©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutational Analysis of Saccharomyces cerevisiae ARF1(*)

(Received for publication, July 21, 1994; and in revised form, October 31, 1994)

Richard A. Kahn (§) Jenny Clark Cherrie Rulka Tim Stearns (1) Chun-jiang Zhang Paul A. Randazzo Takeshi Terui Margaret Cavenagh

From the Laboratory of Biological Chemistry, Developmental Therapeutics Program, Division of Cancer Treatment, NCI, National Institutes of Health, Bethesda, Maryland 20892 and the Department of Biological Sciences, Stanford University, Stanford, California 94305-8309

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Wild type and eight point mutants of Saccharomyces cerevisiae ARF1 were expressed in yeast and bacteria to determine the roles of specific residues in in vivo and in vitro activities. Mutations at either Gly^2 or Asp resulted in recessive loss of function. It was concluded that N-myristoylation is required for Arf action in cells but not for either nucleotide exchange or cofactor activities in vitro. Asp (homologous to Gly of p21) was essential for the binding of the activating nucleotide, guanosine 5`-3-O-(thio)triphosphate. This is in marked contrast to results obtained after mutagenesis of the homologous residue in p21 or G(s)alpha, and suggests a fundamental difference in the guanine nucleotide binding site of Arf with respect to these other GTP-binding proteins.

Two dominant alleles were also identified, one activating dominant ([Q71L]Arf1) and the other ([N126I]) a negative dominant. A conditional allele, [W66R]Arf1, was characterized and shown to have approx300-fold lower specific activity in an in vitro Arf assay. Two high-copy suppressors of this conditional phenotype were cloned and sequenced. One of these suppressors, SFS4, was found to be identical to PBS2/HOG4, recently shown to encode a microtubule-associated protein kinase kinase in yeast.


INTRODUCTION

ADP-ribosylation factors (Arf) (^1)are 21-kDa GTP-binding proteins, originally identified as the protein cofactor required for efficient ADP-ribosylation of the stimulatory, regulatory component of adenylate cyclase, G(s), by cholera toxin ((1) ; for recent reviews, see (2) and (3) ). This cofactor activity can be made linearly related to the formation of the activated (GTP-bound) species and used as a quantitative assay of Arf activity (1) . Arf proteins are ubiquitous in eukaryotes and both structure and function have been very highly conserved throughout eukaryotic evolution ((4) ; human and Xenopus laevis, Schizosaccharomyces pombe, and Saccharomyces cerevisiae Arf1 are 99, 90, and 74% identical, respectively). ARF1 and ARF3 (96% identical) are usually the most abundantly expressed members of the Arf family in mammalian cells and tissues.

A number of functions have been ascribed to Arf proteins, particularly as components of intracellular membrane traffic (for review, see (3) ). Since its original purification, based on its activity as a cofactor for cholera toxin(5) , Arf proteins have also been purified based on their ability to inhibit intra-Golgi transport (6) and nuclear vesicle fusion (^2)in the presence of GTPS. In addition, Arf was purified independently in two different laboratories as the soluble factor required for the stimulation of phospholipase D activity by GTPS(7, 8) . Arf proteins have also been identified as components of nonclathin-coated vesicles(9) , on which much speculation regarding cellular functions have focused.

One of the first indications that Arf proteins act in one or more steps of the protein secretory pathway came from studies in S. cerevisiae(10, 11) which have two ARF genes, ARF1 and ARF2. Deletion of both genes is lethal while deletion of ARF2 is without any defined effect. Disruption of ARF1 (arf1::URA3) was found to slow the rate of cell growth, cause a defect in the processing of invertase, and make cells supersensitive to fluoride ions(11) . Each of these phenotypes could be complemented by transformation with a centromere-containing plasmid carrying wild type ARF1 or ARF2. Thus, yeast offers an attractive system for genetic studies of the role(s) for Arf protein in eukaryotes as phenotypes have already been described, which result from mutation of ARF1.

Mutational studies, using both yeast and mammalian proteins, have been used to define the role of specific residues in the guanine nucleotide binding sites of a number of GTP-binding proteins, including p21, G(s)alpha, SEC4, YPT/RAB, and others. Some recent reports (12, 13, 14) have utilized homologous mutations to study functions of mutant Arf proteins in mammalian cells. However, none of these previous reports characterized the biochemical consequences of these mutations but instead relied upon the assumed conservation of consequences between Arf and Ras or other GTP-binding proteins. With the limited extent of sequence identity between ARF and either the other members of the Ras superfamily or alpha subunits of the heterotrimeric G proteins (approx20-25% identity), it is risky to assume that homologous mutations in one family will have the same consequences on nucleotide handling or protein interactions in a member of another family. Indeed, the solution of the crystal structure of human ARF1, along with some of the data presented below, provide direct demonstrations of the fact that the nucleotide binding site of Ras and Arf have distinctive features.

The availability of a quantitative biochemical assay of Arf activity has proven of great value in the characterization of the protein, in spite of the unclear relevance of ADP-ribosylation to the in vivo action of Arf proteins in cells. Characterization of phenotypes of ARF mutants in yeast allowed the integration of biochemical information with functional characterization in cells.


MATERIALS AND METHODS

Polymerase Chain Reaction Mutagenesis and Plasmid Construction

Mutants were constructed by polymerase chain reaction amplification of the ARF1 open reading frame using synthetic oligonucleotides that incorporated the desired mutations. Products were sequenced to insure that the desired mutations were obtained and that no additional changes were introduced. Restriction sites were added at the 5`- and 3`-ends of the open reading frame to facilitate subcloning into expression vectors, as shown in Fig. 1. BamHI and NdeI sites were added at the initiating methionine to allow subcloning into yeast and bacterial expression vectors, respectively. An XbaI site occurs 6 bp downstream of the stop codon in the ARF1 gene and SalI and BamHI sites were added immediately 3` of the XbaI site. Different combinations of double enzyme digestions allowed subcloning of wild type and mutant Arf constructs into thee different expression plasmids: (i) the NdeI-BamHI fragments were subcloned into pET3C (15) for expression in bacteria; (ii) the BamHI-SalI fragments were ligated into unique sites in pBM272 (the generous gift of Dr. Mark Johnston, Washington University, St. Louis, MO; (16) ), for expression in yeast under control of the inducible GAL1 promoter; and (iii) the NdeI-XbaI fragments were cloned into pJCY1-35, a centromere containing plasmid carrying an ARF1 gene with unique NdeI and XbaI sites at the initiating methionine and 6 bp downstream of the stop codon, respectively, for expression of Arf proteins in yeast under control of the ARF1 promoter.


Figure 1: Sites and cloning strategies for construction of plasmids directing the expression in yeast and bacteria of wild type and mutants of ARF1. The coding region of S. cerevisiae ARF1 is shown with the restrictions sites which were added on each end to facilitate subcloning into the different expression vectors. Details of the construction of mutants and subcloning are described under ``Materials and Methods.'' The different expression systems are indicated below, along with the promoters used in each and the sites used to clone into the parent plasmids.



Tests of Arf Function in Yeast

Yeast were cultured in rich complete medium, YPD (1% yeast extract, 2% peptone, 2% dextrose), or for selection in S.D. (0.15% yeast nitrogen base, 2% dextrose, and the appropriate amino acid or nucleotide) at 30 °C, unless otherwise stated. Transformation of yeast was achieved by using the lithium acetate method of Ito et al.(17) .

Overexpression of ARF1 was previously shown to be harmful or lethal to yeast cells(11) . This deleterious effect on vegetative growth can be seen when overexpression is achieved either by maintenance of an ARF1 gene on a high-copy, 2µ plasmid or by expression from the stronger, GAL1 promoter. The pGAL1-Arf-X plasmids were transformed into both a wild type strain, PSY315, and an arf1 strain, TT104 (isogenic to PSY315 in all other respects), and plated on selective medium using glucose as carbon source. Transformants were replica-plated to selective plates, YPD, and YPGal plates and growth was scored on days 2-4.

Sensitivity to fluoride was tested by plating transformants on selective medium, YPD, and YPD plates containing 40 mM sodium fluoride (YPD/F) and scoring for growth after 2-4 days. YPD/F plates were used within 1 week after pouring as they become toxic to all yeast strains after this time. The reason for this is not understood, although it may involve chelation of one or more metals by these high concentrations of fluoride ions.

Production of Recombinant Arf Proteins

Wild type and mutants of ARF1 were subcloned into the pET3C vector (15) to allow the high level expression of Arf proteins in bacteria, as described previously for mammalian Arf proteins(18, 19) . Each of the yeast Arf proteins was expressed at an estimated 5-10% of bacterial cell protein. Purification was achieved as described previously (19) by sequential batch elution from DEAE-Sephacel and gel filtration chomatography on Ultrogel AcA 54 in a buffer containing 20 mM Tris-Cl, pH 7.4, 100 mM NaCl, 2 mM MgCl(2), and 1 mM dithiotheitol. Purified proteins were concentrated to approximately 1 mg/ml and stored at -80 °C. Yields were typically 2-5 mg of purified protein/liter of bacterial culture.

Biochemical Assays

Assays of Arf activity(20) , GTPS binding(20) , and GDP dissociation (18) were performed as described previously. The Arf assay measures the cholera toxin-dependent ADP-ribosylation of G(s), using [alpha-P]NAD and recombinant G(s)alpha (with added bovine brain Gbeta) as substrates. Specific activities were determined by normalizing Arf activity to GTPS binding sites on Arf; determined independently under identical conditions. Units of Arf activity are picomole of ADP-ribose incorporated into G(s)alpha per picomole of ArfbulletGTPS in a 20-min reaction.

Stoichiometry of GTPS binding was determined by trapping bound nucleotide on BA85 nitrocellulose filters after 20 min at 30 °C in a binding reaction that included 1 µM Arf, 25 mM HEPES, pH 7.4, 100 mM NaCl, 0.5 mM MgCl(2), 1 mM EDTA, 1 mM dithiotheitol, 3 mM dimyristoyl-L-alpha-phosphatidylcholine, 0.1% sodium cholate, and 10 µM [S]GTPS (approx10,000 cpm/pmol).

Dissociation rates of GDP were determined after loading protein with [^3H]GDP. Arf1 (5 µM) was incubated with 5 µM [^3H]GDP (approx20,000 cpm/pmol) for 1 h at 30 °C in a reaction containing 25 mM HEPES, pH 7.4, 100 mM NaCl, 0.5 mM MgCl(2), 1 mM EDTA, 3 mM dimyristoyl-L-alpha-phosphatidylcholine, and 0.1% sodium cholate. Protein was diluted 10-fold into the same buffer but containing 0.5 mM unlabeled GDP and 50 µg/ml bovine serum albumin. Duplicate 10-µl samples were taken at 10 time points between 0 and 20 min by dilution into 2 ml of ice-cold 20 mM Tris-Cl, pH 8.0, 100 mM NaCl, 10 MgCl(2), 1 mM dithiotheitol. Protein bound [^3H]GDP was determined after trapping on nitrocellulose as described previously (21) . Dissociation rates were calculated using the UltraFit program (Biosoft, Cambridge, United Kingdom). Curves were well fitted to equations for single exponential decay with an offset.

Immunoprecipitation of Yeast Arf and Determination of the Bound Nucleotide

Polyclonal antibody, R-40, was raised in a rabbit after injection of purified recombinant S. cerevisiae Arf1, as described in Stearns et al.(10) . This antibody specifically recognized both yeast Arf1 and Arf2 in immunoblotting experiments and immunoprecipitated both Arf proteins metabolically labeled with [S]methionine. Specific immunoprecipitation of Arf proteins was achieved with R-40 in the absence of detergents. Normal rabbit serum was used as a negative control in the immunoprecipitation experiments. Yeast cells were grown in S.D. medium or switched to SGal medium for 6 h. Cells were collected by centrifugation and incubated in phosphate-free S.D. for 30 min before adding 0.1 mCi/ml carrier-free P(i). After 3 h at 30 °C, cells were harvested by centrifugation, washed once with water, resuspended in ice-cold lysis buffer (25 mM Tris-Cl, pH 7.5, 20 mM MgCl(2)), and lysed by vortexing in the presence of glass beads. Lysates were cleared by centrifugation at 14,000 times g for 15 min at 4 °C. Supernatants were incubated for 1 h at 4 °C with antiserum or normal rabbit serum and for an additional 1 h in the presence of protein G-agarose. Precipitates were collected by centrifugation at 14,000 times g and washed three times with lysis buffer before resuspension in 20 µl of 20 mM Tris-Cl, pH 7.5, 20 mM EDTA, 2% SDS. The suspension was heated at 65 °C for 5 min before spotting on polyethyleneimine-cellulose thin-layer plates. Separation was achieved using a mobile phase of 1 M formic acid, 1 M LiCl. The migration of GDP and GTP were determined in each experiment with pure nucleotide standards. Controls were performed to insure that hydrolysis of guanine nucleotides did not occur during the analysis.


RESULTS

Eight point mutations were introduced into S. cerevisiae ARF1 and analyzed using a number of specific tests of in vivo and in vitro activities. The mutations and a brief indication of the rationale behind their construction appear in Fig. 2. More details of the rationale for each mutant appear in the relevant sections below. The strategy used in the construction of plasmids used to express wild type and each of the mutants in yeast, using either the ARF1 promoter or the inducible GAL1 promoter, and bacteria is shown in Fig. 1. Table 1lists each of these plasmids and the yeast strains used in the studies described below.


Figure 2: Point mutations introduced into Arf1. The consensus sequences for GTP-binding proteins are indicated in the first line, with those found in members of the Arf family on the line below. Each of the point mutation analyzed in this work are indicated, along with a brief description of the reason for its construction.





N-Myristoylation Is Required for Arf Activity in Vivo

N-Myristoylation occurs on NH(2)-terminal glycine residues after cleavage of the initiating methionine. Thus, mutation of Gly^2 to Ala^2 should completely prevent the acylation of Arf proteins.

Two of the previously defined phenotypes associated with ARF1 expression, the rescue of supersensitivity to fluoride in arf1 cells and inhibition of cell proliferation when overexpressed, were used as in vivo tests of Arf functions. The haploid arf1 strain, TT104, fails to grow on YPD in the presence of 40 mM NaF (YPD/F; Table 2; (11) ). When TT104 cells carried a copy of the ARF1 gene on a centromere-containing plasmid (strain RT135), growth on YPD/F was restored. In contrast, such a plasmid carrying the ARF1 gene with the [G2A] mutation (RT136) failed to allow growth of arf1 cells on YPD/F (see Table 2).



Overexpression of ARF1 is lethal to yeast cells (Table 2; (11) ). The inducible GAL1 promoter was used to express wild type or mutants of ARF1 to high levels. Cells (RT164) carrying a CEN plasmid, directing the expression of wild type ARF1 under control of the GAL1 promoter, grew well on S.D. (synthetic medium with dextrose) but not on SGal (synthetic medium with galactose), which induces the GAL1 promoter. In contrast, overexpression of the [G2A] mutant had no apparent effect on cell growth (Table 2). Immunoblots of cell lysates with an antibody directed against yeast Arf proteins confirmed that RT164 cells grown on SGal expressed much higher levels of Arf1 than either the RT152 control or any of the strains grown on glucose. These data confirmed the previous observations (11) that overexpression of wild type Arf1 was harmful to cells and sensitivity to 40 mM NaF resulted from loss of Arf1 function and indicate that the [G2A] mutant is a null allele of ARF1.

Yeast Arf proteins were expressed in bacteria and several milligrams of each were obtained, using previously established procedures(19) . N-Myristoylation of the yeast Arf1 in bacteria was achieved by the co-expression of Arf with the yeast N-myristoyltransferase gene, NMT1(22, 23) . The covalent incorporation of myristic acid into Arf proteins expressed in bacteria was monitored by inclusion of [^3H]myristic acid in the culture medium. Although the Arf1 protein was efficiently myristoylated in bacteria expressing N-myristoyltransferase, the [G2A]Arf1 protein failed to incorporate any labeled myristate (data not shown). Thus, the [G2A] point mutation completely prevented N-myristoylation. Biochemical analyses of non-myristoylated wild type and [G2A]Arf1 revealed no differences in the rates of GDP dissociation, the steady-state level of GTPS binding, or specific activities in the cholera toxin-dependent ADP-ribosylation of G(s) (Arf assay; see Table 3). Thus, the only apparent consequence of mutation of Gly^2 to Ala^2 was to prevent N-myristoylation of Arf1. However, this single change was responsible for the complete loss of in vivo activity.



For comparative purposes the non-myristoylated forms of each of the other mutant proteins were analyzed below, as the extent of myristoylation of recombinant proteins expressed in bacteria can vary dramatically.

Mutations of Asp Resulted in Arf Proteins with Decreased Affinity for GTP

The residue in the first consensus GTP binding domain of Arf1, homologous to Gly of Ras, is Asp. We introduced three different point mutations at residue 26; glycine, alanine, or valine. Like the [G2A]Arf1 mutant, each of these three mutants lacked Arf1 activities in cells. Specifically, they neither restored resistance of arf1 disrupted cells to 40 mM NaF when present on a low-copy plasmid, nor inhibited cell proliferation when overexpressed in ARF1 cells (see Table 2).

Each of the Asp mutants was expressed in and the proteins were purified from bacteria, as described under ``Materials and Methods.'' All three mutant proteins were found to have specific activities in the Arf assay that were slightly higher than that of wild type Arf1 (Table 3). The three mutants all behaved very similarly and the range of activities were 200-500% that of wild type, determined in at least four different experiments. It is important to note that activity in the Arf assay is normalized to GTPS binding sites to determine the specific activities.

The binding of radiolabeled GDP to the Asp mutants was also not much different from that of wild type Arf1, although the GDP off-rate from [D26V]Arf1 was found to be slower than wild type and the other Asp mutants (Table 3). However, the slowly hydrolyzable analog of GTP, GTPS, bound very poorly to each of these three Asp mutants ( Table 3and Fig. 3). Thus, Asp is critical for high affinity binding of the activating nucleotide. Determination of specific activity in the Arf assay takes into account this low level of binding of GTPS as the activity is normalized to GTPS binding sites. It is the switch to this active conformation that appears to be impaired by mutations at position 26.


Figure 3: Mutation of Asp results in loss of GTPS but not GDP binding. GDP (upper panel) and GTPS (lower panel) binding to Arf1 (circles), [D26G]Arf1 (squares), [D26A]Arf1 (triangles), and [D26V]Arf1 (inverted triangles) was determined as a function of time at 30 °C, as described under ``Materials and Methods.'' The curves drawn are from first-order rate equations fit to the data by the Marquardt algorithm.



[Q71L] Is a Dominant Activating Mutation

Mutations of glutamine in the consensus GTP binding domain, DXXGQ, in both alpha subunits of heterotrimeric G proteins (24, 25) and in Ras (26) lead to activation of the proteins as a result of decreased intrinsic, or GAP stimulated-GTPase activities, respectively. The corresponding residue in Arf proteins, Gln, was mutated to leucine in Arf1 and subjected to functional analyses. Transformants carrying plasmid pJCY1-40, directing expression of [Q71L]Arf1 under regulation by the GAL1 promoter, grew well on S.D. but were killed when plated on SGal (Table 2). As described above, this is the same result observed with the overexpression of Arf1. However, when the same plasmid was used to transform the arf1 null cells, TT104, it was apparent that overexpression of this allele is more toxic than is the wild type protein. We were unable to obtain transformants of either ARF1 (PSY315) or arf1 (TT104) cells with pArf1-[Q71L]Arf1. Increased lethality over that of wild type Arf1 and failure to obtain transformants that express the mutant under control of the ARF1 promoter are consistent with the [Q71L] mutation being a dominant lethal allele of ARF1.

Nucleotide exchange on [Q71L]Arf1 was comparable to that of Arf1, with an apparent first-order GDP off-rate of 0.535 min (see Table 3). The specific activity in the Arf assay of [Q71L]Arf1 was also quite similar to the wild type protein. It should be noted that hydrolysis of GTP by Arf is not required for activity in the Arf assay(27) . Thus, the dominant activation of the [Q71L]Arf1 in cells is not readily explained by changes in the intrinsic nucleotide handling of the purified protein. It was not possible to test for a decrease in this rate, as has been found for homologous mutations in other GTP-binding proteins, as Arf proteins, including yeast Arfs (data not shown), have no intrinsic GTPase activity(27) .

Expression of [Q71L]Arf1 in Yeast Cells Resulted in the Accumulation of Activated (GTP-bound) Arf

Immunoprecipitation of Arf proteins from yeast was accomplished using an antiserum, R-40, from a rabbit immunized with purified, recombinant S. cerevisiae Arf1, as described under ``Materials and Methods.'' Labeling of yeast cells with 0.1 mCi/ml P(i) resulted in the optimal incorporation of radioactivity into guanine nucleotides bound to Arf proteins, detectable after immunoprecipitation with antibody R-40 and denaturation. Analysis of the guanine nucleotides obtained from immunoprecipitates from wild type cells by thin layer chomatography revealed that virtually all of the nucleotide precipitated with the Arf was GDP. Studies of wild type and point mutants induced for expression by 9 h of growth in SGal revealed almost exclusively GDP bound to the Arf in most cases. Small amounts of GTP were observed in cells overexpressing Arf1 and [W66R]Arf1. Cells expressing excess Arf (e.g. [G2A]Arf1) yielded more detectable P-labeled GDP in immunoprecipitates than that found in control strains. Analysis of bound nucleotides in strain RT132, after induction of [Q71L]Arf1 for 9 h, revealed that over half of the bound nucleotide (60-80%) was GTP. The accumulation of a large fraction of activated Arf in cells expressing the [Q71L] mutant and lack of intrinsic GTPase activity of both wild type and mutant Arf1 are consistent with the presence of an Arf GTPase-activating protein (Arf GAP) in yeast which promotes the hydrolysis of GTP bound to wild type Arf but is much less effective on the [Q71L] mutant, as has been shown for the [Q61L] mutant and Ras GAP(28) .

It is worth noting that ARF, immunoprecipitated from cells labeled with radioactive inorganic phosphate, failed to incorporate any covalently attached phosphate. Thus, if Arf is a substrate for any yeast protein kinase the extent of modification was so low as to be undetectable, or the phosphate linkage was labile under the conditions used in these studies.

[N126I]Arf1 Is a Dominant Lethal Allele

The asparagine residue in the consensus GTP binding domain, NKXD, of regulatory GTP-binding proteins is involved in coordination of the guanine base in both Ras and the alpha subunit of transducin, G(t)alpha(29) . Mutation of this residue to isoleucine results in a dominant loss of function of SEC4(30) . The corresponding mutation in Arf1 is [N126I]. We were unable to obtain strains of yeast carrying [N126I]Arf1 under control of the ARF1 promoter. While other transformants with the homologous plasmid could be seen as colonies within 3-5 days of transformation, the cells transfected with pJCY1-61 grew to very small colonies that could be seen only after a week or more of growth at 30 °C. However, these micro-colonies failed to grow when streaked on fresh plates. Transformants containing [N126I]Arf1 under control of the GAL1 promoter grew well on S.D. but very slowly or not at all on SGal. Thus, induction of the expression of [N126I]Arf1 was lethal and this likely explains our failure to obtain transformants expressing this allele under the constitutive ARF1 promoter.

High level expression of [N126I]Arf1 in bacteria was readily achieved using the same expression system used for the other mutants. However, of the 10 alleles of S. cerevisiae ARF1 expressed in bacteria, this was the only one which was found to be insoluble in bacterial lysates. Solubilization was achieved with 7 M urea, but removal of chaotrope failed to allow refolding of protein into a conformation that bound radionucleotides. Such a procedure has been previously shown to allow renaturation of recombinant human/bovine Arf1(18) . Thus, we have no biochemical data on this mutant.

[W66R]Arf1 Is a Conditional Allele That Is Defective in Arf Activity

One mutation was constructed with the specific goal of making a conditional allele of ARF1 suitable for genetic suppressor analysis. The rationale for this mutation, [W66R], stems from the observation that the corresponding mutation in the structurally related GTP-binding protein from S. cerevisiae, CIN4, was a conditional allele that allowed the isolation of suppressors of the supersensitivity to the antimicrotubule drug, benomyl. (^3)The residue being changed in this case is immediately NH(2)-terminal to the second consensus GTP binding domain, DVGGQ, found in all Arf and heterotrimeric G protein alpha subunits and homologous to DTAGQ in the other families in the Ras superfamily.

Overexpression of the [W66R]Arf1 protein was clearly deleterious to PSY315 cells, although less so than was the wild type protein (see Table 2). The centromere containing plasmid, pJCY1-62, carrying this mutant behind the ARF1 promoter was capable of restoring growth to TT104 cells plated on YPD/F. However, wild type ARF1 rescued growth of TT104 cells at both 30 and 37 °C, but the [W66R]ARF1 allele failed to allow growth on YPD/F at the elevated temperature.

The binding of both GDP and GTPS to [W66R]Arf1 were similar to that of the wild type protein, although the rate of dissociation of GDP from the mutant (0.86 min) was almost twice that from the wild type protein (0.48 min) in submillimolar concentrations of magnesium. A larger difference (approx7-fold) in the rate of GDP dissociation was observed in the presence of 2 mM Mg; 0.0098 min and 0.068 min for wild type Arf1 and [W66R]Arf1, respectively.

Comparison of the specific activities in the Arf assay revealed this mutant to be more than 300-fold less active as cofactor in the cholera toxin assay when compared to the Arf1 (see Table 3). As the stoichiometry of binding of GTPS to [W66R]Arf1 was similar to wild type, the low specific activity reflects a loss of productive protein:protein interaction and not decreased ability to bind the activating guanine nucleotide.

Cloning of High Copy Suppressors of the Temperature-sensitive Lethality of arf1-2 Cells Grown on YPD/F

As described above, the centromere containing plasmid carrying the [W66R]Arf1 mutant under control of the ARF1 promoter was found to be temperature-sensitive for suppression of lethality on YPD/F. A haploid strain, RT269, was constructed in which the [W66R]Arf1 mutation (arf1-2) was introduced at the ARF1 locus by gene replacement. Temperature sensitivity of growth on YPD/F was retained in RT269. Growth of RT269 on YPD/F at 37 °C was tightly linked to ARF1 as it was restored by transformation with a wild type ARF1 gene on the centromere containing plasmid, pJCY1-31. Transformation of RT269 cells with a YCp50-based (CEN-containing) genomic S. cerevisiae library and selection on YPD/F at 37 °C led to the cloning of both ARF1 and ARF2 many times, but no other suppressors were detected by this protocol.

High-copy suppressors of arf1-2 were cloned by transforming RT269 with a YEp24 based (2µ genomic S. cerevisiae) library marked with the URA3 gene, after replica plating transformants onto YPD/F at 37 °C. ARF2 and two extragenic suppressors were isolated from this screen, termed suppressors of fluoride sensitivity (SFS3 and SFS4), are described. Each restored growth to RT269 cells grown at 37 °C on YPD/F. Neither suppressor was able to suppress temperature sensitivity to fluoride when present on a low-copy, centromere containing plasmid. Suppression was tightly linked to the SFS genes as sensitivity was restored when library plasmids were lost, by selection on 5-fluoroorotic acid. The level of expression of ARF2 was also examined to determine if suppression resulted from increased expression of that gene. No differences were observed in the level of ARF2 expression in strains carrying the SFS genes on 2µ plasmids.

The smallest genomic fragment with SFS3 activity was a 3765-bp PvuII-SalI fragment that mapped to chomosome XV. The only open reading frame greater than 400 bp in length encodes a predicted protein of 444 amino acids. A search of the Swiss-Prot data bank with FASTA revealed that this open reading frame was identical in predicted protein sequence to that of STD1, a high-copy suppressor of a negative dominant mutation in the TATA-binding protein and a putative transcription factor(31) .

The other high-copy suppressor of arf1-2, termed SFS4, was located on a 4500-bp SstI-BamHI fragment and mapped to chomosome VII. Translation of this fragment in all six reading frames revealed a single open reading frame of greater than 100 amino acids. An open reading frame of 2004 bp was present, which encodes a predicted protein of 668 amino acids. A search of SWISS-PROT revealed near identity (^4)with the predicted sequence of PBS2/HOG4, originally identified as a high-copy suppressor of sensitivity to the polyamine antibiotic polymyxin B(32) . More recently, this protein has been identified as a member of the microtubule-associated protein kinase kinase family involved in a signal transduction pathway that is sensitive to changes in the osmolarity of the extracellular environment(33) .

Cys Is Not Required for Arf Activity in Cells

Cys is the only cysteine residue in Arf1. The cysteine in the sequence TCAT, conserved in both Arf proteins and G protein alpha subunits, has previously been implicated in coupling the latter to receptors(34) . Conservative substitution of this residue was made to test for the potential role of disulfide bond formation or other interactions involving the sulfhydryl group on Arf activity in vivo. As seen in Table 2, this allele was indistinguishable from wild type ARF1 with respect to the rescue of TT104 cells on fluoride plates and in lethality when overexpressed using the GAL1 promoter. Some differences were noted in the guanine nucleotide exchange of the [C159S]Arf1 protein, when compared to wild type. This may result from local changes in the environment of Ala, which is conserved in all GTP-binding proteins and is important in coordination of the guanine nucleotide.

S. cerevisiae Arf1 binds guanine nucleotides more rapidly and with higher stoichiometry than mammalian Arf proteins.The purified recombinant yeast Arf1 protein has previously been shown to bind guanine nucleotides with high affinity and have activity in the (cholera toxin) Arf assay(4) . Further characterization of the nucleotide exchange rate revealed that the yeast protein has a much higher rate of GDP dissociation (k = 0.48 min) than does human Arf1 (k = 0.023 min). This decreased affinity for GDP likely contributes to the higher stoichiometries of GTPS binding observed for the yeast Arf1 protein (typically 20-60%) compared to the human Arf1 protein (typically 1-5%).


DISCUSSION

Expression and functional analyses of point mutants of Arf1 were undertaken to analyze the properties of an ARF family member both in vivo and in vitro. These analyses revealed the essential nature of N-myristoylation of Arf proteins to their functions in cells. Mutational analysis of the guanine nucleotide binding site revealed some similarities to other GTP-binding proteins but also some differences. A conditional allele, arf1-2, was found which allowed suppressor analysis. Genetic evidence suggesting a common signaling pathway which includes both an Arf and a microtubule-associated protein kinase kinase are intriguing in light of the recent demonstrations of the role Ras proteins play in similar kinase cascades(35, 36) .

N-Myristoylation of Arf was shown to be an essential processing step and required for activity in vivo. In contrast, N-myristoylation is required for only some of the activities of Arf proteins in in vitro assays. The need for N-myristoylation of ARF1 to regulate membrane traffic has previously been described after analyses of transient overexpression of the [G2A]ARF1 mutant in HeLa cells(12) . The ability of Arf to serve as cofactor in the cholera toxin assay and dependence of GTP binding on phospholipids are observed for both the acylated and unmodified proteins(18) . However, activation of phospholipase D activity(7) , guanine nucleotide regulated binding of Arf to membranes and lipid micelles(37) , and GTPS dependent inhibition of ER-Golgi (38) and intra-Golgi transport(39) , and endosome fusion (40) assays are all highly dependent on myristoylation of Arf. As the nucleotide-regulated binding of Arf to micelles and membranes requires N-myristoylation, we conclude that reversible membrane association, regulated by guanine nucleotide binding, plays an essential role in the action of Arf in cells.

What is the role of the covalently attached myristate in Arf action? In the GDP-bound state, it is likely that the myristate moiety is not exposed to solvent as we have been unable to resolve the processed and unprocessed forms of Arf on a variety of chomatography resins. (^5)Such a structural role for myristate has previously been demonstrated for the catalytic subunit of the cAMP-dependent protein kinase, in which the fatty acid is buried inside the protein, resulting in increased thermal stability and ordering of the amino terminus(41, 42) . N-Myristoylation of G(o)alpha was found to increase its affinity for Gbeta(43) . Upon binding GTP the Arf protein acquires increased affinity for micelles and membranes, in a process that is highly dependent on N-myristoylation. Thus, it is reasonable to conclude that the myristate becomes much more exposed and may be directly involved in the membrane association of the active protein. Limiting the protein to the two-dimensional space of the bilayer may be sufficient to increase protein:protein interaction and result in an apparent increase in affinity of Arf for its effector. There is currently no evidence that the myristate is directly involved in the interaction of Arf with any protein effector, receptor, or modulator; e.g. Arf GAP.

Functional effects of mutation of Asp were quite different from predictions, based on homologous mutations made in p21 or alpha subunits of G proteins. The [D26G/A/V]Arf1 proteins display unaltered binding of GDP but a dramatically decreased affinity for GTPS. However, the specific activities of the Asp mutants in the Arf assay are comparable to wild type. Thus, the mutant proteins are less able to bind GTP but once activated, appear to have full activity. In contrast, mutations in the homologous residue of Ras (Gly) increase transforming activity, likely as a result of decreased intrinsic GTPase activity(44) . This allele of Ras binds both guanine nucleotides and GAP with similar affinity to the wild type protein. Similar to Ras, a homologous mutant of the alpha subunit of G(s) ([G49V]) is a potent activator of adenylate cyclase with a 5-fold lower intrinsic GTPase rate and a 7-fold increase in fractional occupancy by GTP; while the GDP dissociation rate is unchanged(24) . Interestingly, bacterial EF-Tu, like Arf, lacks a glycine at the corresponding position (Val) in the first consensus GTP binding domain. However, the [V20G] mutant of EF-Tu has dramatic consequences on nucleotide binding and GTPase rates quite different from what was seen with [D26G/A/V]ARF1. This mutant EF-Tu has 20- and 10-fold increases in GDP dissociation and association rates, respectively, which leads to a higher affinity for GTP, in comparison to wild type(45) . In addition, the intrinsic GTPase activity of the mutant is reduced about 5-fold. Arf1 differs from these other GTP-binding proteins in having severely reduced affinity for GTP after mutation of this residue. The absolute conservation of Asp in this concensus GTP binding domain of members of the Arf family further suggests that this unique property is important to the actions of this subfamily of regulatory proteins.

Changing the conserved glutamine in the consensus GTP binding domain, DXXGQ, to leucine is a dominant activating mutation among most GTP-binding proteins, including Ras (Gln) and G(s)alpha (Gln), but not SEC4(30) . The homologous mutation in Arf1 is a dominant lethal allele which leads to an accumulation of the active form of the protein in yeast cells. The biochemical properties of the purified recombinant [Q71L]Arf1 protein are not much different from wild type. This is similar to what has been observed for the homologous mutation in Ras and suggests that the most important consequence of this point mutation may be increased affinity for, but decreased sensitivity to, Arf GAP(46) . Preliminary evidence supports this conclusion as human [Q71L]Arf3 is a potent inhibitor of partially purified bovine brain Arf GAP. (^6)Thus, the dominant nature of this mutant may be explained by the activity of the mutant protein, by the increased activity of wild type proteins which share a common GAP (and which would become activated as a result of the inhibition of that GAP by the mutant), or by some combination of these two events.

The asparagine in the conserved NKXD motif is known to be involved in binding the guanine base in other GTP-binding proteins, including Ras and G(t)alpha. This is likely true of Arf1 as well, as the homologous Arf mutant was expressed at high levels but unable to bind nucleotides. The [N126I]Arf1 mutant appears to be a partial dominant allele in yeast as it is harmful to the vegetative growth of both wild type and arf1 cells. Either increased or decreased expression of Arf is deleterious to the growth of yeast cells. However, as the effect of this mutant appears more marked in the Arf1 deficient cells, it is a negative dominant allele. Our working hypothesis is that the decreased affinity for guanine nucleotides results in an accumulation of the apo-protein. The nucleotide-free form of other GTP-binding proteins has been proposed to have high affinity for guanine nucleotide exchange factors(47, 48) . Thus, the negative dominance may result from the decrease in activated Arf proteins, resulting from the inhibition of Arf exchange factor(s).

The [W66R]Arf1 mutant was found to have decreased affinity for GDP and Mg, resulting in higher stoichiometries of GTPS binding. Surprisingly, the specific activity of this protein was approx300-fold lower than that of the wild type protein. Thus, in contrast to the Asp mutants which bind GTP poorly but retain wild type specific activities, this mutant binds GTP better than wild type yet remains inactive. Possible explanations for these observations include an essential role for Trp in the effector binding site or in interactions with other parts of the protein that are required for formation of the active conformation. The importance of Trp in the activation process makes it a likely major contributor to the large change in intrinsic fluoresence that has previously been shown to accompany the binding of GTP(27) . The ability to separate GTP binding from formation of the active conformer may prove useful in the further dissection of the molecular mechanisms of Arf regulation.

Suppressor analysis of arf1-2 led to the isolation of genes that permitted growth of RT269 cells on YPD/F at 37 °C. The identification of SFS4 as PBS2/HOG2, a microtubule-associated protein kinase kinase, is particularly intriguing in light of the recent work documenting the role of Ras in triggering the microtubule-associated protein kinase cascade by localizing the kinases to the plasma membrane in higher eukaryotes. Curiously, the roles for RAS genes in S. cerevisiae has been shown to be intimately associated with the regulation of adenylate cyclase and not protein kinase activation. It will likely prove interesting to determine if other similarities exist between the Arf and Ras signaling pathways in mammals and yeast.

The identification of SFS3 as identical to STD1 suggests that this putative transcription factor may regulate the expression of one or more of the components in an Arf signaling pathway. The immunoblotting to test for the level of expression of ARF2 in strains carrying SFS genes is probably not sensitive enough to rule out ARF2 as a possible target for SFS3/STD1. Alternatively, it might prove fruitful to identify other genes whose expression is affected by SFS3/STD1 and test them as potential modulators of Arf action.

Results from these studies further reinforce the conclusion that the Arf proteins have distinctive structural and functional features compared to other members of the RAS superfamily of low molecular weight GTP-binding proteins. The crystal structure of human Arf1/GDP has recently been solved (^7)(49) and will clearly provide a wealth of new information and suggest hypotheses which may be tested using variations of the protocols described in these studies.


FOOTNOTES

*
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: Bldg. 37, Rm. 5D-02, Bethesda, MD 20892.

(^1)
The abbreviations used are: Arf, ADP-ribosylation factor; Arl, Arf-like; GAP, GTPase activating protein; S.D., synthetic medium with 2% dextrose as carbon source; SGal, synthetic medium with 2% galactose as carbon source; bp, base pair(s); GTPS, guanosine 5`-3-O-(thio)triphosphate; SFS, suppressor of fluoride sensitivity.

(^2)
A. Boman and K. Wilson, unpublished observation.

(^3)
T. Stearns, M. A. Hoyt, and D. Botstein, unpublished observation.

(^4)
Two regions of non-identity were discovered when SFS4 was aligned with the published sequence of PBS2. We obtained the original PBS2 plasmid (YEp24.PBS2) from Dr. George Boguslawski, who first described this gene. Sequencing of the regions in dispute revealed errors in the published sequence, resulting in changes in residues 222-223 to Gly-Leu (instead of Ala-Val) and a stop codon after Leu, resulting in a predicted protein that is 42-amino acids shorter (668 residues). The sequence of the 4500-bp SstI-BamHI genomic fragment has been submitted to Genbank and has been assigned the accession number U12237[GenBank].

(^5)
R. A. Kahn, unpublished observation.

(^6)
P. A. Randazzo and R. A. Kahn, unpublished observation.

(^7)
Amor, J. C., Harrison, D. H., Kahn, R. A., and Ringe, D.(1994) Nature, in press.


ACKNOWLEDGEMENTS

We thank Dr. Mark Johnston for his gift of pBM272, and helpful advice on its use in yeast cells during the course of these studies, and Dr. William Studier for his generous gift of pET3C plasmid and BL21(DE3) cells, used for expressing Arf proteins in bacteria. Dr. Jeffrey Gordon and Dr. Jenifer Lodge (Washington University, St. Louis, MO) provided plasmids allowing expression of N-myristoyltransferase in bacteria and invaluable advice. We also thank Dr. George Boguslawski for sharing information concerning PBS2 as well as his clone containing this gene. The continued support of the Developmental Therapeutics Program in the Division of Cancer Treatment is also gratefully acknowledged.


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