ( ) for 60 min at 37 °C before collection of proteins
on nitrocellulose for radioassay. Arfaptin 1 alone did not bind
GTP S.
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Effect of Arfaptin on Release of Bound [3H]GTP
S
from ARF3--
ARF3 (15 pmol) with 2.4 pmol of
[3H]GTP
S bound was incubated for 40 min at 37 °C
with or without arfaptin 1 and several concentrations of GDP or GTP
S
(Table III). Differences in
[3H]GTP
S bound differed <10% under all conditions,
although binding was consistently slightly higher in all samples that
contained arfaptin 1. Thus, arfaptin 1 displayed little, if any, ARF
GEP activity.
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Table III
Effect of arfaptin 1 on release of [35S]GTP S bound to ARF3
Native ARF3 (750 nM) was incubated first with 4 µM [35S]GTP S, 2 mM EDTA, and
1.12 mM MgCl2 for 40 min at 37 °C then cooled on
ice. Samples (20 µl, 15 pmol) were added to 80 µl of solution to
achieve final concentrations of 0.48 mM EDTA and 3.18 mM MgCl2 without or with arfaptin (60 pmol). After
incubation for 40 min at 37 °C with the indicated concentrations of
unlabeled GTP S or GDP, proteins were collected on nitrocellulose for
radioassay. At the beginning of the final 40-min incubation, bound
[35S]GTP S was 2.45 pmol without and 2.43 pmol with
arfaptin. After incubation for 40 min without unlabeled nucleotide,
2.33 pmol were bound in the absence of arfaptin and 2.54 pmol in its
presence.
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Effect of Arfaptin on [35S]GTP
S or
[3H]GDP Binding to ARF3 in the Presence of GEP--
To
determine whether arfaptin interfered with GEP stimulation of
nucleotide binding, ARF3 was incubated with GEP with or without arfaptin 1 and [35S]GTP
S or [3H]GDP.
Binding of [35S]GTP
S or [3H]GDP was
slightly greater in the presence of arfaptin, than in its absence
(Table IV). These small differences could
reflect slowed dissociation of nucleotides.
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Table IV
Effect of arfaptin on [35S]GTP S or [3H]GDP
binding to ARF3 in the presence of GEP
Native ARF3 (0.3 µg, ~15 pmol) was incubated with the indicated
amount of arfaptin in a total volume of 40 µl (3 mM
MgCl2, 1 mM EDTA) for 10 min at 37 °C. Samples
were placed on ice while GEP, 3 mM MgCl2, and 4 µM [35S]GTP S or [3H]GDP (final
concentrations) were added in a volume of 10 µl followed by
incubation at 37 °C for 40 min before collection of proteins on
nitrocellulose for radioassay. GEP had been purified from rat spleen
cytosol through the heparin-agarose step (9).
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ARF3 was incubated with GTP with or without ARF GEP and/or arfaptin,
then assayed for stimulation of CTA activity. Without ARF GEP, ARF1-3
stimulation of CTA activity was inhibited by addition of arfaptin in a
concentration-dependent manner (Table
V) as shown also in Fig. 1 and Table I.
After incubation with ARF GEP, ARF1-3 increased CTA activity ~120%
above the level induced by ARF1-3 alone, and its activity was likewise
inhibited by arfaptin. ARF5 was not activated by ARF GEP and arfaptin
inhibited its activity with or without GEP, as shown also in Table I
and Fig. 1.
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Table V
Effect of arfaptin on CTA activation by ARFs with and without GEP
Native ARF proteins were incubated for 10 min at 37 °C without or
with arfaptin 1 as indicated (total volume 40 µl). After addition of
20 µM GTP and 4 mM MgCl2 (final
concentrations) with or without GEP (final volume 50 µl), incubation
was continued for 40 min at 37 °C. Activated ARF was then assayed by
its effect on CTA ADP-ribosyltransferase activity and is reported as
the increment in CTA activity due to addition of ARF.
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DISCUSSION |
Arfaptin inhibited the actions of all classes of ARFs on CTA and
PLD activities. However, ARF1 and ARF3 (class I) were more sensitive
than ARF5 (class II) and ARF6 (class III) to its inhibitory effects
(Tables I and II). Studies on the interaction of recombinant human
ARF1, -3, -5, and -6 with immobilized GST-arfaptin fusion proteins
previously revealed an extent of stable association decreasing in that
order with negligible binding of ARF6 (15). Because of the demonstrated
importance of the ARF N terminus in its association with arfaptins, the
several differences among ARFs in amino acid sequences of this region
(1) may provide clues to structural requirements for the interaction.
ARF6, in fact, lacks 4 of the first 11 residues present in the other
ARFs. Interaction of ARF with arfaptin was also demonstrated in a more
physiological setting. Brefeldin A inhibited the
GTP
S-dependent association of arfaptin 1 and ARF from
HL-60 cell cytosol with rat liver Golgi membranes (15), consistent with
the demonstrated association of arfaptin with activated ARF and not
ARF-GDP. When ARF-depleted cytosol was used, recombinant ARF3 was able
to support arfaptin binding to Golgi. Arfaptin 1 was initially
described as a widely distributed cytosolic protein target of activated
ARF, which recruited it to Golgi membranes (15). A clone for a second
protein also containing 341 amino acids (60% identical), termed
arfaptin 2, was studied less extensively but has properties similar to
those of arfaptin 1 (15).
Myristoylation was demonstrated as not essential for ARF3 interaction
with arfaptin 1 in the yeast two-hybrid screen, although its influence
was not directly assessed (15). In the functional studies reported
here, no consistent effect of myristoylation on the inhibitory action
of arfaptin was observed (Tables I and II). The activity of CTA was not
inhibited by arfaptin in the absence of ARF, and the effect of arfaptin
on the very low activity of PLD in the absence of ARF is probably due
to ARF contamination of the PLD preparation (data not shown).
Percentage inhibition of PLD activation by arfaptin was, in general,
greater than that of CTA activation. It may be relevant that the
N-terminal 13 amino acids of ARF that were required for PLD activation
were also required for arfaptin inhibition of cholera toxin activation
(Tables I and II). Interpretation of the difference is complicated,
however, by the major differences in assay conditions. It is necessary also to take into account the differences in effects of specific phospholipids on GTP binding by individual ARFs that can influence markedly the rate at (and extent to) which each becomes activated. Differences in behavior of the chimeric proteins are seemingly more
straightforward in determining the structural requirement for
interaction of ARF and arfaptin. As expected, ARF73ARL did not activate
CTA but did activate PLD (4), and its action on the latter was
inhibited by arfaptin (Table II). Neither ARL73ARF nor rARF1
13
significantly activated PLD in these experiments as reported (4). Both
did, however, activate CTA, and arfaptin had no effect on their action,
consistent with the suggestion of Kanoh et al. (15) that the
N terminus of ARF is important for its interaction with arfaptin.
No GEP-like activity of arfaptin was detected. Nor was there any
evidence that arfaptin 1 interfered with the action of a GEP partially
purified from rat spleen. Whether nucleotide binding or release was
measured, the amount of GTP
S or GDP bound to several ARF
preparations was consistently slightly higher in the presence of
arfaptin 1 than in its absence. This was true also when GEP was added
to accelerate guanine nucleotide exchange. Since this effect of
arfaptin 1 was greater with longer times of incubation, it could
reflect stabilization of the ARF protein as a result of its association
with arfaptin 1. The effect was larger with GTP
S than with GDP
(Table IV), which would be consistent with the demonstrated preference
of arfaptin 1 for interaction with activated ARF (15), and the
possibility that the complex of activated ARF and arfaptin 1 is a
functional entity.
Because arfaptin was discovered using the yeast two-hybrid system with
a presumably GTP-liganded ARF mutant as bait, ARF was apparently able
to interact with arfaptin in these cells (15). In addition, arfaptin in
HL-60 cell cytosol was recruited by ARF to Golgi membranes in a
GTP
S-dependent and brefeldin A-sensitive manner (15). It
remained unclear, however, from the prior studies how its interaction
with arfaptin would affect the ability of ARF to activate either PLD or
cholera toxin. The experiments reported here established that, even
though activation of PLD and cholera toxin involves two different
domains of ARF (4), arfaptin interferes with both processes. Based on
these data, it would appear that once ARF recruits arfaptin to a
membrane, dissociation of ARF from arfaptin would need to occur prior
to activation of phospholipase D.
The present findings demonstrate that arfaptin is a potent inhibitor of
ARF actions on CTA and PLD in vitro, but has minimal effects
on GEP-catalyzed guanine nucleotide binding to ARF. Among the questions
arising from this work are how the association of ARF with arfaptin
modifies its interaction with GAPs and the hydrolysis of bound GTP. ARF
plays a major role in the regulation of vesicular trafficking through
the Golgi, and it will be important to demonstrate whether and how this
is modified by arfaptin. These and other questions are subjects of
current study.