(Received for publication, October 10, 1995 )
From the
We have investigated the role of N-myristoylation in
the activation of bovine ADP-ribosylation factor 1 (ARF1). We
previously showed that myristoylation allows some spontaneous
GDP-to-GTP exchange to occur on ARF1 at physiological Mg levels in the presence of phospholipid vesicles (Franco, M.,
Chardin, P., Chabre, M., and Paris, S.(1995) J. Biol. Chem. 270, 1337-1341). Here, we report that this basal nucleotide
exchange can be accelerated (by up to 5-fold) by addition of a soluble
fraction obtained from bovine retinas. This acceleration is totally
abolished by brefeldin A (IC
= 2 µM)
and by trypsin treatment of the retinal extract, as expected for an
ARF-specific guanine nucleotide exchange factor. To accelerate GDP
release from ARF1, this soluble exchange factor absolutely requires
myristoylation of ARF1 and the presence of phospholipid vesicles. The
retinal extract also stimulates guanosine
5`-3-O-(thio)triphosphate (GTP
S) release from ARF1 in the
presence of phospholipids, but in this case myristoylation of ARF is
not required. These observations, together with our previous findings
that both myristoylated and nonmyristoylated forms of ARF
but only the myristoylated form of ARF
bind to
membrane phospholipids, suggest that (i) the retinal exchange factor
acts only on membrane-bound ARF, (ii) the myristate is not involved in
the protein-protein interaction between ARF1 and the exchange factor,
and (iii) N-myristoylation facilitates both spontaneous and
catalyzed GDP-to-GTP exchange on ARF1 simply by facilitating the
binding of ARF
to membrane phospholipids.
ADP-ribosylation factors (ARFs) ()are a family of
small (
20 kDa) guanine nucleotide binding proteins, originally
identified as cofactors of cholera toxin and more recently recognized
as essential participants in intracellular vesicular
transport(1, 2, 3, 4) . All ARFs
contain the amino-terminal myristoylation consensus sequence and are
believed to be myristoylated in vivo(5) . The
functions of ARFs in membrane traffic are linked to their guanine
nucleotide-dependent interactions with membranes. Myristoylation is
also crucial for these interactions, but the precise mechanism for the
attachment of ARF to membranes is still unclear. According to the model
initially proposed by Serafini et al.(6) and at
present most commonly accepted, the GDP-bound form of ARF is cytosolic;
upon interaction with a specific nucleotide exchange protein, described
as membrane-bound by some authors(7, 8, 9) and soluble by others(10, 11) ,
ARF
is converted to ARF
. This conversion
would promote a conformational change of the amino terminus, allowing
exposure of the myristoyl group and its insertion into membranes. This
model, referred to as ``myristoyl-GTP switch'' by analogy
with the Ca
-myristoyl switch model proposed for
recoverin(12) , therefore implies that myristoylation of ARF is
not necessary for its interaction with the nucleotide exchange enzyme (9, 13) but is required after the exchange reaction
for the stable association of ARF
with membranes. Our
recent observations do not support this model. First, we found that
nonmyristoylated ARF
strongly binds to phospholipid
vesicles(14) , indicating that a protein-lipid interaction must
also be involved in the association of ARF
with membranes
rather than just a simple lipid-lipid interaction. Second, by comparing
the properties of nonmyristoylated ARF1 (rARF1) to those of
myristoylated ARF1 (myr-rARF1) both produced in Escherichia
coli, we found that myr-ARF
partially binds to
phospholipid membranes while rARF
is totally water
soluble(15) . We therefore proposed that myr-ARF
is loosely attached to membranes by the myristate chain, whereas
myr-ARF
is strongly bound to phospholipids via both the
fatty acid and hydrophobic or electrostatic interactions between a
protein domain and the membrane bilayer. In other words, we propose
that nucleotide exchange increases the affinity of ARF for
phospholipids through a ``protein switch'' rather than a
``myristoyl switch.''
Moreover, we have reported that
interaction of myr-ARF with phospholipids allows a
significant spontaneous nucleotide exchange at physiological
(mM) levels of Mg
, conditions under which
GDP release from rARF
is undetectable(15) . Here,
we report that a soluble ARF-specific guanine nucleotide exchange
activity can be obtained from bovine retinas and that stimulation of
GDP release from ARF1 by this activity also requires myristoylation of
ARF1 and the presence of phospholipids. In addition, we present
evidence that the myristate is not necessary for the protein-protein
interaction between ARF and the exchange factor but simply facilitates
the binding of ARF
to the phospholipids, where it can
interact with the exchange factor.
Figure 1:
Binding of myr-rARF1 and rARF1, in the
presence of GDP or GTPS, to ROS membranes. Comparison of total ROS
suspensions and washed ROS membranes is shown. A, total ROS (T) and washed ROS membranes (W) were prepared as
described under ``Experimental Procedures'' and were
incubated with myr-rARF1
or rARF1
as
indicated in the presence of 100 µM GDP (lanes 1, 2, 5, and 6) or GTP
S (lanes 3, 4, 7, and 8). After 1 h at 30 °C, the
membranes were sedimented and subjected to SDS-PAGE in a 15% acrylamide
gel and stained with Coomassie Blue. Membrane-bound ARF is indicated
with a closed arrow for myr-rARF1 and an open arrow for rARF1, since the electrophoretic mobilities of the two
proteins are slightly different (15) . B,
myr-rARF1
was incubated in the presence of GTP
S with
total ROS (lane 1), washed ROS membranes (lane 2), or
washed ROS membranes supplemented with the soluble fraction resulting
from the first isotonic wash (lane 3). This soluble fraction
(RIE) was concentrated on a Centricon-10 (Amicon) according to method I
as described under ``Experimental Procedures'' and was added
at a final concentration of 0.15 mg of protein/ml equivalent to the
concentration of the soluble proteins in total ROS at 35 µM rhodopsin (conditions of lane 1). C, compared
analysis of RIE prepared by methods I and II. Lane 1, RIE
obtained by sedimentation of a crude ROS suspension (method II). Lane 2, RIE obtained by washing of freeze-thawed ROS (method
I). Approximately 2 µg of protein was loaded on each lane of a 9%
acrylamide gel, which was subsequently stained with Coomassie Blue.
Some well known soluble retinal proteins are identified on the right.
It was
possible to restore a strong binding of myr-rARF1 to washed ROS
membranes in the presence of GTPS by addition of the soluble
fraction resulting from the first isotonic wash (Fig. 1B, lanes 1-3), while
reconstitution with the subsequent hypotonic washes did not enhance
myr-rARF1 binding (not shown). The simplest interpretation of these
data is that washing of ROS membranes eliminates a soluble factor
required for optimal GTP
S-dependent binding of myr-rARF1 to ROS
membranes. This factor is mostly recovered in the first isotonic wash,
referred to as RIE.
Figure 2:
Effect of ROS isotonic extract on
[S]GTP
S binding to myr-rARF1 and rARF1.
Binding of [
S]GTP
S to 2 µM myr-rARF1 (A) or 1.5 µM rARF1 (B)
was determined as described under ``Experimental Procedures''
at 1 mM Mg
in the presence of phospholipid
vesicles, with (
,
) or without (
,
) RIE (0.6
mg of protein/ml), prepared by method I. Binding of
[
S]GTP
S to RIE alone was also monitored
(
). All values were corrected for nonspecific binding of
[
S]GTP
S to phospholipids and filters,
measured in the absence of any protein.
Addition of the fungal metabolite brefeldin A (BFA) did not affect
the spontaneous phospholipid-dependent GTPS binding to myr-rARF1
but totally abolished the RIE-catalyzed exchange, with half-maximal
inhibition at 2 µM BFA (Fig. 3A). BFA is
known to inhibit a wide variety of membrane traffic pathways (20) and has been reported to inhibit an ARF-specific guanine
nucleotide exchange activity present in Golgi membranes (7, 8, 9) or in brain cytosol(10) .
The exact target of BFA is not yet known. It was proposed to be not the
exchange factor itself but rather an associated protein because the
sensitivity to BFA of the soluble exchange activity from bovine brain
was lost after partial purification(11) . The exact mechanism
notwithstanding, the complete inhibition by BFA strongly suggests that
the retinal extract contains an ARF-specific guanine nucleotide
exchange factor. Moreover, this factor is protease sensitive, since
trypsin treatment of RIE totally abolished its stimulatory effect on
the activation of myr-rARF1 (Fig. 3B). Trypsin
sensitivity was also demonstrated for the Golgi membrane-bound exchange
activity(7, 8, 9) . This trypsin control is
important in light of the recent report (21) that acid
phospholipids such as phosphatidylinositol 4,5-bisphosphate can greatly
increase the rate of GDP dissociation from ARF1. In fact, two
additional observations further argue against a possible role for acid
phospholipids in our RIE effects: (i) the exchange activity remained
soluble after a centrifugation at 400,000
g, which
excludes the presence in RIE of membrane vesicles, and (ii) inclusion
of 20% (w/w) phosphatidylinositol 4,5-bisphosphate in azolectin
vesicles had only a marginal (<2-fold) stimulatory effect on the GDP
dissociation rate from myr-ARF1 at µM levels of
Mg
and no effect at all at mM levels of
Mg
(data not shown), consistent with the prediction
that under the latter conditions most of the negative charges of
phosphatidylinositol 4,5-bisphosphate should be masked(22) .
Thus, the most straightforward interpretation for our data is that a
soluble protein is responsible for the RIE-catalyzed guanine nucleotide
exchange on myr-rARF1.
Figure 3:
Effects of brefeldin A and trypsin
treatment on RIE-stimulated GTPS binding to myr-rARF1. A,
myr-rARF1 (2 µM) was incubated with
[
S]GTP
S, with or without RIE, as described
for Fig. 2A except that increasing concentrations of
brefeldin A were added. Brefeldin A was solubilized in methanol, and
the final methanol concentration in all incubations was 5% (v/v). Data
are means ± S.D. of three independent experiments, with
incubation times varying from 5 to 10 min. Spontaneous binding of
[
S]GTP
S to RIE and to myr-rARF1,
insensitive in both cases to brefeldin A, was subtracted. Results are
thus expressed as RIE-stimulated GTP
S binding (the difference
between the observed binding with ARF + RIE and the expected sum),
with the enhanced binding measured in the absence of brefeldin A taken
as 100%. B, the retinal extract (RIE) was treated with trypsin
(
) or, as control, with trypsin inactivated by preincubation with
trypsin inhibitor (TI) (
), as described under
``Experimental Procedures,'' before being added to 1
µM myr-rARF1 and [
S]GTP
S in
the presence of phospholipid vesicles as in Fig. 2A.
Binding of [
S]GTP
S to trypsinized RIE or to
control RIE was measured in parallel and subtracted (maximal values, at
60 min, were 2 and 11 pmol for trypsinized and mock RIE,
respectively).
It should be noted, however, that this
protein does not necessarily derive from the outer segments of rod
cells. Indeed, the exchange activity was found to be more abundant in a
soluble fraction prepared from a crude ROS suspension just separated
from the rest of the retina by flotation on high density sucrose (see
method II for RIE preparation as described under ``Experimental
Procedures''). Thus, it is very likely that while the exchange
activity is released upon shearing of the outer segments, it in fact
comes from the inner segments, known to be rich in BFA-sensitive
transport vesicles(23) . The preparations obtained by method I
(washing of freeze-thawed ROS fragments) and method II (collection of
the soluble fraction resulting from crude ROS sedimentation) had a
similar specific activity and were therefore both designated as RIE.
The yield of method II, however, was higher. From the same number of
retinas, the total exchange activity recovered by method II was
10-fold higher than by method I. It is noteworthy that the protein
pattern (analyzed by SDS-PAGE) of the two preparations was completely
different (Fig. 1C), which suggests that, in both
preparations, the exchange activity is due to a very minor component.
Figure 4:
Effect of RIE on GDP dissociation from
rARF1 and myr-rARF1 in the presence or absence of phospholipid
vesicles. As described under ``Experimental Procedures,''
rARF1 (,
) or myr-rARF1 (
,
,
) was first
loaded with [
H]GDP in the presence (A)
or absence (B) of phospholipids. Maximal radiolabeling was
obtained after 1 h at 37 °C at 0.1 µM free
Mg
(2 mM EDTA, 0.15 mM MgCl
) for myr-rARF1 in the absence of phospholipids
and for rARF1 with or without phospholipids. For myr-rARF1 in the
presence of phospholipids (A), a 3-h incubation at 0.5 mM Mg
was chosen to prevent labeling of the
contaminating unmyristoylated protein. In all cases, Mg
was then raised to 1 mM, and the dissociation was
initiated (at time 0) by addition of 1 mM GDP with (
,
) or without (
,
) RIE, indifferently prepared by
method I or II (both preparations gave similar results). In a control
experiment, RIE was replaced by bovine serum albumin at the same
protein concentration (
). 100% refers to the amount of
H-labeled ARF at the initiation of the dissociation and
corresponds to 25-35 pmol (per 50-µl aliquot). Dissociation
rates are: 0.022 min
(
) and 0.12
min
(
) in A, 8
10
min
(
) and 4
10
min
(
,
) in B.
In the presence of
phospholipids, very similar results were obtained for
[H]GDP dissociation (Fig. 4A) and
[
S]GTP
S binding (Fig. 2). No
dissociation of GDP could be detected from rARF1, with or without RIE,
whereas the dissociation rate from myr-rARF1 was increased
5-fold
by the retinal exchange protein.
In the absence of phospholipids (Fig. 4B), there was still no measurable dissociation
of [H]GDP from unmyristoylated ARF1 with or
without RIE, but most importantly, there was also no acceleration of
GDP release from myr-rARF1 in the presence of RIE. In fact, the rate of
[
H]GDP dissociation was even decreased (by
2-fold) by addition of the retinal extract, but the same effect was
obtained with bovine serum albumin, which points to a nonspecific
stabilizing effect of RIE proteins. Moreover, addition of BFA to the
release medium did not affect the inhibitory effect of RIE in Fig. 4B, while it totally abolished its stimulatory
effect in Fig. 4A (not shown). Altogether, these data
indicate that the retinal exchange factor can recognize ARF
only if ARF is myristoylated and if phospholipid vesicles are
present. In other words, the catalyzed exchange reaction occurs on the
membrane and not in solution.
In the
experiment described in Fig. 5, myr-rARF1 and rARF1 were
preloaded with [S]GTP
S in the presence of
phospholipids, and a large excess of unlabeled GTP
S was added, at
1 mM Mg
, to initiate the release assay. In
the absence of RIE, spontaneous [
S]GTP
S
dissociation was very slow for myr-rARF1 (Fig. 5A) but
notably faster for rARF1 (Fig. 5B) at the same
concentration of phospholipid vesicles. This difference reflects the
different affinity of the two proteins for phospholipids. Indeed,
increasing the concentration of phospholipids 3-fold reduced the
GTP
S dissociation rate from rARF1 by 2-fold and also increased (up
to 80% at equilibrium) the fraction of rARF1 bound to
[
S]GTP
S in the preloading step (data not
shown). This is consistent with the view that phospholipids are
absolutely required for stabilizing ARF
, whether
myristoylated or not, but with a different concentration dependence for
the two proteins because the myristate increases the affinity of
ARF
for lipids (by at least 10-fold, as judged by
sedimentation experiments, not shown).
Figure 5:
Effects of RIE and brefeldin A on
GTPS dissociation from myr-rARF1 and rARF1 in the presence of
phospholipid vesicles. myr-rARF1 (A) and rARF1 (B)
were first loaded with [
S]GTP
S in the
presence of phospholipids, at 1 µM free Mg
(2 mM EDTA, 1 mM MgCl
), for 20 min (A) or 90 min (B) at 37 °C. Then, Mg
was raised to 1 mM, and the dissociation assay was
initiated by addition of 1 mM GTP
S with (filled
symbols) or without (open symbols) RIE prepared by method
II (0.4 mg of protein/ml) and with 300 µM brefeldin A
(
,
,
,
) or 1% methanol as control (
,
,
,
). Samples of 50 µl were withdrawn at the
indicated times. 100% corresponds to 53 pmol in A and 24 pmol
in B. Dissociation rates in A are 7
10
min
without RIE (
) and
0.02 min
with RIE (
); in B, they are
5
10
min
without RIE
([
) and 0.025 min
with RIE (
).
With brefeldin A, the same results were obtained when BFA was added to
ARF and phospholipids 5 min before RIE instead of together with
RIE.
Addition of RIE caused a
marked acceleration of [S]GTP
S release from
both myr-rARF1 and rARF1 (Fig. 5, A and B).
This indicates that (i) like Ras-specific exchange factors mentioned
above, RIE can also recognize the GTP-bound form of its target protein
and catalyze a GTP release, albeit to a much lesser extent than the GDP
release (compare the dissociation rates of GDP and GTP
S from
myr-rARF1 in the presence of RIE in Fig. 4A and Fig. 5A), and (ii) the retinal exchange factor can
recognize nonmyristoylated about as well as myristoylated
membrane-bound ARF
. Therefore, this result strongly
suggests that in RIE-catalyzed GDP release (Fig. 4), the
myristate was required for binding ARF
to the
phospholipids rather than directly involved in the interaction of
ARF
with the exchange factor.
It is noteworthy that
RIE-catalyzed GTPS release from both ARFs was only partly
inhibited by BFA, even at 300 µM (Fig. 5, A and B), whether BFA was preincubated with RIE or with ARF
and phospholipids. The significance of this result is unclear since the
exact mechanism of action of BFA is not known. The fact that the
spontaneous release of GTP
S was unchanged in the presence of BFA
confirms that BFA does not affect the interaction of ARF
with phospholipids. It remains possible that this lipophilic drug
somehow hinders the attachment of the exchange factor to the lipids,
the inhibition being less pronounced when the target protein
(ARF
) is itself solidly membrane-bound.
Importantly, very similar results were obtained when
GTPS-to-[
S]GTP
S exchange was measured (Fig. 6) instead of
[
S]GTP
S-to-GTP
S exchange (Fig. 5). Again, the spontaneous exchange was faster for
nonmyristoylated ARF1 (Fig. 6B), RIE markedly
accelerated the exchange on both ARFs, and BFA caused a partial
inhibition (Fig. 6, A and B). Together, the
results of Fig. 5and Fig. 6unambiguously demonstrate
that RIE promotes GTP
S-GTP
S exchange on both ARFs, but it
should be noted that measure of both GTP
S release (Fig. 5)
and GTP
S loading (Fig. 6) was necessary to eliminate all
possible artifacts. Indeed, as GTP
S release is tightly correlated
with the dissociation of ARF
from phospholipids,
RIE-stimulated GTP
S release in Fig. 5could have been due
in part to a displacement of ARF
from the lipids,
possibly by proteins of RIE unrelated to the exchange activity. On the
other hand, RIE-catalyzed GTP
S binding in Fig. 6could have
been due to some residual ARF
, at least in the case of
myr-rARF1. The symmetry of the data obtained by the two methods
definitely rules out all of these possibilities.
Figure 6:
Effects
of RIE and brefeldin A on the
GTPS-to-[
S]GTP
S exchange on myr-rARF1
and rARF1. 1 µM myr-rARF1 (A) or 1.3 µM rARF1 (B) was first incubated with 10 µM unlabeled GTP
S in the presence of phospholipids and at 1
µM free Mg
for 20 min (A) or 2
h (B) at 37 °C. Then, 300 µM brefeldin A
(
,
) or 1% methanol was added, and 10 min later
MgCl
was added (to a final concentration of 1 mM free Mg
) together with RIE prepared by method II
(0.4 mg of protein/ml, filled symbols) or the corresponding
buffer (open symbols) and 0.2 mM ATP.
[
S]GTP
S was then added (time 0) to a final
activity of
1000 cpm/pmol. All concentrations are indicated as
final, after dilution with RIE and additions. Samples of 25 µl were
withdrawn at the indicated times. [
S]GTP
S
bound by RIE alone (1.3 pmol with or without BFA) has been subtracted
from the data.
In this study, we have analyzed the role of N-myristoylation of bovine ARF1 in its activation in vitro by a soluble, brefeldin A-sensitive guanine-nucleotide exchange
activity obtained from bovine retinas. We demonstrate that stimulation
by the retinal extract of GDP release from ARF1 strictly requires
myristoylation of ARF1 and the presence of phospholipids. The retinal
extract also stimulates to a lesser extent the release of GTPS
from ARF
in the presence of phospholipids but, in
that case, myristoylation of ARF is not required.
The simplest
interpretation of these data is that the retinal exchange factor acts
only on membrane-bound ARF. Its interaction with ARF requires myristoylation of ARF because only myr-ARF
can significantly bind to phospholipids. In contrast, its
interaction with ARF
does not require
myristoylation of ARF because both myristoylated and nonmyristoylated
ARF
strongly bind to phospholipids. In other words,
the myristate is not directly involved in the interaction of ARF with
the exchange factor; it is simply required to bring ARF
to the membrane where the catalyzed exchange reaction occurs. A
similar role of the myristate in facilitating membrane binding has been
ascribed to several other myristoylated proteins(27) .
How
could one explain that an apparently soluble exchange factor interacts
only with membrane-bound ARF? At least two possibilities can be
entertained. First, it can be questioned whether the exchange protein
is totally cytosolic in the cell. Indeed, it is rather puzzling that in
rat liver Golgi membranes the BFA-sensitive nucleotide exchange
activity was described as tightly bound to membranes, being resistant
to salt extraction but not to alkali extraction(8) . While it
is of course possible that the Golgi-bound exchange activity is
completely different from the soluble enzyme present in our retinal
extract, it may well be also that the two enzymes are related
peripheral proteins more or less tightly bound to membranes depending
on the tissue. Thus, the retinal exchange factor might in fact loosely
bind to membranes like myr-ARF, possibly also through a
lipid modification or via a positively charged structure such as a
pleckstrin homology domain frequently found in small G protein-specific
guanine nucleotide exchange factors (28) and thought to be
involved in interactions with membrane phospholipids(29) .
Accordingly, the probability of interaction between myr-rARF
and the exchange factor would be enhanced on the membrane surface
as a result of an increased local concentration of the two proteins. A
similar mechanism has been recently proposed to explain the
lipid-dependent interactions of transducin
and
subunits(30) .
Alternatively, another possibility is that
the insertion of the myristate chain into the lipid bilayer might
induce a conformational change of the ARF protein, which would properly
expose the interaction domain with the exchange factor. Indeed, support
for a membrane-dependent conformational change of myr-ARF comes from our observation that the rate of GDP release is
increased by phospholipids(15) . The amphipathic amino-terminal
-helix most likely participates in this conformational change.
This helix has been shown to be held by hydrophobic forces in a cleft
of the GDP-bound unmyristoylated form of ARF1(31) , but its
fate upon binding of the myristoyl group to the membrane is not known.
It seems reasonable to predict that the helix could be displaced out of
the cleft and come in contact with the phospholipid bilayer. The
interaction with the exchange factor could somehow accentuate the
conformational change and further open the nucleotide binding site.
These two possible mechanisms are in fact not exclusive. Both an
increased local concentration and a correct orientation of
myr-ARF
and the exchange enzyme might well facilitate
their interaction.
Our finding that only myristoylated ARF can be activated (by GDP-to-GTP exchange) by the retinal exchange factor contradicts the conclusions of two previous studies on Golgi membranes (9, 13) but provides a reasonable explanation for the observation that myristoylation is required for many ARF activities(32, 33, 34) .
It should be
stressed that only the GDP-to-GTP exchange has a functional meaning.
Our observation that in vitro release of GTPS was
stimulated by the retinal extract provides insight into the role of
myristoylation but is of questionable physiological relevance. It is
very likely that in vivo ARF
will not remain
bound to its exchange factor because of the presence of effectors with
higher affinity.
We have observed that brefeldin A totally inhibits
the retinal extract-stimulated GDP release from myr-ARF with an
IC of 2 µM, whereas it inhibits only
partially the stimulated GTP
S release. The significance of this
result is difficult to assess as long as the molecular basis for
brefeldin inhibition is unknown. The exact target of BFA is still not
found, and we cannot exclude that the nucleotide exchange activity of
our retinal extract involves in fact several proteins. Thus, in
addition to the exchange factor itself, an adaptor protein serving as a
membrane anchor might be required, which could be the receptor for BFA.
This would be consistent with the observation that the sensitivity to
BFA is lost during purification of the exchange protein(11) .
Obviously, the exact mechanism underlying ARF activation is far from
being understood, but if the process proves to involve several
components, it may help to first describe the characteristics of a
crude preparation before trying to reconstitute the system with
purified elements.