©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Myristoylated Amino Terminus of ADP-ribosylation Factor 1 Is a Phospholipid- and GTP-sensitive Switch (*)

Paul A. Randazzo (§) , Takeshi Terui , Stacey Sturch , Henry M. Fales (1), Anthony G. Ferrige (2), Richard A. Kahn

From the (1)Laboratory of Biological Chemistry, Developmental Therapeutics Program, Division of Cancer Treatment, NCI and the Laboratory of Biophysical Chemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892 and (2)MaxENT Solutions, Ltd., Cambridge CB75 QS1, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

ADP-ribosylation factor 1 (Arf1) is an essential N-myristoylated 21-kDa GTP-binding protein with activities that include the regulation of membrane traffic and phospholipase D activity. Both the N terminus of the protein and the N-myristate bound to glycine 2 have previously been shown to be essential to the function of Arf in cells. We show that the bound nucleotide affects the conformation of either the N terminus or residues of Arf1 that are in direct contact with the N terminus. This was demonstrated by examining the effects of mutations in this N-terminal domain on guanosine 5`-O-(3-thio)triphosphate (GTPS) and GDP binding and dissociation kinetics. Arf1 mutants, lacking 13 or 17 residues from the N terminus or mutated at residues 3-7, had a greater affinity for GTPS and a lower affinity for GDP than did the wild-type protein. As the N terminus is required for interactions with target proteins, we conclude that the N terminus of Arf1 is a GTP-sensitive effector domain.

When Arf1 was acylated, the GTP-dependent conformational changes were codependent on added phospholipids. In the absence of phospholipids, myristoylated Arf1 has a lower affinity for GTPS than for GDP, and in the presence of phospholipids, the myristoylated protein has a greater affinity for GTPS than for GDP. Thus, N-myristoylation is a critical component in the construction of this phospholipid- and GTP-dependent switch.


INTRODUCTION

ADP-ribosylation factors (Arfs)()are ubiquitous and highly conserved GTP-binding proteins that regulate a number of steps in the exocytic and endocytic pathways (1, 2) and activate of phospholipase D(3, 4) . Other activities ascribed to Arf proteins include cofactor for cholera toxin (5, 6) and regulator of coat protein binding to membranes(7, 8) . In addition to the regulatory ligand GTP, Arf activities require an intact amino terminus (9, 10) and the cotranslational covalent addition of myristate to the amino-terminal glycine(3, 7, 11, 12, 13, 14) .

Activation of Arf is tightly correlated with the high affinity binding of GTP. This was first demonstrated when ArfGTPS was found to be active as a cofactor for cholera toxin-catalyzed ADP-ribosylation of G, whereas ArfGDP was inactive(6) . Other Arf activities, including activation of phospholipase D activity, inhibition of intra-Golgi and endoplasmic reticulum Golgi transport, and inhibition of endosome-to-endosome and nuclear vesicle fusion(3, 4, 8, 15, 16, 17) , are also highly dependent on the binding of GTP or the slowly hydrolyzable analog GTPS. Similarly, ArfGTP was found to have at least a 50-fold greater affinity for Arf GTPase-activating protein than did ArfGDP(18) . Thus, as is true for many, if not all, regulatory GTP-binding proteins, the nucleotide bound has been shown to regulate the affinity of Arf for target proteins.

The specific domains of Arf that interact with target proteins, referred to as effector domains, have not yet been identified. Two criteria have previously been used to define effector domains in other GTP-binding proteins. The conformation of the effector domain should be sensitive to bound nucleotide. For instance, comparison of RasGDP and RasGTP crystal structures has revealed differences in two regions, referred to as switch 1 and switch 2(19) . In addition, certain mutations in the effector region should allow uncoupling of GTP binding and increased binding of target proteins. In Ras, switch 1 also meets this criterion, identifying this as an effector domain(19) . Based on data from previous studies, the N-terminal domain of Arf has been implicated as an effector domain. Deletion of the N-terminal 13 or 17 amino acids results in proteins that can bind GTP but that are inactive as cofactors for cholera toxin-catalyzed ADP-ribosylation of G(9, 10) . These mutant proteins also have decreased affinity for Arf GTPase-activating protein(10) . Thus, the N terminus meets one of the criteria used to define the effector domain in Ras.

A critical aspect of Arf action in cells appears to be its regulated binding to and release from different intracellular membranes(7, 8, 15) . While regulated by GTP, this membrane association is greatly enhanced by myristate bound to the N terminus(7, 8, 20) . N-Myristoylation is a cotranslational covalent modification with an absolute requirement for an amino-terminal glycine, the acceptor site(21, 22) . Residues located immediately C-terminal (positions 3-7) are also critical determinants in whether or not a protein is a substrate for N-myristoyltransferase(23) . As several of the putative Arf effectors in cells (e.g. phospholipase D and G) are membrane-bound, it is important to distinguish between changes in the ability of Arf to associate with membranes and changes in its ability to interact with effectors.

We present evidence that the conformation of the N-terminal domain (or residues in direct contact with the N terminus) of Arf1 depends on the bound nucleotide. Because the affinity of Arf1 for GTP is lower than that for GDP, the conformational changes accompanying GTP binding must consume a large part of the binding energy. Therefore, deleting or mutating a domain that undergoes the GTP-dependent conformational change should reciprocally affect GTP and GDP binding affinities. In contrast, any change in the nucleotide-binding site itself is likely to affect binding of GDP and GTP similarly. As mutations in the N terminus caused an increased GTP affinity and a decreased GDP affinity, we conclude that the N terminus forms at least part of the GTP-sensitive switch. We also demonstrate that N-myristoylation is the predominant factor responsible for regulation of nucleotide binding by phospholipids.


MATERIALS AND METHODS

Proteins

Recombinant Arf1, [17]Arf1 (Arf1 with amino acids 1-17 deleted), and [13]Arf1 (Arf1 with amino acids 1-13 deleted) were prepared as described previously(9, 10) . The N-terminal mutants [6,7SK]Arf1 (Arf1 in which residues Ala and Asn are replaced by Ser and Lys) and [3-7LFASK]Arf1 (Arf1 in which wild-type residues Asn-Ile-Phe-Ala-Asn are replaced by Leu-Phe-Ala-Ser-Lys, as found at residues 3-7 in Saccharomyces cerevisiae Arf1) were constructed by polymerase chain reaction amplification of the human Arf1 coding region with sense primers that include the desired mutations and incorporating an NdeI site at the initiating methionine codon and with an antisense primer that adds a BamHI site 6 base pairs 3` of the stop codon. The resulting open reading frame was subcloned into the NdeI and BamHI sites of the pET3C vector and transfected into BL21(DE3) cells as described(24, 25, 26) . The full open reading frame of each mutant was sequenced to ensure that no additional mutations were introduced in the polymerase chain reactions.

Coexpression of Arf Proteins with N-Myristoyltransferases in Bacteria

The T7 polymerase/promoter system of Studier et al.(24, 25) , for expression of foreign proteins in bacteria, was modified as described by Duronio et al.(27) to allow the coexpression of Arfs with N-myristoyltransferases. Yeast (S. cerevisiae) N-myristoyltransferase expression was achieved in BL21(DE3) cells using plasmid pBB171 (the generous gift of Dr. Jeffrey I. Gordon, Washington University, St. Louis, MO)(27) , which carries the structural gene for yeast N-myristoyltransferase under regulation by the T7 promoter, and a kanamycin resistance selectable marker. Human N-myristoyltransferase expression was achieved with a similar plasmid, pNMT1, which contains the entire open reading frame in an 1.6-kilobase BglII-EcoRI fragment cloned into the same sites of pBB131 (the human N-myristoyltransferase open reading frame was also kindly provided by Dr. Jeffrey I. Gordon)(28) . Doubly transformed BL21(DE3) cells were obtained by plating transformants on LB medium with 100 µg/ml ampicillin and 50 µg/ml kanamycin after transformation of calcium-competent cells with up to 1 µg of each plasmid. Transformants were grown in liquid culture to a density of A = 0.7-1.0 before adding the inducer isopropyl--D-thiogalactopyranoside to a final concentration of 1 mM. Where indicated, 125 µM myristate was added at the time of induction as described(26) . When radiolabeling with [H]myristate was performed, the label was added at the time of induction to 25-75 µCi/ml. Cells were harvested after 90 min, unless otherwise specified.

Nucleotide Binding

Binding reactions contained 1 µM Arf and a 10 µM concentration of the indicated radiolabeled guanine nucleotide (specific activity = 1000-10,000 cpm/pmol) in exchange buffer containing 25 mM HEPES, pH 7.4, 100 mM NaCl, 0.5 mM MgCl, 1 mM EDTA, 1 mM dithiothreitol, 50 µg/ml bovine serum albumin, and, where indicated, 3 mML--dimyristoylphosphatidylcholine (DMPC) and 2.5 mM sodium cholate (DMPC/cholate). Samples (10 µl) were taken at the indicated times, and bound nucleotide was determined by rapid filtration on BA85-nitrocellulose filters (Schleicher & Schuell)(29) . Data were fit to a first-order rate equation using the Marquardt algorithm.

Nucleotide Dissociation

Arf (2-5 µM) was incubated with 50 µM [S]GTPS (specific activity = 20,000-50,000 cpm/pmol), 10 µM [H]GDP (specific activity = 10,000-25,000 cpm/pmol), or 5 µM [-P]GDP (specific activity = 5000-25,000 cpm/pmol) for 2-3 h in exchange buffer. Dissociation was determined as a loss of protein-bound radiolabel following dilution into exchange buffer containing 1 mM unlabeled guanine nucleotide. Samples were withdrawn at 10-15 time points between 0 and 120 min. Data were fit to a single or double exponential decay equation using the Marquardt algorithm.

Determination of Relative Nucleotide Affinities

The relative affinities of Arf for GTPS and GDP were determined by competing the binding of radiolabeled GDP with GTPS. Arf (1 µM) was incubated with 10 µM [-P]GDP or [H]GDP (specific activity = 5000-10,000 cpm/pmol) and unlabeled 0.5-1000 µM GTPS in exchange buffer for 3 h for Arf1 or for 1-2 h for the other proteins at 30 °C. Protein-bound radionucleotide was determined by rapid filtration on nitrocellulose filters. Each nucleotide was present at concentrations greater than the number of available binding sites, and the concentration of GDP was much greater than K, allowing the data to be fit to the following equation: [ArfGDP]/[Arf] = (K/K)[GDP]/((K/K)[GDP] + [GTPS]), where all Ks are equilibrium dissociation constants. For [13]Arf1, a significant decrease in GDP binding occurred when the concentration of GTPS was equal to the concentration of Arf, violating one assumption used in deriving the equation; however, this leads to an underestimate of the protein's affinity for GTPS (30) and does not influence the conclusions of this paper.

HPLC of Acylated Arf

Acylated and unmodified Arf proteins were resolved under denaturing conditions by reverse-phase chromatography on an analytical C column (Dynamax 300Å, Rainin Instrument Co. Inc., Woburn, MA) developed in 0.1% trifluoroacetic acid with a 30-75% acetonitrile gradient over 45 min (31). Proteins were detected by absorption at 280 nm. Peaks were integrated to determine relative abundance of the modified proteins.

Electrospray Ionization-Mass Spectrometry

Mass spectra were determined on a Finnigan TSQ-70 spectrometer. Solutions of protein (5-20 pmol/µl) were prepared in acetic acid/methanol/water (5:20:80, v/v). Approximately 30 µl was infused from a Harvard syringe pump at 1 µl/min, averaging scans in the profile mode for 3 min. After preliminary analysis using the Finnigan deconvolution program, data were downloaded in ASCII format and sent to MaxENT Solutions, Ltd. (Cambridge, United Kingdom) for maximum entropy analysis.

Miscellaneous

[-P]GTP, [S]GTPS, and [H]GDP were purchased from DuPont NEN. [-P]GDP was prepared as described (32). DMPC (P-0888), GTP (G-8877), and ATP (A-7894) were obtained from Sigma. Sodium cholate was purchased from Fluka Chemical Co. (Ronkokoma, NY), and GTPS from Boehringer Mannheim. Protein concentrations were determined using the Amido Black assay(33) . Nucleotide concentrations were determined by UV absorbance.


RESULTS

Modification of the N Terminus of Arf1 Has Opposite Consequences for GTPS and GDP Binding Properties

To determine the influence of the amino terminus on GTPS binding, we compared Arf1 with two N-terminal deletion mutants. For a typical preparation of nonmyristoylated Arf1, steady-state binding of GTPS reached a stoichiometry of 0.01 mol/mol of protein when determined in the absence of phospholipids ( Fig. 1and ). In contrast, [13]Arf1 and [17]Arf1 (mutants in which the N-terminal 13 and 17 amino acids were deleted, respectively), bound 0.92 and 0.75 mol of nucleotide/mol of protein, respectively (see Ref. 10 and ).


Figure 1: Phospholipid dependence of nucleotide binding to Arf. GTPS binding to Arf1 (invertedtriangles), [3-7LFASK]Arf1 (triangles), or acylated [3-7LFASK]Arf1 (circles) was determined in the presence (closedsymbols) or absence (opensymbols) of DMPC/cholate as described under ``Materials and Methods.'' Each point contained 10 pmol of Arf. This is a representative experiment of those used to determine the values presented in Table I.



These differences in equilibrium binding of GTPS result from differences in nucleotide affinities. GTPS dissociated from [13]Arf1 and [17]Arf1 slower than from Arf1 (Fig. 2A and ). In contrast, GDP dissociated faster from [13]Arf1 and [17]Arf1 than from the wild-type protein (Fig. 2B and ). These opposite changes in dissociation rates should reflect reciprocal changes in guanine nucleotide affinity if nucleotide association rates were not affected by the deletions. Unfortunately, determination of nucleotide association rates is inaccurate and technically demanding because the apoprotein is unstable(34) . Instead, the relative affinities for GDP and GTPS were determined by monitoring the binding of GDP when competed with GTPS. Data are expressed as a ratio of the equilibrium dissociation constants K/K as described under ``Materials and Methods.'' A value >1 indicates that the protein has a greater affinity for GTPS than for GDP. The ratio K/K was >1 for [17]Arf1 and [13]Arf1 and <1 for Arf1 as indicated in I. These data reveal that deleting 13 or 17 residues from the N terminus led to coordinate and opposite changes in the binding affinities for GDP and GTPS. If the amino terminus were simply inhibiting GTP binding, one would expect the deletions to have the same effect on both GTP and GDP binding. Mutation of Residues 3-7 Increases the Affinity for GTPS-One mutant ([3-7LFASK]Arf1) designed to be a better substrate for N-myristoyltransferase and to allow the production of extensively modified Arf1 in bacteria (see below) fortuitously provided information about the effect of the bound nucleotide on the N terminus. Other than being a better substrate for N-myristoyltransferase, we did not expect the mutations to affect the biochemical properties of the protein. Indeed, no differences in specific activities as cofactors for cholera toxin-catalyzed ADP-ribosylation of G (data not shown) were observed between [3-7LFASK]Arf1 and Arf1. However, the nucleotide binding properties of this mutant were found to be similar to those of [13]Arf1 and [17]Arf1 ( Fig. 1and ); [3-7LFASK]Arf1 bound more GTPS at equilibrium than did Arf1. The increased binding of GTPS was a result of reciprocal changes in the affinity for GTP and GDP. GTPS dissociated more slowly and GDP dissociated more rapidly from [3-7LFASK]Arf1 than from Arf1. The ratio K/K was >1 for [3-7LFASK]Arf1 (). Thus, this mutant provided further evidence that the conformation of the N-terminal domain differs in the GDP- and GTP-bound states.


Figure 2: Effect of changing the amino terminus on nucleotide dissociation from Arf. The time dependence of GTPS (A) and GDP (B) dissociation from Arf1 (invertedtriangles) [17]Arf1 (squares), [13]Arf1 (diamonds), and [3-7LFASK]Arf1 (triangles) was determined as described under ``Materials and Methods.'' The data are expressed as a fraction of radiolabeled nucleotide bound to Arf at the time the reaction was initiated and were fit to single or double exponential decay equations to derive values for the dissociation rate presented in Table II. This is one of at least three representative experiments.



Preparation of Acylated Arf Protein

To examine the role of myristate in nucleotide exchange, a source for unmodified and acylated Arf1 preparations was sought. Arf preparations from mammalian tissues are composed of a mixture of gene products that cannot be resolved efficiently. Recombinant Arf1 has been produced in bacteria in large amounts and found to yield a homogeneous nonacylated preparation of Arf1(34) . The coexpression of human Arf1 and yeast N-myristoyltransferase has been used previously to produce the properly modified protein(26) . However, the extent of myristoylation, determined by reverse-phase HPLC, was typically only 10-15%(31) . Attempts to resolve the processed and unprocessed forms of Arf1 or Arf3 by chromatography on DEAE-Sephacel, MonoQ, hydroxylapatite, phenyl-Sepharose, heptylamine-agarose, or Ultrogel AcA 44 or 54 resins proved unsuccessful (data not shown). Changing the time of induction (up to 18 h) or the concentration of inducer (isopropyl--D-thiogalactopyranoside as low as 50 µM) did not result in increased extents of acylation of Arf1. Similarly, coexpression of human Arf1 with human N-myristoyltransferase in bacteria was not much different from coexpression with yeast N-myristoyltransferase and did not increase the extent of myristoylated human Arf1.

In contrast to the inefficient acylation of mammalian Arfs in bacteria, the yeast Arf proteins were observed to have a near optimal sequence (residues 2-7) for N-myristoyltransferase, and a peptide derived from the N terminus of S. cerevisiae Arf2 was shown to be an excellent substrate for N-myristoyltransferase (23). When protein expression was induced in the presence of [H]myristic acid, similar amounts of label were incorporated into human Arf1 with either human or yeast N-myristoyltransferase (Fig. 3, first and fifthlanes). In contrast, although yeast Arf1 was expressed at similar levels compared with human Arf1, it incorporated much more [H]myristate, regardless of the source of N-myristoyltransferase (Fig. 3, fourth and eighth lanes). Therefore, S. cerevisiae Arf1 is a much better substrate for either yeast or human N-myristoyltransferase than is human Arf1.


Figure 3: Yeast Arf1 and amino-terminal mutants of human Arf1 are highly N-myristoylated when coexpressed in bacteria with human or yeast N-myristoyltransferase. Human Arf1 (hARF1), [6,7SK]Arf1, [3-7LFASK]Arf1, and S. cerevisiae Arf1 (ScARF1) were coexpressed in bacteria with either human (hNMT) or yeast (ScNMT) N-myristoyltransferase. Culturing, induction, and labeling with [H]myristate were performed as described under ``Materials and Methods.'' Approximately 25 µg of total bacterial protein was loaded on each lane of the gel, which was subsequently stained with Coomassie Blue (A) or developed for fluorography (B). The film was exposed overnight at -80 °C. Only the 20-kDa region of the fluorogram is shown in B as no other bands were detected elsewhere on the gel.



Guided by the extensive characterization of sequence specificities in substrates for N-myristoyltransferase(22, 23) , we constructed two mutants of human Arf1 in which the corresponding residues of S. cerevisiae Arf1 were substituted in efforts to allow acylation with high efficiency. Changing only residues 6 and 7 ([6,7SK]Arf1) led to an increase in the level of N-myristoylation of mutant human Arf1 coexpressed with S. cerevisiaeN-myristoyltransferase, but decreased that coexpressed with human N-myristoyltransferase (Fig. 3, second and sixthlanes). Changing residues 3-7 ([3-7LFASK]Arf1) further increased the extent of acylation of human Arf1 achieved in the presence of S. cerevisiaeN-myristoyltransferase and also increased that obtained with human N-myristoyltransferase (Fig. 3, third and seventhlanes). Expression of this mutant allowed a level of myristoylation comparable to that observed when S. cerevisiae Arf1 was coexpressed in bacteria with S. cerevisiaeN-myristoyltransferase. [3-7LFASK]Arf1 purified from bacteria expressing S. cerevisiaeN-myristoyltransferase is referred to as acylated [3-7LFASK]Arf1.

Reverse-phase HPLC and electrospray ionization-mass spectrometry revealed multiple chemical forms present in the preparation of N-acylated [3-7LFASK]Arf1. A series of peaks were observed (Fig. 4) that differed in molecular mass by 27.2 ± 1.6 Da. These molecular masses, determined using a maximum entropy algorithm to deconvolute the data from electrospray ionization-mass spectrometry(35) , differ by the mass of a CH group and are consistent with the different forms being the result of heterogeneous acylation. The species with a mass consistent with the myristoylated protein was 18% of the total protein if no myristate was added at the time of induction and 40% of the total protein if myristate was added. The remainder of the acyl groups were predicted to be saturated acyl chains of 4-24 carbons (Fig. 4). This heterogeneity in acyl groups covalently bound to Arf1 likely results from the forced overexpression in bacteria as Arf proteins purified from bovine brain did not appear to be heterogeneously acylated(36) . This recombinant preparation allowed us to examine the consequence of acylation on nucleotide binding kinetics by comparison with nonacylated [3-7LFASK]Arf1.


Figure 4: Electrospray ionization-mass spectrum and normal and maximum entropy deconvolution of partially myristoylated [3-7LFASK]Arf1. Acylated [3-7LFASK]Arf1 expressed in Escherichia coli with no added myristic acid was dissolved in acetic acid/methanol/water (5:20:80), and the sample was admitted by infusion from a syringe pump at 1 µl/min as described under ``Methods and Materials.'' The MaxENT spike/error plot shows peak widths corresponding to the relative errors.



Acylation Decreases the Affinity of Arf1 for GTPS in the Absence of Phospholipids

Acylated [3-7LFASK]Arf1 bound 90% less GTPS at equilibrium than did the nonacylated protein ( Fig. 1and ) when assayed in the absence of added phospholipids. GTPS dissociated from the acylated protein faster than from the nonacylated protein (Fig. 5A and ). In contrast, acylation had little effect on the rate of GDP dissociation (Fig. 5B and ). Consistent with the changes in the dissociation rates, the K/K ratio was <1 for acylated [3-7LFASK]Arf1 and >1 for [3-7LFASK]Arf1 (I). Thus, acylation decreases the affinity of Arf1 for GTPS when measured in the absence of phospholipids.


Figure 5: Phospholipid dependence of nucleotide dissociation from Arf. GTPS (A) and GDP (B) dissociation from [3-7LFASK]Arf1 (triangles) and acylated [3-7LFASK]Arf1 (circles) in the presence (closedsymbols) or absence (opensymbols) of DMPC/cholate was determined. The data are expressed as a fraction of radiolabeled nucleotide bound to Arf at the time the reaction was initiated. The experiment is representative of three.



N-Myristate Is a Major Determinant of Phospholipid-dependent GTPS Binding to Arf1

Binding of GTPS to Arf proteins purified from mammalian tissues is highly dependent on added phospholipids(6) . Similarly, GTPS binding to acylated [3-7LFASK]Arf1 was highly dependent on DMPC/cholate ( Fig. 1and ). In contrast, GTPS binding to [3-7LFASK]Arf1 is largely independent of added DMPC/cholate ( Fig. 1and ). In the absence of DMPC/cholate, acylated [3-7LFASK]Arf1 bound less GTPS than did the nonacylated protein. In the presence of DMPC/cholate, acylated [3-7LFASK]Arf1 bound GTPS faster and to a 50-80% higher stoichiometry than did the nonacylated protein ( Fig. 1and ). The DMPC/cholate dependence was similar to that of Arf purified from bovine brain(6) .

The effects of phospholipids on GTPS binding were the result of changes in nucleotide affinities. DMPC/cholate slowed the GTPS dissociation rate and accelerated the GDP dissociation rate from acylated [3-7LFASK]Arf1 ( Fig. 5and ). The net effect was a 14-fold increase in the K/K ratio from 0.35 to 4.8 (I). Thus, the addition of phospholipids to acylated Arf1 resulted in increased and decreased affinities for GTPS and GDP, respectively. The effect of phospholipids on nucleotide binding, nucleotide dissociation, and relative nucleotide affinities was minimal for the nonacylated proteins used in these studies. Among this group of proteins, the largest effect observed was seen for binding of GTPS to Arf1, which was 5.5 ± 2.2-fold (n = 3) greater in the presence of DMPC/cholate than in its absence.


DISCUSSION

These studies were undertaken to test the model that there are conformational differences in the N termini of ArfGDP and ArfGTP, which could provide evidence for this region of the protein acting as an effector domain. From this model, mutations that affect the conformation of this domain should have different consequences for GTP and GDP binding. This prediction was borne out by the data. The amino terminus of Arf1 is a GTP-sensitive switch. As previous studies have shown that the amino terminus interacts with target proteins(9, 10, 13, 14) , it may be considered an effector domain of Arf1. We have further demonstrated that myristoylation is the predominant (although not sole) determinant of the phospholipid-dependent transition of Arf1 to the active conformer. Thus, the myristoylated N terminus of Arf1 is a GTP- and phospholipid-sensitive switch.

Conformational differences in the N termini of Arf1GDP and Arf1GTPS were demonstrated by comparing the nucleotide binding properties of Arf1 with those of N-terminal mutants. Two characteristics of Arf1 allowed this approach. First, the conformational change in Arf that accompanied the exchange of GTP for GDP consumed enough energy to result in a large difference in binding affinities for the nucleotides(34) . Second, the N terminus does not contribute directly to the nucleotide-binding pocket(37) . Therefore, if the GTP-sensitive conformational change were localized to the N terminus or to residues in direct contact with the N terminus, removing or altering this domain should result in an increased affinity for GTP. Furthermore, depending on whether the region also contributed to stabilizing the GDP-bound form of the protein, mutations of the N terminus should either have no effect on or decrease the protein's affinity for GDP. Thus, the finding that [3-7LFASK]Arf1, [13]Arf1, and [17]Arf1 have lower affinities for GDP and higher affinities for GTPS than does wild-type Arf1 is consistent with the N terminus stabilizing the GDP-bound form of the protein and undergoing a conformational change when GTP exchanges for GDP.

Our conclusion that the N terminus is an effector domain of Arf1 does not preclude the presence of a second effector domain. Arf has been shown to have at least two distinct binding sites. One site is for G and requires an intact N terminus of Arf. A second site binds cholera toxin independently of the N terminus(9, 10, 38, 39) . As binding to cholera toxin appears to be GTP-dependent(38, 39) , the presence of a second effector domain seems likely. A likely candidate for this second domain is -sheet 2 and loop 4 identified in the crystal structure of Arf1GDP described by Amor et al.(37) . The residues composing this putative effector domain are found between the two GTP-binding consensus sequences GXXXXGK and DXXG (where X is any amino acid), as is true for the effector domain (loop 2) of Ras. This domain is hydrophobic and highly exposed to solvent, but in the crystal, forms the interface for a dimer that is proposed to be a site of protein-protein interaction(37) .

A number of roles for covalently bound myristate have been ascribed to proteins, including Arf. These studies provide evidence for a role of the acyl group in integration of ligand (GTP) and phospholipid binding. N-Myristoylation has been found to play a structural role (e.g. cAMP-dependent protein kinase(40) ) and a role in membrane anchoring (e.g. viral group-specific antigen proteins(41) ) and in ligand-sensitive membrane association (e.g. recoverin, Arf(7, 12, 42, 43, 44) ). We found that the interaction of the acyl chain with Arf1 is sensitive to the guanine nucleotide bound. We also showed that the myristate interacts with phospholipids to help drive Arf1 into the active conformer. These data are in concordance with those published by Franco et al. (45) using myristoylated wild-type Arf1.

The above data show that the acyl group (myristate in native Arf proteins) is more important than the amino-terminal domain as a determinant of interaction with phospholipids. This is consistent with previous work showing that GTP-dependent association with phospholipids and membranes is highly dependent on Arf being myristoylated(7, 8, 12, 20, 44) . These data also help explain the differences in binding stoichiometries reported for Arfs purified from bovine brain and recombinant Arfs(6, 34) .

Studies in the laboratory of Dr. Jeffrey I. Gordon have demonstrated the importance of the amino terminus in determining both specificity and rate constants for the acylation of peptides by a yeast N-myristoyltransferase(47) . Mammalian Arf proteins are poor substrates for N-myristoyltransferase, while Arfs found in the yeast S. cerevisiae are quite good substrates. Mutagenesis of human Arf1 provided additional evidence of the importance of residues 3-5 as well as 6 and 7 in substrate recognition by N-myristoyltransferases, further defined differences in fungal and mammalian N-myristoyltransferase specificities, and allowed the production of recombinant proteins that are highly processed and suitable for structural and functional studies of the myristoylated Arf protein.

Myristoylated proteins purified from mammalian tissues, with the exception of the retina(48, 49) , have been found to be homogeneously modified with myristate(21, 22, 36, 49) . In characterizing recombinant Arfs that are good substrates for N-myristoyltransferase in the bacterial coexpression system (S. cerevisiae Arf1 and [3-7LFASK]Arf1), we found them to be heterogeneously acylated. Deconvolution of the mass spectrometry data revealed a series of peaks differing by 26-28 atomic mass units, consistent with the addition or deletion of an ethylene group (CH). Thus, fatty acid chain lengths from 6 to 16 carbons in length were observed on Arf proteins. While >95% of the recombinant Arfs were acylated, in the preparation used for the kinetic studies reported here, 40% were myristoylated. This heterogeneity is greater than that reported for recoverin or transducin in the retina. This heterogeneity likely results from the forced overexpression of myristoylated proteins in an organism (bacteria) that does not normally produce myristoylated proteins, lacks N-myristoyltransferase, and maintains lower levels of N-myristoylated CoA. We believe that the differences described between the acylated and nonacylated proteins are at least qualitatively the same as those between unmodified and myristoylated Arfs. Furthermore, some of our observations have recently been verified(45) . Other means of preparing a fully myristoylated single Arf gene product are currently being tested, including those methods described by Franco et al.(45) .

For some of the proteins, particularly nonmyristoylated Arf1, the effect of phospholipids on affinities was less than that anticipated based on the phospholipid effect on binding stoichiometry. Also, the difference in affinities for GTP and GDP was not as great as previously reported(34) . There are a number of possible reasons for the apparent discrepancy. The previous study determined association rates using nucleotide-free proteins, being renatured out of 7 M urea. This preparation is both very unstable and proper, and complete refolding could not be asserted. In the studies reported above, the concentrations of both protein and nucleotides were greater than those used in previous studies. Both the stability of Arf proteins in solution and the determined binding stoichiometries have been reported to be sensitive to the concentration of Arf in the binding reaction(46) . This still cannot completely explain the binding data. Indeed, the complexities of nucleotide exchange on Arf are illustrated in the double exponential decay kinetics of ArfGTPS dissociation, suggesting that two populations of Arf1 exist. We currently have no good model to explain this observation.

These studies have provided evidence for the identification of the amino terminus of Arf as an effector domain and provide support for the conclusion that the role of the myristate is to integrate phospholipid binding and GTP binding with protein activation. We are currently using these results to further define specific residues involved in interaction with Arf GTPase-activating protein and other target proteins.

  
Table: Myristoylation confers increased phospholipid dependence, while loss of the N terminus is associated with decreased phospholipid dependence of GTPS binding

Binding in the presence (+DMPC) or absence (-DMPC) of DMPC/cholate was determined as described under ``Materials and Methods.'' The binding rates, k (min), and maximum binding stoichiometry, max (mol of nucleotide/mol of Arf), were determined by fitting the data to the equation ArfGXP/Arf = max (1 - e). [13] and [17] refer to [13]Arf1 and [17]Arf1, respectively. 37Arf1 and myr37 refer to [3-7LFASK]Arf1 and acylated [3-7LFASK]Arf1, respectively. The data are the means ± S.D. of three experiments for Arf1 and the means ± range of two experiments for all other proteins.


  
Table: Differences in steady-state binding of GTPS reflect changes in nucleotide dissociation rates

Nucleotide dissociation rates from the indicated proteins in the presence (+DMPC) or absence (-DMPC) of DMPC/cholate were determined as described under ``Materials and Methods.'' [13] and [17] refer to [13]Arf1 and [17]Arf1, respectively. 37Arf1 and myr37 refer to [3-7LFASK]Arf1 and acylated [3-7LFASK]Arf1, respectively. The data are the means ± S.D. of three experiments. GTPS dissociation from Arf1 could not be fit to a single exponential, but did fit a double exponential decay equation. The fast component composed 0.51 ± 0.09 of the signal. The decay rates are given as k and k, and a weighted average of the two is given for the sake of comparison with the other proteins, whose dissociation time courses fit single exponential decay equations.


  
Table: Effects of myristoylation, mutation, and phospholipids on the relative nucleotide affinities of four mutant Arfs

K/K was determined in the presence (+DMPC) or absence (-DMPC) of DMPC/cholate as described under ``Materials and Methods.'' K and K are the dissociation constants for ArfGDP and ArfGTPS, respectively. 37Arf1 and myr37 refer to [3-7LFASK]Arf1 and acylated [3-7LFASK]Arf1, respectively. The data are the means ± S.D. of three experiments.



FOOTNOTES

*
This work was supported by the Developmental Therapeutics Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: NIH, Bldg. 37, Rm. 5D-02, Bethesda, MD 20892. Tel.: 301-496-3788; Fax: 301-480-2514.

The abbreviations used are: Arfs, ADP-ribosylation factors; Arf1, human ADP-ribosylation factor 1; GTPS, guanosine 5`-O-(3-thio)triphosphate; DMPC, L--dimyristoylphosphatidylcholine; HPLC, high pressure liquid chromatography.


ACKNOWLEDGEMENTS

We thank Dr. John K. Northup for helpful discussions throughout the work and preparation of the manuscript and Juan Carlos Amor and Drs. Dagmar Ringe, Chun-jiang Zhang, Annette Boman, and Anne G. Rosenwald for comments on the manuscript. Drs. Jeffrey I. Gordon and Jennifer Lodge generously provided the structural genes for the human and yeast N-myristoyltransferases as well as advice on use in bacteria.


REFERENCES
  1. Kahn, R. A., Yucel, J. K., and Malhotra, V.(1993) Cell75, 1045-1048 [CrossRef][Medline] [Order article via Infotrieve]
  2. Rosenwald, A. G., and Kahn, R. A.(1994) in GTPase-controlled Molecular Machines (Corda, D., Hamm, H., and Luini, A., eds) pp. 143-155, Ares-Serono Symposium Publications, Rome
  3. Brown, H. A., Gutowski, S., Moomaw, C. R., Slaughter, C., and Sternweis, P. C.(1993) Cell75, 1137-1144 [Medline] [Order article via Infotrieve]
  4. 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) Science263, 523-526 [Medline] [Order article via Infotrieve]
  5. Kahn, R. A., and Gilman, A. G.(1984) J. Biol. Chem.259, 6228-6234 [Abstract/Free Full Text]
  6. Kahn, R. A., and Gilman, A. G.(1986) J. Biol. Chem.261, 7906-7911 [Abstract/Free Full Text]
  7. Donaldson, J. G., Kahn, R. A., Lippincott-Schwartz, J., and Klausner, R. D.(1991) Science254, 1197-1199 [Medline] [Order article via Infotrieve]
  8. Donaldson, J. G., Cassel, D., Kahn, R. A., and Klausner, R. D.(1992) Proc. Natl. Acad. Sci. U. S. A.89, 6408-6412 [Abstract]
  9. 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]
  10. Randazzo, P. A., Terui, T., Sturch, S., and Kahn, R. A.(1994) J. Biol. Chem.269, 29490-29494 [Abstract/Free Full Text]
  11. Kahn, R. A., Clark, J., Rulka, C., Stearns, T., Zhang, C., Randazzo, P. A., Terui, T., and Cavanagh, M.(1995) J. Biol. Chem.270, 143-150 [Abstract/Free Full Text]
  12. Randazzo, P. A., Yang, Y. C., Rulka, C., and Kahn, R. A.(1993) J. Biol. Chem.268, 9555-9563 [Abstract/Free Full Text]
  13. Balch, W. E., Kahn, R. A., and Schwaninger, R.(1992) J. Biol. Chem.267, 13053-13061 [Abstract/Free Full Text]
  14. Lenhard, J. M., Kahn, R. A., and Stahl, P. D.(1992) J. Biol. Chem.267, 13047-13052 [Abstract/Free Full Text]
  15. Donaldson, J. G., Lippincott-Schwartz, J., and Klausner, R. D.(1991) J. Cell Biol.112, 579-588 [Abstract]
  16. Boman, A. L., Taylor, T. C., Melancon, P., and Wilson, K. L.(1992) Nature358, 512-514 [Medline] [Order article via Infotrieve]
  17. Taylor, T. C., Kahn, R. A., and Melancon, P.(1992) Cell70, 69-79 [Medline] [Order article via Infotrieve]
  18. Randazzo, P. A., and Kahn, R. A.(1994) J. Biol. Chem.269, 10758-10763 [Abstract/Free Full Text]
  19. Lowy, D. R., and Willumsen, B. M.(1993) Annu. Rev. Biochem.62, 851-891 [CrossRef][Medline] [Order article via Infotrieve]
  20. Haun, R. S., Tsai, S. C., Adamik, R., Moss, J., and Vaughan, M.(1993) J. Biol. Chem.268, 7064-7068 [Abstract/Free Full Text]
  21. Gordon, J. I., Duronio, R. J., Rudnick, D. A., Adams, S. P., and Gokel, G. W.(1991) J. Biol. Chem.266, 8647-8650 [Free Full Text]
  22. Johnson, D. R., Bhatnagar, R. S., Knoll, L. J., and Gordon, J. I. (1994) Annu. Rev. Biochem.63, 869-914 [CrossRef][Medline] [Order article via Infotrieve]
  23. Rocque, W. J., McWherter, C. A., Wood, D. C., and Gordon, J. I.(1993) J. Biol. Chem.268, 9964-9971 [Abstract/Free Full Text]
  24. Studier, F. W., and Moffatt, B. A.(1986) J. Mol. Biol.189, 113-130 [Medline] [Order article via Infotrieve]
  25. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Methods Enzymol.185, 60-89 [Medline] [Order article via Infotrieve]
  26. Randazzo, P. A., Weiss, O., and Kahn, R. A.(1992) Methods Enzymol.219, 362-369 [Medline] [Order article via Infotrieve]
  27. Duronio, R. J., Jackson-Machelski, E., Heuckeroth, R. O., Olins, P. O., Devine, C. S., Yonemoto, W., Slice, L. W., Taylor, S. S., and Gordon, J. I.(1990) Proc. Natl. Acad. Sci. U. S. A.87, 1506-1510 [Abstract]
  28. Duronio, R. J., Reed, S. I., and Gordon, J. I.(1992) Proc. Natl. Acad. Sci. U. S. A.89, 4129-4133 [Abstract]
  29. Northup, J. K., Smigel, M. D., and Gilman, A. G.(1982) J. Biol. Chem.257, 11416-11423 [Free Full Text]
  30. Cha, S.(1970) J. Biol. Chem.245, 4814-4818 [Abstract/Free Full Text]
  31. Randazzo, P. A., and Kahn, R. A.(1995) Methods Enzymol., in press
  32. Hamel, E., and Lin, C. M.(1984) J. Biol. Chem.259, 11060-11069 [Abstract/Free Full Text]
  33. Schaffner, W., and Weissman, C.(1973) Anal. Biochem.56, 502-514 [Medline] [Order article via Infotrieve]
  34. Weiss, O., Holden, J., Rulka, C., and Kahn, R. A.(1989) J. Biol. Chem.264, 21066-21072 [Abstract/Free Full Text]
  35. Ferrige, A. G., Seddon, M. J., Jarvis, S., Skilling, J., and Welch, J. (1991) in Maximum Entropy and Bayesian Methods (Smith, C. R., ed) pp. 327-335, Kluwer Academic Publishers, Dordrecht, The Netherlands
  36. Kahn, R. A., Goddard, C., and Newkirk, M.(1988) J. Biol. Chem.263, 8282-8287 [Abstract/Free Full Text]
  37. Amor, J. C., Harrison, D., Kahn, R. A., and Ringe, D.(1994) Nature372, 704-708 [CrossRef][Medline] [Order article via Infotrieve]
  38. Hong, J.-X., Haun, R. S., Tsai, S.-C., Moss, J., and Vaughan, M.(1994) J. Biol. Chem.269, 9743-9745 [Abstract/Free Full Text]
  39. Price, S. R., Nightingale, M., Tsai, S.-C., Williamson, K. C., Adamik, R., Chen, H.-C., Moss, J., and Vaughan, M.(1988) Proc. Natl. Acad. Sci. U. S. A.85, 5488-5491 [Abstract]
  40. Zheng, I., Knighton, D. R., Xuong, N. H., Taylor, S. S., Sowadski, J. M., and Ten Eyck, L. F.(1993) Protein Sci.2, 1559-1573 [Abstract/Free Full Text]
  41. Hayakawa, T., Miyazaki, T., Misumi, Y., Kobayashi, M., and Fujisawa, Y. (1992) Gene (Amst.) 119, 273-277 [Medline] [Order article via Infotrieve]
  42. Dizhoor, A. M., Chen, C.-K., Olshevskaya, E., Sinelnikova, V. V., Phillipov, P., and Hurley, J. B.(1993) Science259, 829-832 [Medline] [Order article via Infotrieve]
  43. Zozulya, S., and Stryer, L.(1992) Proc. Natl. Acad. Sci. U. S. A.89, 11569-11573 [Abstract]
  44. Kahn, R. A.(1992) J. Biol. Chem.266, 15595-15597 [Abstract/Free Full Text]
  45. Franco, M., Chardin, P., Chabre, M., and Paris, S.(1995) J. Biol. Chem.270, 1337-1341 [Abstract/Free Full Text]
  46. Randazzo, P. A., Northup, J. K., and Kahn, R. A.(1992) J. Biol. Chem.267, 18182-18189 [Abstract/Free Full Text]
  47. Lodge, J. K., Johnson, R. L., Weinberg, R. A., and Gordon, J. I.(1994) J. Biol. Chem.269, 2996-3009 [Abstract/Free Full Text]
  48. Dizhoor, A. M., Ericsson, L. H., Johnson, R. S., Kumar, S., Olshevskaya, E., Zozulya, S., Neubert, T. A., Stryer, L., Hurley, J. B., and Walsh, K. A.(1992) J. Biol. Chem.267, 16033-16036 [Abstract/Free Full Text]
  49. Johnson, R. S., Ohguro, H., Palczewski, K., Hurley, J. B., Walsh, K. A., and Neubert, T. A.(1994) J. Biol. Chem.269, 21067-21071 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.