From the Institut de Pharmacologie Moléculaire et Cellulaire, CNRS-UMR 6097, 660 Route des Lucioles, 06560 Valbonne Sophia-Antipolis, France
Received for publication, April 12, 2001, and in revised form, May 3, 2001
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ABSTRACT |
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ARF1 and ARF6 are distant members of the
ADP-ribosylation factor (ARF) small G-protein subfamily. Their distinct
cellular functions must result from specificity of interaction with
different effectors and regulators, including guanine nucleotide
exchange factors (GEFs). ARF nucleotide-binding site opener
(ARNO), and EFA6 are analogous ARF-GEFs, both comprising a
catalytic "Sec7" domain and a pleckstrin homology domain. In
vivo ARNO, like ARF1, is mostly cytosolic, with minor
localizations at the Golgi and plasma membrane; EFA6, like ARF6, is
restricted to the plasma membrane. However, depending on conditions,
ARNO appears active on ARF6 as well as on ARF1. Here we analyze the
origin of these ARF-GEF selectivities. In vitro, in the
presence of phospholipid membranes, ARNO activates ARF1 preferentially
and ARF6 slightly, whereas EFA6 activates ARF6 exclusively; the
stimulation efficiency of EFA6 on ARF6 is comparable with that of ARNO
on ARF1. These selectivities are determined by the GEFs Sec7 domains
alone, without the pleckstrin homology and N-terminal domains, and by
the ARF core domains, without the myristoylated N-terminal
helix; they are not modified upon permutation between ARF1 and ARF6 of
the few amino acids that differ within the switch regions. Thus
selectivity for ARF1 or ARF6 must depend on subtle folding differences
between the ARFs switch regions that interact with the Sec7 domains.
The small GTP-binding protein ADP-ribosylation factors
(ARFs),1 originally
identified as cofactors for the ADP-ribosyltransferase activity of
cholera toxin, form a distinct six-member subgroup within the small G
proteins (1). Like all G proteins, the ARFs cycle between inactive
GDP-bound and active GTP-bound states, which differ mainly by the
conformation of two regions called switch I and switch II. These two
switches constitute a major site of interaction with regulator and
effector proteins, including the guanine nucleotide exchange factors.
But the ARFs differ from all other small G proteins by a myristoylated
N-terminal amphipathic helix, which also changes
conformation between the ARF-GDP and ARF-GTP states and is responsible
for the GTP-dependent binding of ARF to the phospholipid
membrane (2-4). The ARFs are key regulators of vesicular trafficking
in eukaryotic cells. The best characterized members of the family, ARF1
and ARF6, are the most distant and appear to have very distinct
functions. ARF1, which is mostly cytosolic, is clearly involved in
intra-Golgi transport by regulating coat protein recruitment onto the
Golgi membranes (5, 6); an additional possible role of ARF1 in focal
adhesion assembly in regulating paxillin recruitment onto the plasma
membrane has recently been suggested (7). ARF6, which localizes mostly
at the cell periphery, has been implicated in regulating endocytosis and the organization of the cortical actin cytoskeleton (8-10).
Guanine nucleotide exchange factors (GEFs) mediate the activation of
G-proteins by stimulating the dissociation of the bound GDP that
rate-limits the binding of GTP. About 10 ARF-GEFs have been identified.
They all share a conserved domain of about 200 amino acids, homologous
to a portion of the Sec7 protein (11), which bears the nucleotide
exchange catalytic activity (12, 13). The ARF GEFs can be divided into
two major classes based on their size and primary sequence; one is a
high molecular weight class composed of the GEA/GBF and Sec7/BIG
subgroups, and the other is a low molecular weight class. This latter
class shares a common structure organization in which the catalytic
Sec7 domain is always linked to a PH domain specifically interacting
with membrane-embedded polyphosphoinositides. This small ARF-GEF class can be further divided in the ARNO/cytohesin and EFA6 subclasses. The
human ARNO/cytohesin subclass includes four members that are more than
77% identical: ARNO, cytohesin-1, ARNO3, and cytohesin-4; their
N-terminal region contains a coiled-coil domain. The EFA6 subclass
includes three members: EFA6 and two close analogs, Tic and the coding
sequence KIAA0942, which have been deposited in data bank but not yet
characterized. The N-terminal domain of EFA6 contains a proline-rich
motif, and the long C-terminal extension to the PH domain contains a
coiled-coil and two proline-rich motifs.
Within one cell several ARF proteins coexist with several GEFs, raising
the question of the different functions of the various ARFs and of the
specificities of their interactions with the various GEFs. We and
others (14, 15) observe that overexpression of ARNO leads to
fragmentation and loss of function of the Golgi complex, which suggests
a role for ARNO in ARF1 activation in the Golgi. This suggestion was
reinforced by the observation of a Golgi localization for the
N-terminal coiled-coil domain of ARNO fused to green fluorescent
protein (16). But another experiment indicated a recruitment to the
plasma membrane of adipocytes of full-length ARNO fused to green
fluorescent protein and of a green fluorescent protein-PH domain of
ARNO upon insulin stimulation of phosphatidylinositol 3-kinase (17);
moreover, a small fraction of overexpressed ARNO and ARF6 appeared in
epifluorescence microscopy to partially colocalize at the plasma
membrane (18). Overall these results suggest that the ARNO/cytohesins
are able to localize at and probably act on both Golgi and plasma
membranes, but their ARF substrates at these two localizations are not
clearly identified. On the other hand EFA6 localizes at the plasma
membrane, where it couples actin cytoskeleton remodeling with endocytic
trafficking (19). This is compatible with a role of EFA6 in ARF6
activation. Indeed, exogenous expression of EFA6 or of ARF6-Q67L, a
constitutively active ARF6 mutant, induces large plasma membrane
invaginations enriched in polymerized actin where both proteins
localize (9, 19). Likewise, both EFA6 and ARF6-Q67L decrease the rate
of transferrin uptake. This phenotype is dependent on the nucleotide exchange activity of EFA6, as a mutant devoid of exchange activity has
no effect on transferrin internalization (19).
In the present study, we analyze in vitro, but in the
presence of lipid membrane, the specificities for ARF1
versus ARF6 of ARNO and EFA6 constructs. We also study the
influence of the PH domain of ARNO on the substrate specificity.
Finally, we try to determine by site-directed mutagenesis within the
switch regions of the ARFs the molecular basis of their specificity of
interaction with their GEF.
Materials--
Egg phosphatidylcholine, egg
phosphatidylethanolamine, brain phosphatidylserine, and azolectin
were purchased from Sigma. Brain phosphatidyl-4,5-bisphosphate
((4,5)PIP2) was from Avanti Polar Lipids (Birmingham, AL).
[3H]GDP was from Amersham Pharmacia Biotech, and
unlabeled nucleotides were from Sigma.
Expression and Purification of Recombinant
Proteins--
Myristoylated ARF1 and ARF6 were expressed in
Escherichia coli by co-expression with a yeast
N-myristoyltransferase (20) and purified as previously
described (3, 21). The myrARF1 protein is essentially purified as a
GDP-bound form, whereas myrARF6 is totally in the GTP-bound form.
Before use, the concentrated myrARF6 was loaded with GDP by a 45-min
incubation at 37 °C in a buffer containing 2 mM GDP and
1 µM free Mg2+.
N-terminal-truncated [
For production of full-length EFA6, BL21(DE3) bacteria were transformed
with the pET3a-EFA6 plasmid. Transformed cells were grown at 37 °C
to A600 = 0.6. The expression was induced with 0.1 mM
isopropyl-1-thio- Preparation of Phospholipid Vesicles--
Large unilamellar
vesicles of azolectin were prepared as described in Szoka and
Papahadjopoulos (22) and extruded through a 0.1 µM pore
polycarbonate filter (Isopore, Millipore). Vesicles of defined
composition were prepared as described (23). Concentration of each
phospholipid is indicated for each experiment in the legend.
Fluorescence Measurements--
The large increase in intrinsic
fluorescence of ARF-GTP over ARF-GDP (24) was used to monitor in real
time ARF activation upon GDP to GTP exchange. All measurements were
performed at 37 °C in HKM buffer (50 mM Hepes, pH 7.5, 100 mM KCl, 1 mM MgCl2, 1 mM dithiothreitol) supplemented with azolectin vesicles
(final lipid concentration 0.4 g/liter). The protein was diluted into the cuvette (0.5 µM myrARF with 50 µM GDP
or 1 µM GDP Dissociation Assay--
MyrARF protein ( ARF6-GDP Is Fully Soluble in Vitro, like ARF1-GDP, and Has a Higher
Spontaneous GDP Exchange Rate--
Upon production in bacteria and
purification as described under "Experimental Procedures," the
full-length myristoylated ARF1 protein was obtained as a GDP-bound
form, whereas myrARF6 was totally in the GTP-bound form. Before use the
concentrated myrARF6 was fully loaded with GDP by incubation at
37 °C for 45 min in a buffer containing 2 mM GDP and 1 µM free Mg2+ (1 mM total
Mg2+ and 2 mM EDTA). Returned to a medium with
a physiological magnesium concentration, myrARF6-GDP remained fully
soluble in vitro in the presence of phospholipid vesicles.
This contrasts with the in vivo situation where ARF6 appears
mainly membrane bound.
The kinetics of GDP/GTP exchange in myrARF1 and myrARF6 were followed
in real time by fluorescence (see "Experimental Procedures"); the
rates of nucleotide exchange are monitored as the rates of tryptophan
fluorescence increase in ARF upon its trans-conformation from ARF-GDP
to ARF-GTP. In the absence of GEF in vitro at millimolar magnesium concentration and in the presence of azolectin vesicles, myrARF1-GDP spontaneously exchanged its bound nucleotide very slowly
(Fig. 1, A and B).
Under the same conditions the spontaneous GDP/GTP exchange in ARF6 was
about 15 times faster than in ARF1 (Fig. 1, C and
D). Altogether these results might indicate that in cells
ARF6 is primarily in its GTP-bound form associated to the membrane or
that there exists an ARF6-GDI (GDP dissociation inhibitor) that would
retain ARF6-GDP at the membrane.
ARNO Preferentially Activates myrARF1 but Also myrARF6, whereas
EFA6 Exclusively Activates myrARF6--
We then compared the activity
of the purified full-length exchange factors, ARNO and EFA6, also
produced in bacteria (see "Experimental Procedures"), on
full-length myristoylated ARF1 and ARF6. The catalytic efficiency of a
GEF is best expressed by the fold stimulation of the spontaneous
nucleotide exchange rate of the substrate protein. 50 nM
ARNO increased about 20-fold the nucleotide exchange rate in ARF1 but
only 5-fold the nucleotide exchange rate in ARF6 (Fig. 1, A
and C). Conversely, 50 nM EFA6 increased the
rate of nucleotide exchange in ARF6 about 17-fold, whereas it had no
detectable effect on the rate of nucleotide exchange in ARF1 (Fig. 1,
B and D). Thus ARNO is rather selective for ARF1,
and EFA6 is strictly specific for ARF6. Furthermore, in terms of
fold-stimulation of the spontaneous exchange rate, the efficiency of
ARNO on ARF1 is about the same as that of EFA6 on ARF6.
ARNO PH Domain Interaction with (4,5)PIP2 Increases the
Activity of ARNO on both ARF1 and ARF6 and Does Not Change Its
Selectivity--
We have previously shown that the nucleotide exchange
activity of ARNO on myrARF1 is increased in the presence of
(4,5)PIP2-containing vesicles, and that this stimulation is
strictly dependent on the PH domain of ARNO (12). In addition, we have
observed that (4,5)PIP2 had no effect on the nucleotide
exchange activity of ARNO when tested on a soluble mutant of ARF1
lacking the first 17 amino acids, [ The Specificities of ARNO and EFA6 for Distinct ARFs Lie within the
Sec7 Domain of the GEFs and the Core Domain of the ARFs--
We had
previously shown that the isolated Sec7 domain of ARNO was sufficient
to catalyze the guanine nucleotide exchange reaction on myristoylated
ARF1 in the presence of azolectin vesicles (12). Moreover, in the
absence of any phospholipids, the Sec7 domain of ARNO could stimulate
nucleotide exchange in a soluble [ Residues That Differ between ARF1 and ARF6 within the Switch I and
II Regions Do Not Determine the Specificities of the ARF-Sec7 Domain
Interaction--
The ARFs switch I and II regions are the principal
sites of interaction with the GEFs. The first step of an ARF
interaction with its GEF is the docking of ARF-GDP onto the Sec7
domain, as modeled in Fig. 4 (26). Upon
conformational change of the switch I and II of ARF, which interact
with the hydrophobic groove region of Sec7, the Glu-156 "glutamic
finger" of Sec7 dislodges the GDP from ARF, resulting in a high
affinity nucleotide-free complex (25-27). Within the switch I and II
regions, ARF1 and ARF6 have identical primary sequences except for
seven residues exposed on the surface of the molecule. To test if these
seven residues account for the selectivity of the interaction between
ARF1-ARNO and ARF6-EFA6, we produced mutants of [ Determining the specificities of the various ARF-GEFs for their
substrates is fundamental for deciphering the cellular functions of the
different ARF proteins. The very different cellular functions of ARF1
and ARF6 must be related to their distinct cellular localizations and
to specificities of interactions with different GEFs. In contrast with
ARF1 and all the other ARFs, ARF6 appears mainly membrane-bound in the
cells (28-30). The cellular localizations of the ARF-GEFs of the small
ARNO/cytohesin and EFA6 types seemed to coincide, respectively, with
that of ARF1 and ARF6. This strongly suggested that ARNO would be
specific to ARF1 and EFA6 specific to ARF6, as was proposed in the
initial studies characterizing ARNO and EFA6 (14, 19). However,
depending on the cellular context ARNO has been found to also partially
localize with and activate ARF6 (17, 18, 31). In this study we have
further analyzed in vitro the ARF1 versus ARF6
specificities of ARNO and EFA6. We first noticed that, at physiological
magnesium concentration and in the presence of phospholipid vesicles,
myristoylated ARF6-GDP is soluble and that its spontaneous GDP/GTP
exchange rate is 15 times higher than that of ARF1-GDP. We then
demonstrated that the full-length ARNO catalyzes the GDP-GTP exchange
reaction preferentially for ARF1, and the full-length EFA6 catalyzes
the GDP-GTP exchange reaction exclusively for ARF6. It is also worth
noting that the two GEFs have very comparable activities on their
specific substrate.
Next, we attempted to determine whether the PH domains of ARNO and EFA6
are implicated in the GEF specificities for the ARFs. The PH domain of
ARNO has been shown to mediate membrane association via specific
interaction with polyphosphoinositides (12, 23, 32). Here we observed
that (4,5)PIP2 incorporated into lipid vesicles does not
modify the selectivity of ARNO towards ARF proteins. These data
demonstrate that the PH domain participates in the membrane
localization of ARNO but is not involved in the specificity of the
interaction of ARNO with ARF1. Recently, Knorr et al.
proposed that the interaction of cytohesin-1 with phosphoinositides
could regulate its GEF activity and control its ARF specificity (33). They observed that the addition of PIP2 or phosphatidyl
inositol 1,4,5-trisphosphate (PIP3) into liposomes strongly
suppressed the activity of cytohesin-1 on ARF6, whereas it increased
its activity on ARF1. Since ARNO and cytohesin-1 are 82% identical and
90% in their PH domain, it is surprising that they behave differently
in the presence of PIPs. The differences may arise from the methods of
purification and the preparation of the ARF proteins used in the assay.
We further confirmed that the catalytic Sec7 domains of ARNO and EFA6
alone, without their adjacent PH domain and N- and C-terminal
extensions, contain all the determinants of their specificities for
ARF1 and ARF6.
The coiled-coil motif of ARNO seems to be a Golgi-targeting signal
(16). Indeed, a green fluorescent protein fusion of the coiled-coil
domain predominantly localizes to the Golgi apparatus. A single
substitution of one of the hydrophobic residues is sufficient to alter
this localization. These results suggest that the localization at the
Golgi apparatus of ARNO is regulated by the coiled-coil domain, whereas
its localization at the plasma membrane is controlled by the PH domain
via phosphatidyl inositol 1,4,5-trisphosphate (PIP3)
binding. EFA6 also contains both a PH and a coiled-coil domain. It has
been clearly demonstrated that the PH domain is responsible for the
strong association of EFA6 to the plasma membrane (19). However, no
information is available regarding the precise function of the
coiled-coil domain. In addition, EFA6 contains some other adaptor
modules such as a few proline-rich regions. All these domains may be
involved in the formation of a multi-protein complex serving as a
central platform to coordinate the plasma membrane dynamics with the
actin cytoskeleton remodeling.
As for the ARF1 and ARF6 substrates, their differential recognition by
the GEF Sec7 domains is not related to their very different N-terminal
amphipathic helix motifs. Thus the specificity determinants must be
contained within the "core" of the ARFs, most probably within the
effector region, including the switch I and switch II domains that are
known to strongly interact with the GEF (27). The primary sequence of
these two switch regions is strictly identical in ARF1 and ARF6, except
for seven residues. We investigated whether these few differences were
sufficient to account for the different substrate specificities we
observed. Individual substitutions of each of these different residues
between ARF1 and ARF6 do not affect the specificity of the ARF1-Sec7
interaction. Thus these seven distinct residues do not define the
different selectivities of interaction of the ARFs with the Sec7
domains. The recently resolved crystallographic structure of ARF6 in
the GDP-bound form (34) appears similar to that of ARF1-GDP, except in
the switch regions; the two switch I regions have comparable
conformations but are markedly displaced by several Angströms,
and the switch II of ARF6-GDP is structurally ordered, in contrast to
the switch II of ARF1-GDP, which is flexible. Thus the specificity of
the ARF-GEF interaction does not result from primary sequence within the switch regions but rather from a folding difference dictated to the
switch regions from elsewhere in the protein.
In situ ARF6 appears mainly membrane-bound, in contrast with
the other ARFs, which appear mainly cytosolic (28-30). But in our
hands, in vitro myristoylated ARF6 remained fully soluble when fully converted to the GDP form, even in the presence of lipid
membrane. This could suggest that in situ the
membrane-associated ARF6 is primarily in its GTP-bound form. However,
it seems unlikely that ARF6 could be constitutively activated in
vivo. Indeed, we have previously shown that the effects of
overexpression of EFA6 are mediated through the activation of ARF6 and
can be mimicked by the expression of ARF6-Q67L, a constitutively
activated form of ARF6 (19). Therefore, we rather propose that in
vivo a large pool of ARF6-GDI (GDP dissociation inhibitor) is
responsible for retaining ARF6-GDP at the membrane and slowing down the
GDP dissociation rate.
Our in vitro studies provide information as to how the GEFs
could control in vivo the specific functions mediated by
their cognate ARF. Our previous studies demonstrated that the formation of the complex between ARF1 and ARNO, which precedes the activation of
ARF1 by GDP/GTP exchange, occurs at the membrane (35, 36). In
vitro and in vivo ARF proteins appear soluble in their
inactive GDP-bound state, whereas the GTP form is strongly associated
with the phospholipid membranes, suggesting that the specific
localization of the GEF determines the membrane site where the ARF
protein will be activated and consequently anchored to the membrane.
Regarding ARNO, different laboratories using various cellular models
and assays led to controversial conclusions as to its role of GEF for
ARF6 (14, 18, 19, 31, 37, 38). Although it is well accepted that
in vitro ARNO works better on ARF1, two observations on the
effects of the fungus toxin brefeldin A (BFA) have been used to support
its role on ARF6; it was observed long ago that the Golgi localization
of ARF1 is BFA-sensitive, whereas ARF6 localization at the cell
periphery is not affected by BFA (9). Then, in vitro studies
demonstrated that the GEF activity of ARNO was not sensitive to BFA
(12, 39, 40). The conjunction of these two observations suggested that
ARNO is the BFA-insensitive ARF6 GEF. However, a high molecular weight
BFA-insensitive ARF-GEF, GBF1, which does not localize at the periphery
but to the Golgi apparatus, has recently been found (41). Thus
BFA-insensitive GEFs are probably not restricted to the activation of ARF6.
Several groups have detected by subcellular fractionation or
immunofluorescence a fraction of ARNO at the plasma membrane together
with ARF6 (18, 31, 37, 42). However, although ARF1 is enriched in the
Golgi area, it is predominantly a soluble protein. Therefore, one
cannot exclude that the fraction of ARNO detected at the plasma
membrane could recruit ARF1 onto it as well. In agreement with this
hypothesis, it has been recently described a role for ARF1 in the
recruitment of paxillin to the focal adhesion complexes (7). A thorough
localization analysis of ARF1 at the cell periphery is needed.
In conclusion, our results suggest that EFA6 and/or its two close
analogs Tic and the coding sequence KIAA0942 are the major GEFs for
ARF6 and that ARNO is mostly involved with ARF1 activation. The first
indication is that in vitro but in the presence of
phospholipid membranes at a physiological magnesium concentration,
full-length ARNO is less efficient on ARF6 than on ARF1. This
specificity of ARNO for ARF1 is not affected by the interaction of its
PH domain with (4,5)PIP2, which implies that in
vivo it would not depend on the phosphoinositide composition of
the membrane. The specificity of ARNO for ARF1 is entirely contained
within the Sec7 domain of ARNO, and the Sec7 domain of EFA6, which is
very different from that of ARNO (only 30% identity), achieves a
markedly better activity on ARF6. Moreover, the analysis of this
in vitro study appears consistent with our earlier results
on the effects of the overexpression of these two GEFs in
vivo (14, 19). Exogenous expression of EFA6, but not of ARNO,
mimics the effects of expression of constitutively activated ARF6,
i.e. cortical actin reorganization and inhibition of
transferrin uptake. Conversely, ARNO, but not EFA6, affects the
morphology and the function of the Golgi apparatus where ARF1 plays a
major role.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
17]ARF1 and [
13]ARF6 mutants, which
lack, respectively, the first 17 and 13 amino acids, were expressed in
E. coli, purified on an ACA44 (Ultrogel) gel filtration and GDP-loaded before storage at
70 °C (4). Full-length ARNO
and ARNO-Sec7 domain were expressed in E. coli and isolated
by anion exchange on Q-Sepharose and gel filtration on Sephacryl S-100 HR (Amersham Pharmacia Biotech) as previously described (12).
-D-galactopyranoside, and the
temperature was reduced to 27 °C to increase the yield of proteins
in the supernatant. After 3 h, the cells were harvested and lysed
using a French press in 50 mM Tris/HCl, pH 8.0, 1 mM EDTA, 0.15% sodium cholate, 1 mM
dithiothreitol and a tablet of protease mixture inhibitors (Roche
Molecular Biochemicals). After sequential centrifugation at 8,000 × g and at 150,000 × g, the supernatant
was applied to a Q-Sepharose column equilibrated with 20 mM
Tris/HCl, pH 8.0, 1 mM MgCl2, 0.15% sodium
cholate, and 0.5 mM dithiothreitol. The flow-through
fractions containing EFA6 were pooled and applied directly to a Mono S
column (Amersham Pharmacia Biotech) equilibrated with 10 mM
MES, pH 6.0, 1 mM MgCl2, and 0.5 mM
dithiothreitol (Mono S buffer). The bound protein was eluted with a
gradient of NaCl (0-1 M). Fractions containing EFA6
(eluted between 350 and 450 mM NaCl) were pooled,
concentrated on a Centricon 30 (Amicon Corp., Beverly MA), diluted 3 times in Mono S buffer, and stored at
70 °C. Several EFA6-Sec7
constructs were tested before obtaining a stable and active protein.
EFA6-Sec7 domain (from His-125 to Asp-347) was generated by high
fidelity polymerase chain reaction and inserted into pET11a expression
vector. The resulting construct was sequenced and used to transformed
BL21(DE3) bacteria. After protein expression and lysis of the bacteria,
purification was achieved by separation on Q-Sepharose, concentration
on Centricon 10 (Amicon Corp.), gel filtration on Sephacryl S-200
(Amersham Pharmacia Biotech), concentration, and purification by
Mono S column. As judged by Coomassie Blue staining after
SDS-polyacrylamide gel electrophoresis, final purity was >90% for ARF
proteins, 50-60% for full-length ARNO and EFA6, and 90% for the
two isolated Sec7 domains.
-ARF with 1 µM GDP) to perform
fluorescence measurements. Then, GDP/GTP exchange was allowed by the
addition of GTP as indicated.
20
µM) was first loaded with ([3H]GDP) by
incubating 45 min at 37 °C in a buffer containing
[3H]GDP (500 µM, 500 dpm/pmol) with 1 µM free Mg2+ (1 mM
MgCl2 and 2 mM EDTA). When maximal loading was
reached, the concentration of free Mg2+ was raised to 1 mM to stop the reaction. This solution was diluted 10 times
(2 µM final) in HKM buffer supplemented with lipid
vesicles, and nucleotide dissociation was monitored as a loss of
protein-bound radiolabel after the addition of 2 mM GTP.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
ARNO preferentially activates myrARF1, and
EFA6 exclusively activates myrARF6. Kinetics of GDP to GTP
exchange in the ARFs were monitored by the correlated variation in
tryptophan fluorescence (Fluo.) as detailed under
"Experimental Procedures." The purified proteins myrARF1
(A and B) and myrARF6 (C and
D) were preloaded with GDP and injected at 0.5 µM in a fluorescence cuvette containing azolectin
vesicles (0.4 g/liter) in HKM buffer. As indicated by the
arrows, the purified GEF protein (50 nM) was
injected (black line) or not (gray line) followed
by the addition of GTP (250 µM). GTP activation kinetics
were fitted to single exponentials. The value of the GDP/GTP exchange
rate (kexch) for each experiment is shown in the
inset. Fold stimulation is the ratio of the GEF-catalyzed
over the spontaneous exchange rates. A.U., units.
17]ARF1 (25). These results
suggest that (4,5)PIP2, by recruiting ARNO onto the
vesicles, increases its probability of interaction with the
vesicle-bound myrARF1 but has no effect on the intrinsic catalytic
activity of the Sec7 domain. Now we address the role of
(4,5)PIP2 on the specificity of ARNO for ARF1 versus ARF6. The composition in phospholipids, and
particularly in phosphoinositides of azolectin vesicles being unknown,
we tested the effect of (4,5)PIP2 on ARNO activity on
purified myrARF1 and myrARF6 using pure phospholipid vesicles
(phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine: 35/35/30%) supplemented or not with 2% (4,5)PIP2 (Fig.
2). This (4,5)PIP2 addition
did not modify the spontaneous nucleotide exchange rate of either
myrARF, but it increased the nucleotide exchange rate on both myrARF1
and myrARF6 in the presence of ARNO. The PIP2-induced
increase of ARNO activity appears larger on ARF1 than on ARF6,
suggesting that in the presence of (4,5)PIP2, the specificity of ARNO for ARF1 is conserved or even increased. We conclude that (4,5)PIP2 does not affect either the
intrinsic catalytic activity nor the selectivity of ARNO.
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Fig. 2.
Dependence on (4,5)PIP2 of the
activity of ARNO on ARF1 and ARF6. As described under
"Experimental Procedures," the kinetics of GDP/GTP exchange in ARF1
and ARF6 were measured by following the dissociation of
[3H]GDP from [3H]GDP-ARF (2 µM) upon the addition of an excess nonradioactive GTP (2 mM). The measurements were performed in the presence or not
of ARNO (20 nM) and in the presence of 0.4 g/liter
phospholipid vesicles of defined composition, 35% phosphatidylcholine,
35% phosphatidylethanolamine, 30% phosphatidylserine, supplemented or
not with 2% (4,5)PIP2 (A). The dissociation
kinetics were fitted to single exponentials. Values of the GDP/GTP
exchange rate under the various conditions are represented in
B. PIP2 is seen to increase the activity of ARNO
on both ARF proteins, not changing significantly its specificity for
ARF1.
17]ARF1 mutant deleted of its
amphipathic N-terminal helix. In the absence of GTP, these two proteins
formed a stoichiometric nucleotide-free [
17]ARF1· Sec7
domain complex (25, 26). This minimal complex between ARNO-Sec7 and the
ARF1 core, without its N-terminal helix, includes all the structural
components required to catalyze the nucleotide exchange. We now ask
whether the specificity of a given GEF for a particular ARF is
maintained within this minimal complex? We produced and purified
isolated Sec7 domains of ARNO and EFA6 (that are less that 30%
identical) and ARF proteins deleted from their N-terminal myristoylated
amphipathic helix, [
17]ARF1 and [
13]ARF6, that remain soluble
in their GTP-bound state. Using the tryptophan fluorescence method, we
analyzed the effect of increasing concentrations of ARNO-Sec7 (Fig.
3A) and of EFA6-Sec7 (Fig.
3B) on the rate of GDP/GTP exchange by [
17]ARF1 and
[
13]ARF6 and in the absence of lipid vesicles. We observed that
the selectivity of the isolated Sec7 domains was conserved or even
increased; ARNO-Sec7 (50 nM) stimulated the GDP
dissociation rate on [
17]ARF1 50-fold and [
13]ARF6 only
2.7-fold (Fig. 3) as compared with 21- and 5-fold, respectively, for
the full-length ARNO (Fig. 1). EFA6-Sec7 (800 nM)
stimulated the rate of GDP dissociation on [
13]ARF6 8-fold, and as
full-length EFA6, did not display any activity on [
17]ARF1. Thus
the Sec7 domains of ARNO and EFA6 contain all the determinants of
specificity for distinct ARFs.
View larger version (31K):
[in a new window]
Fig. 3.
The specificity of ARNO and EFA6 for distinct
ARF proteins lies within their Sec7 domain. Kinetics of GDP to GTP
exchange were measured by fluorescence as in Fig. 1. The
N-terminal-deleted ARFs, [ 17]ARF1 and [
13]ARF6, were
injected (1 µM) in HKM buffer in the absence of lipid
vesicles. The rates of exchange were measured as a function of the
added concentrations of ARNO-Sec7 domain (panel A) or
EFA6-Sec7 domain (panel B). Fold stimulation
indicates the ratio of the Sec7 catalyzed over the spontaneous exchange
rate for [
17]ARF1 (
) or [
13]ARF6 (
). The
upper insets show the exchange rate (kexch) on
the N-terminal-deleted ARFs in the presence (black bar) or
not (gray bar) of ARNO-Sec7 (50 nM) or EFA6-Sec7
(400 nM).
17]ARF1 in which
each one of these seven residues was substituted by their equivalent found in ARF6. We constructed 3 point mutants in the switch I region
([
17]ARF1-E41Q, [
17]ARF1-I42S, [
17]ARF1-E57T), one point mutant in the interswitch region ([
17]ARF1-S62K), and a triple mutant in the switch II region ([
17]ARF1-F82Y-Q83T-N84G). Using the tryptophan fluorescence method we tested their sensitivity to the
Sec7 domain of ARNO and EFA6. We observed that ARNO-Sec7 activated the
mutants about as much as the [
17]ARF1 wild type, whereas EFA6-Sec7
was not much more active on the mutants than on the [
17]ARF1 wild
type (Fig. 5). Hence, none of the
ARF1/ARF6 point mutations in the switch I and II domains affected the
specificity of the ARNO and EFA6 Sec7 domains.
View larger version (102K):
[in a new window]
Fig. 4.
Schematic representation for the docking
complex between ARF1GDP and ARNO-Sec7 domain. ARF1 interacts with
the Sec7 domain via its switch I and II regions. In this
Sec7-interacting surface, the seven residues represented in the figure
are different between ARF1 and ARF6. We produced mutants of
[ ]17ARF1 in which each one of these residues was replaced by the
equivalent found in ARF6. The catalytic glutamic residue (Glu-156) of
ARNO-Sec7 is also shown.
View larger version (21K):
[in a new window]
Fig. 5.
Permutation of the distinct amino acids
between ARF6 and ARF1 in the switch regions of ARF1 does not change the
specificity of the ARF1-Sec7 domain interaction. Kinetics of the
GDP to GTP exchange were monitored by the correlated variation in
tryptophan fluorescence. Measurements were performed on
[ 17]ARF1-WT (1 µM) and the various [
17]ARF1
mutants with the indicated ARF1->ARF6
mutations in the presence of 25 nM ARNO-Sec7 (panel
A) and 400 nM EFA6-Sec7 (panel B). The GEF
activities, deduced from the activation rates, are normalized to
that observed for WT [
17]ARF1. Relative activities of ARNO-Sec7
and of EFA6-Sec7 on wild-type (WT) [
13]ARF6 are also
shown for comparison. TM corresponds to the
[
17]ARF1-F82Y-Q83T-N84G triple mutant.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Mariagrazia Partisani for skillful technical assistance. We are grateful to Drs. F. Luton, K. Singer, and C. Favard for very helpful comments on this manuscript.
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FOOTNOTES |
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* This study was supported by CNRS institutional funding and a specific grant from the Groupement des Entreprises Françaises dans la Lutte contre le Cancer (to M. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a doctoral fellowship from the Association pour la
Recherche sur le Cancer.
§ To whom correspondence should be addressed. Tel.: 33 4 93 95 77 70; Fax: 33 4 93 95 77 10; E-mail: franco@ipmc.cnrs.fr.
Published, JBC Papers in Press, May 7, 2001, DOI 10.1074/jbc.M103284200
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ABBREVIATIONS |
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The abbreviations used are: ARF, ADP-ribosylation factor; myrARF, myristoylated ARF; GEF, guanine nucleotide exchange factor; PH, pleckstrin homology; (4, 5)PIP2, phosphatidyl-4,5-bisphosphate; MES, 4-morpholineethanesulfonic acid; BFA, brefeldin A.
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