Vol. 269, No. 5, Issue of February 4, pp. 3135-3138, 1994
Printed an U.S.A.
An Activating Mutation in ARFl
Stabilizes Coatomer Binding to
(Received for publication, November 1, 1993, and in revised form,
which in its GTP state supports the binding of coatomer,
a cytosolic coat protein complex, to Golgi membranes.
To create an "active"
ARF, we constructed a point muta-
tion inARF1, Q711, which was predicted to slow the rate
of GTP hydrolysis. W e demonstrate that 6711, in con-
trast to wild type ARF1, exhibits a =-fold increase in
the half-life of ARF-GTP and is able to promote stable
coatomer binding to Golgi membranes in the presence
GTP i n vitro. Additionally, Q71I is able to support the
binding of a significant amount of coatomer to mem-
branes in the absence of added nucleotides, effectively
bypassing the brefeldin A (BFA)-sensitive exchange ac-
tivity. Furthermore, transfection of cells with Q711, but
not -1, renders the Golgi association of coatomer re-
sistant to the effects of BFA in vivo.
provide compelling evidence that A R F l is a necessary
GTP binding protein that regulates the reversible bind-
ing of coat proteins to Golgi membranes and that the
effects of BFA on this process in living cells must be a
consequence of BFA's inhibition of guanine nucleotide
exchange onto ARF1.
December 2, 1993)
Stephanie B. Teal$, Victor W. Hsu,
Peter J. Peters$, Richard D. Klausner, and
Julie G. Donaldsonll
From the Cell Biology and Metabolism Branch,
National Institute of Child Health and Human
Development, National Institutes of Health,
Bethesda, Maryland 20892
Ras-related protein ADP-ribosylation factor
is a low molecular weight GTP binding protein,
The reversible binding of cytosolic coat proteins (coatomer) to
Golgi membranes is believed to function in the maintenance of
structure and regulation of membrane traffic in the Golgi com-
plex (1). The assembly of coatomer onto Golgi membrane re-
quires GTP and
a small GTP binding protein, ADP-ribosylation
factor 1 (ARF1)l (2, 3). The ability of ARFl to mediate the
assembly of coatomer onto Golgi membranes is believed to re-
quire the activation of ARFl by a membrane nucleotide ex-
change protein (4-6) which results in the association of A R F 1 -
GTP with the membrane followed by coatomer binding (2, 3).
* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"aduertisement" in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
$ Supported by the Howard Hughes Medical Institute-National Insti-
tutes of Health Research Scholars Program.
5 Visiting Fellow from
School, University of Utrecht, The Netherlands.
ll To whom correspondence should be addressed: CBMB, Bldg. 18Tl
the Department of Cell Biology, Medical
101, NIH, Bethesda, MD 20892. Tel.: 301-402-0360; Fax: 301-402-0078.
The abbreviations used are: ARF, ADP ribosylation factor; BFA,
brefeldin A, GTPyS, guanosine 5'-0-(3-thiotriphosphate); COP,
coatomer protein; HA, influenza hemagglutinin.
Hydrolysis of the ARF-bound GTP is proposed to be coupled
to the release of both ARF-GDP and coatomer from
Pharmacologic reagents that perturb the activation/
inactivation cycle of GTPases have proved to be valuable tools
for dissecting their functions. Addition of the nonhydrolyzable
GTP analogue GTPyS, which persistently activates GTPases,
results in enhanced and irreversible binding
coatomer to Golgi membranes (8-10). In contrast, addition of
brefeldinA(BFA1 inhibits the activation ofARF catalyzed by
Golgi-associated nucleotide exchange protein and prevents the
binding ofARF and, therefore, coatomer to membrane (8,9,11).
While it is tempting to conclude that ARF is the sole GTP bind-
ing protein required for coatomer binding, previous experiments
do not exclude a requirement for other GTP binding proteins
known to be associated with the Golgi membrane (12-14).
To determine whether ARFl is both the sole, GTP-requiring
component for coatomer binding and the sole target of BFA in
this process, we constructed a point mutation in human ARFl
that was predicted to inhibit the rate of GTP hydrolysis, creat-
ing a persistently activeARF1. Characterization of this mutant
AFtF is presented below and confirms that ARFl is the key
BFA-sensitive component required for coat protein binding to
of ARF and
Purification ofARE: Golgi Membranes, and Cytosol-Wild type ARFl
and the mutantARF1, Q711, were co-expressed in Escherichia coli with
N-myristoyltransferase and isolated as previously described (15, 16).
Golgi-enriched membrane fractions from rat liver (17) or Chinese ham-
ster ovary cells (18) were obtained as described. Cytosol was isolated
from bovine brains (19).
Nucleotide Exchange Assays-Nucleotide exchange was assessed by
measuring nucleotide binding to ARF andor rat liver Golgi membranes
using a filter binding assay as described previously (4). Incubations
were carried out in a 0.1-ml reaction volume containing 25 m Hepes-
KOH, pH 7.0, 25 m KC1, 2.5 m MgC12, 0.2 M sucrose, 1 m dithio-
threitol, 1 m~ ATP (Sigma A2383),2-5 pg of recombinant ARF protein,
and 4-6 pg of Golgi membranes and [a-32P1GTP or [y-32PlGTP (1 m,
0.7-1.4 Ci mmol-') at 30 "C for the indicated times. The reaction was
stopped by the addition of cold buffer, the mixture was filtered on BAS5
nitrocellulose filters and washed with five 2-ml volumes of wash buffer,
and the amount of radionucleotide trapped to the filter was measured as
described (4). The amount of membrane-catalyzed nucleotide bound to
ARF was calculated by subtracting the nucleotide bound after incuba-
tions ofARF alone and Golgi membranes alone from the amount bound
after incubations of ARF plus Golgi membranes (4).
Coatomer Binding Assay-Membranes from Chinese hamster ovary
cells (8 pg of protein) and ARFl or Q71I (10 pg of protein each) were
incubated under conditions described above for nucleotide exchange
assays with and without activators prior to the addition of cytosol (600
pg of protein) and further incubation to allow for coatomer
membranes and bound material were pelleted at 14,000 x g, and the
amount of p-COP associated with the pellet was analyzed by SDS-
polyacrylamide gel electrophoresis and immunoblot, and quantitated by
PhosphorImager (Molecular Dynamics) (2). The background amount of
p-COP binding to membranes that occurred in the absence of added
ARF was subtracted from values obtained with ARFl and 6711.
Dansfection of Cells and Immunofluorescence Assays-ARF1 and
6711 containing the hemagglutinin epitope at the C terminus were
subcloned into the expression vector PCDLSRa (20). COS-1 cells grown
on coverslips were transiently transfected by the calcium phosphate
precipitation method and were analyzed 40 h later to assess sensitivity
to BFA. For immunofluorescence, cells on coverslips were fixed in 2%
formaldehyde and double labeled with a monoclonal antibody to the HA
epitope (12CA5) to detect transfected HA-tagged ARF protein and a
polyclonal anti-p-COP peptide (EAGE) antiserum as described (9).
ARFl and Golgi Membrane Coat Proteins
.1 1 10 1000 100
(open squares) and [y-3zPlGTP (solid squares) bound to A R F l (A) or Q71I (B) after incubation ofARF and membranes for the indicated times at
30 "C. C, nucleotide binding associated with AFtFl (open squares) and 6711 (solid squares) after incubation of A R F l and 6711 with Golgi
membranes in the presence of 1 w [C~-~~PIGTP for 20 min at 30 "C in the presence of increasing amounts of BFA. D,
prebound onto A R F l (open squares) and Q71I (solid sqwres) after incubation ofARF with membranes in the presence of [y32P]GTP for 20 min
followed by the addition of 100 w unlabeled GTP; the loss of y-phosphate bound to ARF was assayed with time after addition of unlabeled GTP.
All experiments were repeated at least 3 times. For C and D,
the mean & S.E. of triplicate samples is shown.
FIG. 1. Golgi membrane-catalyzed GTP exchange and hydrolysis onto ARFl and Q7lI. A and B, comparison between [or-32P]GTP/GDP
hydrolysis of [ys2P]GTP
RESULTS AND DISCUSSION
To create a persistently active ARF1, we selected a point
mutation based upon the homology of ARFl protein sequence
domains with Ras (21). In Ras the conserved glutamine at
position 61 is required for efficient GTP hydrolysis; mutations
at this position stabilize the Ras-GTP state, resulting in a per-
sistently activated protein (22, 23). The analogous position in
ARFl is glutamine 71, and this was changed to an isoleucine.
This mutant, called Q711, was co-expressed in E. coli with
N-myristoyltransferase in order to produce the biologically ac-
tive N-myristoylated form of ARF (15). The recombinant pro-
tein was isolated and its activity monitored in nucleotide ex-
change, GTP hydrolysis, and coatomer binding assays in uitro,
as well as in transfected cells.
Isolated Golgi membranes catalyzed nucleotide exchange
onto both ARFl and Q71I with similar kinetics using a-labeled
L3'P1GTP (Fig. 1, A and B, open squares). This exchange activ-
ity was inhibited for both ARFl and Q71I in a dose-dependent
manner by BFA (Fig. 1C). During the membrane-catalyzed
nucleotide exchange assay, GTP is bound to ARF and is then
hydrolyzed to GDP (4,7). Thus, using a-labeled [32P]GTP, total
exchange events onto ARF are recorded even if the ARF-bound
GTP has been hydrolyzed to GDP. When y-labeled [32P]GTP
was used in the exchange assay to assess the amount of GTP
exchanged onto ARF that was not hydrolyzed (Fig. 1, A and B,
solid squares), a larger fraction of nucleotide bound to Q71I
remained as GTP as compared with ARF1. To further assess
this distinction, we measured the rate of hydrolysis of
membranes by A R F l and Q71I. Golgi membranes and A R F l (A) or
Q71I (B) were preincubated with no activator (open bars), 10 w GTP
(hatched bars), or 10
GTPyS (solid bars) for 5 min at 37 "C, followed
by the addition of BFA to all samples (300 w). Then, cytosol was added,
and samples were incubated for an additional 5 min to allow coatomer
FIG. 2. Nucleotide requirements for preactivation of Golgi
binding. To some samples, during the first incubation BFA was added
before the addition of activators to inhibit activation (BFAbefore). After
incubation the amount of pC0P bound to membranes was measured as
described under "Experimental Procedures."
[y-32PlGTP that had been preloaded onto A R F l and Q71I by
monitoring the loss of ARF-bound radioactivity after the addi-
tion of unlabeled GTP. GTP that was bound to ARF was hydro-
lyzed with a tM of less than 2 min for A R F l and approximately
5 min for Q71I (Fig.
lD). The addition of BFA, in place of
unlabeled GTP, yielded similar results. Thus, while there was
no observed change in the BFA-sensitive nucleotide exchange
onto Q711, there was a 2-3-fold decrease in the rate of mem-
brane-dependent GTP hydrolysis associated with the Q71I mu-
tation in vitro.
ARFl and Golgi Membrane Coat Proteins
HA-tagged ARFl or 6711 were either not treated or treated with BFA (10 pdml) for 10 min at 37 “C prior to double labeling with antibodies to the
HA epitope and B-COP.
n ~ .
3. 6711 confers BFA resistance onto the membrane association of the coatomer protein &COP. COS-I cells transfected with
Incubation of Golgi membranes with ARFl in the presence of
GTPyS is sufficient to preactivate the membranes, making
them competent for binding of subsequently added coatomer in
the absence of free nucleotide (2). While BFA inhibits the initial
activation step, it has no effect on subsequent coatomer binding
to such “preactivated” membranes. Importantly, this preactiva-
tion is not observed with hydrolyzable GTP. The relative sta-
bility of GTP bound to Q71I suggested that the mutant might,
in contrast, preactivate the membrane for coatomer binding
with GTP alone. Indeed, incubation of Q71I with Golgi mem-
branes in the presence of GTP allowed maximal levels of bind-
ing of the coatomer protein pC0P to occur during a second
incubation in the presence of coatomer (Fig. 2 B ) . The level of
PCOP binding achieved was nearly that
In contrast, as observed previously (3, preactivation of Golgi
membranes by wild type ARFl was only observed if GTPyS was
included in the first incubation (Fig. 2A 1.
Another difference between Q71I and wild type ARFl was
the significant level of BFA-resistant activation of membranes
by Q71I that was observed in the absence of any added nucleo-
tide (Fig. 2 B , open bars). This activity of Q71I increased with
longer incubation but was never observed with ARF1. Possible
explanations for this observation are that the preparation of
Q71I contains “activated” ARF that effectively bypasses any
BFA-sensitive step in the membrane. Thus, 6711 appears to
have the characteristics of both a longer lived, persistent ARF-
GTP as well as a constitutively active ARF1.
Preincubation of permeabilized cells with GTPyS blocks the
effects of subsequent addition of BFA on coatomer dissociation
from the Golgi apparatus (8). Since Q71I has the characteristic
of acting in vitro like ARF-GTPyS in the absence of added
GTPyS, we could now test whether the expression of this “ac-
tive” ARFl in cells would confer resistance to BFA’s effects on
coatomer association with the Golgi apparatus. COS-1 cells
were transfected with either ARFl or Q71I containing an HA
epitope at the carboxyl terminus so that the transfected
could be immunologically detected. In cells transfected with
either ARFl or Q711, the HA tagged ARFs were localized to a
perinuclear region, which co-localized, by immunofluorescence,
with the coatomer component p-COP (Fig. 3) and Golgi resident
markers (not shown). p-COP localization in untransfected cells
was indistinguishable from that in the transfected
BFA was added to cells transfected with wild type ARF1, both
ARFl and p-COP rapidly became diffusely distributed to the
achieved with GTPyS.
cytosol, effects indistinguishable from those seen in untrans-
fected cells. The effects of BFA on cells transfected with Q71I
were quite different. The distribution of neither the introduced
Q71I nor p-COP changed in response to the addition of the
drug. Even after extended incubations of 1 h in the presence of
10 pg/ml BFA, @COP and Q71I remained co-localized to peri-
nuclear structures. Other markers of the Golgi complex also
remained in these p-COP-labeled structures (not shown). The
presence of the HA epitope had no effect on the phenotype since
transfection of untagged ARFs resulted in similar effects (not
shown). Myristoylation of the proteins was required for their
biological function, since cells transfected with ARF sequences
that contained an additional mutation at position 2 from a
glycine to an alanine abolished myristoylation and Golgi local-
ization for both ARFl and Q71I and eliminated BFA protection
in cells transfected with Q71I. The BFA-resistant phenotype of
Q71I cannot be explained by overexpression of ARF proteins
per se since it was observed in all cells expressing variable
amounts of Q71I and never observed in cells overexpressing
wild type ARF1.
Our expectation in creating Q71I was that this mutation in
ARF, analogous to QSlL in Ras, would inhibit hydrolysis of
GTP bound to the protein, creating a persistently active GTP-
ARF. If the inhibition of hydrolysis was sufficiently strong,
resistance to the effects of BFA on coatomer binding would be
expected. We measured, however, only a 2-%fold increase in
the half-life of GTP bound to Q71I in our in vitro assays, which
would predict a delay in, but not the complete inhibition of, the
effects of BFA on coatomer binding that was observed in cells
transfected with Q71I. Although it is possible that the half-life
of Q71I-GTP in vivo may be longer, studies with the analogous
mutation in Rab3A have shown that the rates of GTP hydroly-
sis as measured in vivo are actually similar for the wild type
and mutant proteins, and these authors caution that the anal-
ogy made to the observation with Ras mutations may not al-
ways be valid (24). This raises the
in ARF1, while it does affect to some extent GTP hydrolysis,
may also result in a conformational change in the protein that
effectively makes it “active” regardless of the nucleotide bound.
In this regard, it might be significant that the site of this
mutation is adjacent to a critical glycine (at position 70 in
ARF1, 60 in Ras), which is believed to make contact with the
y-phosphate and be involved in the GDP-GTP-induced confor-
mational change in the protein (21).
possibility that this mutation
ARFl and Golgi Membrane Coat Proteins
Regardless of the mechanism, the effect of expressing this
activated ARF is comparable with selectively activating ARF
with GTPyS, rendering the cell resistant to the effects of BFA
on both coat assembly and redistribution of Golgi membrane
into the endoplasmic reticulum. Furthermore, these results
demonstrate that the only process inhibited by BFA in the
ARF-coatomer binding reaction is the nucleotide exchange ac-
tivity. ARF thus becomes one of the few small GTPases for
which an effector function is identifie4 its cycle of activation
and inactivation regulates the binding of coatomer to Golgi
Acknowledgments-We thank J. Bonifacino, A. Finegold, and C. Ooi
for critical reading of the manuscript.
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