(Received for publication, September 10, 1996, and in revised form, December 5, 1996)
From the Department of Medicine, Washington University, St. Louis, Missouri 63110
We have compared the abilities of mammalian
ADP-ribosylation factors (ARFs) 1, 5, and 6 and Saccharomyces
cerevisiae ARF2 to serve as substrates for the rat liver Golgi
membrane guanine nucleotide exchange factor and to initiate the
formation of clathrin- and coatomer protein (COP) I-coated vesicles on
these membranes. While Golgi membranes stimulated the exchange of
GTPS for GDP on all of the ARFs tested, mammalian ARF1 was the best
substrate, with an apparent Km of 5 µM. In all cases myristoylation of ARF was required for
stimulation. Agents that inhibit the Golgi membrane guanine nucleotide
exchange factor (the fungal metabolite brefeldin A and trypsin
treatment) selectively inhibited the guanine nucleotide exchange on
mammalian ARF1. Taken together, these data indicate that of the ARFs
tested, only mammalian ARF1 is activated efficiently by the Golgi
guanine nucleotide exchange factor. The other ARFs are activated mainly
by another mechanism, possibly phospholipid-mediated. Once activated,
all of the membrane-associated, myristoylated ARFs promoted the
recruitment of coatomer to about the same extent. Mammalian ARFs 1 and
5 were the most effective in promoting the recruitment of the AP-1
adaptor complex, whereas yeast ARF2 was the least active. These data
indicate that the specificity for ARF action on the Golgi membranes is
primarily determined by the Golgi guanine nucleotide exchange factor,
which has a strong preference for myristoylated mammalian ARF1.
ADP-ribosylation factors (ARFs)1 are
small GTP-binding proteins that function in the assembly of coated
vesicles that serve to transport proteins between intracellular
organelles (1). In the Golgi, ARF is required for the formation of at
least two types of transport vesicles, coatomer protein (COP) I-coated
and clathrin-coated vesicles (2-5). Golgi membranes contain a guanine nucleotide exchange factor (GEF) that catalyzes the exchange of GTP for
GDP on ARF (6-8). Once it has bound GTP or its slowly hydrolyzable
analog, GTPS, ARF facilitates the recruitment of the
heterotetrameric AP-1 adaptor complex and clathrin onto the Golgi
membranes (2, 3). COPI-coated vesicle formation also proceeds following
the initial recruitment of ARF onto the Golgi (4, 5).
The family of mammalian ARFs (mARFs) has been grouped into three classes based on amino acid sequence (9). Class I is composed of mARFs 1-3, class II of mARFs 4 and 5, and class III has a single member, mARF6. All of the ARFs contain a glycine at position 2 that is a site for N-terminal myristoylation (10).
The majority of in vitro studies of the role of ARF in coat formation on the Golgi have utilized recombinant mARF1 or a mixture of cytosolic ARFs purified from various tissues. Consequently, little is known about the ability of the other ARFs to interact with the Golgi GEF and to facilitate the recruitment of AP-1 and coatomer, the protomer of the COPI coat, onto Golgi membranes. We have tested the ability of one mammalian ARF from each class, mARFs 1, 5, and 6, and Saccharomyces cerevisiae ARF2 (yARF2) to perform a number of the known functions required for coat formation on the Golgi membranes using an in vitro assay system. Our results indicate that myristoylated mARF1 has the highest affinity for the Golgi GEF. The other ARFs are activated by the Golgi membranes to some extent, mainly by a GEF-independent mechanism. Once activated, all of the ARFs tested promote the binding of coatomer and AP-1 but with variable efficiencies.
Brefeldin A (BFA), protease inhibitors, trypsin,
DTT, antibiotics, 20 cetyl ether (Brij 58), ATP, and GDP were obtained
from Sigma; GTP and GTPS from Boehringer Mannheim;
[35S]GTP
S was from DuPont NEN;
[
-32P]GTP and [3H]myristic acid were
from ICN Biomedicals Inc (Irvine, CA); Superdex-75 prep grade,
DEAE-Sepharose Fast Flow, SP-Sepharose Fast Flow, and the SDS-PAGE low
molecular weight markers were purchased from Pharmacia Biotech Inc.;
nitrocellulose was from Schleicher & Schuell; enhanced
chemiluminescence reagents for chemiluminescence were purchase from
Amersham Corp.; isopropyl-1-thio-
-D-galactopyranoside was from Amresco (Solon, OH); myristic acid was from Nu Chek Prep (Elysian, MN). All other reagents were the highest grade available.
The Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (NMT) expression vector pBB131 (11), the S. cerevisiae ARF2 expression vector precA-ARF2p (12), and the human NMT expression vector pHS7-1 were provided by J. Gordon, Washington University. J. Lodge constructed pHS7-1 by inserting the BglII-EcoRI fragment containing the human cDNA from pBB218 (13) into pBB131. The bovine ARF1 expression vector pOW12 (14) was a gift from Richard Kahn (Emory University, Atlanta). pET vectors (Stratagene, La Jolla, CA) containing the cDNAs for human ARFs 5 and 6 (15) were a gift from Richard Klausner (National Institutes of Health, Bethesda).
AntibodiesThe polyclonal antibody AE/1 to -adaptin was
provided by Linton Traub of our laboratory (16) and was used at a
concentration of 1:5000 for immunoblotting. The monoclonal antibody
M3A5 against
-COP was a gift from Thomas Kreis (University of
Geneva, Geneva, Switzerland) (17) and was used at a dilution of 1 µg/ml. Horseradish peroxidase-conjugated antibodies against mouse and
rabbit immunoglobins were purchased from Amersham Corp.
For the
production of the mammalian ARFs, competent BL21(DE3) Escherichia
coli were transformed with an ARF expression vector. When
myristoylated ARF was made, the BL21 cells were co-transformed with
either the S. cerevisiae or human NMT expression vector. In
a typical preparation, 1-3 liters of bacterial culture were grown at
37 °C until A600 = 0.8-1.2 and then
isopropyl-1-thio--D-galactopyranoside (1 mM)
was added. When myristoylated ARFs were prepared, myristic acid (500 µM) and Brij 58 (0.5%) were also added. The culture was
then grown overnight at 27-30 °C. The cells were collected by
centrifugation at 5000 × g for 15 min at 4 °C.
Myristoylated or unmyristoylated yARF2 was produced as described
previously (18). The bacterial pellets were either stored at
80 °C
or used immediately.
The bacteria were lysed by repeated cycles of freezing and thawing (19)
and resuspended in column buffer (50 mM Tris-HCl, 1 mM DTT, 1 mM magnesium acetate, 10 µM GDP, 0.02% sodium azide) plus protease inhibitors (1 µg/ml leupeptin, 2 µg/ml antipain, 10 µg/ml benzamidine, 1 µg/ml Trasylol, 1 µg/ml chymostatin, 1 µg/ml pepstatin) at pH 8.0 for mARFs 1 and 5 and yARF2 and at pH 7.0 for mARF6. The lysate was
cleared by centrifugation at 10,000 × g for 15 min at
4 °C. The supernatant was collected and loaded onto a 20-ml
DEAE-Sepharose column (mARF1 and 5 and yARF2) or a 20-ml SP-Sepharose
column (mARF6) equilibrated with column buffer. The column was eluted
at 90 ml/h with a 30-ml gradient from 0 to 200 mM NaCl for
mARFs 1 and 5 and yARF2 and from 0 to 500 mM NaCl for
mARF6. Aliquots of the column fractions were subjected to 15% SDS-PAGE
followed by Coomassie Blue staining. The ARFs were detected as
prominent 20-kDa bands, as confirmed by ligand blotting with
[-32P]GTP (data not shown). All of the ARFs except
mARF6 were recovered primarily in the flow-through fractions; mARF6
eluted at approximately 400 mM NaCl. ARF-containing
fractions were pooled, concentrated to about 3 ml using a Centriprep-10
(Amicon, Beverly, MA), and loaded onto a Superdex-75 column (1.5 × 55 cm) equilibrated in column buffer, pH 7.5, adjusted to 10%
sucrose. Column fractions were monitored for ARF content as described
previously. ARF-containing fractions were pooled and concentrated to
>2 mg of protein/ml. The sample was separated into small aliquots,
frozen on dry ice/methanol, and stored at
80 °C. Protein
concentration was determined with the Bio-Rad (Bradford) Protein Assay,
using standard I (Bio-Rad). The percentage of ARF in the final product
was determined by densitometry scanning of a Coomassie Blue-stained gel
using a Personal Densitometer (Molecular Dynamics Inc., Sunnyvale, CA)
with Image-Quant software.
A 2-ml culture of bacteria expressing both ARF and NMT was grown as described above except that 25 µl/ml [3H]myristic acid (14 mCi/ml) was added instead of the unlabeled myristic acid. The bacteria were lysed as described above, resuspended in 20-40-µl column buffer at pH 7.5, and the lysate was cleared by centrifugation at 13,700 × g for 5 min at 4 °C in a microcentrifuge. The supernatant fraction was used without further purification.
Determination of ARF Myristoylation2.5-5-µg aliquots of the various ARFs were loaded alongside 5 µg of [3H]myristate-labeled lysate on a 13% (mARFs 1, 5, and yARF2) or a 15% (mARF6) SDS-polyacrylamide gel, measuring 25 cm long. The gels were electrophoresed until the 17-kDa See Blue prestained marker (Novex, San Diego, CA) was near the bottom of the gel. The gel was stained with Coomassie Blue, incubated in Amplify (Amersham Corp.), dried, and analyzed by autoradiography. The bands corresponding to myristoylated protein were identified by their co-migration with [3H]myristate-labeled ARF and by their absence in unmyristoylated ARF preparations. The ratio of myristoylated ARF to total ARF in each preparation was determined by densitometry scanning of the Coomassie Blue-stained gel.
Preparation of Golgi-enriched Membranes and Cytosolic FractionsRat liver Golgi membranes and an AP-1/COPI-enriched
fraction of rat liver cytosol were prepared as described previously
(3), except that the latter was concentrated approximately 10-fold using a centriprep-10, frozen on dry ice in small aliquots, and stored
at 80 °C. The protein concentration of these fractions was
determined with the Bio-Rad protein assay, using standard II. The final
coat protein-enriched fraction had a concentration of AP-1 that was
about 3 times that in cytosol, as determined by quantitative
immunoblotting. There was no ARF detectable in this fraction as
analyzed by ligand blotting with [
-32P]GTP.
A guanine nucleotide
exchange assay was developed based on the assays of Northrup et
al. (20) and Kahn and Gilman (21). Except where indicated,
recombinant ARF (2.5 µM) was added with or without Golgi
membranes (50 µg/ml) to a 100-µl reaction mixture containing 25 mM Hepes, pH 7.0, 50 mM potassium acetate, 2.5 mM magnesium acetate, 1 mM DTT, 1 mM ATP, 1 mg/ml BSA, and 1 µM
[35S]GTPS (3 × 105-1 × 106 cpm). The reaction mixture was incubated for 10 min at
37 °C, and then 1 ml of 25 mM Hepes, pH 7.0, 50 mM potassium acetate, 2.5 mM magnesium acetate,
1 mM DTT, at 4 °C, was added to stop the reaction. The
sample was then passed over a nitrocellulose filter in a sampling
manifold (Model 3025 from Millipore, Bedford, MA), and the filter was
washed 10 × 1 ml with the buffer used to stop the reaction. Each
experimental point was assayed in triplicate. The radioactivity bound
to the filter was measured, and the amount of GTP
S bound to protein
was calculated. Background binding of [35S]GTP
S to the
nitrocellulose, determined either by adding 5 mM GTP to a
standard reaction mixture containing both ARF and Golgi membranes or by
filtering a sample containing only [35S]GTP
S and
buffer, was less than 0.1% of the radioactivity added.
BFA, when included, was added to 200 µg/ml from a 10 mg/ml stock solution made fresh in ethanol. In these experiments, an equal volume of ethanol was added to the control reaction mixtures.
Protease-treated Golgi membranes were prepared as follows. Aliquots of Golgi membranes (2 mg/ml) in 25 mM Hepes, pH 7.0, 50 mM potassium acetate, 2.5 mM magnesium acetate, 1 mM DTT, 1 mM ATP, 1 mg/ml BSA were treated in one of three ways. One aliquot was incubated with trypsin (50 µg/ml), another with trypsin inhibitor (2.5 mg/ml), and the third with both together for 15 min at room temperature. Trypsin inhibitor (2.5 mg/ml) was then added to the reaction containing trypsin alone, and the Golgi membranes were incubated for an additional 10 min at room temperature. The Golgi membranes were then used directly in the assay.
For the assays to determine the concentration dependence of guanine
nucleotide exchange on mARFs 1 and 6 and yARF2, the Multiscreen Filtration System Vacuum Manifold (Millipore) was used. In these assays, the BSA concentration was reduced to 100 µg/ml, and the [35S]GTPS concentration was increased to 10 µM. The reactions were stopped by immediately filtering
50 µl of a 100-µl reaction mixture onto a HA-High Protein and
Nucleic Acid Binding Plate (Millipore). The filters were washed 7 × 200 µl with 25 mM Hepes, pH 7.0, 50 mM
potassium acetate, 2.5 mM magnesium acetate, 1 mM DTT, at 4 °C.
The recruitment
assays were performed in a total volume of 400 µl in 1.5-ml
presiliconized tubes to reduce background. The AP-1/COPI-enriched
cytosolic fraction was precleared by centrifugation at 220,000 × g for 20 min at 4 °C before use in the assay. Golgi membranes (50 µg/ml), recombinant myristoylated ARF to the
concentration indicated in the figure legends, the AP-1/COPI-enriched
cytosolic fraction (5.6 mg/ml), and either GTPS (100 µM) or GDP (1 mM) were mixed together on ice
in assay buffer (25 mM Hepes, pH 7.0, 250 mM
potassium acetate, 2.5 mM magnesium acetate, 1 mM DTT, 1 mg/ml BSA). The reaction mixtures were incubated
for 15 min at 37 °C, and the assay was stopped by returning the
tubes to ice. The reaction mixtures were then transferred to fresh
1.5-ml presiliconized tubes to further reduce the background. The Golgi membranes were reisolated as described previously (3) except that the
final Golgi pellet was washed two times with assay buffer. The
membranes were then solubilized in SDS-PAGE sample buffer (2.3% SDS,
62.5 mM Tris-HCl, pH 6.8, 5%
-mercaptoethanol, 10% sucrose), and analyzed by 13% SDS-PAGE followed by immunoblotting or
Coomassie Blue staining. ARF binding was detected by Coomassie Blue
staining; AP-1 binding was detected by immunoblotting using AE/1, a
polyclonal antibody to
-adaptin, and coatomer binding was detected
by immunoblotting using M3A5, a monoclonal antibody to
-COP. The
immunoblots or Coomassie Blue-stained gels were quantified by
densitometry scanning.
Electrophoresis and immunoblotting were performed as described previously (3). Gels to be analyzed by fluorography were incubated in gel dry (1% glycerol, 25% methanol) for at least 1 h, and then incubated in Amplify for 30 min. The gels were dried onto paper and exposed to X-Omat AR film (Eastman Kodak) using one or two intensifying screens.
Since it is
difficult to fractionate tissue-purified ARF into individual family
members, we used a bacterial expression system to produce homogeneous
populations of individual, recombinant ARFs (11, 22). Myristoylated
ARFs were prepared by co-expressing the various ARFs with NMT, the
enzyme that catalyzes the addition of myristate onto proteins (23).
However, since the efficiency of myristoylation of the different ARFs
varied considerably and one goal of this study was to determine the
role of myristoylation in several functions mediated by the ARFs, it
was important to determine the extent of myristoylation of each
recombinant ARF preparation. ARF preparations can contain three types
of molecules as follows: ARF that still contains its N-terminal
methionine, which blocks myristoylation; unmyristoylated ARF that has
its N-terminal methionine cleaved; and ARF that is myristoylated on its
N-terminal glycine. For mARF1, these three forms can be clearly resolved by SDS-PAGE, as shown in Fig. 1, lanes
2 and 3 (24). In all cases, ARF made in bacteria
co-expressing NMT had a faster migrating ARF band that was absent in
unmyristoylated ARF preparations (Fig. 1, compare lanes 1 and 2, 5 and 6, 9 and 10, and
13 and 14) (18, 24, 25). This faster migrating
band co-migrated with [3H]myristate-labeled ARF
(Fig. 1, lanes 2-4, 6-8, 10-12, and 14-16). The ratio of myristoylated ARF to total ARF in each preparation could
be determined by quantitating the Coomassie Blue-stained bands using
densitometry scanning. The various mARF1 preparations were
myristoylated between 10 and 50%, mARF6 preparations between 30 and
50%, and mARF5 and yARF2 preparations were completely
myristoylated.
Effect of Myristoylation of ARF on Golgi Membrane-stimulated Guanine Nucleotide Exchange
We first tested the ability of rat
liver Golgi membranes to stimulate guanine nucleotide exchange on
myristoylated and unmyristoylated mARFs 1, 5, and 6, and yARF2. As
summarized in Table I, each ARF species exhibited some
spontaneous guanine nucleotide exchange. The Golgi membranes also bound
a small amount of GTPS in the absence of added ARF. When these
background values were taken into account, it was apparent that
incubation of the unmyristoylated ARFs with Golgi membranes did not
result in an increase in GTP
S binding. By contrast, each
myristoylated ARF species gave rise to a stimulation in GTP
S binding
over the background values. This increase has been shown to be due to
increased GTP
S binding to ARF, not to an ARF-induced increase in
GTP
S binding to another Golgi protein (6, 8). However, the magnitude
of this stimulation varied considerably among the various ARFs, with
the exchange on mARF1 being by far the greatest. The effect of ARF
concentration on the extent of stimulation was determined for the
myristoylated forms of mARF1, mARF6, and yARF2 (Fig. 2).
The apparent Km values for activation by the Golgi
membranes were 5 µM for mARF1 and 12.5 µM
for mARF6. An accurate Km value for the activation of yARF2 could not be determined, but it was greater than 20 µM. The apparent Vmax values for
myristoylated mARF1 and mARF6 were 400 and 250 pmol of GTP
S
bound/min/mg Golgi membranes, respectively. Thus the catalytic
efficiency (Vmax/Km) was 4 times greater for myristoylated mARF1 than for myristoylated mARF6. The
apparent Vmax for yARF2 could not be
calculated.
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Since the preparation of myristoylated mARF1 used in most of the experiments contained a significant fraction of the unmyristoylated species (80%), we tested whether the unmyristoylated mARF1 was an inhibitor of the Golgi membrane-stimulated guanine nucleotide exchange on the myristoylated protein. To do this, a large excess of unmyristoylated mARF1 was added to an assay containing myristoylated mARF1 and Golgi membranes. As shown in Table II, unmyristoylated mARF1 did not inhibit the Golgi membrane-stimulated guanine nucleotide exchange on myristoylated mARF1.
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The effect of BFA on the Golgi membrane-stimulated guanine nucleotide exchange on the various myristoylated ARFs was tested next. This fungal metabolite inhibits the activity of the Golgi GEF and thereby provides a means to determine the role of the Golgi GEF in these exchange reactions (6-8). The results of a typical experiment are shown in Table III. BFA inhibited the Golgi-stimulated guanine nucleotide exchange on myristoylated mARF1 by 72% while inhibiting the exchange on myristoylated mARFs 5 and 6 by only 20%. The exchange on myristoylated yARF2 was not inhibited. Similar results were obtained when Golgi membranes were treated with trypsin (Table IV). This protease has been shown to inactivate the Golgi GEF (7, 8). In this representative experiment, trypsin treatment of the Golgi membranes inhibited guanine nucleotide exchange on myristoylated mARF1 by 92%, whereas the exchange on myristoylated mARFs 5 and 6 was only inhibited 19 and 30%, respectively. The exchange on myristoylated yARF2 was not inhibited. Golgi membranes treated with trypsin in the presence of trypsin inhibitor remained fully active, showing that the effect on the GEF activity requires protease action.
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The data in Tables I, III, and IV are summarized graphically in Fig.
3, which presents the extent of Golgi-stimulated guanine nucleotide exchange activity for each ARF, expressed as a percent of
the activity of myristoylated mARF1. The figure shows that myristoylated mARF5, mARF6, and yARF2 differ both quantitatively and
qualitatively from myristoylated mARF1 in undergoing much less guanine
nucleotide exchange which is almost completely insensitive to BFA or
trypsin treatment of the Golgi membranes. The sensitivity to BFA and
protease treatment displayed by myristoylated mARF1 is a hallmark of
Golgi GEF activity.
Binding of ARFs to Golgi Membranes
Each of the myristoylated
ARFs was tested for its ability to bind to Golgi membranes and to
promote coat protein recruitment using an in vitro
recruitment assay (3). In this assay, the recombinant myristoylated ARF
is incubated with Golgi membranes, a rat liver cytosolic fraction
enriched in AP-1 and COPI but depleted of ARF, and either GTPS or
GDP. After incubation at 37 °C for 15 min, the Golgi membranes are
reisolated and analyzed for ARF binding and coat protein
recruitment.
The binding of the various ARFs to the Golgi membranes as a function of
myristoylated ARF concentration is shown in Fig. 4. A shows the Coomassie Blue staining pattern of Golgi
membranes incubated with increasing concentrations of myristoylated ARF in the presence of GTPS. In all cases there was increased staining of a band at 20 kDa as the concentration of myristoylated ARF in the
reaction increased. The identity of this band as ARF was confirmed by
demonstrating that it bound [
-32P]GTP in a ligand
blotting assay (data not shown). Very little ARF was found on the Golgi
membranes when GDP was present instead of GTP
S. When recombinant ARF
was omitted, the Coomassie Blue-stained 20-kDa bands were not
observed.
When the Coomassie Blue-stained bands migrating at 20 kDa were quantitated by densitometry scanning, it was possible to plot the amount of ARF bound as a function of myristoylated ARF added (Fig. 4B). It is apparent that myristoylated mARF1 bound with the highest efficiency, myristoylated mARF6 and yARF2 bound significantly less well, and myristoylated mARF5 bound poorly.
Effect of ARFs on Coat Protein Recruitment onto Golgi MembranesAfter mARF1 binds to Golgi membranes, it promotes the
recruitment of AP-1 and coatomer onto these membranes (2-5). To
evaluate the ability of the other ARFs to mediate these processes, the Golgi membranes used in the ARF binding experiments were assayed for
their content of AP-1 and coatomer by immunoblotting. AP-1 recruitment
was detected using an antibody to the -adaptin subunit, whereas
coatomer recruitment was detected with an antibody to the
-COP
subunit. The resulting bands were quantitated by densitometry scanning.
These values were compared with those obtained with myristoylated mARF1
assayed in the same experiment. The mARF1 curves were also used to
normalize the values obtained in several independent experiments. When
the data were plotted as the amount of
-adaptin or
-COP recruited
versus the concentration of myristoylated ARF present in the
reaction, it was apparent that mARF1 was much more effective than the
other ARFs in promoting the binding of the coat proteins to the Golgi
membranes (Figs. 5 and 6,
A-C). These data reflect both the efficiency of the Golgi
GEF in activating the various ARFs as well as the ability of the bound
ARFs to facilitate coat protein recruitment. By plotting the data as
coat protein recruited as a function of the amount of myristoylated ARF
actually bound to the Golgi membranes in the same assay, it was
possible to analyze the steps that occur after the ARFs bind to the
Golgi membranes. When considered in this way, it was apparent that
mARF5, once bound to the membranes, was somewhat more active than mARF1 in promoting
-adaptin recruitment, whereas mARF6 was slightly less
active and yARF2 was much less active (Fig. 5, D-F). In
contrast, mARF5 and yARF2 promoted
-COP recruitment to about the
same extent as mARF1, and mARF6 appeared to be slightly less active
(Fig. 6, D-F). It is also evident that
-COP binding to
the Golgi membranes reaches saturation at a lower level of bound mARF1
than that required for maximal
-adaptin binding. This has been
observed previously by Stamnes and Rothman (2).
During the formation of coated vesicles on Golgi membranes, ARF is
activated by an as yet unidentified, membrane-associated GEF which
catalyzes the exchange of GTP for GDP. The activated, membrane-bound
ARF in turn facilitates the recruitment of the AP-1 adaptor complex and
the COPI coat complex onto the Golgi membranes. By comparing the
ability of the Golgi membrane GEF to activate mARFs 1, 5, and 6, we
have determined that it has specificity for mARF1. Several lines of
evidence lead to this conclusion. First, although the Golgi membranes
were able to stimulate the activation of all of the ARFs tested, the
stimulation of mARF1 was much greater. Consistent with this, the
apparent Km for activation of mARF1 by the Golgi
membranes was much lower than for mARF6 or yARF2. In addition, we found
that the Golgi membrane-dependent activation of mARF1, but
not that of the other ARFs tested, was greatly inhibited by the
addition of BFA, a compound known to inhibit the Golgi guanine
nucleotide exchange activity in vitro (6-8), and the
binding of ARF, -COP, and
-adaptin to the Golgi membranes
in vivo (26-29). Furthermore, trypsin treatment of the
Golgi membranes, which is also known to destroy the activity of the
Golgi GEF (7, 8), markedly inhibited the activation of mARF1 but had
only a small effect on the activation of mARFs 5 and 6 and no effect on
yARF2 activation. Thus, for the following reasons we believe mARF1 (or
class I ARFs) to be the most likely physiological mediator of coated
vesicle formation on the Golgi membranes: 1) the Golgi membrane GEF has
specificity for mARF1, or potentially for class I ARFs; 2) the
cytosolic concentration of mARFs 1 and 3 is high relative to the other
mammalian ARFs (30); and 3) mARF1 has been localized to Golgi membranes
in vivo (15).
Because trypsin treatment almost completely destroyed the activity of
the BFA-sensitive Golgi GEF, we think it unlikely that a protein on the
Golgi membranes is responsible for the activation of the
BFA-insensitive ARFs. Instead, we hypothesize that the Golgi membrane
phospholipids are responsible. Phospholipids have been shown to affect
the nucleotide state of ARF1 in vitro, stabilizing the
GTPS-bound form of the myristoylated protein and slightly destabilizing the GDP-bound form (24, 31). Consistent with this
hypothesis, activation of yARF2 (which was completely insensitive to
inhibition by BFA and trypsin) was unsaturable over the range of ARF
concentrations tested, as might be expected for a nonenzymatic, phospholipid-mediated process.
Comparison of the ability of the Golgi GEF to activate myristoylated and unmyristoylated mARF1 showed that myristoylation is absolutely required for mARF1 activation. Addition of an excess of unmyristoylated mARF1 did not inhibit activation of myristoylated mARF1, suggesting that myristoylation is required for its recognition by the GEF. There are several possible roles for the myristoyl group in this process. The myristoyl group may interact directly with the Golgi GEF, facilitating the protein-protein interaction. Alternatively, the myristoyl group may be required to maintain a conformation of ARF competent to interact with the Golgi GEF. Finally, recent work has suggested that ARF may need to associate with a membrane before it interacts with a GEF (32) and that the myristoyl group gives the GDP-bound form of the protein the required affinity for phospholipids (24).
These findings are consistent with the results obtained in overexpression studies in vivo (33). When ARF1(T31N), a mutant of mARF1 deficient in GTP binding, was overexpressed, it had a BFA-like effect on the cells. It has been hypothesized that this protein acts by binding irreversibly to the Golgi GEF, preventing activation of the wild type protein. However, overexpression of ARF1(G2A,T31N), a double mutant also lacking the site of myristoylation, had no effect on the cells, suggesting that it was unable to block the active site of the Golgi GEF. It would be interesting to test whether these mARF1 mutants can act as inhibitors of the Golgi GEF in vitro.
In contrast to our results, others have found that unmyristoylated mARF1 can be activated by Golgi membranes in vitro, but its activation was much less efficient than that of the myristoylated protein (34). In addition, it has been reported that unmyristoylated mARF3 can be activated by a trypsin-sensitive factor on the Golgi membranes (8). The reason for these discrepancies is unclear but may be due to differences in assay conditions.
While our results demonstrate that the Golgi GEF is specific for mARF1, we also found that all of the ARFs tested could bind to the Golgi membranes in vitro, as had been shown earlier for mARFs 1, 3, and 5 (35, 36). Therefore, the Golgi membranes themselves have no specificity for a single ARF. In addition, all of the mARFs tested could promote binding of AP-1 and coatomer, although to different extents. In particular, mARF6 promoted recruitment of the coat proteins less well than mARF1 and mARF5. Yeast ARF2 promoted the recruitment of AP-1 very poorly although it mediated the recruitment of coatomer reasonably well. These results suggest that there may be some specificity for activating the downstream events that result in coat recruitment.
Several models have been proposed for how ARF promotes coated vesicle formation on the Golgi membranes. In the simplest, ARF acts directly as a receptor for the vesicle coat protein (5). However, this model does not explain how coatomer is specifically recruited onto the membranes of the Golgi stack whereas AP-1 recruitment is confined to the trans-Golgi network (TGN). To address the localization of AP-1 recruitment, it has been proposed that ARF activates a putative docking protein present in the TGN (3). Upon activation, the docking protein would undergo a conformational change resulting in high affinity binding of AP-1. A variant of this model is that ARF activates a receptor present on the TGN, such as the mannose-6-P/IGF II receptor, to allow it to interact with AP-1 with greater affinity (37). It should be noted that in the latter two models, the effects of ARF on the target proteins could be direct or indirect.
In this regard, ARF has been identified as an activator of
membrane-associated phospholipase D (PLD), including phospholipase D
localized to the Golgi membranes (38-44). Furthermore, the Golgi membranes of PtK1 and Madin-Darby canine kidney cells, which have high
levels of endogenous PLD activity, form COPI-coated vesicles in the
absence of added ARF and GTP (45) and bind -COP in a BFA-insensitive
manner (27, 46, 47). To explain this, Ktistakis and co-workers (45)
have proposed that PLD acts as a downstream effector of ARF in
COPI-coated vesicle formation and that the activated PLD in the Golgi
membranes of the PtK1 and Madin-Darby canine kidney cells allows the
requirement for exogenous ARF activation to be bypassed. They further
suggest that the activated PLD degrades phosphatidylcholine in the
Golgi membranes to phosphatidic acid which in turn brings about the
recruitment of coatomer either directly or indirectly. Interestingly,
-adaptin binding to the Golgi membranes of these cells remains
sensitive to BFA, consistent with ARF acting via two pathways to
recruit these two types of coat proteins (27, 47). The finding that
maximal recruitment of coatomer and AP-1 in our assays is achieved at
different levels of mARF1 binding (Figs. 5 and 6) is consistent with
there being two activation pathways. Clearly, additional studies will
be necessary to sort out these fundamental issues concerning the role
of ARF in coat protein recruitment.
We thank J. Gordon, R. Kahn, and R. Klausner
for the ARF and NMT expression vectors. The -COP antibody was
generously provided by T. Kreis. R. Kornfeld, M. Wilson, Y. Zhu, and L. Traub gave helpful comments on the manuscript.