Brefeldin A Inhibited Activity of the Sec7 Domain of p200, a Mammalian Guanine Nucleotide-exchange Protein for ADP-ribosylation Factors*

Naoko MorinagaDagger , Ronald Adamik, Joel Moss, and Martha Vaughan

From the Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ARFs are ~20-kDa GTPases originally isolated as activators of cholera toxin-catalyzed ADP-ribosylation of purified Galpha 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 Delta 13ARF1.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Materials-- [35S]GTPgamma S (1,250 Ci/mmol) was purchased from NEN Life Science Products Inc., BFA from Epicenter, phosphatidylserine and GTPgamma 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 Delta 13ARF1 (20) and cytohesin-1 (13) are published.

Preparation of p200 Mutants-- Structures of p200 mutants are shown schematically in Fig. 1. The Delta 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). Delta CDelta 520 was made similarly by ligation of two DNA fragments using the SpeI site. One fragment was amplified from plasmid Delta 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 Delta C plasmid with SpeI and NotI. The two fragments were ligated into pAcHLT-C, which had been digested with NdeI and NotI. Delta CDelta 570 was amplified from Delta 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.

Delta CDelta 594, Delta CDelta 630, Delta CDelta 660, and Delta CDelta 697 were amplified by the same procedure as Delta CDelta 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 Delta CDelta 570 migrated a ~39-41-kDa doublet on SDS-PAGE.

Gel Filtration Analysis of Delta 13ARF1 and GEP Interaction-- To determine whether stable complexes were formed, Delta 13ARF1 and Delta CDelta 697, Delta CDelta 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 GTPgamma 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 GTPgamma 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]GTPgamma 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 GTPgamma 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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

GEP Activity of p200 and Mutants-- Recombinant His6-tagged p200 and mutant proteins (Fig. 1) were initially evaluated by their effects on GTPgamma 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 Delta 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 Delta C of the indicated numbers of amino acids at the N terminus yielded proteins Delta CDelta 520, Delta CDelta 570, Delta CDelta 594, Delta CDelta 630, Delta CDelta 660, and Delta CDelta 697 (the Sec7 domain). The activity of Delta CDelta 570 was quite similar to those of p200 and Delta C, reaching a maximum with ~1 pmol (Fig. 2A). Although Delta CDelta 520 was not dramatically less active, the activity of two different preparations was consistently less than that of Delta CDelta 570. The activity of Delta CDelta 594 was also distinctly less than that of Delta CDelta 570 and similar to that of Delta CDelta 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, Delta C, Delta CDelta 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 GTPgamma S was 2.0 ± 0.28 pmol with 400 pmol of Delta CDelta 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. Delta C indicates deletion of 964 amino acids C-terminal to the Sec7 domain. The number following Delta  indicates the number of amino acids deleted from N terminus of Delta 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 GTPgamma S binding to native ARF1/ARF3. A, the indicated amounts of p200 (black-triangle), Delta C (), Delta CDelta 520 (down-triangle), Delta CDelta 570 (open circle ), or Delta CDelta 594 (diamond ) proteins were incubated at 30 °C for 20 min with 10 pmol of native ARF1/ARF3 in the presence of 4 µM [35S]GTPgamma S, 20 µg of phosphatidylserine, and 5 mM MgCl2 before collection of proteins on nitrocellulose filters for radioassay. GTPgamma S bound to ARF alone, in the absence of p200 or mutant (0.21 pmol), was subtracted to yield values that represent picomoles of GTPgamma 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 Delta CDelta 630 (), Delta CDelta 660 (Delta ), and Delta CDelta 697 (black-square). Binding to ARF alone was 0.10 pmol.

The low rate of binding of GTPgamma S to ARF alone in the absence of GEP was essentially constant for 60 min at 30 °C (Fig. 3). Addition of p200 or Delta C (0.4 pmol) increased the initial rate similarly, but the magnitude of the effect began to diminish before 20 min. Both Delta CDelta 630 and Delta CDelta 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 GTPgamma S binding by ARF with p200 and mutants. Binding of [35S]GTPgamma S to 10 pmol of native ARF1/ARF3 with 0.4 pmol of p200 (black-triangle) or Delta C (), 40 pmol of Delta CDelta 630 () or Delta CDelta 697 (black-square), or no GEP (black-diamond ) 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.

Relative GEP activities of p200 and several mutants are summarized in Table I. GEP activity is expressed as GTPgamma 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 Delta CDelta 594 to yield Delta CDelta 630. Further truncation, producing Delta CDelta 630, Delta CDelta 660, and the Sec7 domain itself (Delta CDelta 697), had no further effect on activity (Table I).

                              
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Table I
Effect of p200 and mutants on GTPgamma S binding to ARF1 and ARF3
Stimulation of binding of [35S]GTPgamma S to 10 pmol of ARF by each GEP protein was determined as described under "Experimental Procedures." GEP activity is the increment in GTPgamma S binding to ARF induced by the GEP protein expressed as picomoles of GTPgamma 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.

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 Delta CDelta 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 Delta CDelta 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 Delta CDelta 570 on GTPgamma S binding to ARF proteins
Stimulation of [35S]GTPgamma S binding to the indicated amount of ARF protein by recombinant GEP was assessed in the presence of 4 µM GTPgamma 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 Delta CDelta 570 in experiment 3, and 0.5 pmol of Delta CDelta 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 GTPgamma 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.

Effect of BFA on GEP Activity of p200 and Mutants-- Decreasing the incubation temperature to 22 °C increased the stability of both Delta CDelta 697 (Fig. 4A) and Delta CDelta 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 (, black-square) 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, Delta CDelta 697 (diamond , black-diamond ); B, Delta CDelta 570 (open circle , ); C, cytohesin-1 (down-triangle, black-down-triangle ); C-1 Sec7 (Delta , black-triangle).

As shown in Fig. 5, the activities of p200, Delta C, Delta CDelta 570, and Delta CDelta 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 Delta CDelta 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]GTPgamma 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 (black-triangle), 0.5 pmol of Delta C (), Delta CDelta 520 (down-triangle), Delta CDelta 570 (open circle ), or Delta CDelta 594 (diamond ), 50 pmol of Delta CDelta 630 (), 40 pmol of Delta CDelta 697 (black-square), or 20 pmol of cytohesin-1 (black-down-triangle ). 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 Delta CDelta 697 (triangle ). Data are mean ± S.E. of values from triplicate assays. GEP activities in the absence of BFA were 0.96 (p200), 0.62 (Delta C), 0.48 (Delta CDelta 520), 1.2 (Delta CDelta 570), 0.55 (Delta CDelta 594), 0.54 (Delta CDelta 630), and 1.3 (Delta CDelta 697) pmol of GTPgamma S bound.

Physical Interaction of Delta 13ARF1 and p200 Mutants-- Because the ARNO Sec7 domain had been reported to form a stable complex with Delta 17ARF1 (22), we investigated the association of Delta 13ARF1 with the p200 deletion mutants Delta CDelta 570 and Delta CDelta 697 (the putative p200 s7 domain). GTPgamma S binding to 13Delta ARF1 was stimulated ~8-fold by C-1Sec7 independent of PS, but not by Delta CDelta 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 Delta CDelta 697 increased GTPgamma S binding to 62 pmol of Delta 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 Delta CDelta 697, consistent with the absence of phospholipid effects on the function of ARF mutants lacking the N-terminal alpha -helix (22). In the same experiment, with a relatively high Mg2+ concentration that was reported to be unfavorable for ARF-GEP complex formation (22), Delta 13ARF1 with [35S]GTPgamma S bound eluted in a single symmetrical peak from Ultrogel AcA54 at the position expected for monomeric ARF and the amount of bound [35S]GTPgamma S was increased after incubation with Delta CDelta 697 (Fig. 6).

                              
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Table III
Effect of p200 mutant, C-1Sec7, and phosphatidylserine on GTPgamma S binding to Delta 13ARF1 and native ARF1/ARF3
In experiment 1, GEP and 50 pmol of Delta 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 Delta CDelta 697 on [35S]GTPgamma S binding to Delta 13ARF1. Delta 13ARF1 (320 pmol) was incubated for 1 h at 37 °C with 5 mM MgCl2, 1 mM EDTA, and 4 µM [35S]GTPgamma S (3.5 × 106 cpm) without (open circle ) or with (black-square) 50 pmol of Delta CDelta 697 (total volume 250 µl). After transfer of a sample (50 µl) to a nitrocellulose filter for determination of protein-bound [35S]GTPgamma 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.

When Delta CDelta 697 and Delta 13ARF1 were incubated at the lower Mg2+ concentration (Fig. 7A), Delta 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 GTPgamma S (Fig. 7B), and Delta 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, Delta CDelta 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 Delta 13ARF1 and Delta CDelta 697. Delta 13ARF1 ((40 µg, 2.1 nmol) was incubated with Delta CDelta 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 GTPgamma 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 Delta CDelta 697 (black-square) or Delta 13ARF1 (open circle ) is reported as percentage of the total recovered in all fractions = 100%.

For comparison, the same kind of experiment was performed with C-1Sec7 replacing the p200 mutants. After incubation of Delta 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 GTPgamma S, the amount of apparent complex was much less, appearing as a shoulder on the Delta 13ARF1 peak (Fig. 8B). The stability of the association of C-1Sec7 with Delta 13ARF1 was clearly much greater than that of the p200 mutants with Delta 13ARF1, although some weak association of Delta 13ARF1 with Delta CDelta 697 was detected at the lower Mg2+ concentration.


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Fig. 8.   Effect of Mg2+ concentration on association of Delta 13ARF1 and C-1Sec7. Delta 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 GTPgamma S (B) for 1 h at 37 °C before gel filtration and analysis of column fractions. Optical density of C-1Sec7 () and Delta 13ARF1 (open circle ), is reported as described in the legend to Fig. 7.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 GTPgamma 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 Delta 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 Delta 17ARF1 (22). It was more difficult, however, to document complex formation with Delta 13ARF1 and Delta CDelta 570 or Delta CDelta 697. By comparing their behaviors on gel filtration at different concentrations of Mg2+, we obtained unequivocal evidence of a relatively weak interaction of Delta CDelta 697 with Delta 13ARF1 at a low Mg2+ concentration. The data with Delta CDelta 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 Delta CDelta 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.

Dagger 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; GTPgamma S, guanosine 5'-(gamma -thio)triphosphate; C-1Sec7, His6-tagged Sec7 domain of cytohesin-1; Delta 13ARF1, mutant ARF1 that lacks the first 13 amino acids; PS, L-alpha -phosphatidylserine; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; PH, pleckstrin homology.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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