Functional Interaction of ADP-ribosylation Factor 1 with Phosphatidylinositol 4,5-Bisphosphate*

(Received for publication, September 9, 1996, and in revised form, December 10, 1996)

Paul A. Randazzo

From the Laboratory of Biological Chemistry, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The relationship between ADP-ribosylation factor (Arf) 1 and phosphoinositides, which have been independently implicated as regulators of membrane traffic, was examined. Because both Arf-dependent phospholipase D and Arf1 GTPase-activating protein (GAP) require phosphatidylinositol 4,5-bisphosphate (PIP2), Arf1 complexed with PIP2 has been proposed to interact with target proteins. This hypothesis was tested using Arf1 GAP as a model system. Arf1 was shown to bind to PIP2 in Triton X-100 micelles with a Kd of 45 ± 13 µM. Arf1 also bound phosphatidic acid but with 10-fold lower affinity. PIP2 binding was specifically disrupted by mutating lysines 15, 16, and 181 and arginine 178 to leucines. Decreased PIP2 binding resulted in an increased EC50 of PIP2 for activation of Arf GAP. None of the mutations that decreased PIP2 binding affected binding to or activation of GAP by phosphatidic acid, which acts at a functionally distinct site. These data support the hypothesis that PIP2 binding to Arf1 promotes interaction with Arf GAP. The implications of lipid-directed protein-protein interactions for membrane traffic are discussed.


INTRODUCTION

ADP-ribosylation factors (Arf)1 are a family of GTP-binding proteins first identified and purified as a cofactor for cholera toxin-catalyzed ADP-ribosylation of the heterotrimeric GTP-binding protein Gs (1). This pathophysiologic activity has been useful for functionally defining members of the Arf family (2); however, the physiologic role of Arf is thought to be as a regulator of membrane traffic (for reviews, see Refs. 3-6). In eukaryotes, membrane traffic is mediated by protein-coated vesicles (3). With GTP bound, Arf1 recruits proteins of two classes of coats, coatomer and clathrin assembly protein-1, to Golgi-enriched membranes (7-10). This event facilitates the assembly of the coat and drives the budding of the vesicles (3). The hydrolysis of GTP by Arf1 is necessary for the uncoating and fusion of the vesicles (3-6). Arf1 has also been purified as an activator of phospholipase D (PLD) (11, 12) which has been proposed to mediate Arf-stimulated coated vesicle assembly (13).

Phosphoinositides have been independently implicated as regulators of membrane traffic (14). In yeast, mutants of phosphatidylinositol transfer protein and phosphatidylinositol 3-kinase are deficient in protein secretion and vacuolar sorting (15, 16). Phosphoinositides play a role in membrane traffic in mammalian cells as well (14). For example, exocytosis in PC12 cells requires a number of soluble proteins including phosphatidylinositol transfer protein and phosphatidylinositol 4-phosphate 5-kinase (PIP kinase) (17, 18). Similarly, phosphatidylinositol transfer protein has been purified as a required soluble factor for the formation of secretory vesicles in adrenal medulla (19).

Arf1 is a possible site at which phosphoinositides may play a role in membrane traffic. The interaction of Arf1 with two different GTPase-activating proteins (GAP)2 (20, 21) and PLD (12, 22) has been shown to be PIP2-dependent. On further characterization, the Arf1 GAP activity from bovine brain was found to be coordinately regulated by PA and PIP2 (21). Although PA did not activate GAP activity by itself, it lowered the EC50 of PIP2 required for activation by 90%. These data functionally define two lipid binding sites (21, 23). GAP activity depends on occupancy of both sites. Site 1 is specific for PIP2. Site 2 may be occupied by either PIP2 or PA.

Although two lipid binding sites have been functionally defined for the Arf1-GAP interaction, the proteins on which the binding sites reside have not been identified. Arf1 has been speculated to contain an acid lipid binding site. The effect of PIP2 on nucleotide dissociation from Arf1 has been taken as evidence for PIP2 binding to Arf1 (23). Furthermore, the crystal structure of Arf1 has revealed that basic residues from positions 10, 15, 16, 59, 178, and 181 cluster to form a solvent-exposed patch of positive potential (24, 25). This polarized surface is reminiscent of pleckstrin homology domains (26, 27) and could be a site of binding to PIP2. These data, with the functional data, have led to the suggestion that Arf1, in addition to GTP, must bind PIP2 to interact with target proteins such as GAP or PLD (21, 23).

The hypothesis that PIP2, by binding to Arf1, directs Arf1-protein interactions was tested using Arf1 GAP as a model system. PIP2 binding to Arf1 was demonstrated using a direct assay. To specifically disrupt PIP2 binding to Arf1, basic residues that comprise the positively charged surface-exposed patch on Arf1 were mutated to neutral residues. The mutant Arf1 proteins had reduced affinity for PIP2 and required greater concentrations of PIP2 for productive interaction with Arf GAP than did the wild type protein. The mutations had no effect on either PA binding or PA-dependent GAP activation. These data support the hypothesis that Arf1 with PIP2 bound is the substrate for GAP. The ability of specific lipids to direct Arf1-protein interactions could play a role in spatially and temporally separating events in membrane traffic.


EXPERIMENTAL PROCEDURES

Materials

Triton X-100, phosphatidylcholine (PC), 1-stearoyl-2-arachidonoyl, phosphatidic acid (PA), phosphatidylserine (PS), and PIP2 were from Sigma. [alpha -32P]GTP, [gamma -32P]GTP, and [35S]GTPgamma S were from DuPont NEN. AcA44 was from Biosepra Inc. (Marlborough, MA). Bovine brains were obtained from a local slaughter house. Arf1 GTPase-activating protein (Arf1 GAP) was purified as described (21). Recombinant Gsalpha was expressed in and purified from bacteria as described (28). Gbeta gamma was purified from bovine brain as described (29). Cholera toxin was purchased from Calbiochem and was preactivated by incubation in 25 mM potassium Pi, pH 7.4, 20 mM dithiothreitol for 15 min at 37 °C.

Mutagenesis

Site-directed mutagenesis was performed by polymerase chain reaction amplification of the open reading frame of human Arf1 using synthetic oligonucleotides incorporating the desired mutations. NdeI and BamHI restriction sites were added at the 5' and 3' ends to allow subsequent subcloning into pET3C bacterial expression vector (30, 31). The sequence of the entire open reading frame was determined by dye-terminator sequencing using an ABI instrument (model 373A) and reagents from Perkin-Elmer. Wild type and mutant proteins were expressed in BL21 (DE3) (30, 31) and purified (32) as previously described.

Nucleotide Binding

Binding reactions contained 1 µM Arf1 and 10 µM [35S]GTPgamma S (specific activity, 1000-5000 cpm/pmol) in exchange buffer of 25 mM HEPES, pH 7.4, 100 mM NaCl, 0.5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 3 mM L-alpha -dimyristoylphosphatidylcholine, and 2.5 mM sodium cholate. Samples (10 µl) were taken 12 times ranging from 2 to 120 min, and bound nucleotide was determined by rapid filtration on BA85 nitrocellulose (Schleicher and Schuell) (33). Data were fit to a first order rate equation using the Marquardt algorithm.

ADP-ribosylation Assay

Arf-dependent cholera toxin-catalyzed ADP-ribosylation of Gs was determined as described previously (34). Incubations containing 25 mM HEPES, pH 7.4, 100 mM NaCl, 0.5 mM MgCl2, 1 mM EDTA, 0.25 µM concentration of the indicated Arf protein, 2.2 µM recombinant Gsalpha , 20 µM Gbeta gamma , 40 µg/ml cholera toxin, 5 µM NAD (approximately 20,000 cpm/pmol), 3 mM L-alpha -dimyristoylphosphatidylcholine, and 2.5 mM cholate were for 20 min at 30 °C.

GTPase-activating Protein Assay

GAP activity was determined as previously described (21). Lipids were added in mixed micelles with Triton X-100. The incubations contained 8 µg/ml GAP, 0.1% Triton X-100, and the indicated concentrations of PIP2 and PA. As discussed in Ref. 21, the concentration of Arf1·GTP was much less than the Km. Under this condition, substrate is consumed with a first order rate equal to Vmax/Km. This rate is expressed here as the fraction of Arf1·GTP hydrolyzed/min.

Arf1 Binding to Triton X-100 Micelles and Phospholipid Liposomes

Phospholipids in chloroform/methanol, 95:5, were taken to dryness under a stream of nitrogen. To prepare liposomes, the lipids were then resuspended in 25 mM Tris, pH 8.0, to a final lipid concentration of 13 mM. The suspension was sonicated for 5 min in a G112SP1 ultrasonic cleaner (Laboratory Supplies Co., Hicksville, NY). To prepare micelles, the lipids were solubilized in 1% Triton X-100. Arf1 was loaded with [gamma -32P]GTP by incubation with 10 µM [gamma -32P]GTP (25,000-50,000 cpm/pmol) in 20 mM potassium Pi, pH 7.4, 100 mM NaCl, 0.5 mM MgCl2, 1 mM EDTA, and 0.1% Triton X-100 for 20 min at 30 °C, and the MgCl2 concentration was adjusted to 2 mM. Arf1·GTP was diluted 1:19 into either mixed micelles of phospholipids and 0.1% Triton X-100 or 650 µM phospholipid in 25 mM HEPES, pH 7.4, 100 mM NaCl, 3.5 mM MgCl2, 1 mM ATP, 1 mM GTP, and 1 mM dithiothreitol, incubated at 30 °C for 2 min, and then chilled. Samples (100 µl) were then applied to 1 ml of AcA44 (Biosepra) in a Bio-Rad polyprep disposable column. The column was centrifuged at 200 × g for 2 min, and the amount of radiolabeled nucleotide in the eluate was determined. 67 ± 5% of the phospholipids but less than 10% of the Arf1 (in the absence of acid phospholipids) applied to the column were recovered in the eluate. Phospholipid composition did not affect lipid recovery. To determine the fraction of Arf1 recovered, total protein-bound radiolabeled nucleotide was determined in samples from the same incubations (33).

Miscellaneous

Protein concentrations were estimated by the Amido Black assay using bovine serum albumin as a standard (35). Total lipid phosphorus was determined as described (36).


RESULTS

Arf1 Binds to Acid Phospholipids

The effects of PIP2 on nucleotide dissociation from Arf1 has been taken as evidence for PIP2 binding to Arf1 (23). To directly demonstrate this interaction, spin columns constructed of AcA44 (an acrylamide-agarose gel filtration matrix with a Mr cut off of 130,000) were used to rapidly separate Arf1·GTP bound to micelles from free protein (Fig. 1). Less than 10% of Arf1·GTP bound to micelles of Triton X-100. The binding of Arf1 was increased by including PIP2 in the micelles with a half-maximal effect occurring at 45 ± 13 µM. Maximal binding was 60 ± 8% of the total Arf1. Arf1·GTP also bound to PA- and PS-containing micelles but with lower affinity than to PIP2 (Fig. 1). Arf1 binding to PIP2 was independent of lipid presentation. Binding to mixed liposomes of PC and PIP2 was dependent on PIP2 concentration with half-maximal binding occurring at 30 ± 11 µM PIP2.


Fig. 1. Arf1 binds to acid phospholipids. Arf1 was loaded with [gamma -32P]GTP and incubated with Triton X-100 micelles containing the indicated concentrations of either PIP2, PA, or PS. Arf1 bound to the vesicles was rapidly separated from free Arf1 using spin columns as described under "Experimental Procedures." The data are presented as Arf1·GTP bound to the micelles/total Arf1·GTP. The average ± the range for duplicate determinations is shown.
[View Larger Version of this Image (17K GIF file)]


Arf1 Binding to Acid Phospholipids Was Decreased by Neutralizing Positively Charged Solvent-exposed Amino Acids

Using the crystal structure of Arf1·GDP as a guide, 5-point mutants of Arf1 were generated by polymerase chain reaction mutagenesis. Each mutation neutralized either one or two of the basic residues that contribute to the polarized patch on the surface of Arf1 (Table I). All mutants were expressed in Escherichia coli and purified by the same methods used to purify recombinant Arf1. Two quantitative functional assays were used to determine if the mutations had global conformational effects. First, GTPgamma S binding was determined (Table I). Of the mutants, only [K59L]Arf1 did not bind nucleotide. The reason for this has not yet been determined, but the lack of binding precluded the use of the mutant for this study. The other four mutants, [K16L]Arf1, [K15L,K16L]Arf1, [R178L]Arf1, and [R178L,K181L]Arf1, were similar to wild type protein in both rate and extent of binding. Thus, the mutations had not disrupted the nucleotide binding pocket. Second, activity as a cofactor for cholera toxin-catalyzed ADP-ribosylation of Gs was determined. This activity requires that Arf1 bind GTP (1), cholera toxin (38, 39), and Gs (1, 39) and, therefore, was felt to be a test of preservation of global conformation. All mutants that could bind nucleotide were indistinguishable from the wild type protein (Table I).

Table I.

Properties of Arf1 mutants

GTPgamma S binding to Arf1 with the indicated mutations was performed as described under "Experimental Procedures." The amount of protein-bound nucleotide was determined at 12 time points for 10-µl samples from incubations containing 1 µM protein and 10 µM GTPgamma S. The data were fit to a first order rate equation using the Marquardt algorithm, and the estimated parameters ± S.E. are presented. Cholera toxin-catalyzed ADP-ribosylation of Gs in the presence of a 0.25 µM concentration of the indicated Arf1 protein as a cofactor was determined for a 20-min incubation. Triplicate samples were taken. The data are expressed ± S.E. for the number of independent experiments indicated in parentheses. The mutations are indicated as follows: K16L, lysine 16 to leucine ([K16L]Arf1); K15L,K16L, lysine 15 and lysine 16 to leucines ([K15L,K16L]Arf1); K59L, lysine 59 to leucine ([K59L]Arf1); R178L, arginine 178 to leucine ([R178L]Arf1); R178L, K181L, arginine 178 and lysine 181 to leucines ([R178L,K181L]Arf1).
Mutant GTPgamma S binding
Arf activity
k Maximum

min-1 pmol pmol ADP-ribose/pmol Arf
Wild type 0.060  ± 0.011 0.89  ± 0.04 2.8  ± 0.4 (11)
K16L 0.051  ± 0.007 0.80  ± 0.04 3.1  ± 0.9 (3)
K15L,K16L 0.050  ± 0.005 1.33  ± 0.04 3.5  ± 0.5 (6)
K59L No binding detected Not done
R178L 0.077  ± 0.010 0.88  ± 0.03 3.5  ± 0.5 (4)
R178L,K181L 0.064  ± 0.011 0.49  ± 0.03 2.9  ± 0.8 (2)

Neutralizing the residues from the positively charged cluster on Arf1 specifically affected PIP2 binding. The PIP2 dependence for binding to Triton micelles was determined for wild type Arf1 and the four mutants that bound nucleotide. The concentration of PIP2 for half-maximal binding was 2-fold greater for [K16L]Arf1 and [R178L]Arf1 and more than 4-fold greater for [K15L,K16L]Arf1 and [R178L,K181L]Arf1 than for wild type Arf1 (Fig. 2 and Table II). The relative affinities were independent of phospholipid presentation. Similar results were obtained using liposomes of PC and PIP2 (not shown). In contrast, the mutations did not detectably affect binding to PA (Fig. 3 and Table II).


Fig. 2. Neutralizing basic residues 15, 16, 178, and 181 of Arf1 reduces binding to PIP2. The binding of wild type Arf1 (bullet ), [K16L]Arf1 (black-square), [K15L,K16L]Arf1 (black-triangle), [R178L]Arf1 (black-down-triangle ), and [R178L,K181L]Arf1 (black-diamond ) to Triton X-100 micelles containing the indicated concentrations of PIP2 was determined. The data are presented as the Arf1·GTP bound to the vesicles/total Arf1·GTP. The average ± the range for duplicate determinations is shown.
[View Larger Version of this Image (22K GIF file)]


Table II.

Kinetic constants for lipid binding to Arf and activation of Arf1 GAP

Kinetic constants were obtained by fitting the data from Figs. 3, 4, 5, and 6 to a hyperbolic single ligand binding site equation. The mutants are indicated as in Table I.
Binding, Kd
GAP activation, EC50
PIP2a PAb PIP2b PAc

µM µM
Wild type 45  ± 13 508  ± 84 18  ± 4 407  ± 7
K16L 102  ± 27 448  ± 172 38  ± 8 335  ± 40
K15L,K16L >200d 434  ± 65 >100e 332  ± 12
R178L 78  ± 8 728  ± 173 42  ± 8 372  ± 40
R178L,K181L 216  ± 38 533  ± 21 >100e 288  ± 93

a Average value ± S.D. for 3 experiments.
b Value ± S.E. from experiment shown (Figs. 3 and 4).
c Average ± range for two experiments.
d Binding was linear with lipid concentration to 180 µM; therefore, binding constants could not be estimated (see Fig. 2).
e Activity was linear with phospholipid concentration to 90 µM (see Fig. 4).


Fig. 3. Binding of mutant proteins to phosphatidic acid. The binding of wild type Arf1 (bullet ), [K16L]Arf1 (black-square), [K15L,K16L]Arf1 (black-triangle), [R178L]Arf1 (black-down-triangle ), and [R178L,K181L]Arf1 (black-diamond ) to Triton X-100 micelles containing the indicated concentrations of PA was determined. The average ± the range for duplicate determinations is shown.
[View Larger Version of this Image (21K GIF file)]


Mutations That Decreased PIP2 Binding to Arf1 Also Affected the PIP2 Dependence of Arf1 GAP Activity

Two phospholipid binding sites have been functionally defined for Arf1-Arf1 GAP interactions. One site binds PIP2 specifically. The second site binds both PIP2 and PA. To examine site 1, PIP2 dependence in the presence of a fixed concentration of PA (700 µM was chosen because it is saturating for functionally defined site 2, see Fig. 5) was determined for the wild type protein and the mutants (Fig. 4). The EC50 of PIP2 in the GAP-catalyzed reaction correlated with the relative affinities of the mutants for PIP2. The EC50 of PIP2 was approximately 2-fold greater for [K16L]Arf1 and [R178L]Arf1 and more than 5-fold greater for the mutants with two residues neutralized, [K15L,K16L]Arf1 and [R178L, K181L]Arf1, than for wild type Arf1 (Fig. 4 and Table II).


Fig. 5. PA dependence of GAP activity using wild type Arf1 and four mutant Arf1s as substrates. The PA dependence of GAP activity in the presence of 50 µM PIP2 using Arf1 (bullet ), [K16L]Arf1 (black-square), [K15L,K16L]Arf1 (black-triangle), [R178L]Arf1 (black-down-triangle ), and [R178L,K181L]Arf1 (black-diamond ) as substrates was determined as described. The data are expressed as the fraction of GTP on Arf1 hydrolyzed/min.
[View Larger Version of this Image (20K GIF file)]



Fig. 4. PIP2 dependence of GAP activity using wild type Arf1 and four mutant Arf1s as substrates. The PIP2 dependence of GAP activity in the presence of 700 µM PA using wild type Arf1 (bullet ), [K16L]Arf1 (black-square), [K15L,K16L]Arf1 (black-triangle), [R178L]Arf1 (black-down-triangle ), and [R178L,K181L]Arf1 (black-diamond ) was determined as described under "Experimental Procedures." The data are expressed as the fraction of GTP on Arf1 hydrolyzed/min.
[View Larger Version of this Image (22K GIF file)]


To examine the effect of the mutations on the functionally defined site 2, the PA concentration dependence, at a fixed concentration of PIP2 (50 µM, which is greater than the EC50 for site 1 but less than the EC50 for functionally defined site 2 using wild type Arf1), was determined for the wild type protein and each mutant. If the mutations had affected site 2, then the EC50 of PA should be affected. If the mutations had an isolated effect on functionally defined site 1, then the EC50 of PA would be unaffected but the maximal velocity would be decreased (because site 1 would not be completely occupied at the fixed concentration of PIP2 used). The latter was the case (Fig. 5 and Table II). The maximum velocity observed with both [K16L]Arf1 and [R178L]Arf1 was approximately 70% that of the wild type protein. The maximum velocity observed with the mutants with two residues neutralized, [K15L,K16L]Arf1 and [R178L,K181L]Arf1, was 15-30% that of wild type. However, the EC50 for PA was the same for the mutants as for the wild type protein. Thus, the mutations had an isolated effect on the functionally defined PIP2 binding site 1. Taken together, these data support the hypothesis that Arf1 with PIP2 bound is the substrate for GAP.


DISCUSSION

Both Arf1 and phosphoinositides are required for efficient vesicular transport (14). Potential relationships between these required factors have been discussed previously (22, 23, 40). Here, we show that Arf1 binds PIP2. Furthermore, disrupting PIP2 binding to Arf1 is shown to increase the EC50 of PIP2 for Arf1 GAP activation. These data support the hypothesis that PIP2 binding to Arf1 directs the interaction with GAP and suggest a model in which Arf1 may "sense" lipids, interacting with specific proteins depending on the lipid environment. This mechanism could provide a means of ordering events in membrane traffic.

Arf1 binds to phospholipid vesicles containing acid phospholipids. Prior to this report, the effects of PIP2 on nucleotide exchange on Arf1 suggested that Arf1 could bind phosphoinositides (23). Here, a spin column-based assay was used to directly demonstrate Arf1 binding to PIP2-containing micelles and liposomes. Arf1 bound to other acid phospholipids but with lower affinity. The specificity of Arf binding to PIP2 was further tested by determining if any mutations in Arf1 had an isolated effect on PIP2 binding. Arf1 contains a surface-exposed cluster of basic amino acids that could play a role in binding acid phospholipids such as PIP2 (26, 27). Consistent with specificity for PIP2, neutralizing residues from this cluster affected binding of PIP2 but not PA.

Two lipid binding sites have been functionally defined for Arf1 GAP activation. One site was proposed to be on Arf1. Thus, Arf1·PIP2 would be acting as the substrate for Arf1 GAP. The identification of Arf1 mutants with reduced affinities for PIP2 allowed this to be tested. As predicted by the model, the concentration of PIP2 required for activation of GAP was found to depend on the affinity of the particular Arf1 mutant for PIP2. These data demonstrate that Arf1 is the previously functionally defined PIP2 binding site. The role of PIP2 was more than simply targeting Arf1 to the membranes. Both PA and PS promote Arf1 binding to vesicles, but neither activated GAP in the absence of PIP2. Thus, PIP2 must be inducing a conformational change necessary for the interaction with GAP.

The second functionally defined lipid binding site is structurally distinct from site 1. This site, which could be occupied by either PIP2 or PA, was unaffected by the mutations in Arf1 and, therefore, could be either a distinct site on Arf1 or on GAP. As was the case for PIP2, the role of PA is not simply recruiting Arf1 to the phospholipid surface. PA did not increase the fraction of Arf1 bound to vesicles to an extent greater than PIP2 alone did. This phenomenon is similar to that seen with the kinetics of PIP2-phospholipase C-gamma (41). PA converts a sigmoid PIP2 dependence to a hyperbolic dependence and lowers the Km 10-fold. However, PA does not affect the association constant of the enzyme with the detergent micelles used in the study. Therefore, PA is acting as an allosteric modifier for both phospholipase C and GAP. The nature of this allosterism may be of general interest in understanding phosphoinositide metabolism since another enzyme of this pathway, PIP kinase, is also activated by PA (42, 43).

The ability of phospholipids to direct Arf1-protein interactions may order events in membrane traffic. Several activities have been ascribed to Arf1 including activity as a cofactor for cholera toxin, vesicle coat protein recruitment (5), PIP kinase activation (44), and PLD activation (11, 12, 40). The lipid requirements of two of these have been examined. PLD requires PIP2 but not PA, the product of the reaction catalyzed by PLD. Activity as a cofactor for cholera toxin is inhibited by PIP2.2 Thus, three different Arf1-protein interactions have different lipid requirements. Since Arf1 also affects lipid composition of membranes by activating both PIP kinase (44) and PLD (11, 12), these observations have led to suggestions that Arf1 may help order events in membrane traffic by responding to and affecting changes in phospholipid composition (21-23, 40). With low concentrations of PIP2 and PA present, Arf1 might initiate assembly of protein-coated vesicles. In the vesicle, Arf1 activates PIP kinase. This produces PIP2 which allows Arf1 to activate PLD. In turn, PLD generates PA which together with PIP2 activates GAP and, consequently, terminates the assembly signal. A combination of biochemistry and genetics using mutant proteins similar to those described in this paper should allow this and similar models to be tested.


FOOTNOTES

*   This work was supported by the Division of Basic Sciences, NCI, National Institutes of Health.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: Laboratory of Biological Chemistry, Division of Basic Sciences, Bldg. 37, Rm. 5D-02, NCI, NIH, Bethesda, MD 20892. Tel.: 301-496-3788; Fax: 301-496-5839; E-mail: randazzo{at}helix.nih.gov.
1   The abbreviations used are: Arf, ADP-ribosylation factor; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; GAP, GTPase-activating protein; PA, phosphatidic acid; PC, phosphatidylcholine; PI, phosphatidylinositol; PIP kinase, PI 4-phosphate 5-kinase; PIP2, PI 4,5-bisphosphate; PS, phosphatidylserine; PLD, phospholipase D.
2   P. A. Randazzo, unpublished data.

Acknowledgments

I thank John K. Northup, Ronald Felsted, and Julie Donaldson for helpful discussions, Jenny Clark, Stacey Sturch, and Sandor Berghoffer for technical assistance, and Albert Fornace and Richard Kahn, for support.


REFERENCES

  1. Kahn, R. A., and Gilman, A. G. (1986) J. Biol. Chem. 261, 7906-7911 [Abstract/Free Full Text]
  2. Kahn, R. A., Kern, F. G., Clark, J., Gelmann, E. P., and Rulka, C. (1991) J. Biol. Chem. 266, 2606-2614 [Abstract/Free Full Text]
  3. Rothman, J. E. (1994) Nature 372, 55-63 [CrossRef][Medline] [Order article via Infotrieve]
  4. Donaldson, J. G., and Klausner, R. D. (1994) Curr. Opin. Cell Biol. 6, 527-532 [Medline] [Order article via Infotrieve]
  5. Schekman, R., and Orci, L. (1996) Science 271, 1526-1533 [Abstract]
  6. Moss, J., and Vaughan, M. (1995) J. Biol. Chem. 270, 12327-12330 [Free Full Text]
  7. Donaldson, J. G., Cassel, D., Kahn, R. A., and Klausner, R. D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6408-6412 [Abstract]
  8. Stamnes, M. A., and Rothman, J. E. (1993) Cell 73, 999-1005 [Medline] [Order article via Infotrieve]
  9. Traub, I. M., Ostrom, J. A., and Kornfeld, S. (1993) J. Cell. Biol. 123, 561-573 [Abstract]
  10. Palmer, D. J., Helms, J. B., Beckers, C. J. M., Orci, L., and Rothman, J. E. (1993) J. Biol. Chem. 268, 12083-12089 [Abstract/Free Full Text]
  11. Cockcroft, S., Thomas, G. M. H., Fensome, A., Geny, B., Cunningham, E., Gout, I., Hiles, I., Totty, N. F., Truong, O., and Hsuan, J. J. (1994) Science 263, 523-526 [Medline] [Order article via Infotrieve]
  12. Brown, H. A., Gutowski, S., Moomaw, C. R., Slaughter, C., and Sternweis, P. C. (1993) Cell 75, 1137-1144 [Medline] [Order article via Infotrieve]
  13. Ktistakis, N. T., Brown, H. A., Waters, M. G., Sternweis, P. C., and Roth, M. G. (1996) J. Cell Biol. 134, 295-306 [Abstract]
  14. De Camilli, P., Emr, S. D., McPherson, P. S., and Novick, P. (1996) Science 271, 1533-1539 [Abstract]
  15. Schu, P. V., Takegawa, K., Fry, M. J., Stack, J. H., Waterfield, M. D., and Emr, S. D. (1993) Science 260, 88-91 [Medline] [Order article via Infotrieve]
  16. Bankaitis, V. A., Aitken, J. R., Cleves, A. E., and Dowhan, W. (1990) Nature 347, 561-562 [CrossRef][Medline] [Order article via Infotrieve]
  17. Hay, J. C., and Martin, T. F. J. (1993) Nature 366, 572-575 [CrossRef][Medline] [Order article via Infotrieve]
  18. Hay, J. C., Fisette, P. L., Fukami, K., Takenawa, T., Anderson, R. A., and Martin, T. F. J. (1995) Nature 374, 173-177 [CrossRef][Medline] [Order article via Infotrieve]
  19. Ohashi, M., de Vries, K. J., Frank, R., Snoek, F., Bankaitis, V., Wirtz, K., and Huttner, W. B. (1995) Nature 377, 544-547 [CrossRef][Medline] [Order article via Infotrieve]
  20. Makler, V., Cukierman, E., Rotman, M., Admon, A., and Cassel, D. (1995) J. Biol. Chem. 270, 5232-5237 [Abstract/Free Full Text]
  21. Randazzo, P. A., and Kahn, R. A. (1994) J. Biol. Chem. 269, 10758-10763 [Abstract/Free Full Text]
  22. Liscovitch, M., Chalifa, V., Pertile, P., Chen, C.-S., and Cantley, L. C. (1994) J. Biol. Chem. 269, 21403-21406 [Abstract/Free Full Text]
  23. Terui, T., Kahn, R. A., and Randazzo, P. A. (1994) J. Biol. Chem. 269, 28130-28135 [Abstract/Free Full Text]
  24. Amor, J. C., Harrison, D., Kahn, R. A., and Ringe, D. (1994) Nature 372, 704-708 [CrossRef][Medline] [Order article via Infotrieve]
  25. Greasely, S. E., Jhoti, H., Teahan, C., Solari, R., Fensome, A., Thomas, G. M. H., Cockcroft, S., and Bax, B. (1995) Nat. Struct. Biol. 2, 797-806 [Medline] [Order article via Infotrieve]
  26. Harlan, J. E., Yoon, H. S., Hajduk, P. J., and Fesik, S. W. (1995) Biochemistry 34, 9859-9864 [Medline] [Order article via Infotrieve]
  27. Lemmon, M. A., Ferguson, K. M., O'Brien, R., Sigler, P. B., and Schlessinger, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10472-10476 [Abstract]
  28. Graziano, M. P., Freissmuth, M., and Gilman, A. G. (1989) J. Biol. Chem. 264, 409-418 [Abstract/Free Full Text]
  29. Sternweis, P. C., and Robishaw, J. D. (1984) J. Biol. Chem. 259, 13806-13813 [Abstract/Free Full Text]
  30. Studier, F. W., and Moffatt, B. A. (1986) J. Mol. Biol. 189, 113-130 [Medline] [Order article via Infotrieve]
  31. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Methods Enzymol. 185, 60-89 [Medline] [Order article via Infotrieve]
  32. Randazzo, P. A., Weiss, O., and Kahn, R. A. (1992) Methods Enzymol. 219, 362-369 [Medline] [Order article via Infotrieve]
  33. Northup, J. K., Smigel, M. D., and Gilman, A. G. (1982) J. Biol. Chem. 257, 11416-11423 [Free Full Text]
  34. Kahn, R. A., Randazzo, P., Serafini, T., Weiss, O., Rulka, C., Clark, J., Amherdt, M., Roller, P., Orci, L., and Rothman, J. E. (1992) J. Biol. Chem. 267, 13039-13046 [Abstract/Free Full Text]
  35. Schaffner, W., and Weissman, C. (1973) Anal. Biochem. 56, 502-514 [Medline] [Order article via Infotrieve]
  36. Bartlett, G. R. (1959) J. Biol. Chem. 234, 466-469 [Free Full Text]
  37. Deleted in proofDeleted in proof
  38. Tsai, S.-C., Noda, M., Adamik, R., Moss, J., and Vaughan, M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5139-5142 [Abstract]
  39. Randazzo, P. A., Terui, T., Sturch, S., and Kahn, R. A. (1994) J. Biol. Chem. 269, 29490-29494 [Abstract/Free Full Text]
  40. Liscovitch, M., and Cantley, L. C. (1995) Cell 81, 659-662 [Medline] [Order article via Infotrieve]
  41. Jones, G. A., and Carpenter, G. (1993) J. Biol. Chem. 268, 20845-20850 [Abstract/Free Full Text]
  42. Jenkins, G. H., Fisette, P. L., and Anderson, R. A. (1994) J. Biol. Chem. 269, 11547-11554 [Abstract/Free Full Text]
  43. Moritz, A., De Graan, P. N. E., Gispen, W. H., and Wirtz, K. W. A. (1992) J. Biol. Chem. 267, 7207-7210 [Abstract/Free Full Text]
  44. Martin, A., Brown, F. D., Hodgkin, M. N., Bradwell, A. J., Cook, S. J., Hart, M., and Wakelam, M. J. O. (1996) J. Biol. Chem. 271, 17397-17403 [Abstract/Free Full Text]

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