Brefeldin A Inhibited Activity of the Sec7 Domain of p200, a
Mammalian Guanine Nucleotide-exchange Protein for ADP-ribosylation
Factors*
Naoko
Morinaga
,
Ronald
Adamik,
Joel
Moss, and
Martha
Vaughan
From the Pulmonary-Critical Care Medicine Branch, NHLBI, National
Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
A brefeldin A (BFA)-inhibited guanine
nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARF)
was purified earlier from bovine brain cytosol. Cloning and expression
of the cDNA confirmed that the recombinant protein (p200) is a
BFA-sensitive ARF GEP. p200 contains a domain that is 50% identical in
amino acid sequence to a region in yeast Sec7, termed the Sec7 domain. Sec7 domains have been identified also in other proteins with ARF GEP
activity, some of which are not inhibited by BFA. To identify structural elements that influence GEP activity and its BFA
sensitivity, several truncated mutants of p200 were made. Deletion of
sequence C-terminal to the Sec7 domain did not affect GEP activity. A
protein lacking 594 amino acids at the N terminus, as well as sequence following the Sec7 domain, also had high activity. The mutant lacking
630 N-terminal amino acids was, however, only 1% as active, as was the
Sec7 domain itself (mutant lacking 697 N-terminal residues). It appears
that the Sec7 domain of p200 contains the catalytic site but additional
sequence (perhaps especially that between positions 595 and 630)
modifies activity dramatically. Myristoylated recombinant ARFs were
better than non-myristoylated as substrates; ARFs 1 and 3 were better
than ARF5, and no activity was detected with ARF6. Physical interaction
of the Sec7 domain with an ARF1 mutant was demonstrated, but it was
much weaker than that of the cytohesin-1 Sec7 domain with the same ARF
protein. Effects of BFA on p200 and all mutants with high activity were
similar with ~50% inhibition at
50 µM. The
inactive BFA analogue B36 did not inhibit the Sec7 domain or p200.
Thus, the Sec7 domain of p200, like that of Sec7 itself (Sata, M.,
Donaldson, J. G., Moss, J., and Vaughan, M. (1998) Proc.
Natl. Acad. Sci. U. S. A. 95, 4204-4208), plays a role in BFA
inhibition as well as in GEP activity, although the latter is markedly
modified by other structural elements.
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INTRODUCTION |
ARFs are ~20-kDa GTPases originally isolated as activators of
cholera toxin-catalyzed ADP-ribosylation of purified G
s and now
known to play a critical role in trafficking of coatomer-coated vesicles between endoplasmic reticulum
(ER)1 and Golgi and between
Golgi compartments, as well as clathrin-coated vesicles (1-3). They
can also activate phospholipase D and might thereby function
additionally in signal transduction (4-6). Six mammalian ARFs have
been cloned and grouped in three classes based on deduced amino acid
sequence, size, gene structure, and phylogenetic analysis: class I,
ARFs 1, 2, and 3; class II, ARFs4 and 5; class III, ARF6 (3).
ARFs are inactive when GDP is bound. Activation, which requires
replacement of bound GDP with GTP, is accelerated by guanine nucleotide
exchange proteins or GEPs that can serve as regulators of ARF activity
(3). We had purified p200 from bovine brain cytosol as a GEP for ARF1
and ARF3 (7, 8) and found that it is 33% identical in deduced amino
acid sequence to Sec7 from Saccharomyces cerevisiae (9),
which is a component of ER to Golgi transport vesicles (10). Yeast Sec7
contains a so-called Sec7 domain of ~190 amino acids that are 50%
identical to an analogous sequence in the bovine p200 GEP. Other ARF
GEPs known to contain Sec7 domains include Gea1 and 2 from S. cerevisiae (11) as well as human ARNO (12) and cytohesin 1 (13).
The ARNO Sec7 domain itself (12), as well as that of Sec7 (14),
exhibited GEP activity.
Two types of ARF GEP have been distinguished by their susceptibility to
inhibition by brefeldin A (BFA), a macrocyclic lactone synthesized from
palmitate by a variety of fungi (15) and initially identified as an
antiviral agent (16). After it was found that BFA inhibits an early
step in secretion causing retention of secretory and membrane proteins
in the ER of many cells (17), it became an important tool for the study
of intracellular vesicular trafficking. These BFA effects result from
its interference with ARF activation as a consequence of inhibition of
ARF GEP (18, 19). The purified bovine GEP p200 is BFA-sensitive (7, 8),
as are Gea1 and 2 (11), whereas ARNO (12) and cytohesin 1 (13) are not. The BFA-sensitive GEPs are all relatively larger molecules than those
that are insensitive, but the only apparent sequence similarity among
all of the GEPs is in the Sec7 domains. To identify structural determinants of GEP activity and BFA sensitivity, several truncated mutants of p200 were assayed, and the physical interaction of two of
them with a mutant ARF1 was evaluated by gel filtration. As reported
here, the Sec7 domain itself exhibited GEP activity, albeit, 2 orders
of magnitude lower than that of intact p200, and the activity was
inhibited by BFA. All observations were consistent with the conclusion
that elements of amino acid sequence N-terminal to the Sec7 domain
enhance its GEP activity and its ability to form a stable complex with
13ARF1.
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EXPERIMENTAL PROCEDURES |
Materials--
[35S]GTP
S (1,250 Ci/mmol) was
purchased from NEN Life Science Products Inc., BFA from Epicenter,
phosphatidylserine and GTP
S from Sigma, and Pfu polymerase from
Stratagene. The baculovirus expression kit was from PharMingen.
Rapid-Ag-Stain Kit from ICN was used for silver staining. The Sec7
domain of cytohesin-1 (Arg62-Asp249) was
synthesized as a His6 fusion protein and purified as
described by Meacci et al. (13). Details of preparation of
13ARF1 (20) and cytohesin-1 (13) are published.
Preparation of p200 Mutants--
Structures of p200 mutants are
shown schematically in Fig. 1. The
C cDNA was made by ligation
of two DNA fragments that result from cleavage at a SpeI
restriction site at position 1737. One fragment was made by polymerase
chain reaction amplification of 200A (8), using forward primer
5'-CAAACATATTCATATGACTAGTAAATGATCTGT and reverse primer (G54)
5'-GATTGTCATCGCGGCCGCTCATTTCATTGATATCTTTTTTC, followed by digestion of the product with SpeI and
NotI. G54 introduced a NotI site (underlined)
after the stop codon (bold). The other fragment was excised from
plasmid A (8) with NdeI and SpeI. The two
fragments were ligated in-frame through their SpeI sites into NdeI- and NotI-digested baculovirus transfer
vector (pAcHLT-C).
C
520 was made similarly by ligation of two DNA
fragments using the SpeI site. One fragment was amplified
from plasmid
C using forward primer
5'-CAAAACACACATATGGATGCAGATTGAGGT and reverse primer 5'-TTGACACTAAGCATTCTAAACC (G53). The product was digested with NdeI (underlined site in forward primer) and SpeI
(internal site in amplified DNA). The other DNA fragment was made by
digestion of
C plasmid with SpeI and NotI. The
two fragments were ligated into pAcHLT-C, which had been digested with
NdeI and NotI.
C
570 was amplified from
C
plasmid by polymerase chain reaction using forward primer
5'-AAACATATGTTGTGACTTAAATG (NdeI site
underlined) and reverse primer G54. The amplified DNA was first
subcloned into pCRscript Amp SK(+) and, after verification of the
sequence by automatic sequencing, the DNA was excised with
NotI and NdeI and subcloned into baculovirus
transfer vector, pAcHLT-C, which had been digested with NdeI
and NotI.
C
594,
C
630,
C
660, and
C
697 were amplified by
the same procedure as
C
570 using reverse primer G54, with
forward primers 5'-CCCAAGGCATATGGAGTCAGGAACTTGGTA,
5'-GGAGCAACATATGGTATGTGAATCCCAACTC, 5'-CCAGAGACCATATGCAGATACGGAAGTTTAAA, and
5'-TCGAAGTCCTCATATGGCAGAAAGAAATAATAG, respectively.
Underlined sequence is an NdeI site.
Plasmid Purification and DNA Sequencing--
Plasmid DNA was
purified using Wizard Plus SV Minipreps (Promega) from 10 ml of LB
broth culture containing ampicillin (50 µg/ml) that had been
incubated overnight at 37 °C. An ABI373 DNA sequencer was used for
DNA sequencing.
Protein Expression and Purification--
p200 and mutant
proteins were synthesized in Sf9 cells as
His6-tagged fusion proteins and purified using Ni-NTA
(nickel-nitrilotriacetic acid-agarose). Sf9 cells (1 × 106/3 ml medium) transfected with 1 µg of Baculogold DNA
and 3 µg of each recombinant baculovirus transfer vector were
incubated at 27 °C for 5 days before 1 ml of the supernatant was
transferred to fresh Sf9 cells (1-2 × 107/15
ml medium) and incubated for 3 days. Cells were collected, washed with
phosphate-buffered saline, and suspended in 1 ml of ice-cold 10 mM sodium phosphate, pH 8.0, containing 100 mM
NaCl and protease inhibitors. Cells were lysed by freezing and thawing twice and cellular debris was removed by centrifugation (16,000 × g, 15 min). Presence of recombinant protein was verified by SDS-PAGE and the clear lysate was incubated for 1 h with 0.5 ml of
Ni-NTA, which was then transferred to a column and washed with 30 ml of
20 mM imidazole, 50 mM sodium phosphate, pH
8.0, 300 mM NaCl, 10% glycerol, 0.5 mM
Pefabloc (Roche Molecular Biochemicals) to remove loosely bound
proteins. His6-tagged proteins were eluted either with 80 mM Tris-HCl, pH 8.0, 2 M NaCl, 100 mM EDTA or with wash buffer containing 400 mM
imidazole, pH 6.0, immediately neutralized, and dialyzed against 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 1 mM NaN3, 0.25 M sucrose, 5 mM MgCl2, 0.5 mM Pefabloc, 30 mM NaCl (or without
MgCl2 for experiments like those in Figs. 7 and 8).
Proteins (~4-120 µg/ml) were stored in small portions at
20 °C in the same medium. Purity of the mutant proteins ranged
from 60 to 90%. The purified
C
570 migrated a ~39-41-kDa
doublet on SDS-PAGE.
Gel Filtration Analysis of
13ARF1 and GEP Interaction--
To
determine whether stable complexes were formed,
13ARF1 and
C
697,
C
570, or C-1Sec7 were incubated for 1 h at
37 °C in TNDSA buffer (20 mM Tris-Cl, pH 8.0, 1 mM NaN3, 1 mM DTT, 0.25 M sucrose, 1 mM aminoethylbenzenesulfonyl
fluoride) containing either 1 mM MgCl2 plus 2 mM EDTA or 2 mM MgCl2 plus 0.1-0.5
mM EDTA and 4 µM GTP
S with 140 µg of
bovine serum albumin in a total volume of 250-500 µl. The mixture
was then applied to a column (0.9 × 44 cm) of Ultrogel AcA54
equilibrated and eluted with TNDSA buffer containing 10 µg/ml bovine
serum albumin, 0.1 M NaCl and either low MgCl2
buffer (1 mM MgCl2 and 2 mM EDTA)
or high MgCl2 buffer (2 mM MgCl2
with 1 mM EDTA), depending on the prior incubation conditions. After elution of 11.5 ml, fraction collection was begun.
Samples (45 µl) of fractions (0.33 ml) were subjected to SDS-polyacrylamide gel electrophoresis in 14 or 16% gels, followed by
silver staining and quantification of stained bands by densitometry (Molecular Dynamics Personal Densitometer S1). Amount of each protein
is reported as percentage of the total recovered in all fractions. All
observations were replicated with at least two preparations of
recombinant protein.
Assay of GEP Activity--
GEP activity of mutant proteins was
assayed by quantifying GTP
S binding to a partially purified mixture
of native ARFs (predominantly ARFs 1 and 3) or other ARF preparation,
as indicated. ARF and purified His6-tagged recombinant
proteins were incubated with 4 µM
[35S]GTP
S (~5 × 106 cpm) in 20 mM Tris-HCl, pH 8.0, 1 mM DTT, 1 mM
EDTA, 5 mM MgCl2 with 50 µg of bovine serum
albumin, and 20 µg of phosphatidylserine at 30 °C for 20 min
unless otherwise indicated, in a total volume of 100 µl. Samples were
transferred to nitrocellulose filters followed by washing six times (1 ml each) with ice-cold buffer (25 mM Tris-HCl, pH 8.0, 2.5 mM DTT, 5 mM MgCl2, 100 mM NaCl) and 5 ml of scintillation fluid were added to each
filter for radioassay. Routine assays contained phosphatidylserine
because without it GTP
S binding to ARF was very low and GEP activity of p200 was not detected, as reported for a ~55-kDa GEP purified from
rat spleen cytosol (21).
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RESULTS |
GEP Activity of p200 and Mutants--
Recombinant
His6-tagged p200 and mutant proteins (Fig.
1) were initially evaluated by their
effects on GTP
S binding to a mixture of native ARF1 and ARF3. As the
Sec7 domain of the BFA-insensitive ARNO had been shown to have ARF GEP
activity (12), the mutant
C was constructed to eliminate the 964 amino acids C-terminal to the Sec7 domain. Its activity was almost
identical to that of p200 (Fig.
2A). Because the yield of p200
was always much lower than that of the mutants, 0.7 pmol was the
largest amount used for the experiment in Fig. 2. Deletion from
C of
the indicated numbers of amino acids at the N terminus yielded proteins
C
520,
C
570,
C
594,
C
630,
C
660, and
C
697 (the Sec7 domain). The activity of
C
570 was quite
similar to those of p200 and
C, reaching a maximum with ~1 pmol
(Fig. 2A). Although
C
520 was not dramatically less
active, the activity of two different preparations was consistently
less than that of
C
570. The activity of
C
594 was also
distinctly less than that of
C
570 and similar to that of
C
520 (Fig. 2A). The three mutants with N-terminal deletions of 630, 660, and 697 amino acids (Fig. 2B),
however, had activities less than 1% those of the most active
constructs (p200,
C,
C
570). The GEP activity was
concentration-dependent, although maximal effects of the
mutants with very low activity were not established even with amounts
of >100 pmol (Fig. 2B). In the experiment shown in Fig.
2B, binding of GTP
S was 2.0 ± 0.28 pmol with 400 pmol of
C
697 (the Sec7 domain).

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Fig. 1.
Deletion mutants of p200. The 188 amino
acids between 697 and 885 constitute the Sec7 domain. C indicates
deletion of 964 amino acids C-terminal to the Sec7 domain. The number
following indicates the number of amino acids deleted from N
terminus of C. In parentheses, is molecular mass of the
His6 fusion proteins; that of p200 is 212 kDa.
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Fig. 2.
Effect of deletion mutants of p200 on
GTP S binding to native ARF1/ARF3.
A, the indicated amounts of p200 ( ), C ( ),
C 520 ( ), C 570 ( ), or C 594 ( ) proteins were
incubated at 30 °C for 20 min with 10 pmol of native ARF1/ARF3 in
the presence of 4 µM [35S]GTP S, 20 µg
of phosphatidylserine, and 5 mM MgCl2 before
collection of proteins on nitrocellulose filters for radioassay.
GTP S bound to ARF alone, in the absence of p200 or mutant (0.21 pmol), was subtracted to yield values that represent picomoles of
GTP S bound due to the indicated amount (pmol) of p200 or mutant
protein. Data are mean ± S.E. of values from triplicate assays.
Error bars smaller than symbols are not shown. B,
assays were carried out as in A with the indicated amounts
of C 630 ( ), C 660 ( ), and C 697 ( ). Binding to
ARF alone was 0.10 pmol.
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The low rate of binding of GTP
S to ARF alone in the absence of GEP
was essentially constant for 60 min at 30 °C (Fig.
3). Addition of p200 or
C (0.4 pmol)
increased the initial rate similarly, but the magnitude of the effect
began to diminish before 20 min. Both
C
630 and
C
697 (40 pmol) had lesser effects on rate that were essentially terminated in 20 min (Fig. 3).

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Fig. 3.
Time course of GTP S
binding by ARF with p200 and mutants. Binding of
[35S]GTP S to 10 pmol of native ARF1/ARF3 with 0.4 pmol
of p200 ( ) or C ( ), 40 pmol of C 630 ( ) or C 697
( ), or no GEP ( ) was carried out as described in the legend to
Fig. 2 except that samples were incubated at 30 °C for indicated
times before proteins were collected on nitrocellulose for radioassay.
Data are reported as in Fig. 2.
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Relative GEP activities of p200 and several mutants are summarized in
Table I. GEP activity is expressed as
GTP
S binding (to 10 pmol of the native ARF1/ARF3 mixture) per pmol
of each GEP protein, based on data from experiments like those in Fig. 2 in the range in which binding was proportional to the amount of
protein added. Most striking was the decrease in activity, 2 orders of
magnitude, occasioned by the removal of 36 N-terminal amino acids from
C
594 to yield
C
630. Further truncation, producing
C
630,
C
660, and the Sec7 domain itself (
C
697), had no
further effect on activity (Table I).
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Table I
Effect of p200 and mutants on GTP S binding to ARF1 and ARF3
Stimulation of binding of [35S]GTP S to 10 pmol of ARF by
each GEP protein was determined as described under "Experimental
Procedures." GEP activity is the increment in GTP S binding to ARF
induced by the GEP protein expressed as picomoles of GTP S per pmol
of GEP protein, based on data from experiments in which binding was
proportional to the amount of GEP used. Data are mean ± S.E. (or
1/2 the range when n = 2) of values from the
indicated number (n) of separate experiments. In
parentheses, number of different protein preparations used.
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ARF Specificity of p200 by GEP Activity--
We initially reported
that native p200 purified from bovine brain cytosol failed to activate
recombinant myristoylated ARF5 (7). In subsequent studies, recombinant
p200, as well as
C
570, consistently exhibited GEP activity with
mARF5 (Table II), although it appeared to
be not as good a substrate as class I ARFs 1 and 3. None of four
different preparations of recombinant myristoylated ARF6, including one
synthesized in Sf9 cells that was a substrate for C-1Sec7 (29),
appeared to be a substrate for p200 or
C
570 (Table II). Thus, we
may conclude that under the assay conditions used, p200 is a GEP for
class I ARFs 1 and 3, and might also function with ARF5 (class II), but
seemingly not with ARF6 (class III). Non-myristoylated ARFs 1, 3, and 5 were always utilized less effectively than their myristoylated
counterparts (Table II), as has been noted for other GEP preparations
(21).
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Table II
Effect of recombinant p200 or C 570 on GTP S binding to ARF
proteins
Stimulation of [35S]GTP S binding to the indicated amount
of ARF protein by recombinant GEP was assessed in the presence of 4 µM GTP S and 20 µg of phosphatidylserine. Binding in
the absence of ARF has been subtracted. Two different preparations of
recombinant p200 (0.4 pmol) were used as GEP in experiments 1 and 2, 2 pmol of C 570 in experiment 3, and 0.5 pmol of C 570 in
experiment 4. Native (n) ARF1 and ARF3 or a mixture of nARF1
and 3 (nARF1/3) or recombinant (r) or recombinant
myristoylated (m) ARFs were used. Different preparations are
identified by letters in parentheses. Data (pmol) are mean ± S.E.
of values from triplicate assays. Binding of GTP S to 15 pmol of
rARF6 (B) was increased from 0.12 ± 0.02 to 1.05 ± 0.03 pmol (20 min, 37 °C) by 22 pmol of C-1Sec7.
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Effect of BFA on GEP Activity of p200 and Mutants--
Decreasing
the incubation temperature to 22 °C increased the stability of both
C
697 (Fig. 4A) and
C
570 (Fig. 4B), facilitating demonstration of the
major difference in their specific activities and the ~90%
inhibition by BFA, which did not inhibit cytohesin-1 or its Sec7 domain
(Fig. 4C). As noted before (29), the specific activity of
C-1Sec7 was significantly greater than that of intact cytohesin-1, and
was also less stable.

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Fig. 4.
Effect of BFA on GEP activity. Native
ARF1/3 (A, 10 pmol; B and C, 20 pmol)
was incubated without ( , ) or with 10 pmol of GEP without
(open symbols) or with (solid symbols) 0.4 mM BFA at 22 °C for the indicated time (50 µl total
volume). Binding in the absence of ARF has been subtracted.
A, C 697 ( , ); B, C 570 ( ,
); C, cytohesin-1 ( , ); C-1 Sec7 ( , ).
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As shown in Fig. 5, the activities of
p200,
C,
C
570, and
C
594 were inhibited
50% by 50 µM BFA. Although certain mutants appeared to be somewhat
less sensitive (likely because of instability), all were inhibited by
BFA (Fig. 5). Percentage inhibition by BFA, which does not affect basal
exchange, is lower when the fractional contribution of non-catalyzed
exchange to the total is increased, e.g. when inactivation
of GEP occurs during an assay. Inhibition by BFA was specific inasmuch
as 200 µM B36, an inactive analogue, failed to inhibit
p200 or
C
697, whereas 200 µM BFA caused 100% inhibition of p200 and ~40% inhibition of the mutant (Fig. 5).

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Fig. 5.
Effect of BFA on GEP activity of p200 and
mutants. Binding of [35S]GTP S to 10 pmol of
native ARF1/ARF3 was carried out at 30 °C for 20 min with the
indicated concentration of BFA and 0.4 pmol of p200 ( ), 0.5 pmol of
C ( ), C 520 ( ), C 570 ( ), or C 594 ( ), 50 pmol of C 630 ( ), 40 pmol of C 697 ( ), or 20 pmol of
cytohesin-1 ( ). BFA was dissolved in methanol at a concentration of
71 mM; all assays contained 0.6% methanol. Binding to ARF
alone in the absence of GEP was subtracted from that with ARF plus GEP
before percentage inhibition was calculated. The inactive BFA analogue
B36 (0.1 and 0.2 mM) was used instead of BFA with p200 (*)
and at 0.2 mM with C 697 ( ). Data are mean ± S.E. of values from triplicate assays. GEP activities in the absence of
BFA were 0.96 (p200), 0.62 ( C), 0.48 ( C 520), 1.2 ( C
570), 0.55 ( C 594), 0.54 ( C 630), and 1.3 ( C 697) pmol
of GTP S bound.
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Physical Interaction of
13ARF1 and p200 Mutants--
Because
the ARNO Sec7 domain had been reported to form a stable complex with
17ARF1 (22), we investigated the association of
13ARF1 with the
p200 deletion mutants
C
570 and
C
697 (the putative p200 s7
domain). GTP
S binding to 13
ARF1 was stimulated ~8-fold by
C-1Sec7 independent of PS, but not by
C
570 (Table III). Both did activate native ARF1/ARF3
in the presence of PS, but not in its absence (Table III). By
prolonging the incubation (without PS) to 1 h with 2 mM MgCl2 and 0.5 mM EDTA it was
shown that 10 pmol of
C
697 increased GTP
S binding to 62 pmol
of
13ARF1 from 2.4 ± 0.05 to 3.9 ± 0.15 pmol (mean ± one-half the range, n = 2). Similarly, in parallel
assays that included PS, binding was increased from 2.4 ± 0.11 to
3.8 ± 0.03 pmol by
C
697, consistent with the absence of
phospholipid effects on the function of ARF mutants lacking the
N-terminal
-helix (22). In the same experiment, with a relatively
high Mg2+ concentration that was reported to be unfavorable
for ARF-GEP complex formation (22),
13ARF1 with
[35S]GTP
S bound eluted in a single symmetrical peak
from Ultrogel AcA54 at the position expected for monomeric ARF and the
amount of bound [35S]GTP
S was increased after
incubation with
C
697 (Fig. 6).
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Table III
Effect of p200 mutant, C-1Sec7, and phosphatidylserine on GTP S
binding to 13ARF1 and native ARF1/ARF3
In experiment 1, GEP and 50 pmol of 13ARF1 or 20 pmol of native
ARF1/ARF3 were incubated for 20 min at 37 °C with 2 mM
MgCl2 and 0.5 mM EDTA with or without GEP and/or 20 µg of PS as indicated. Experiment 2 was identical except that 10 pmol
of ARF1/ARF3 were used with 4 mM MgCl2 and 0.4 mM EDTA. Data are mean ± one-half the range of values
from duplicate samples.
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Fig. 6.
Effect of
C 697 on
[35S]GTP S binding to
13ARF1. 13ARF1 (320 pmol) was incubated for
1 h at 37 °C with 5 mM MgCl2, 1 mM EDTA, and 4 µM [35S]GTP S
(3.5 × 106 cpm) without ( ) or with ( ) 50 pmol
of C 697 (total volume 250 µl). After transfer of a sample (50 µl) to a nitrocellulose filter for determination of protein-bound
[35S]GTP S, the remainder was applied to a column
(0.9 × 44 cm) of Ultrogel AcA54 equilibrated and eluted with
TNDSA buffer containing bovine serum albumin, 10 µg/ml, 0.1 M NaCl, 2 mM MgCl2, and 1 mM EDTA. After elution of 11.5 ml, collection of fractions
(0.33 ml) was begun. Samples (300 µl) of fractions were transferred
to vials for radioassay. Positions of elution of molecular size
standards are blue dextran (D) exclusion volume, ovalbumin
(O) 43 kDa, cytochrome c (C) 12.5 kDa.
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When
C
697 and
13ARF1 were incubated at the lower
Mg2+ concentration (Fig.
7A),
13ARF1 was eluted in
an asymmetric peak with a shoulder preceding the major peak, consistent
with some degree of association of the two proteins. This was not seen
when the experiment was carried out at a higher Mg2+
concentration with GTP
S (Fig. 7B), and
13ARF1 was
eluted in a narrower, more symmetric peak at the position of an ARF
monomer. In similar experiments at both low and high Mg2+
concentrations,
C
570 was eluted from Ultrogel AcA54 in the exclusion volume of the column, i.e. as a multimer or
aggregate.

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Fig. 7.
Effect of Mg2+ concentration on
association of 13ARF1 and
C 697. 13ARF1 ((40
µg, 2.1 nmol) was incubated with C 697 (40 µg, 1.6 nmol) in
the presence of 1 mM MgCl2 plus 2 mM EDTA (A) or 2 mM
MgCl2 plus 0.1 mM EDTA and GTP S
(B) for 1 h at 37 °C before gel filtration and
analysis of column fractions by SDS-PAGE, silver stain, and
densitometry was carried out as described under "Experimental
Procedures." Optical density of C 697 ( ) or 13ARF1 ( )
is reported as percentage of the total recovered in all fractions = 100%.
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For comparison, the same kind of experiment was performed with C-1Sec7
replacing the p200 mutants. After incubation of
13ARF1 and C-1Sec7
at a relatively low Mg2+ concentration, the proteins
co-eluted from Ultrogel AcA54 in a position consistent with the size of
a heterodimer (Fig. 8A). At
the higher Mg2+ concentration with GTP
S, the amount of
apparent complex was much less, appearing as a shoulder on the
13ARF1 peak (Fig. 8B). The stability of the association
of C-1Sec7 with
13ARF1 was clearly much greater than that of the
p200 mutants with
13ARF1, although some weak association of
13ARF1 with
C
697 was detected at the lower Mg2+
concentration.

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|
Fig. 8.
Effect of Mg2+ concentration on
association of 13ARF1 and C-1Sec7.
13ARF1 (540 pmol) was incubated with C-1Sec7 (450 pmol) in the
presence of 1 mM MgCl2 plus 2 mM
EDTA (A) or 2 mM MgCl2 plus 0.1 mM EDTA and GTP S (B) for 1 h at 37 °C
before gel filtration and analysis of column fractions. Optical density
of C-1Sec7 ( ) and 13ARF1 ( ), is reported as described in the
legend to Fig. 7.
|
|
 |
DISCUSSION |
To date, almost all descriptions of ARF GEP activity have involved
class I ARFs, probably at least in part because they are the most
abundant and most studied of the mammalian ARFs. In addition, it seems
not unlikely that because conditions used for in vitro assays have frequently been developed for use with ARFs 1 and 3, they
are less favorable for other ARFs, which, are known to differ in their
responses to certain phospholipids and detergents. In the cell,
individual ARFs are undoubtedly localized differently, dependent on
their specific interactions with numerous proteins, e.g.
phospholipase D, coatomer, GTPase-activating proteins, and GEPs, as
well as specific phospholipids. A previously recognized functional
interaction of ARF1 with phosphoinositide 4,5-bisphosphate was shown to
require specifically four amino acids (lysines 15, 16, and 181 and
arginine 178) that in the crystal structure are part of a cluster of
basic residues on the surface of the molecule (23). All of these are
present in class I, but not in class II or class III human ARFs.
Recognizing the importance of phosphoinositide 3-kinases in so many
regulatory pathways, Klarlund et al. (24) undertook to
identify by expression cloning proteins that bind specifically 3-phosphoinositides. They termed these "general receptors for phosphoinositides" or GRPs. GRP1 clones from both mouse brain and fat
cells encode a 399-amino acid protein remarkably similar in sequence to
cytohesin-1 with a central Sec7 domain and a C-terminal pleckstrin
homology (PH) domain (24). Specific binding of 3-phosphoinositides to
the PH domains from both proteins, but not to PH domains from IRS-1 or
SOS, was demonstrated. A role for phosphoinositide 3-kinase, via its
p110 subunit, and the PH domain of GRP1 or cytohesin-1 in linking
tyrosine kinase receptors to ARF activation was suggested and the
similarity of cytohesin-1 and GRP-1 to ARNO was noted (24).
ARNO (12) and cytohesin-1 (13) are much smaller (~47 kDa) than p200
and have BFA-insensitive GEP activity. The report (25) that ARNO is a
GEP for ARF6 is consistent with earlier observations that BFA had no
effect on the subcellular localization of ARF6 (26). In
vitro, ARNO enhanced guanine nucleotide exchange on ARF1, as
previously reported (12), as well as on ARF6 (25). After density
gradient fractionation of BHK cell membranes, endogenous ARNO was
recovered with the plasma membrane marker enzyme
Na+,K+-ATPase (and in adjacent fractions); it
was not, however, detected in fractions containing Golgi membranes
(25), as might be expected for an ARF1 GEP. Several earlier studies had
implicated ARF6 in plasma membrane dynamics related to function of the
actin cytoskeleton (27, 28). Transient expression of Myc-tagged ARNO in
BHK or HeLa cells revealed sites of localization at the plasma membrane along with co-expressed ARF6, whereas transiently expressed ARF1 exhibited a Golgi-like distribution (25), as expected. The authors suggested that cytohesin-1, GRP-1, and ARNO might all be involved in
the regulation of ARF6 activity, in response to extracellular stimuli
(25). It will be important to learn whether by modifying conditions for
the assay of GEP activity, perhaps by using 3-phosphoinositides to
replace other lipids, a preference of ARNO, cytohesin-1, and GRP-1 for
ARF6 over ARF1 and ARF3 might be shown. In assays containing phosphatidylserine, His-tagged cytohesin-1 had been found to accelerate GTP
S binding to native ARF1 and ARF3, but not to recombinant myristoylated ARF5 (13). Subsequently, activity toward ARF5, but not
ARF6, was shown (29).
Formation of a stable complex between
13ARF1 and the Sec7 domain of
cytohesin-1 was readily demonstrated in buffer with a low
Mg2+ concentration, consonant with the earlier report of
stable interaction of the ARNO Sec7 domain with
17ARF1 (22). It was
more difficult, however, to document complex formation with
13ARF1
and
C
570 or
C
697. By comparing their behaviors on gel
filtration at different concentrations of Mg2+, we obtained
unequivocal evidence of a relatively weak interaction of
C
697
with
13ARF1 at a low Mg2+ concentration. The data with
C
570 remain ambiguous, despite experiments with six different
preparations of the recombinant protein, since each was eluted in the
exclusion volume as an aggregate or multimer, independent of
Mg2+ concentration.
Deletion of the PH domain had essentially no effect on the GEP activity
of ARNO, but abolished the >10-fold stimulation of wild type activity
induced by the addition of phosphatidylinositol 4,5-bisphosphate
to phospholipid vesicles used in the standard GEP assays (12). The
additional deletion of 67 amino acids from the N terminus, leaving only
the Sec7 domain, had little further effect on activity or
phosphatidylinositol 4,5-bisphosphate binding. It did, however,
apparently alter self-association, since both the intact ARNO and that
lacking the PH domain behaved on gel filtration as dimers, whereas the
Sec7 domain was monomeric (12). The GEP activity of the Sec7 domain of
p200 was also clearly influenced by other parts of the molecule. The
decrease in activity associated with deletion of 520 amino acids from
the N terminus of a p200 molecule that lacks sequence following the
Sec7 domain was not very large but was seemingly consistent and
reversed with the removal of 50 more amino acids to produce
C
570.
Further analysis of structure in this region and characterization of
the activity of other mutants may provide clues to the mechanism of its
influence on GEP activity. The drastic loss of activity associated with removal of 36 amino acids between positions 595 and 630 would seem to
dictate intensive investigation of this region also.
Although the determinants of GEP activity and its susceptibility to BFA
inhibition lie within the Sec7 domains of p200, as well as Sec7 (13),
other regions of p200 clearly influence those properties, as shown
here. It is notable that removal of p200 sequence near the N terminus
of the Sec7 domain caused a dramatic decrease in specific activity,
whereas the specific activity of C-1Sec7 under the same conditions was
clearly greater than that of the intact cytohesin-1. Identification of
the structural elements responsible for these effects on activity of
the Sec7 domain is, of course, of considerable interest and importance.
To ascertain whether substrate specificity is also modified by
structure outside of the Sec7 domain, the activities of cytohesin-1 and
C-1Sec7 toward several different ARFs and related molecules were
compared. C-1Sec7 was active with a much broader range of substrates
than was the intact molecule (29), indicating that some determinants of
specificity do lie outside the Sec7 domain. Two amino acids in the Sec7
domain of yeast Sec7 that are responsible for BFA inhibition have
recently been identified (30).
 |
ACKNOWLEDGEMENTS |
We thank Dr. Su-Chen Tsai for providing
native ARF1, ARF3, and mixed ARF1/ARF3, recombinant myristoylated and
unmyristoylated ARF3 and ARF5, and unmyristoylated ARF6, Dr. Gustavo
Pacheco-Rodriguez for additional preparations of recombinant ARF1, -5, and -6, and Drs. Nicolas Vitale, Walter Patton, and Elisabetta Meacci
for preparations of recombinant myristoylated ARF6. We thank Dr.
Gustavo Pacheco-Rodriguez also for recombinant His6-tagged
C-1Sec7, Dr. Julie G. Donaldson for the BFA analogue B36, and Carol
Kosh for expert secretarial assistance.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Rm. 5N-307, Bldg. 10, 10 Center Dr. MSC 1434, National Institutes of Health, Bethesda, MD
20892-1434. Tel.: 301-496-4554; Fax: 301-402-1610.
 |
ABBREVIATIONS |
The abbreviations used are:
ER, endoplasmic
reticulum;
ARF, ADP-ribosylation factor;
GEP, guanine
nucleotide-exchange proteins;
BFA, brefeldin A;
GTP
S, guanosine
5'-(
-thio)triphosphate;
C-1Sec7, His6-tagged Sec7 domain
of cytohesin-1;
13ARF1, mutant ARF1 that lacks the first 13 amino
acids;
PS, L-
-phosphatidylserine;
PAGE, polyacrylamide
gel electrophoresis;
DTT, dithiothreitol;
PH, pleckstrin
homology.
 |
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