From the
ADP-ribosylation factor 1 (Arf1) is an essential N-myristoylated 21-kDa GTP-binding protein with activities
that include the regulation of membrane traffic and phospholipase D
activity. Both the N terminus of the protein and the N-myristate bound to glycine 2 have previously been shown to
be essential to the function of Arf in cells. We show that the bound
nucleotide affects the conformation of either the N terminus or
residues of Arf1 that are in direct contact with the N terminus. This
was demonstrated by examining the effects of mutations in this
N-terminal domain on guanosine 5`-O-(3-thio)triphosphate
(GTP
When Arf1 was acylated,
the GTP-dependent conformational changes were codependent on added
phospholipids. In the absence of phospholipids, myristoylated Arf1 has
a lower affinity for GTP
ADP-ribosylation factors (Arfs)
Activation of Arf is tightly correlated with the high affinity
binding of GTP. This was first demonstrated when Arf
The
specific domains of Arf that interact with target proteins, referred to
as effector domains, have not yet been identified. Two criteria have
previously been used to define effector domains in other GTP-binding
proteins. The conformation of the effector domain should be sensitive
to bound nucleotide. For instance, comparison of Ras
A critical aspect of Arf
action in cells appears to be its regulated binding to and release from
different intracellular membranes(7, 8, 15) .
While regulated by GTP, this membrane association is greatly enhanced
by myristate bound to the N terminus(7, 8, 20) . N-Myristoylation is a cotranslational covalent modification
with an absolute requirement for an amino-terminal glycine, the
acceptor site(21, 22) . Residues located immediately
C-terminal (positions 3-7) are also critical determinants in
whether or not a protein is a substrate for N-myristoyltransferase(23) . As several of the putative
Arf effectors in cells (e.g. phospholipase D and
G
We present
evidence that the conformation of the N-terminal domain (or residues in
direct contact with the N terminus) of Arf1 depends on the bound
nucleotide. Because the affinity of Arf1 for GTP is lower than that for
GDP, the conformational changes accompanying GTP binding must consume a
large part of the binding energy. Therefore, deleting or mutating a
domain that undergoes the GTP-dependent conformational change should
reciprocally affect GTP and GDP binding affinities. In contrast, any
change in the nucleotide-binding site itself is likely to affect
binding of GDP and GTP similarly. As mutations in the N terminus caused
an increased GTP affinity and a decreased GDP affinity, we conclude
that the N terminus forms at least part of the GTP-sensitive switch. We
also demonstrate that N-myristoylation is the predominant
factor responsible for regulation of nucleotide binding by
phospholipids.
In contrast to the
inefficient acylation of mammalian Arfs in bacteria, the yeast Arf
proteins were observed to have a near optimal sequence (residues
2-7) for N-myristoyltransferase, and a peptide derived
from the N terminus of S. cerevisiae Arf2 was shown to be an
excellent substrate for N-myristoyltransferase (23). When
protein expression was induced in the presence of
[
Reverse-phase HPLC and
electrospray ionization-mass spectrometry revealed multiple chemical
forms present in the preparation of N-acylated
[3-7LFASK]Arf1. A series of peaks were observed (Fig. 4) that differed in molecular mass by 27.2 ± 1.6 Da.
These molecular masses, determined using a maximum entropy algorithm to
deconvolute the data from electrospray ionization-mass
spectrometry(35) , differ by the mass of a C
The effects of phospholipids on GTP
These studies were undertaken to test the model that there
are conformational differences in the N termini of Arf
Conformational differences in the N
termini of Arf1
Our
conclusion that the N terminus is an effector domain of Arf1 does not
preclude the presence of a second effector domain. Arf has been shown
to have at least two distinct binding sites. One site is for G
A number of roles for
covalently bound myristate have been ascribed to proteins, including
Arf. These studies provide evidence for a role of the acyl group in
integration of ligand (GTP) and phospholipid binding. N-Myristoylation has been found to play a structural role (e.g. cAMP-dependent protein kinase(40) ) and a role in
membrane anchoring (e.g. viral group-specific antigen
proteins(41) ) and in ligand-sensitive membrane association (e.g. recoverin,
Arf(7, 12, 42, 43, 44) ). We
found that the interaction of the acyl chain with Arf1 is sensitive to
the guanine nucleotide bound. We also showed that the myristate
interacts with phospholipids to help drive Arf1 into the active
conformer. These data are in concordance with those published by Franco et al. (45) using myristoylated wild-type Arf1.
The above
data show that the acyl group (myristate in native Arf proteins) is
more important than the amino-terminal domain as a determinant of
interaction with phospholipids. This is consistent with previous work
showing that GTP-dependent association with phospholipids and membranes
is highly dependent on Arf being
myristoylated(7, 8, 12, 20, 44) .
These data also help explain the differences in binding stoichiometries
reported for Arfs purified from bovine brain and recombinant
Arfs(6, 34) .
Studies in the laboratory of Dr.
Jeffrey I. Gordon have demonstrated the importance of the amino
terminus in determining both specificity and rate constants for the
acylation of peptides by a yeast N-myristoyltransferase(47) . Mammalian Arf proteins are
poor substrates for N-myristoyltransferase, while Arfs found
in the yeast S. cerevisiae are quite good substrates.
Mutagenesis of human Arf1 provided additional evidence of the
importance of residues 3-5 as well as 6 and 7 in substrate
recognition by N-myristoyltransferases, further defined
differences in fungal and mammalian N-myristoyltransferase
specificities, and allowed the production of recombinant proteins that
are highly processed and suitable for structural and functional studies
of the myristoylated Arf protein.
Myristoylated proteins purified
from mammalian tissues, with the exception of the
retina(48, 49) , have been found to be homogeneously
modified with
myristate(21, 22, 36, 49) . In
characterizing recombinant Arfs that are good substrates for N-myristoyltransferase in the bacterial coexpression system (S. cerevisiae Arf1 and [3-7LFASK]Arf1), we
found them to be heterogeneously acylated. Deconvolution of the mass
spectrometry data revealed a series of peaks differing by 26-28
atomic mass units, consistent with the addition or deletion of an
ethylene group (C
For some of
the proteins, particularly nonmyristoylated Arf1, the effect of
phospholipids on affinities was less than that anticipated based on the
phospholipid effect on binding stoichiometry. Also, the difference in
affinities for GTP and GDP was not as great as previously
reported(34) . There are a number of possible reasons for the
apparent discrepancy. The previous study determined association rates
using nucleotide-free proteins, being renatured out of 7 M urea. This preparation is both very unstable and proper, and
complete refolding could not be asserted. In the studies reported
above, the concentrations of both protein and nucleotides were greater
than those used in previous studies. Both the stability of Arf proteins
in solution and the determined binding stoichiometries have been
reported to be sensitive to the concentration of Arf in the binding
reaction(46) . This still cannot completely explain the binding
data. Indeed, the complexities of nucleotide exchange on Arf are
illustrated in the double exponential decay kinetics of
Arf
These studies have provided evidence for the
identification of the amino terminus of Arf as an effector domain and
provide support for the conclusion that the role of the myristate is to
integrate phospholipid binding and GTP binding with protein activation.
We are currently using these results to further define specific
residues involved in interaction with Arf GTPase-activating protein and
other target proteins.
Binding in the presence
(+DMPC) or absence (-DMPC) of DMPC/cholate was determined as
described under ``Materials and Methods.'' The binding rates, k (min
Nucleotide dissociation rates from the indicated proteins in the
presence (+DMPC) or absence (-DMPC) of DMPC/cholate were
determined as described under ``Materials and Methods.''
[
K
We thank Dr. John K. Northup for helpful discussions
throughout the work and preparation of the manuscript and Juan Carlos
Amor and Drs. Dagmar Ringe, Chun-jiang Zhang, Annette Boman, and Anne
G. Rosenwald for comments on the manuscript. Drs. Jeffrey I. Gordon and
Jennifer Lodge generously provided the structural genes for the human
and yeast N-myristoyltransferases as well as advice on use in
bacteria.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
S) and GDP binding and dissociation kinetics. Arf1 mutants,
lacking 13 or 17 residues from the N terminus or mutated at residues
3-7, had a greater affinity for GTP
S and a lower affinity
for GDP than did the wild-type protein. As the N terminus is required
for interactions with target proteins, we conclude that the N terminus
of Arf1 is a GTP-sensitive effector domain.
S than for GDP, and in the presence of
phospholipids, the myristoylated protein has a greater affinity for
GTP
S than for GDP. Thus, N-myristoylation is a critical
component in the construction of this phospholipid- and GTP-dependent
switch.
(
)are
ubiquitous and highly conserved GTP-binding proteins that regulate a
number of steps in the exocytic and endocytic pathways (1, 2) and activate of phospholipase
D(3, 4) . Other activities ascribed to Arf proteins
include cofactor for cholera toxin (5, 6) and regulator
of coat protein binding to membranes(7, 8) . In addition
to the regulatory ligand GTP, Arf activities require an intact amino
terminus (9, 10) and the cotranslational covalent
addition of myristate to the amino-terminal
glycine(3, 7, 11, 12, 13, 14) .
GTP
S was
found to be active as a cofactor for cholera toxin-catalyzed
ADP-ribosylation of G
, whereas Arf
GDP was
inactive(6) . Other Arf activities, including activation of
phospholipase D activity, inhibition of intra-Golgi and endoplasmic
reticulum Golgi transport, and inhibition of endosome-to-endosome and
nuclear vesicle
fusion(3, 4, 8, 15, 16, 17) ,
are also highly dependent on the binding of GTP or the slowly
hydrolyzable analog GTP
S. Similarly, Arf
GTP was found to
have at least a 50-fold greater affinity for Arf GTPase-activating
protein than did Arf
GDP(18) . Thus, as is true for many,
if not all, regulatory GTP-binding proteins, the nucleotide bound has
been shown to regulate the affinity of Arf for target proteins.
GDP and
Ras
GTP crystal structures has revealed differences in two
regions, referred to as switch 1 and switch 2(19) . In addition,
certain mutations in the effector region should allow uncoupling of GTP
binding and increased binding of target proteins. In Ras, switch 1 also
meets this criterion, identifying this as an effector
domain(19) . Based on data from previous studies, the N-terminal
domain of Arf has been implicated as an effector domain. Deletion of
the N-terminal 13 or 17 amino acids results in proteins that can bind
GTP but that are inactive as cofactors for cholera toxin-catalyzed
ADP-ribosylation of G
(9, 10) . These mutant
proteins also have decreased affinity for Arf GTPase-activating
protein(10) . Thus, the N terminus meets one of the criteria
used to define the effector domain in Ras.
) are membrane-bound, it is important to distinguish
between changes in the ability of Arf to associate with membranes and
changes in its ability to interact with effectors.
Proteins
Recombinant Arf1,
[17]Arf1 (Arf1 with amino acids 1-17 deleted), and
[
13]Arf1 (Arf1 with amino acids 1-13 deleted) were
prepared as described previously(9, 10) . The N-terminal
mutants [6,7SK]Arf1 (Arf1 in which residues Ala
and Asn
are replaced by Ser
and
Lys
) and [3-7LFASK]Arf1 (Arf1 in which
wild-type residues Asn
-Ile-Phe-Ala-Asn
are
replaced by Leu
-Phe-Ala-Ser-Lys
, as found at
residues 3-7 in Saccharomyces cerevisiae Arf1) were
constructed by polymerase chain reaction amplification of the human
Arf1 coding region with sense primers that include the desired
mutations and incorporating an NdeI site at the initiating
methionine codon and with an antisense primer that adds a BamHI site 6 base pairs 3` of the stop codon. The resulting
open reading frame was subcloned into the NdeI and BamHI sites of the pET3C vector and transfected into BL21(DE3)
cells as described(24, 25, 26) . The full open
reading frame of each mutant was sequenced to ensure that no additional
mutations were introduced in the polymerase chain reactions.
Coexpression of Arf Proteins with N-Myristoyltransferases
in Bacteria
The T7 polymerase/promoter system of Studier et
al.(24, 25) , for expression of foreign proteins in
bacteria, was modified as described by Duronio et al.(27) to allow the coexpression of Arfs with N-myristoyltransferases. Yeast (S. cerevisiae) N-myristoyltransferase expression was achieved in BL21(DE3)
cells using plasmid pBB171 (the generous gift of Dr. Jeffrey I. Gordon,
Washington University, St. Louis, MO)(27) , which carries the
structural gene for yeast N-myristoyltransferase under
regulation by the T7 promoter, and a kanamycin resistance selectable
marker. Human N-myristoyltransferase expression was achieved
with a similar plasmid, pNMT1, which contains the entire open reading
frame in an 1.6-kilobase BglII-EcoRI fragment
cloned into the same sites of pBB131 (the human N-myristoyltransferase open reading frame was also kindly
provided by Dr. Jeffrey I. Gordon)(28) . Doubly transformed
BL21(DE3) cells were obtained by plating transformants on LB medium
with 100 µg/ml ampicillin and 50 µg/ml kanamycin after
transformation of calcium-competent cells with up to 1 µg of each
plasmid. Transformants were grown in liquid culture to a density of A
= 0.7-1.0 before adding the
inducer isopropyl-
-D-thiogalactopyranoside to a final
concentration of 1 mM. Where indicated, 125 µM myristate was added at the time of induction as
described(26) . When radiolabeling with
[
H]myristate was performed, the label was added
at the time of induction to 25-75 µCi/ml. Cells were
harvested after 90 min, unless otherwise specified.
Nucleotide Binding
Binding reactions contained 1
µM Arf and a 10 µM concentration of the
indicated radiolabeled guanine nucleotide (specific activity =
1000-10,000 cpm/pmol) in exchange buffer containing 25 mM HEPES, pH 7.4, 100 mM NaCl, 0.5 mM
MgCl, 1 mM EDTA, 1 mM dithiothreitol, 50
µg/ml bovine serum albumin, and, where indicated, 3 mML-
-dimyristoylphosphatidylcholine (DMPC) and 2.5 mM sodium cholate (DMPC/cholate). Samples (10 µl) were taken at
the indicated times, and bound nucleotide was determined by rapid
filtration on BA85-nitrocellulose filters (Schleicher &
Schuell)(29) . Data were fit to a first-order rate equation
using the Marquardt algorithm.
Nucleotide Dissociation
Arf (2-5
µM) was incubated with 50 µM [S]GTP
S (specific activity =
20,000-50,000 cpm/pmol), 10 µM
[
H]GDP (specific activity =
10,000-25,000 cpm/pmol), or 5 µM [
-
P]GDP (specific activity =
5000-25,000 cpm/pmol) for 2-3 h in exchange buffer.
Dissociation was determined as a loss of protein-bound radiolabel
following dilution into exchange buffer containing 1 mM unlabeled guanine nucleotide. Samples were withdrawn at
10-15 time points between 0 and 120 min. Data were fit to a
single or double exponential decay equation using the Marquardt
algorithm.
Determination of Relative Nucleotide
Affinities
The relative affinities of Arf for GTPS and GDP
were determined by competing the binding of radiolabeled GDP with
GTP
S. Arf (1 µM) was incubated with 10 µM [
-
P]GDP or
[
H]GDP (specific activity =
5000-10,000 cpm/pmol) and unlabeled 0.5-1000 µM GTP
S in exchange buffer for 3 h for Arf1 or for 1-2 h
for the other proteins at 30 °C. Protein-bound radionucleotide was
determined by rapid filtration on nitrocellulose filters. Each
nucleotide was present at concentrations greater than the number of
available binding sites, and the concentration of GDP was much greater
than K
, allowing the data to be fit to the
following equation:
[Arf
GDP]/[Arf
] = (K
/K
)[GDP]/((K
/K
)[GDP]
+ [GTP
S]), where all Ks are equilibrium
dissociation constants. For [
13]Arf1, a significant
decrease in GDP binding occurred when the concentration of GTP
S
was equal to the concentration of Arf, violating one assumption used in
deriving the equation; however, this leads to an underestimate of the
protein's affinity for GTP
S (30) and does not
influence the conclusions of this paper.
HPLC of Acylated Arf
Acylated and unmodified Arf
proteins were resolved under denaturing conditions by reverse-phase
chromatography on an analytical C column (Dynamax
300Å, Rainin Instrument Co. Inc., Woburn, MA) developed in 0.1%
trifluoroacetic acid with a 30-75% acetonitrile gradient over 45
min (31). Proteins were detected by absorption at 280 nm. Peaks were
integrated to determine relative abundance of the modified proteins.
Electrospray Ionization-Mass Spectrometry
Mass
spectra were determined on a Finnigan TSQ-70 spectrometer. Solutions of
protein (5-20 pmol/µl) were prepared in acetic
acid/methanol/water (5:20:80, v/v). Approximately 30 µl was infused
from a Harvard syringe pump at 1 µl/min, averaging scans in the
profile mode for 3 min. After preliminary analysis using the Finnigan
deconvolution program, data were downloaded in ASCII format and sent to
MaxENT Solutions, Ltd. (Cambridge, United Kingdom) for maximum entropy
analysis.
Miscellaneous
[-
P]GTP,
[
S]GTP
S, and [
H]GDP
were purchased from DuPont NEN. [
-
P]GDP was
prepared as described (32). DMPC (P-0888), GTP (G-8877), and ATP
(A-7894) were obtained from Sigma. Sodium cholate was purchased from
Fluka Chemical Co. (Ronkokoma, NY), and GTP
S from Boehringer
Mannheim. Protein concentrations were determined using the Amido Black
assay(33) . Nucleotide concentrations were determined by UV
absorbance.
Modification of the N Terminus of Arf1 Has Opposite
Consequences for GTP
To
determine the influence of the amino terminus on GTPS and GDP Binding Properties
S binding, we
compared Arf1 with two N-terminal deletion mutants. For a typical
preparation of nonmyristoylated Arf1, steady-state binding of GTP
S
reached a stoichiometry of
0.01 mol/mol of protein when determined
in the absence of phospholipids ( Fig. 1and ). In
contrast, [
13]Arf1 and [
17]Arf1 (mutants
in which the N-terminal 13 and 17 amino acids were deleted,
respectively), bound 0.92 and 0.75 mol of nucleotide/mol of protein,
respectively (see Ref. 10 and ).
Figure 1:
Phospholipid
dependence of nucleotide binding to Arf. GTPS binding to Arf1 (invertedtriangles), [3-7LFASK]Arf1 (triangles), or acylated [3-7LFASK]Arf1 (circles) was determined in the presence (closedsymbols) or absence (opensymbols) of
DMPC/cholate as described under ``Materials and Methods.''
Each point contained 10 pmol of Arf. This is a representative
experiment of those used to determine the values presented in Table
I.
These differences in
equilibrium binding of GTPS result from differences in nucleotide
affinities. GTP
S dissociated from [
13]Arf1 and
[
17]Arf1 slower than from Arf1 (Fig. 2A and ). In contrast, GDP dissociated faster from
[
13]Arf1 and [
17]Arf1 than from the
wild-type protein (Fig. 2B and ). These
opposite changes in dissociation rates should reflect reciprocal
changes in guanine nucleotide affinity if nucleotide association rates
were not affected by the deletions. Unfortunately, determination of
nucleotide association rates is inaccurate and technically demanding
because the apoprotein is unstable(34) . Instead, the relative
affinities for GDP and GTP
S were determined by monitoring the
binding of GDP when competed with GTP
S. Data are expressed as a
ratio of the equilibrium dissociation constants K
/K
as described
under ``Materials and Methods.'' A value >1 indicates that
the protein has a greater affinity for GTP
S than for GDP. The
ratio K
/K
was >1
for [
17]Arf1 and [
13]Arf1 and <1 for
Arf1 as indicated in I. These data reveal that deleting 13
or 17 residues from the N terminus led to coordinate and opposite
changes in the binding affinities for GDP and GTP
S. If the amino
terminus were simply inhibiting GTP binding, one would expect the
deletions to have the same effect on both GTP and GDP binding. Mutation of Residues 3-7 Increases the Affinity for
GTP
S-One mutant ([3-7LFASK]Arf1) designed to
be a better substrate for N-myristoyltransferase and to allow
the production of extensively modified Arf1 in bacteria (see below)
fortuitously provided information about the effect of the bound
nucleotide on the N terminus. Other than being a better substrate for N-myristoyltransferase, we did not expect the mutations to
affect the biochemical properties of the protein. Indeed, no
differences in specific activities as cofactors for cholera
toxin-catalyzed ADP-ribosylation of G
(data not shown) were
observed between [3-7LFASK]Arf1 and Arf1. However, the
nucleotide binding properties of this mutant were found to be similar
to those of [
13]Arf1 and [
17]Arf1 ( Fig. 1and ); [3-7LFASK]Arf1 bound
more GTP
S at equilibrium than did Arf1. The increased binding of
GTP
S was a result of reciprocal changes in the affinity for GTP
and GDP. GTP
S dissociated more slowly and GDP dissociated more
rapidly from [3-7LFASK]Arf1 than from Arf1. The ratio K
/K
was >1 for
[3-7LFASK]Arf1 (). Thus, this mutant
provided further evidence that the conformation of the N-terminal
domain differs in the GDP- and GTP-bound states.
Figure 2:
Effect of
changing the amino terminus on nucleotide dissociation from Arf. The
time dependence of GTPS (A) and GDP (B)
dissociation from Arf1 (invertedtriangles)
[
17]Arf1 (squares), [
13]Arf1 (diamonds), and [3-7LFASK]Arf1 (triangles) was determined as described under ``Materials
and Methods.'' The data are expressed as a fraction of
radiolabeled nucleotide bound to Arf at the time the reaction was
initiated and were fit to single or double exponential decay equations
to derive values for the dissociation rate presented in Table II. This
is one of at least three representative
experiments.
Preparation of Acylated Arf Protein
To examine the
role of myristate in nucleotide exchange, a source for unmodified and
acylated Arf1 preparations was sought. Arf preparations from mammalian
tissues are composed of a mixture of gene products that cannot be
resolved efficiently. Recombinant Arf1 has been produced in bacteria in
large amounts and found to yield a homogeneous nonacylated preparation
of Arf1(34) . The coexpression of human Arf1 and yeast N-myristoyltransferase has been used previously to produce the
properly modified protein(26) . However, the extent of
myristoylation, determined by reverse-phase HPLC, was typically only
10-15%(31) . Attempts to resolve the processed and
unprocessed forms of Arf1 or Arf3 by chromatography on DEAE-Sephacel,
MonoQ, hydroxylapatite, phenyl-Sepharose, heptylamine-agarose, or
Ultrogel AcA 44 or 54 resins proved unsuccessful (data not shown).
Changing the time of induction (up to 18 h) or the concentration of
inducer (isopropyl--D-thiogalactopyranoside as low as 50
µM) did not result in increased extents of acylation of
Arf1. Similarly, coexpression of human Arf1 with human N-myristoyltransferase in bacteria was not much different from
coexpression with yeast N-myristoyltransferase and did not
increase the extent of myristoylated human Arf1.
H]myristic acid, similar amounts of label were
incorporated into human Arf1 with either human or yeast N-myristoyltransferase (Fig. 3, first and fifthlanes). In contrast, although yeast Arf1 was
expressed at similar levels compared with human Arf1, it incorporated
much more [
H]myristate, regardless of the source
of N-myristoyltransferase (Fig. 3, fourth and eighth lanes). Therefore, S. cerevisiae Arf1 is a
much better substrate for either yeast or human N-myristoyltransferase than is human Arf1.
Figure 3:
Yeast Arf1 and amino-terminal mutants of
human Arf1 are highly N-myristoylated when coexpressed in
bacteria with human or yeast N-myristoyltransferase. Human
Arf1 (hARF1), [6,7SK]Arf1,
[3-7LFASK]Arf1, and S. cerevisiae Arf1 (ScARF1) were coexpressed in bacteria with either human (hNMT) or yeast (ScNMT) N-myristoyltransferase. Culturing, induction, and labeling
with [H]myristate were performed as described
under ``Materials and Methods.'' Approximately 25 µg of
total bacterial protein was loaded on each lane of the gel, which was
subsequently stained with Coomassie Blue (A) or developed for
fluorography (B). The film was exposed overnight at -80
°C. Only the 20-kDa region of the fluorogram is shown in B as no other bands were detected elsewhere on the
gel.
Guided by the
extensive characterization of sequence specificities in substrates for N-myristoyltransferase(22, 23) , we constructed
two mutants of human Arf1 in which the corresponding residues of S.
cerevisiae Arf1 were substituted in efforts to allow acylation
with high efficiency. Changing only residues 6 and 7
([6,7SK]Arf1) led to an increase in the level of N-myristoylation of mutant human Arf1 coexpressed with S.
cerevisiaeN-myristoyltransferase, but decreased that
coexpressed with human N-myristoyltransferase (Fig. 3, second and sixthlanes). Changing residues
3-7 ([3-7LFASK]Arf1) further increased the extent
of acylation of human Arf1 achieved in the presence of S.
cerevisiaeN-myristoyltransferase and also increased that
obtained with human N-myristoyltransferase (Fig. 3, third and seventhlanes). Expression of this
mutant allowed a level of myristoylation comparable to that observed
when S. cerevisiae Arf1 was coexpressed in bacteria with S. cerevisiaeN-myristoyltransferase.
[3-7LFASK]Arf1 purified from bacteria expressing S.
cerevisiaeN-myristoyltransferase is referred to as
acylated [3-7LFASK]Arf1.
H
group and are consistent with the different forms being the
result of heterogeneous acylation. The species with a mass consistent
with the myristoylated protein was 18% of the total protein if no
myristate was added at the time of induction and 40% of the total
protein if myristate was added. The remainder of the acyl groups were
predicted to be saturated acyl chains of 4-24 carbons (Fig. 4). This heterogeneity in acyl groups covalently bound to
Arf1 likely results from the forced overexpression in bacteria as Arf
proteins purified from bovine brain did not appear to be
heterogeneously acylated(36) . This recombinant preparation
allowed us to examine the consequence of acylation on nucleotide
binding kinetics by comparison with nonacylated
[3-7LFASK]Arf1.
Figure 4:
Electrospray ionization-mass spectrum and
normal and maximum entropy deconvolution of partially myristoylated
[3-7LFASK]Arf1. Acylated [3-7LFASK]Arf1
expressed in Escherichia coli with no added myristic acid was
dissolved in acetic acid/methanol/water (5:20:80), and the sample was
admitted by infusion from a syringe pump at 1 µl/min as described
under ``Methods and Materials.'' The MaxENT spike/error plot
shows peak widths corresponding to the relative
errors.
Acylation Decreases the Affinity of Arf1 for GTP
Acylated
[3-7LFASK]Arf1 bound 90% less GTPS in
the Absence of Phospholipids
S at equilibrium
than did the nonacylated protein ( Fig. 1and ) when
assayed in the absence of added phospholipids. GTP
S dissociated
from the acylated protein faster than from the nonacylated protein (Fig. 5A and ). In contrast, acylation had
little effect on the rate of GDP dissociation (Fig. 5B and ). Consistent with the changes in the
dissociation rates, the K
/K
ratio was <1
for acylated [3-7LFASK]Arf1 and >1 for
[3-7LFASK]Arf1 (I). Thus, acylation
decreases the affinity of Arf1 for GTP
S when measured in the
absence of phospholipids.
Figure 5:
Phospholipid dependence of nucleotide
dissociation from Arf. GTPS (A) and GDP (B)
dissociation from [3-7LFASK]Arf1 (triangles)
and acylated [3-7LFASK]Arf1 (circles) in the
presence (closedsymbols) or absence (opensymbols) of DMPC/cholate was determined. The data are
expressed as a fraction of radiolabeled nucleotide bound to Arf at the
time the reaction was initiated. The experiment is representative of
three.
N-Myristate Is a Major Determinant of
Phospholipid-dependent GTP
Binding of
GTPS Binding to Arf1
S to Arf proteins purified from mammalian tissues is highly
dependent on added phospholipids(6) . Similarly, GTP
S
binding to acylated [3-7LFASK]Arf1 was highly dependent
on DMPC/cholate ( Fig. 1and ). In contrast, GTP
S
binding to [3-7LFASK]Arf1 is largely independent of
added DMPC/cholate ( Fig. 1and ). In the absence of
DMPC/cholate, acylated [3-7LFASK]Arf1 bound less
GTP
S than did the nonacylated protein. In the presence of
DMPC/cholate, acylated [3-7LFASK]Arf1 bound GTP
S
faster and to a 50-80% higher stoichiometry than did the
nonacylated protein ( Fig. 1and ). The DMPC/cholate
dependence was similar to that of Arf purified from bovine
brain(6) .
S binding
were the result of changes in nucleotide affinities. DMPC/cholate
slowed the GTP
S dissociation rate and accelerated the GDP
dissociation rate from acylated [3-7LFASK]Arf1 ( Fig. 5and ). The net effect was a 14-fold increase
in the K
/K
ratio
from 0.35 to 4.8 (I). Thus, the addition of phospholipids
to acylated Arf1 resulted in increased and decreased affinities for
GTP
S and GDP, respectively. The effect of phospholipids on
nucleotide binding, nucleotide dissociation, and relative nucleotide
affinities was minimal for the nonacylated proteins used in these
studies. Among this group of proteins, the largest effect observed was
seen for binding of GTP
S to Arf1, which was 5.5 ± 2.2-fold (n = 3) greater in the presence of DMPC/cholate than in
its absence.
GDP and
Arf
GTP, which could provide evidence for this region of the
protein acting as an effector domain. From this model, mutations that
affect the conformation of this domain should have different
consequences for GTP and GDP binding. This prediction was borne out by
the data. The amino terminus of Arf1 is a GTP-sensitive switch. As
previous studies have shown that the amino terminus interacts with
target proteins(9, 10, 13, 14) , it may
be considered an effector domain of Arf1. We have further demonstrated
that myristoylation is the predominant (although not sole) determinant
of the phospholipid-dependent transition of Arf1 to the active
conformer. Thus, the myristoylated N terminus of Arf1 is a GTP- and
phospholipid-sensitive switch.
GDP and Arf1
GTP
S were demonstrated by
comparing the nucleotide binding properties of Arf1 with those of
N-terminal mutants. Two characteristics of Arf1 allowed this approach.
First, the conformational change in Arf that accompanied the exchange
of GTP for GDP consumed enough energy to result in a large difference
in binding affinities for the nucleotides(34) . Second, the N
terminus does not contribute directly to the nucleotide-binding
pocket(37) . Therefore, if the GTP-sensitive conformational
change were localized to the N terminus or to residues in direct
contact with the N terminus, removing or altering this domain should
result in an increased affinity for GTP. Furthermore, depending on
whether the region also contributed to stabilizing the GDP-bound form
of the protein, mutations of the N terminus should either have no
effect on or decrease the protein's affinity for GDP. Thus, the
finding that [3-7LFASK]Arf1, [
13]Arf1,
and [
17]Arf1 have lower affinities for GDP and higher
affinities for GTP
S than does wild-type Arf1 is consistent with
the N terminus stabilizing the GDP-bound form of the protein and
undergoing a conformational change when GTP exchanges for GDP.
and requires an intact N terminus of Arf. A second site binds
cholera toxin independently of the N
terminus(9, 10, 38, 39) . As binding to
cholera toxin appears to be GTP-dependent(38, 39) , the
presence of a second effector domain seems likely. A likely candidate
for this second domain is
-sheet 2 and loop 4 identified in the
crystal structure of Arf1
GDP described by Amor et
al.(37) . The residues composing this putative effector
domain are found between the two GTP-binding consensus sequences
GXXXXGK and DXXG (where X is any amino
acid), as is true for the effector domain (loop 2) of Ras. This domain
is hydrophobic and highly exposed to solvent, but in the crystal, forms
the interface for a dimer that is proposed to be a site of
protein-protein interaction(37) .
H
). Thus, fatty acid chain
lengths from 6 to 16 carbons in length were observed on Arf proteins.
While >95% of the recombinant Arfs were acylated, in the preparation
used for the kinetic studies reported here,
40% were
myristoylated. This heterogeneity is greater than that reported for
recoverin or transducin in the retina. This heterogeneity likely
results from the forced overexpression of myristoylated proteins in an
organism (bacteria) that does not normally produce myristoylated
proteins, lacks N-myristoyltransferase, and maintains lower
levels of N-myristoylated CoA. We believe that the differences
described between the acylated and nonacylated proteins are at least
qualitatively the same as those between unmodified and myristoylated
Arfs. Furthermore, some of our observations have recently been
verified(45) . Other means of preparing a fully myristoylated
single Arf gene product are currently being tested, including those
methods described by Franco et al.(45) .
GTP
S dissociation, suggesting that two populations of
Arf1 exist. We currently have no good model to explain this
observation.
Table: Myristoylation confers increased phospholipid
dependence, while loss of the N terminus is associated with decreased
phospholipid dependence of GTPS binding
), and maximum binding stoichiometry,
max (mol of nucleotide/mol of Arf), were determined by fitting the data
to the equation Arf
GXP/Arf
= max (1
- e). [
13] and [
17] refer
to [
13]Arf1 and [
17]Arf1, respectively.
37Arf1 and myr37 refer to [3-7LFASK]Arf1 and acylated
[3-7LFASK]Arf1, respectively. The data are the means
± S.D. of three experiments for Arf1 and the means ±
range of two experiments for all other proteins.
Table: Differences in steady-state binding of
GTPS reflect changes in nucleotide dissociation rates
13] and [
17] refer to
[
13]Arf1 and [
17]Arf1, respectively.
37Arf1 and myr37 refer to [3-7LFASK]Arf1 and acylated
[3-7LFASK]Arf1, respectively. The data are the means
± S.D. of three experiments. GTP
S dissociation from Arf1
could not be fit to a single exponential, but did fit a double
exponential decay equation. The fast component composed 0.51 ±
0.09 of the signal. The decay rates are given as k
and k
, and a weighted average of the two is
given for the sake of comparison with the other proteins, whose
dissociation time courses fit single exponential decay equations.
Table: Effects of
myristoylation, mutation, and phospholipids on the relative nucleotide
affinities of four mutant Arfs
/K
was
determined in the presence (+DMPC) or absence (-DMPC) of
DMPC/cholate as described under ``Materials and Methods.'' K
and K
are the
dissociation constants for Arf
GDP and Arf
GTP
S,
respectively. 37Arf1 and myr37 refer to [3-7LFASK]Arf1
and acylated [3-7LFASK]Arf1, respectively. The data are
the means ± S.D. of three experiments.
S, guanosine 5`-O-(3-thio)triphosphate; DMPC, L-
-dimyristoylphosphatidylcholine; HPLC, high pressure
liquid chromatography.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.