(Received for publication, September 13, 1994; and in revised form, November 14, 1994)
From the
Recombinant N-myristoylated bovine ADP-ribosylation
factor 1 (myr-rARF1) has been expressed in bacteria and purified to
near homogeneity with a high (85%) myristoylation efficiency. Myr-rARF1
and nonmyristoylated rARF1 have been compared with respect to their
kinetics of guanine nucleotide exchange and their interactions with
phospholipids. Myristoylation is shown to allow the release of bound
GDP at physiological (mM) concentrations of
Mg. GDP dissociation is slow in the absence of
phospholipids but is accelerated 2-fold in the presence of phospholipid
vesicles. On the contrary, myristoylation decreases 10-fold the rate of
dissociation of GTP or guanosine 5`-O-(thiotriphosphate)
(GTP
S) in the presence of phospholipids. As a result, myr-ARF1 can
be spontaneously activated by GTP or GTP
S (t
30 min at 37 °C) at 1 mM Mg
, in the sole presence of phospholipid
membranes without the need for a nucleotide exchange factor.
In
contrast to the nonacylated protein, the GDP-bound form of myr-ARF1
interacts with phospholipids, as demonstrated by its cosedimentation
with phospholipid vesicles and its comigration with
phospholipid/cholate micelles on gel filtration. The interaction is,
however, weaker than for the GTP-bound form, suggesting that only the
myristate in myr-ARF1 interacts with phospholipids,
whereas both the myristate and the amino-terminal hydrophobic residues
in myr-ARF1
bind to phospholipids.
ADP-ribosylation factors (ARFs) ()are a family of
20-kDa guanine nucleotide-binding proteins. ARFs were originally
discovered as cofactors for cholera toxin-catalyzed ADP-ribosylation of
G
(1) , but, as a result of cDNA cloning, they now
appear to constitute a large family that includes both the ARF
proteins, acting as cofactors for cholera toxin activity, and the
ARF-like proteins, which lack this activity(2) . All known
members of the ARF family have a glycine residue at position 2, which
is the site of N-myristoylation, a cotranslational
modification.
ARFs have been recognized as regulators of
intracellular vesicular transport and more recently as activators of
phospholipase D (for review, see (3) ). Both GTP binding and
myristoylation appear to be required for ARF
activities(4, 5, 6, 7) , but the
precise role of myristoylation is not clear. Myristoylation was found
to be necessary for GTP-dependent binding of ARF to Golgi membranes (8, 9) and to phospholipid vesicles(10) , and
some authors favor the view that binding of GTP to ARF induces a
conformational switch that exposes the N-myristoyl group,
enabling its binding to the lipid bilayer (9, 11, 12) according to the model of the
Ca-myristoyl switch proposed for
recoverin(13) . However, our previous report (14) that
nonmyristoylated rARF1 strongly binds to phospholipids once it is
activated by GTP
S does not support this hypothesis. Instead, we
believe that myr-ARF
interacts with lipid bilayers
mostly through the amphiphatic
helix found at the amino terminus (15) and that the GTP-induced conformational change primarily
affects that
helix rather than the fatty acid.
In an attempt,
therefore, to understand at which step of ARF activation myristoylation
plays a role, we have compared in this study the myristoylated and
nonmyristoylated forms of rARF1 for their interactions with
phospholipids and their nucleotide exchange properties. We demonstrate
that myristoylation of ARF allows GDP dissociation at physiological
Mg concentrations, especially in the presence of
phospholipids, because myr-rARF1
interacts with lipid
membranes in marked contrast to rARF1
. This interaction
appears to further open the nucleotide binding site and facilitate
nucleotide exchange. Moreover, myristoylation enhances the
phospholipid-dependent stabilization of the GTP- or GTP
S-bound
form.
Oligonucleotide 1 (5`-TGCACCATGGGGAATATCTTTGCAAACCTCTTC-3`) introduced a NcoI site at the 5` end without changing the coding sequence.
Oligonucleotide 2 (5`-ATGAGGATCCTCATTTCTGGTTCCGGAGCT-3` introduced a BamHI site right after the stop codon. The NcoI-BamHI fragment was inserted in the pET11d expression vector (Novagen Inc.). Saccharomyces cerevisiaeN-myristoyltransferase expression vector (pBB131) was kindly provided by J. I. Gordon(16) .
For
production of myristoylated ARF1 (myr-rARF1), BL21(DE3) bacteria were
cotransformed with the pET11d/Gly-2 ARF1 and pBB131 (yeast N-myristoyltransferase) plasmids and selected for both
ampicillin and kanamycin resistance. Transformed cells were grown at 37
°C to A = 0.6. Myristate (50
µM) bound to bovine serum albumin (6 µM) was
added as a 100-fold concentrated solution prepared by slowly adding
warm sodium myristate 100 mM, pH 9, to fatty acid-free bovine
serum albumin. After 10 min, the coexpression of ARF1 and N-myristoyltransferase was induced with 0.3 mM isopropyl-1-thio-
-D-galactopyranoside, and the
temperature was reduced to 27 °C to increase the yield of
myristoylation (see ``Results''). After 3 h, the cells were
harvested and lysed(17) . The supernatant was precipitated at
35% saturation of ammonium sulfate and centrifuged at 8,000
g for 30 min. The pellet was dissolved in 50 mM Tris/HCl, pH 8, 1 mM MgCl
, 1 mM DTT,
5 µM GDP, dialyzed against the same buffer, and applied to
a DEAE-Sepharose column equilibrated with 50 mM Tris/HCl, pH
8, 1 mM MgCl
, 1 mM DTT. The flow-through
fractions containing ARF1 were pooled, concentrated on a Centricon-10
(Amicon) in 10 mM MES, pH 5.7, 1 mM MgCl
,
1 mM DTT, and applied to a Mono S column (Pharmacia)
equilibrated in the same buffer. The bound proteins were eluted with a
gradient of NaCl (0-500 mM). Fractions containing
myr-rARF1 (eluted between 130 and 170 mM NaCl) were pooled,
concentrated on a Centricon-10 (Amicon) in 50 mM Tris/HCl, pH
8, 1 mM MgCl
, 1 mM DTT, 2 µM GDP, to a protein concentration of
0.8 mg/ml and stored at
-70 °C.
Figure 1:
Expression and
purification of myr-rARF1. A, coexpression of rARF1 and yeast N-myristoyltransferase was induced at 27 °C, and myr-rARF1
was purified from the cytosolic fraction as described under
``Experimental Procedures.'' Samples were subjected to
SDS-PAGE in a 15% gel and stained with Coomassie Blue. Lane 1,
molecular mass markers; lane 2, total cytosol; lane
3, proteins precipitated at 35% saturation of ammonium sulfate; lane 4, pooled DEAE-Sepharose fractions; lane 5,
pooled Mono S fractions. Arrows on the right indicate
the positions of nonmyristoylated rARF1 (upper) and
myristoylated rARF1 (lower). B, compared
electrophoretic mobilities of the three forms of rARF1: Met-1 ARF (M), nonmyristoylated Gly-1 ARF (G
), and myristoylated Gly-1 ARF (myrG
). Lane 1,
nonmyristoylated rARF1 obtained without expression of yeast N-myristoyltransferase and purified on QAE-Sepharose; lane
2, cytosolic extract after coexpression of rARF1 and N-myristoyltransferase at 37 °C; lane 3,
cytosolic extract after coexpression of rARF1 and N-myristoyltransferase at 27 °C; lane 4,
myr-rARF1 purified on Mono S column. Arrows on the right indicate the positions of Met-1 ARF1 (upper) and
myr-rARF1 (lower).
Figure 2:
Time course of
[S]GTP
S binding to rARF1 and myr-rARF1 in
the presence of phospholipid vesicles at 1 µM and 1 mM Mg
. Binding of
[
S]GTP
S to 1 µM rARF1 (
)
or myr-rARF1 (
) was determined as described under
``Experimental Procedures'' in the presence of phospholipid
vesicles. A,1 µM free Mg
(2
mM EDTA, 1 mM MgCl
); B, 1 mM MgCl
. Values are means of two or three independent
experiments and were normalized relative to the maximum binding
observed for each protein at 1 µM Mg
measured after 30 min for myr-rARF1 and 150 min for rARF1. The
maximum binding corresponded to 50% of the concentration of rARF1, and
to 60-90% of the concentration of myr-rARF1, depending on the
preparation used. The curves (except for
in B) were obtained by
fitting the data to the model y = 100
(1 - e
) with k = 0.68
min
for myr-rARF1 and 0.03 min
for rARF1 at 1 µM Mg
, and k = 0.02 min
for myr-rARF1 at 1 mM Mg
.
Figure 3:
Dissociation of GDP and GTPS from
rARF1 and myr-rARF1. A and B, rARF1 (1
µM) was loaded for 60 min at 37 °C with 10 µM [
H]GDP in the presence of 0.1 µM Mg
(1 mM EDTA, 0.1 mM MgCl
), with (
) or without (
) phospholipid
vesicles; myr-rARF1 (1 µM) was loaded with 10 µM [
H]GDP for 60 min in the presence of 100
µM MgCl
and phospholipid vesicles (
) or
for 30 min at 0.1 µM Mg
without
phospholipids (
). [
H]GDP dissociation was
initiated by adding 1 mM unlabeled GDP with MgCl
± EDTA to give a final concentration of 1 µM (A) or 1 mM (B) free
Mg
. The concentration of bound
[
H]GDP just prior to the initiation of the
exchange reaction (taken as 100%) was 0.60 and 0.50 µM for
rARF1, and 0.46 and 0.40 µM for myr-rARF1 with and without
phospholipids, respectively. The curves are best fits assuming
first-order kinetics for nucleotide dissociation. At 1 µM Mg
in the presence of phospholipids k
= 0.05 min
for rARF1
and 0.91 min
for myr-rARF1; at 1 mM Mg
, there was no detectable dissociation from
rARF1, with or without phospholipids, whereas k
values were 15
10
and 8
10
min
for myr-rARF1 with and
without phospholipids, respectively. C and D, in the
presence of phospholipid vesicles, rARF1 (
) and myr-rARF1 (
)
were loaded for 90 and 30 min, respectively, with 10 µM [
S]GTP
S (900 cpm/pmol) at 1 µM free Mg
. In the absence of phospholipids,
loading was performed with 2 µM [
S]GTP
S (6000 cpm/pmol) for 1 h at 1
µM Mg
for rARF1 (
) and for 3 h
at 1 mM Mg
for myr-rARF1 (
).
[
S]GTP
S exchange was initiated by the
addition of 1 mM unlabeled GTP
S either alone (C)
or with 1 mM Mg
, final concentration (D). 100% corresponds to 27 and 31 pmol (per 50-µl
aliquot) in the presence of lipids, and to 6 and 1 pmol in the absence
of lipids, for rARF1 and myr-rARF1, respectively. Dissociation rates
are: 0.074 min
(
) and 0.027 min
with a maximal exchange of 77% (
) in C and 5
10
min
(
), 5
10
min
(
), 0.043
min
(
), and 0.54 min
(
) in D.
We
next compared the rates of dissociation of
[S]GTP
S from rARF1 and myr-rARF1 at low and
high Mg
(Fig. 3, C and D).
We previously reported that phospholipids stabilize the GTP
S-bound
form of rARF1 by decreasing the k
rate of
GTP
S(14) . Myristoylation of rARF1 accentuates this
effect, since in the presence of phospholipids the dissociation of
[
S]GTP
S was notably slower from myr-rARF1
than from rARF1, both at low and at high Mg
concentrations. In contrast, in the absence of lipids, GTP
S
dissociated very rapidly from myr-rARF1, even at 1 mM Mg
(Fig. 3D). For that reason,
it was difficult to load myr-rARF1 with
[
S]GTP
S in the absence of phospholipids. In
the experiment described in Fig. 3D, only 2% of total
ARF had bound [
S]GTP
S at time 0 of the
dissociation, but we are confident that this fraction represented only
myr-rARF1 because GTP
S loading was performed at 1 mM Mg
, which precluded nucleotide exchange on the
contaminating rARF1.
Thus altogether, the effect of phospholipids on
the dissociation of GTPS is much more dramatic for myr-rARF1 than
for rARF1, since for the myristoylated protein the k
rate was
1000-fold lower in the presence of lipids.
myr-rARF1 can also be loaded with GTP at 1 mM Mg in the presence of phospholipids but less
efficiently than with GTP
S (Fig. 4A). This
difference is not due to the hydrolysis of GTP by myr-rARF1 but to a
faster dissociation of GTP than of GTP
S (Fig. 4B).
Two observations demonstrate that myr-rARF1 lacks any GTPase activity:
1) similar kinetics were obtained with
[
-
P]GTP and
[
-
P]GTP both for GTP binding and
dissociation (Fig. 4); 2) no liberation of
P
from [
-
P]GTP could be detected with
either myr-rARF1 or rARF1 (not shown) provided that 3 mM phosphate was included in the assay to inhibit a nucleotidase (or
phosphatase) activity that frequently contaminated the ARF preparations
to a varying degree. This contaminant, which appeared only slightly
smaller than ARF by gel filtration, was very difficult to separate from
rARF1 (myristoylated or not). It hydrolyzed ATP as well as GTP and
could be completely inhibited by a few mM P
or by
AlF
(5 mM F
and 10 µM AlCl
). We cannot exclude that it might be a
degradation product of ARF.
Figure 4:
Comparison of rARF1 and myr-rARF1 for the
binding and the dissociation of GTP at 1 mM Mg in the presence of phospholipids. A, 1 µM of rARF1 (
) or myr-rARF1 (
,
,
) was
incubated in the presence of phospholipid vesicles and 1 mM Mg
with 10 µM [
-
P]GTP (
,
),
[
-
P]GTP (
) or
[
S]GTP
S (
), and 3 mM phosphate (see ``Results''). Values are expressed as
pmol of bound nucleotide/50 pmol of ARF. Fitting the data to the model y = A
(1
-e
) gave the following
parameters: A
= 45 pmol, k = 0.021 min
(
), and A
= 26 pmol, k = 0.022
min
(
,
). B, 1 µM of rARF1 (
) or myr-rARF1 (
,
) was loaded for 30
min or 5 min, respectively, with 10 µM [
-
P]GTP (
,
) or
[
-
P]GTP (
) in the presence of
phospholipids and 1 µM free Mg
.
Dissociation was initiated by the addition of 1 mM unlabeled
GTP and 1 mM Mg
(final concentration).
Nucleotide bound at the initiation of the exchange (100%) was 32 pmol
for myr-rARF1 and 15 pmol for rARF1/50 pmol ARF. Dissociation rates are
5
10
min
(
,
)
and 0.042 min
(
).
The rate of dissociation of GTP from
myr-rARF1 at 1 mM Mg in the presence of
lipids (Fig. 4B) was intermediate between the rates of
dissociation of GTP
S measured at 1 µM and 1 mM Mg
(Fig. 3, C and D).
This suggests that the interaction of Mg
is stronger
with GTP
S than with GTP on myr-rARF1. The same is true for rARF1,
since GTP dissociates
8-fold more rapidly than GTP
S at 1
mM Mg
(Fig. 3D and Fig. 4B).
Figure 5:
Binding of myr-rARF1 to
phospholipid vesicles. rARF
or myr-rARF
was
incubated with phospholipid vesicles as described under
``Experimental Procedures.'' After centrifugation, pellet (P) and supernatant (S) were analyzed by SDS-PAGE and
Coomassie Blue staining. Arrows on the right indicate
the positions of Met-1 rARF1 (openarrow) and
myr-rARF1 (closedarrow). The figures below indicate the distribution (in percent) of ARF between pellet and
supernatant.
Figure 6:
Binding of myr-rARF1 to
phospholipid-cholate micelles. rARF1
or myr-rARF1
was incubated with DMPC/cholate micelles, and the mixture was
analyzed by gel filtration as described under ``Experimental
Procedures.'' The solid curves indicate protein absorbance at 280
nm, and the fractions were analyzed by SDS-PAGE, as shown below. A, myr-rARF1
and DMPC/cholate micelles.
[
H] DPPC (0.2 µM, 40,000 dpm/µl)
was included to indicate the elution volume of the micelles
(
-
). Arrows indicate the positions on the
gel of myr-rARF1 (closedarrow) and nonmyristoylated
rARF1 (openarrow). B, nonmyristoylated
rARF1
and DMPC/cholate
micelles.
Recombinant bovine ARF1 has been expressed in bacteria as
unmodified ARF1 or N-myristoylated ARF1. In contrast to
Randazzo et al.(10) who could not separate the two
forms, we found that myr-rARF1 and rARF1 migrate differently on
SDS-polyacrylamide gels and can be efficiently separated in a few
steps. Obtention of myr-ARF1 that is at least 85% myristoylated allowed
us to compare the properties of the two forms with regard to their
interactions with phospholipids and the Mg dependence
of nucleotide exchange. Two major differences between myristoylated and
nonmyristoylated ARF1 are described.
The first one concerns the
interaction of the GDP-bound form with phospholipids. Nonmyristoylated
ARF1 binds to phospholipids exclusively when it is in the GTP-bound
form. We confirm here with wild-type Gly-2 ARF1 what we previously
reported for Ile-2 ARF1(14) . In fact, we have not detected any
difference between nonmyristoylated Gly-2 and Ile-2 ARF1 proteins in
their interactions with phospholipids or in the kinetics of nucleotide
binding, despite the fact that the amino-terminal Met was partially
cleaved in Gly-2 ARF1 and totally conserved in Ile-2 ARF1. In contrast
with the nonmyristoylated protein, the GDP-bound form of myr-rARF1 also
interacts with phospholipids, either as phospholipid vesicles or as
phospholipid/cholate micelles. However, the interaction is weaker than
for the GTP-bound form, consistent with the idea that in
myr-rARF1 only myristate interacts with lipids with a
rather low affinity(19) , while in myr-rARF1
both
the myristate chain and the amino-terminal hydrophobic residues of the
protein moiety interact with phospholipids. Thus, myr-ARF
should not be considered as totally cytosolic as it is commonly
described (20, 21, 22) but more likely as
predominantly membrane-bound in intact cells due to the high cellular
ARF concentration. Cell fractionation might cause its release into the
soluble fraction as a consequence of the resulting dilution.
Another
salient difference between rARF1 and myr-rARF1 concerns the
Mg dependence of nucleotide exchange. GDP can be
released from nonmyristoylated ARF1 only at low (µM)
concentrations of free Mg
(14) . No
significant nucleotide exchange occurs at physiological (mM)
Mg
concentrations, which led us to hypothesize that a
guanine nucleotide-exchange factor is required for ARF activation in vivo(14) . However, we show in the present study
that, in marked contrast to the nonmyristoylated protein, myr-rARF1 can
release its GDP at high Mg
concentrations. GDP
dissociation is slow in the absence of phospholipids (t
= 90 min at 37 °C) but is accelerated by
about 2-fold in the presence of lipid vesicles. This suggests that
hydrophobic interaction of the myristate chain with either the protein
(in the absence of lipids) or, more efficiently, with the phospholipid
bilayer somehow results in the opening of the nucleotide binding site.
A possible mechanism is the displacement of the amino-terminal peptide,
since its removal was shown to greatly increase the release of bound
GDP(15) .
If GDP can dissociate from myr-rARF1 with or
without phospholipids, it should be noted that its replacement by GTP
or GTPS will efficiently occur only in the presence of lipids,
because in their absence GTP or GTP
S dissociate much faster than
GDP. However, if as discussed above myr-ARF
remains
essentially membrane-bound in cells, the GTP-bound form should be
immediately stabilized by interaction with the bilayer. Myristoylation
appears to be crucial for the lipid-dependent stabilization of the
GTP-bound form (Fig. 4B). It should be noted that with
GTP
S, the importance of myristoylation is less obvious, as
Mg
seems to stabilize GTP
S more strongly than
GTP in the nucleotide binding site and therefore GTP
S dissociates
notably more slowly than GTP from both rARF1 and myr-rARF1 at 1 mM Mg
.
Thus, in summary, myr-ARF1 can be
spontaneously activated by GTP or GTPS at physiological
concentrations of Mg
in the sole presence of
phospholipids without any nucleotide exchange factor. However, the
activation remains rather slow (t
30 min),
which does not preclude the existence in cells of such factors, which
would accelerate the exchange and might also account for the specific
localization of the various ARFs. Indeed, several groups have reported
evidence of an ARF-specific guanine nucleotide-exchange activity in
Golgi membranes (10, 22, 23) , and a partial
purification of this activity from bovine brain cytosol has been
recently described(24) . Brefeldin A inhibits the exchange
activity in the crude preparation but no longer in the partially
purified fraction(24) . It is noteworthy that brefeldin A has
no effect on the spontaneous lipid-dependent activation of myr-ARF1
described here (not shown).
Nevertheless, in most cell-free systems
in which myristoylation has been reported to be essential for ARF
functions, including its role in intra-Golgi (4, 6) or
endoplasmic reticulum to Golgi (5) transports, in
endosome-endosome fusion(25) , and in phospholipase D
activation(7, 26) , it is not necessary to postulate
the involvement of a specific nucleotide exchange factor to account for
the higher activity of myr-ARF. Our present finding that at mM Mg concentrations only the myristoylated protein
can be spontaneously activated provides a reasonable explanation for
the observed requirement for myristoylation.