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INTRODUCTION |
Small G proteins of the Rho family, including Rho, Rac, and
Cdc42p, undergo two interdependent cycles. First, they cycle between inactive (GDP) and active (GTP) conformations through the catalytic action of guanine exchange factors
(GEF)1 and GTPase-activating
proteins. Second, they cycle between cytosolic and membrane-associated
forms through the action of GDI proteins (1-3). How these two cycles
are coupled is critical for the proper interaction of Rho proteins with
their targets. It is generally assumed that Rho proteins must be in the
GTP conformation and associated to membranes to trigger a cellular response.
Dbl homology (DH) domains catalyze the exchange of guanine nucleotides
on small G proteins of the Rho family (4, 5). The DH domain is an
all-
-helix fold that is systematically followed by a pleckstrin
homology (PH) domain. The DH-PH tandem is thus the hallmark of Rho
GEFs, which otherwise display variable domain composition and
organization. Recent structural and mutagenesis studies on DH-PH
domains, either isolated or in complex with Rho proteins, have given
insights into the mechanism by which these domains promote the release
of the bound nucleotide (6-10). One of the best-studied examples is
Tiam, a GEF for Rac. The DH domain of Tiam makes extensive contacts
with the switch I and II regions of Rac and modifies the magnesium,
sugar, and guanine base-binding regions to destabilize the bound
nucleotide (9). In addition, structure-based mutagenesis studies show
that the specificity between functional GEF/Rho pairs relies on a few
residues at the DH/switch II interface (11-13). Binding to switch I
and II regions is a general property of GEFs for small G proteins
(14).
Despite these spectacular progresses, one aspect of the activation of
Rho proteins by DH domains remains obscure: how does the nucleotide
exchange reaction accommodate with the interaction of Rho proteins with
membrane lipids or with GDI proteins? Rho proteins contain a
geranyl-geranyl group at their C termini. This C20 branched isoprene
has a high lipid/water partition coefficient and anchors Rho proteins
on lipid membranes (15). On the other hand, GDI proteins solubilize Rho
proteins in the cytosol by shielding the geranyl-geranyl group from the
solvent (2, 16-18). The GDI/Rho interaction is based on two contacts:
the isoprene group inserts into a deep pocket made by the
immunoglobulin-like
sandwich of GDI and the switch I and II regions
of Rho interact with the regulatory arm of GDI, a small
helix-loop-helix motif (18). The latter interaction makes the Rho/GDI
interaction partially dependent on the conformation of Rho. Thus GDI
interacts preferentially with the GDP-bound form of Rho
proteins (19), although equal interactions with the GDP- and GTP-bound
forms have been reported in some cases (20, 21). Consequently, Rac-GDP
is generally found in complex with GDI in the cytosol, whereas Rac-GTP
is preferentially associated with membranes (2, 3, 22).
Overall, the structure of nucleotide-free Rac in complex with the DH-PH
region of Tiam1 and that of prenylated Cdc42-GDP in complex with GDI
show a strong overlap of the interacting regions, particularly at the
level of the switch I and II regions (9, 18). This strongly suggests
that the two interactions are mutually exclusive and consequently that
Rho-GDP must be released from GDI before being activated by the DH-PH
module. However, given the bipartite nature of the Rho/GDI interface,
the formation at early stages of the exchange reaction of a ternary
complex, where GDI interacts only with the prenyl group of Rho, leaving
the switch regions accessible for the exchange factor, cannot be
excluded. Schematically, two opposite models have been proposed for
GEF-catalyzed nucleotide exchange on Rho proteins coupled to membrane
translocation (1, 2, 5). In the first model, the GEF acts on the
cytosolic complex between Rho-GDP and GDI. After nucleotide exchange,
Rac-GTP translocates to membranes. In the second model, membrane
translocation precedes GEF action: Rac-GDP dissociates from GDI and
translocates to membranes; then GEF promotes GDP-to-GTP exchange on
membrane-bound Rac. As a first step toward understanding the sequence
of events that, from soluble GDI-RacGDP complex, leads to membrane
bound Rac-GTP, we have studied the catalysis of nucleotide exchange on
geranyl-geranylated Rac by the DH-PH region of Tiam1 in the presence of
GDI and liposomes. Our results demonstrate that dissociation of GDI and
membrane translocation is a prerequisite for the efficient activation
of Rac by a DH-PH domain.
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EXPERIMENTAL PROCEDURES |
Purification of the Rac-RhoGDI Complex (Saccharomyces cerevisiae
Expression)--
Constructions of Human Rac1 with an N-terminal
His6 tag and RhoGDI-1 with an N-terminal FLAG cloned in the
yeast 2µ shuttle vector YEpPGAL were a gift from R. Nakamoto (23, 24). These constructs were transformed in S. cerevisiae SY1 strain (MATa, ura3-52,
leu2-3,112,his4-619,sec6-4,GAL) and selected for leucine/uracil independence. Transformed yeast cells were grown in a 5-liter fermenter. After cell wall lysis and fractionation, the Rac1-RhoGDI complex was purified from the cytosol as described for the RhoA-GDI complex using two sequential affinity chromatography assays on a TALON
metal affinity resin column (Clontech) and an
M2-anti-FLAG antibody column (Sigma Chemical) (23, 24). About 100 µg
of Rac1-RhoGDI complex (95% purity) was obtained from one fermenter. The Rac-GDP-GDI complex was stored at
80 °C in 25 mM
Tris, pH 8, 100 mM NaCl, 2 mM
MgCl2, 10% glycerol, and 2 µM GDP.
Purification of C-terminally Truncated Rac1 and Tiam1
DH-PH--
Full-length human Rac1 was cloned in a PGEX-T vector. The
pProEX-Hta vector containing the DH-PH fragment of mouse Tiam1
(residues 1033-1406) was provided by J. Sondek (9). GST-Rac1 and
His6-Tiam1 fragment were purified by affinity
chromatography on glutathione-agarose (Amersham Biosciences) or
Ni2+-agarose beads (QIAGEN) respectively according to the
manufacturer procedures. GST from the Rac chimera was removed by
cleavage with thrombin. All constructs were sequence-proofed. The
purity of proteins was assessed by Coomassie Blue-stained SDS-PAGE, and the protein concentration was estimated by a protein assay
(Bio-Rad).
Azolectin Liposomes--
Large unilamellar liposomes made of
unpurified soybean phospholipids (azolectin type IIS; Sigma Chemical)
were prepared by the reversed-phase method (25). Azolectin (20 mg) was
dissolved in 6 ml diethylether in a 100-ml round-bottomed flask. One
milliliter of buffer A (20 mM HEPES, pH 7.5, and 180 mM sucrose (iso-osmotic with 100 mM NaCl)) was
added and the two-phase mixture was sonicated at 4 °C for
1-2 min in a bath sonicator to make an emulsion. The solvent was
slowly evaporated in a rotary evaporator (100 rpm) under moderate
vacuum at 20-25 °C. After 45 min, the liposome suspension was
collected in an Eppendorf tube and was further incubated in a vacuum
chamber for 15 min to eliminate traces of ether and completed up to 1 ml with buffer A. The liposome suspension was frozen in liquid nitrogen
and stored at
20 °C. Liposomes were extruded before use through
0.4-µm pore-size polycarbonate filters (Millipore). The suspension
was diluted five times in buffer B (20 mM HEPES, pH 7.5, and 100 mM NaCl) to dilute the external sucrose solution.
The liposomes were collected by centrifugation in a TL100 Beckman
centrifuge at 400,000 × g, 18 °C, for 25 min and
resuspended in buffer B.
Sedimentation Assay--
Prenylated Rac-GDP in complex with GDI
was incubated for 20 min at 30 °C with sucrose-loaded azolectin
liposomes in 20 mM HEPES, pH 7.5, 100 mM NaCl,
1 mM MgCl2, 1 mM
dithiothreitol (buffer C) in a final volume of 80 µl.
Nucleotides (50 µM final of GDP-Mg or GTP
S-Mg) were
added as indicated. When indicated, magnesium was chelated by the
addition of 2 mM EDTA (1 µM free
Mg2+) and alkaline phosphatase (Sigma; final concentration,
180 units/ml) was added to hydrolyze guanine nucleotides. After
incubation, vesicles were recovered by centrifugation at 400,000 × g, 24 °C, for 25 min. The pellet was resuspended in
the same volume of buffer, and the amount of prenylated Rac and Tiam1
in the pellet and in the supernatant was determined by densitometry
after protein separation by SDS-PAGE and Coomassie Blue staining.
GDI Removal Protocol--
The RacGDP-GDI complex (1.0 µM) was incubated at 30 °C for 30 min with azolectin
liposomes (5 mg/ml) in buffer C supplemented with 2 mM EDTA
and 50 µM GTP. Liposomes and bound proteins were collected by centrifugation (400,000 × g, 25 min,
18 °C) and washed one time by resuspension/centrifugation in buffer
C. The final liposome suspension containing liposome-translocated
RacGTP was incubated for 30 min at room temperature to promote GTP hydrolysis.
Nucleotide Binding Assay--
The binding of
[35S]GTP
S to Rac was measured as described by Franco
et al. (26) for Arf1. Briefly, the Rac1-GDP-GDI complex was
incubated at 30 °C with [35S]GTP
S (20 µM, 2000 cpm/pmol) in the presence of azolectin liposomes (0 to 3 mg/ml) in buffer C. At the indicated times, 20-µl
aliquots were removed, diluted into 2 ml of ice-cold buffer (buffer D; 20 mM HEPES, pH 7.5, 100 mM NaCl, and 10 mM MgCl2), and filtered on nitrocellulose
filter discs (Schleicher & Schüll). Filters discs were washed
twice with 2 ml of buffer D, dried, and counted. The same protocol was
used to measure the binding of [35S]GTP
S on Rac-GDP
bound to liposomes and on unprenylated Rac in solution or in the
presence of liposomes.
GTPase Assay--
Unprenylated Rac-GDP (0.8 µM) or
prenylated RacGTP bound to liposomes (see the GDI removal protocol) was
incubated at room temperature for 15 min in buffer C with 2 mM EDTA (1 µM free MgCl2) and
with [
-32P]GTP (10 µM). The GTPase
reaction was initiated by the addition of 2 mM
MgCl2 (1 mM free MgCl2). At the
indicated times, 20 µl were removed, diluted into 2 ml of ice-cold
buffer D, and counted on nitrocellulose filter as described for
nucleotide binding.
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RESULTS |
Reconstitution of the GTP-dependent Translocation of
Rac to Liposomes--
We purified recombinant geranylgeranylated
Rac-GDP from yeast cells as a stoichiometric complex with RhoGDI-1
(GDI) according to the protocol developped by Read et al.
(23, 24) for the complex between RhoA-GDP and GDI (Fig.
1). We first assessed the ability of
Rac-GDP, initially associated with GDI, to interact with membrane
lipids and undergo GDP to GTP exchange. To this end, we incubated the
complex between Rac-GDP and GDI with radiolabeled GTP
S and with
liposomes made from soybean lipids (azolectin). Because GDI strongly
inhibits the spontaneous release of GDP from Rho proteins, incubations
were performed at low Mg2+ concentration (1 µM) to accelerate nucleotide exchange. At the end of the
incubation, the concentration of free Mg2+ was set back at
1 mM. The binding of GTP
S to Rac was determined by a
filtration assay, and the membrane partitioning of Rac and GDI was
determined by separating the soluble proteins and the liposome-bound
proteins by centrifugation. Fig.
2B shows that the extent of
GTP
S binding on Rac strongly increased with the liposome
concentration. Notably, no detectable binding of GTP
S was observed
in the absence of membrane lipids (Fig. 2B, open circles). Fig. 2A shows that in all incubations
with GTP
S GDI remained essentially soluble, whereas the amount of
membrane-bound Rac increased with liposome concentration. In contrast,
in the presence of GDP, Rac remained essentially soluble as a complex with GDI. However, a fraction of Rac-GDP bound to liposomes could be
observed at high liposome concentration (> 1 mg/ml) (Fig.
2A). Note a slight contamination of the pellet by soluble
GDI at increasing liposome concentration caused by the increase in the
volume of the pellet. A slight contamination of the pellet with soluble Rac could also have occurred but this could not account for the full
Rac signal in the pellet. These experiments show that one can
reconstitute on liposomes the GTP-dependent translocation of Rac to membrane lipids starting from a soluble Rac-GDP-GDI complex.

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Fig. 1.
Purification of prenylated Rac in complex
with GDI. Human Rac1 and GDI-1 were co-expressed in S. cerevisiae and purified from the cytosol. Fractions were analyzed
by SDS-PAGE and Coomassie Blue staining. Lane 1, elution
from the metal affinity resin; lane 2, elution from the
M2-anti-FLAG antibody column.
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Fig. 2.
GTP-dependent translocation of
prenylated Rac from GDI to liposomes. A, prenylated Rac-GDP in
complex with GDI (0.8 µM) was incubated with
sucrose-loaded azolectin liposomes (from 0.2 to 3 mg/ml) at 30 °C
with 50 µM GTP S at 1 µM free
Mg2+ (2 mM EDTA) or with 50 µM
GDP at 1 mM free Mg2+. After incubation, 2 mM MgCl2 was added back in the samples with
GTP S to get 1 mM free Mg2+. The samples were
centrifuged and the supernatant (S) and the liposome pellet
(P) were analyzed by SDS-PAGE and Coomassie Blue staining.
B, time course of GTP S binding at 30 °C on incubations containing
0.2 µM Rac-GDP-GDI complex, 20 µM
[35S]GTP S, 1 µM free Mg2+
and 0 (open circles), 0.05 (filled
circles), 0.2 (filled squares), or 3 mg/ml (filled triangles) azolectin
vesicles.
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Tiam-catalyzed GDP/GTP
S Exchange on Rac-GDP in
Complex with GDI and in the Presence of Liposomes--
Next, we
studied the influence of liposomes on the kinetics of Tiam1-catalyzed
nucleotide exchange on Rac-GDP, initially in complex with GDI, at
physiological (1 mM) Mg2+ concentration. We did
not observe any significant GDP-to-GTP
S exchange when the complex
was incubated in the absence of liposomes with or without Tiam (Fig.
3A). In contrast, in the
presence of azolectin liposomes, both spontaneous and Tiam-catalyzed
GDP-to-GTP
S exchange on Rac could be detected (Fig. 3, B
and C). Interestingly, increasing liposome concentration had
two effects: it accelerated the initial rate of the reaction and
increased the amount of Rac-GTP that formed at equilibrium. The second
effect resembles that observed at low magnesium concentrations in the
absence of Tiam (see Fig. 2B) and may reflect the opposite
mass action effects of liposomes and GDI on the balance between the
GTP- and the GDP-bound forms of Rac. More surprising is the effect of
liposome concentration on the initial rate of GTP
S
binding. Insofar as only Rac-GDP was present initially, an increase in
the initial rate of GTP
S binding must reflect a positive effect of
liposomes on the ability of Rac-GDP to undergo spontaneous or
Tiam-catalyzed nucleotide exchange. At first glance, this does not seem
compatible with the fact that Rac-GDP is essentially soluble and in
complex with GDI. However, because sedimentation experiments revealed
that a small amount of Rac, bound to liposomes and devoid of GDI, could be detected in incubations conducted in the presence of GDP (Fig. 2A), we suspected that this fraction of Rac could be a
better substrate for Tiam-catalyzed nucleotide exchange than Rac-GDP in
complex with GDI.

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Fig. 3.
Liposomes facilitate Tiam1-catalyzed
nucleotide exchange on prenylated Rac-GDP initially in complex with
GDI. Time course of GTP S binding at 30 °C on incubations
containing 0.2 µM Rac-GDP-GDI complex, 20 µM [35S]GTP S, 1 mM free
Mg2+ with (filled circles) or without
(open circles) 0.2 µM Tiam DH-PH
and with 0 (A), 0.5 (B), or 3 mg/ml
(C) azolectin liposomes.
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A GDI-release Protocol That Produces Liposome-bound Rac-GDP and
Facilitates Rac Activation by Tiam--
We reasoned that if Rac-GDP
bound to liposome was more readily activated by Tiam than soluble
Rac-GDP in complex with GDI, one should change dramatically the
kinetics of GDP-to-GTP
S exchange by modifying the partitioning of
Rac-GDP between the soluble complex with GDI and the liposome-bound
form. To get rid of GDI and obtain Rac-GDP bound to liposomes, we
devised a three-stage protocol based on a single round of Rac-GTP
translocation to liposomes and GTP hydrolysis (Fig.
4A). During stage 1, the
Rac-GDP-GDI complex was incubated with liposomes and with GTP and EDTA
to promote the formation of membrane bound Rac-GTP. Liposomes were then
collected by centrifugation and washed to separate membrane bound
Rac-GTP from soluble GDI and excess GTP. In a second stage, the
lipid pellet was resuspended in a buffer containing 1 mM
MgCl2 and incubated at 30 °C to allow Rac to hydrolyze
the bound GTP. Rac displays a fast intrinsic GTP hydrolysis activity,
and control experiments with [
-32P]GTP showed that GTP
in liposome-bound prenylated Rac was completely hydrolyzed within 30 min with a kinetics similar to that observed with the soluble
unprenylated form of Rac (Fig. 4B). In a third stage, the
resulting liposome suspension containing membrane-bound Rac-GDP (Fig.
4C) was assayed for nucleotide exchange with GTP
S with or
without Tiam1 DH-PH (Fig. 5B).
The time course of GTP
S binding was compared with that observed on
the initial mixture containing liposomes (1 mg/ml) and Rac-GDP in
complex with GDI (Fig. 5A). The GDI removal protocol
dramatically potentiated the rate of GDP-to-GTP
S exchange on Rac, in
both the absence and the presence of Tiam1 DH-PH. This effect was
reversible because restoring purified GDI inhibited the nucleotide
exchange reaction (data not shown). For each time-course experiment,
the apparent rate constant of the exchange reaction was determined and
plotted as a function of Tiam1 DH-PH concentration (Fig.
5E). With GDI present, apparent rate constants in the range
of 10
3 s
1 were observed. In the absence of
GDI, the apparent rate constant of nucleotide exchange was about 1 order of magnitude higher than that observed for the Rac-GDP-GDI
complex. Therefore, these experiments demonstrate that Tiam1 DH-PH acts
preferentially, if not exclusively, on prenylated Rac-GDP dissociated
from GDI and bound to membrane lipids.

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Fig. 4.
GDI-removal protocol. A,
experimental strategy. B, time course of GTP hydrolysis on
unprenylated Rac-GTP in solution (open circles)
or on prenylated Rac-GTP bound to liposomes and prepared as described
in A (filled circles). C,
SDS-Page analysis of the GDI removal protocol. Lane a, the
Rac-GDP-GDI complex; lane b, supernatant from the incubation
of Rac-GDP-GDI with GTP, EDTA, and liposomes; lane c,
supernatant from the subsequent washing step; Lane d,
liposome-bound proteins after the GTP hydrolysis step.
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Fig. 5.
Membrane translocation of prenylated Rac-GDP
facilitates its activation by Tiam DH-PH. Comparison between
prenylated Rac-GDP initially in complex with GDI (A), or
prenylated Rac-GDP associated with azolectin liposomes (B),
or unprenylated Rac-GDP in solution (C) for Tiam1
DH-PH-catalyzed GDP-to-GTP S exchange. All experiments were performed
at 1 mM free Mg2+ with 20 µM
[35S]GTP S and with 0 (open
circles), 0.04 (inverted filled triangles), 0.08 (filled triangles), 0.2 (filled
diamonds), 1 (filled squares), 3 (filled circles), or 4 µM (×) Tiam1 DH-PH.
A, the experiments were performed with 0.2 µM
Rac-GDP-GDI complex in the presence of 1 mg/ml azolectin liposomes.
B, the experiments were performed with prenylated Rac-GDP
(estimated at 0.1 µM) bound to liposomes, which was
prepared according to the GDI-removal protocol (see Fig. 4).
C, the experiments were performed with 0.2 µM
unprenylated Rac-GDP in buffer. D, apparent rate constant of
[35S]GTP S binding on soluble unprenylated Rac (0.5 µM) in the presence of 1 µM Tiam1 DH-PH as
a function of azolectin liposome concentration. E, the
apparent rate constants of nucleotide exchange
(kapp) for prenylated Rac in complex with GDI
(open circles), liposome-bound prenylated Rac
(filled circles), or unprenylated Rac
(filled triangles) were determined from single
exponential fits of the time courses shown in A,
B, and C and plotted as a function of Tiam1 DH-PH
concentration on a linear or log/log scale. Shown are means ± S.E. of two independent experiments.
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Membrane Localization Potentiates the Exchange Activity of Tiam1
DH-PH on Rac--
Next, we wished to compare liposome-bound prenylated
Rac-GDP with soluble non-prenylated Rac-GDP for their responsiveness to
the exchange activity of Tiam DH-PH. This should allow us to assess the
effect of the membrane colocalization of Rac-GDP and Tiam1 DH-PH on the
nucleotide exchange reaction per se (i.e.
independently of the effect of GDI on Rac). Indeed, besides being
required for the dissociation of prenylated Rac from GDI, liposomes
may also positively or negatively influence the interaction between
Rac-GDP and Tiam DH-PH by effects such as reduction of dimensionality (liquid volume versus membrane surface) or conformational change.
GDP-to-GTP
S exchange reactions on non-prenylated Rac-GDP were
conducted in solution at various Tiam1 DH-PH concentration (Fig.
5C). Importantly, azolectine liposomes had no effect on the
reaction (Fig. 5D). The apparent rate constant was plotted as a function of Tiam DH-PH concentration and was compared with that
observed on liposomes-bound Rac-GDP (Fig. 5E). Remarkably, the DH-PH region of Tiam1 was 10 to 20 times more active on prenylated Rac-GDP bound to membrane lipids than on soluble non-prenylated Rac-GDP. The comparison between these two forms was made easy by the
fact that both underwent similar spontaneous exchange kinetics in the
absence of exchange factor. The maximal exchange activity (kcat) of Tiam DH-PH on Rac-GDP bound to
liposomes could not be determined because we could not resolve the
kinetics of GDP/GTP
S exchange at saturating DH-PH concentration.
However, from a rough extrapolation of the data, one can estimate that
there a difference of 1 to 2 orders of magnitude in the maximal
exchange activity (Fig. 5E). We conclude from these
experiments that colocalization at a lipid membrane surface strongly
favors the nucleotide exchange activity of the DH-PH domain of Tiam
toward Rac.
Formation of a Liposome-bound and Nucleotide-free Complex between
Rac and Tiam DH-PH--
The above experiments suggest that the DH-PH
domain of Tiam acts preferentially on prenylated Rac-GDP associated
with membrane lipids. If this assessment is correct, catalytic
intermediates of the nucleotide exchange reaction should be found also
associated with membrane lipids. The essential intermediate of the
catalysis of nucleotide exchange on G protein by GEFs is a
nucleotide-free G protein-GEF complex. We assessed the membrane
partitioning of such a complex in liposome sedimentation experiments.
Prenylated Rac-GDP in complex with GDI (0.8 µM) and Tiam
DH-PH (0.3 µM) were incubated with liposomes. To favor
the stabilization of the nucleotide-free complex, incubations were
performed at low Mg2+ (2 mM EDTA) in the
presence of alkaline phosphatase, which hydrolyzes GDP as it
dissociates from Rac. As shown in Fig. 6,
the DH-PH domain alone of Tiam was more than 50% soluble
(lane 3) although a significant amount of protein (30%) was
associated with liposomes. However, after incubation with prenylated
Rac, initially in the GDP-bound form and complexed to GDI but under
conditions that favor the formation of the nucleotide-free form of Rac,
the amount of membrane-bound Tiam DH-PH increased 2.5-fold (lane
2). Rac was also found to be almost completely associated with
liposomes, whereas GDI remained in the supernatant (lanes 2 and 4). Importantly, no cotranslocation of Rac and Tiam
DH-PH to liposomes was observed in a control incubation conducted in
the presence of GDP and in the absence of EDTA and alkaline phosphatase
(lane 1). We conclude from these experiments that the
nucleotide-free complex between prenylated Rac and Tiam DH-PH is a
membrane-bound complex from which GDI is excluded.

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Fig. 6.
A liposome-bound and nucleotide-free complex
between prenylated Rac and Tiam1 DH-PH domain. Prenylated
Rac-GDP in complex with GDI (0.8 µM) and Tiam DH-PH (0.3 µM) were incubated for 30 min at 30 °C with 1.5 mg/ml
azolectin liposomes. When indicated, EDTA (to obtain 1 µM
free Mg2+), alkaline phosphatase (AP; to
hydrolyze free nucleotides) or GDP (20 µM) were added.
After incubation, the samples were centrifuged. The supernatant
(S) and the liposome pellet (P) were analyzed by
SDS-PAGE, Coomassie Blue staining and densitometry. The percentage of
liposome-bound proteins is indicated in the bottom
graph.
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DISCUSSION |
With the notable exception of Ran, most small G proteins interact
with lipid membranes at some steps of the GDP/GTP cycle. In many cases,
the interconversion between GDP- and GTP-bound forms is directly or
indirectly coupled to the translocation of the protein between the
cytosol and membranes, making the catalysis of GDP to GTP exchange by
GEFs a complex issue. In Arf1, the coupling is direct: a conformational
change in the myristoylated N-terminal helix of Arf1 strengthens its
interaction with membrane lipids and is required for GEF-catalyzed
GDP-to-GTP exchange (27, 28). In Rho proteins, the coupling is
indirect. The GDP- and GTP-bound forms display the same avidity for
membrane lipids, but Rho-GDP is more cytosolic because of its
preferential interaction with GDI (19; see, however, Ref. 20). The
sequence of events that leads, starting from Rho-GDP in a cytosolic
complex with GDI, to membrane anchored Rho-GTP is not well understood
(1). It has been suggested that exchange factors act on the soluble
GDI-Rho-GDP complex and that after nucleotide exchange, Rho-GTP
dissociates from the tripartite complex and translocates to membranes
(24, 29). Alternatively, the dissociation of GDI and the membrane translocation of the GDP-bound form may precede or accompany the functional interaction of Rho with GEF (22). Here, we have taken advantage of a recently described overexpression system for prenylated Rho proteins in complex with GDI (24) to explore the nucleotide exchange reaction on the small G protein Rac in a minimal system with liposomes.
To reconstitute the GDP/GTP switch of Rac on liposomes, one faces a
dilemma. To handle prenylated Rac easily, the protein must be purified
in complex with GDI. In that case, however, Rac-GDP remains essentially
associated with GDI in solution even after incubation with a large
amount of liposomes (Fig. 2). Alternatively, detergents may be used
during the purification procedure to dissociate Rac-GDP from GDI.
However, this will make the reconstitution with liposomes difficult.
The group of Takai (1, 30) reported interesting observations on the
effects of both GDI and prenylation of Rho/Rac proteins on the
catalysis of guanine nucleotide exchange, but the influence of
detergent and lipids in the reaction was not appreciated and thus
prenylation was considered to participate directly in the interaction
of Rho/Rac proteins with exchange factors. To overcome these
difficulties, we devised a protocol whereby, starting from Rac-GDP in
complex with GDI, we recapitulated a complete cycle of nucleotide
exchange and GTP hydrolysis to get prenylated Rac-GDP bound to
liposomes and devoid of GDI (Fig. 4). This allows us to establish an
elementary scheme for the GDI and DH-PH-dependent
activation of Rac at the lipid surface.
In solution, the DH-PH domain of Tiam was unable to promote GDP-to-GTP
exchange on prenylated Rac-GDP in complex with GDI (Fig.
3A). In sharp contrast, robust exchange activity was
observed on prenylated Rac-GDP that was bound to azolectin liposomes
(Fig. 5B). Starting from the Rac-GDP-GDI complex, the
exchange rate increases with liposome concentration, and this
correlates with the appearance of a small fraction of Rac-GDP
associated with liposomes and dissociated from GDI (Figs. 2 and 3).
Last, sedimentation experiments revealed the formation of a
liposome-bound complex between nucleotide-free Rac and the DH-PH domain
of Tiam1, from which GDI was excluded (Fig. 6). These four observations
exclude the formation of a functional tripartite complex between Rac, GDI, and DH-PH in solution and favor a two-step mechanism. First, Rac-GDP dissociates from GDI and translocates to membrane lipids. Second, DH-PH catalyzes the replacement of GDP by GTP on Rac anchored at the membrane surface. Although these two steps may be functionally coupled, they are mechanistically different. In the cell, membrane translocation of Rac can occur even with a catalytically inactive form
of the exchange factor Vav, suggesting that there is a mechanism that
controls the membrane recruitment of Rac before the nucleotide exchange
reaction (31).
One important finding is that the DH-PH module of Tiam1 displays a much
better activity on prenylated RacGDP bound to liposomes (Fig.
5B) than on soluble unprenylated Rac-GDP (Fig.
5C). Because the two forms display the same rate of
spontaneous GDP release (compare Fig. 5, B and
C), it is unlikely that the difference in DH-PH-catalyzed
nucleotide exchange arises from differences in the conformation of Rac.
One explanation for the catalytic advantage provided by membrane
localization is reduction of dimensionality: the lipid membrane acts as
a template that concentrates and better orients the small G protein and
the DH-PH module, hence favoring their mutual interaction. Because both
Rac and the DH-PH module have elements permitting membrane attachment
(prenylation and PH domain), this mechanism is likely to contribute to
the effects observed here. However additional mechanisms may play a
role. If membranes solely concentrated the proteins, the maximal
activities at saturating concentration (kcat)
should be similar in solution and in the presence of liposomes.
However, extrapolation of the dose response curves shown in Fig.
5E suggests different kcat, implying
that membrane colocalization favors the catalytic mechanism per
se. One recurrent observation on DH-PH domains is that their activity on non-prenylated Rho proteins in solution is very weak compared with other catalytic domains that promote nucleotide exchange
on small G proteins (discussed in Ref. 32). This raised the possibility
that the DH-PH module in solution may be somehow autoinhibited. Because
the structures of DH-PH modules show great variations in the relative
orientation of the DH and PH domains, it is tempting to assume that
membrane binding of the PH domain may favor structural rearrangements
at the interface with the DH domain that impact on the nucleotide
exchange activity (4, 10). If so, membrane-bound prenylated Rac-GDP
should be in a better position to detect membrane-induced changes in
the DH-PH module than soluble unprenylated Rac-GDP. Notably, we did not observe any effect of liposomes on the nucleotide exchange activity of
Tiam1 DH-PH on soluble unprenylated Rac (Fig. 5D).
It has been shown recently that the PH domain of Tiam1
interacts preferentially with phosphatidylinositol 3-phosphate but that
this interaction had no effect on the activity of Tiam1 on unprenylated
Rac in solution (33). It will be interesting to assess the effect of
phosphoinositides on nucleotide exchange reactions performed in the
presence of liposomes of defined lipid composition and with prenylated
Rac. In addition, it should be noted that the membrane localization of
full-length Tiam1 has been shown to depend on domains upstream of the
DH-PH module, including a second PH domain, and can be regulated by
phosphorylation (34-37). These domains may thus control the proper
interaction of the DH-PH module with the lipid surface. The
reconstitution of the GTPase cycle of Rac on liposomes should help to
define the molecular mechanisms by which the activation of this small G
protein is regulated.