©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure and Function of ARF Proteins: Activators of Cholera Toxin and Critical Components of Intracellular Vesicular Transport Processes (*)

Joel Moss , Martha Vaughan

From the (1) Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

INTRODUCTION
ARF Structure
ARF Function in Vesicular Transport
Afterword
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


INTRODUCTION

During the 1980s, several cellular factors that enhanced cholera toxin-catalyzed ADP-ribosylation of G or activation of adenylyl cyclase were described. Kahn and Gilman (1) reported the first purification of an 20-kDa membrane-associated protein that enhanced ADP-ribosylation of G and named it ADP-ribosylation factor or ARF.() Two soluble ARFs purified later stimulated toxin-catalyzed ADP-ribosylation of G and simple guanidino compounds (e.g. arginine) as well as toxin auto-ADP-ribosylation in a GTP-dependent manner (2) . It was shown in kinetic studies that ARF interacted with the toxin catalytic subunit to lower the apparent K for both ADP-ribose donor (NAD) and acceptor (agmatine) (3) . Thus, ARF seemed to be an allosteric activator of the toxin, active with GTP bound, and inactive with bound GDP. Whether ARF has an effect on cholera toxin activity in intoxicated cells remains unknown, but its more recently recognized physiological actions are clearly quite different.

Because of limitations of space, this Minireview deals only with the functions of ARF in eukaryotic cells, where these proteins have a critical role in vesicular membrane trafficking in both exocytic and endocytic pathways, and the relationship of ARF structure to its biochemical actions. Recent, more complete reviews (e.g. Ref. 4) consider also ARF cDNA and genomic sequences, as well as data on distribution and expression of ARF proteins and mRNAs.


ARF Structure

ARFs are a multigene family, structurally related to ARF-like proteins (ARLs) that do not activate cholera toxin (4, 5) . Based on size, amino acid sequences (deduced from cDNA sequences), phylogenetic analysis, and gene structure (4) , mammalian ARFs (Fig. 1)() fall into three classes, ARF1, ARF2, and ARF3 in class I, ARF4 and ARF5 in class II, and ARF6 in class III (6) . In vitro, mammalian ARFs from all three classes and ARFs from all eukaryotic organisms activated cholera toxin, which has been, by definition, a requirement for the designation ``ARF.'' Class I ARFs are the best known and most closely resemble ARFs identified in other species. Relatively recently, however, evidence for the presence of class II and III ARFs in Drosophila melanogaster(7) and a possible class III ARF in Saccharomyces cerevisiae(8) has been published.


Figure 1: Deduced amino acid sequences of human ARL1 and mammalian ARFs. Sources of ARF sequences are in Ref. 4; human ARL1 (hARL1) sequence is in Ref. 28. The amino acid number is above the hARL1 sequence. Amino acids identical in hARL1 and hARF1 are indicated with a verticalline; those amino acids in other ARFs identical with hARF1 are indicated with an asterisk. Four consensus sequences (I-IV) believed to be involved in guanine nucleotide binding and hydrolysis are shaded yellow; G2, the site of N-terminal myristoylation after co-translational removal of methionine, is shaded blue.



All ARFs contain consensus amino acid sequences (Fig. 1) involved in GTP binding and hydrolysis, critical functions that determine ARF activity. These are identical in the mammalian ARFs and have very few, for the most part conservative, differences in yeast or Giardia ARFs (4) . It has often been noted that in these ``signature'' sequences, as well as in the myristoylation of an N-terminal glycine, the ARFs are more similar to heterotrimeric G protein subunits than they are to other 20-kDa Ras-like proteins (9). It has also been suggested that ARF may be an ancestor of both of those protein families, as it is present in the primitive parasite Giardia lamblia that apparently lacks G subunits (4) .

Mutations in GTP-binding Sequences

Mutations in several of the consensus sequences for GTP binding and hydrolysis have been introduced to evaluate their effects on ARF functions. Replacement of Gln-71 with Leu produces a mutant ARF1(Q71L) that lacks the ability to hydrolyze GTP and is thus permanently active. Overexpression of ARF1(Q71L) in HeLa (10) or normal rat kidney (11) cells inhibited endoplasmic reticulum to Golgi and intra-Golgi transport, respectively. In normal rat kidney cells, it also inhibited endocytosis (11) . Inhibition by the activated ARF mutant presumably resulted from accumulation of vesicles unable to fuse with target membranes, because the failure to inactivate ARF prevented uncoating (Fig. 2). This was shown in vitro using Golgi membranes and ARF1(Q71L) synthesized in Escherichia coli, as well as in COS-1 cells expressing the mutant (12) . Myristoylation, believed necessary for membrane association, was required for inhibition by the mutant; expression of a double mutant that could not be myristoylated (ARF1(G2A,Q71L)) was without effect (10, 12) .


Figure 2: Role of ARF in vesicular transport. This simplified scheme is discussed, with references, in the text. Because of space limitation, we have not considered the so-called SNARE complexes (35), although it seems widely accepted that this sort of molecular machinery, including the N-ethylmaleimide-sensitive ATPase, functions in many types of vesicular transport systems. Likewise omitted is discussion of the other 20-kDa GTP-binding proteins (e.g. Rabs) and heterotrimeric G proteins (46), which are almost surely involved in specific transport processes, as are cytoskeletal elements (e.g. microtubules) and the obviously necessary processes of retrograde transport for membrane retrieval. PLD, phospholipase D; PC, phosphatidylcholine; PIP, phosphatidylinositol 4-phosphate; PIPK, phosphatidylinositol 4-phosphate 5-kinase; PA, phosphatidic acid.



The mutant ARF1(Q71L) was prepared because replacement of the apparently analogous Gln (in the sequence DXXGQ) in Ras and G subunits yielded constitutively activated proteins. Based on the crystal structure of ARF1-GDP (13) , however, Gln-71 does not correspond in a three-dimensional position to Gln-61 of Ras, with which it is aligned in the linear sequence. ARF-GDP, which crystallized as a dimer, differs in this and several striking ways from the other structures (13) . It is clear that extrapolation of structure-function relationships from Ras, for example, to ARF should be approached with caution.

Other mutants expressed in HeLa cells (10) were designed either to exist chiefly in the GDP-bound state, ARF1(T31N), or to have an altered rate of nucleotide exchange due to replacements in the third nucleotide-binding sequence, ARF1(N126I) or ARF1(D129N). It was suggested that ARF1(T31N), which preferentially binds GDP, prevents activation of endogenous ARF by interacting with an ARF-GEP (guanine nucleotide-exchange protein), effectively inactivating it (10) . Myristoylation was again required for this effect (10) . Mutations in the third nucleotide-binding sequence, which also result in preferential GDP binding, similarly produced proteins with dominant negative activity, although the effects on cell morphology differed from those of the T31N mutant (10) .

Myristoylation is clearly important for ARF function, although non-myristoylated ARF proteins can be active in several in vitro assays. Recently, improved conditions for synthesis of myristoylated recombinant ARF1 in E. coli by co-expression of yeast N-myristoyltransferase and its purification to yield relatively homogeneous myr-rARF1 facilitated evaluation of effects of myristoylation (14) . Comparison of rARF1 (non-myristoylated) and myr-rARF1 revealed that the modification markedly affects guanine nucleotide exchange. It was concluded that myr-rARF1-GDP associated with phospholipid more weakly than did myr-rARF1-GTP because it interacted only via myristate, whereas myr-rARF1-GTP interacted through both the fatty acid and the N-terminal amphipathic helix (14) .

N-terminal Mutations

Sequence differences among ARFs tend to be concentrated near the N terminus, and when sequences of the six mammalian ARFs are aligned, ARF6 has a gap corresponding to amino acids 8-11 in the other ARFs (Fig. 1). It has been reported, however, that the ARF N terminus is critical for its activity in vitro and probably in vivo(15) . A recombinant myristoylated ARF1 mutant lacking the first 17 amino acids failed to activate cholera toxin-catalyzed ADP-ribosylation of G or to rescue the yeast arf1arf2 lethal double mutant; it also differed from the native protein in guanine nucleotide-exchange properties (15) . A chimeric protein containing the first 18 amino acids of human ARF1 and amino acids 19-180 of Drosophila ARL1 (ARF18/ARL) was reported to stimulate cholera toxin-catalyzed modification of G(15) . ARL1, first identified in Drosophila by cDNA cloning, is an essential protein of unknown function, very similar in structure to ARF (Fig. 1) but possessing measurable GTPase activity and lacking ARF activity (5) .

To extend these studies, a peptide with the sequence of ARF1 amino acids 2-17 was prepared (15) . With or without myristoylation, it inhibited ARF activation of cholera toxin and Golgi transport (15, 16, 17) as well as endocytosis (18) . It appears, however, that the effects of this peptide may not be as specific as initially believed. The peptide and mastoparan, which also forms an amphipathic helix, inhibited phospholipase D and exocytosis, as well as a G protein-regulated phospholipase C (19) , and demonstrably damaged Golgi membranes (20) . Thus, conclusions about the function of the ARF N terminus, based on experiments with the peptide, appear to be subject to question.

Other mutations of the ARF1 N terminus yielded somewhat different findings. Recombinant ARF1 lacking the first 13 amino acids (r13ARF1) and rPKA14ARF1 (in which the first 14 amino acids were replaced by the first 7 amino acids of cAMP-dependent protein kinase catalytic subunit) activated cholera toxin-catalyzed ADP-ribosylation of agmatine as effectively as did rARF1 (21) . Unlike rARF1, however, their activity was not dependent on added GTP; bound GDP and GTP were identified, thus explaining the activity (22) . After prolonged dialysis against 7 M urea and renaturation, GTP dependence was demonstrated if SDS was present in the assay (21) . In the absence of detergent, the nucleotide-free r13ARF1 was active, i.e. it appeared to assume an active conformation in the absence of bound nucleotide (22) .

It is unclear just how GTP bound to recombinant ARF is hydrolyzed in E. coli, as ARFs lack intrinsic GTPase activity (23) and the bacteria, which lack ARFs, presumably also lack ARF-GAP (GTP-ase-activating protein). Perhaps GTP bound to the mutant ARF is protected from bacterial enzymes that could otherwise degrade it. This explanation was considered after the earlier isolation of a recombinant ARF6 fusion protein in an active state with GTP bound (24) . The stability of the N-terminal deletion mutants to 7 M urea, which partially inactivated rARF1, was consistent with a role for that part of the molecule in overall structure or conformation (21) . Further study of r13ARF1 provided evidence for an additional effect of the N terminus on specificity of nucleotide binding (22) .

Recently, Randazzo et al.(25) compared activities of r13ARF1 and r17ARF1 in the cholera toxin assay with G as substrate and in their responses to ARF-GAP, a partially purified protein (23) that accelerates hydrolysis of ARF-bound GTP. r13ARF1, like r17ARF1, failed to activate the toxin in this assay, although it did, in contrast to r17ARF1, inhibit activation by rARF1. The authors concluded that amino acids 14-17 are involved in binding of ARF1 to the toxin and play a small but significant role in the interaction of ARF1 with its GAP. They also reported that >80% of the nucleotide bound to both mutant proteins was GTP; a marked effect of the N terminus on GDP dissociation was noted (25) .

Structural Requirements for Activation of Cholera Toxin and Phospholipase D

After reports of ARF activation of phospholipase D (26, 27) , structural requirements for activation of toxin and phospholipase D were compared using chimeric proteins based on deduced amino acid sequences of ARF1 and ARL1 (Fig. 1). Recombinant proteins, in which the first 73 amino acids of ARF1 and ARL1 were exchanged (rF73/L and rL73/F), were compared in assays with cholera toxin and phospholipase D under conditions that differed only in the identity of the radiolabel added (28) . ARF1 activated both enzymes; ARL1 did not activate toxin and only slightly stimulated phospholipase D. rF73/L, in which the N-terminal 73 amino acids of ARF replaced those of ARL, failed to activate the toxin, although it activated phospholipase D as effectively as rARF1. Conversely, rL73/F activated phospholipase D only slightly but was much more effective with cholera toxin. Thus, activation domains for phospholipase D and toxin appear to lie, respectively, in the N- and C-terminal portions of ARF. Effects of GTPS concentration on toxin activation by rL73/F and rARF1 with different phospholipids were quite different, perhaps related to the role of the N terminus in nucleotide exchange and binding or in association with phospholipids (28) .

Further comparisons of mutant and native proteins in specific assays of individual interactions are needed to define more precisely the effects of ARF molecular structure on function. Availability of the recently published crystal structure of non-myristoylated ARF1-GDP (13) should facilitate the design and interpretation of these kinds of studies.


ARF Function in Vesicular Transport

It was in S. cerevisiae that ARF was first implicated in Golgi function (9) , and a large part of the information on ARF function in cells is related to vesicular transport in the Golgi (29) , probably involving class I ARFs. ARF has been implicated also in intravesicular acidification and fusion of microsomal vesicles (30), endosome fusion (18) , nuclear membrane assembly (31) , and formation of clathrin-coated vesicles (32) . In vesicular transport from endoplasmic reticulum to Golgi, ARFs are required for binding of -COP, a component of the ``coatomers'' (33) that cover non-clathrin-coated vesicles (32) . In the formation of Golgi-derived clathrin-coated vesicles, ARF is required for binding of the AP-1 adaptor protein (-adaptin), an analogue of -COP (32) . The report of COP II-coated vesicles, in which ARF is replaced by Sar1 (34) , adds to the evidence that multiple pathways of intracellular vesicular transport utilize analogous protein molecules and mechanisms. This is emphasized in an excellent review by Rothman (35) , whose contributions, experimental and conceptual, to understanding these processes have been major. We rely heavily on that, and several other key articles from the Rothman laboratory, for the current view of intracellular vesicular transport, which is summarized in a simplified fashion in Fig. 2.

Vesicle Formation

Soluble ARF proteins are isolated with tightly bound GDP that exchanges relatively slowly with GTP, even in the presence of phospholipids or detergents that accelerate the process when concentrations of Mg are submicromolar. ARF binding to membranes is enhanced when bound GDP is exchanged for GTP. In several systems, ARF binding to membranes is prevented by brefeldin A, a fatty acid derivative that in intact cells causes apparent disintegration of the Golgi (reversibly) and association of Golgi enzymes with endoplasmic reticulum (36). Brefeldin A inhibition of a Golgi-associated activity (37-39), termed here GEP for guanine nucleotide-exchangeprotein, that accelerates replacement of ARF-bound GDP with GTP has been reported. GEP activity in brain cytosol (40) , which was also inhibited by brefeldin A, stimulated binding of ARF1 and ARF3, but not ARF5, to crude Golgi membranes. It also increased binding of GTPS to ARF1 and ARF3. With partial purification of this soluble GEP, its apparent molecular size decreased from >600 to 60 kDa and susceptibility to inhibition by brefeldin A was lost (41) , consistent with the conclusion that some component of the larger complex confers sensitivity to brefeldin A.

Although ARFGTP (but not ARFGDP) will bind to pure phospholipids as well as to cell membranes, its physiological function presumably requires interaction with a particular site at which vesicle formation will take place. Whether this site involves an ARF ``receptor'' protein or is defined in some other way has not been established. Interaction with a specific Golgi protein was suggested after demonstration of a saturable, liposome-resistant pool of bound ARF, in addition to a pool of liposome-extractable ARF (42) . A recent report of ARF binding to G(43) is consistent with a role for the latter as a membrane receptor. The existence of ARF receptor proteins could provide for specificity of binding of individual ARF proteins to the ``correct'' sites for initiation of vesicle formation.

The mechanism by which ARFGTP bound to its putative receptor causes binding of large protein complexes termed coatomers (33) , whether by interacting with a coatomer protein or by an effect on composition of the adjacent membrane, is unknown. ARF and coatomer were, however, the only soluble components necessary for bud formation on Golgi membranes (35); membrane bilayer fusion to close off the base of the bud required only the addition of fatty acyl-CoA (Fig. 2). The resulting vesicles were then capable of fusing with acceptor Golgi in the presence of cytosol (to provide components of the fusion machinery), ATP, and an ATP-regenerating system (35) . Fusion did not occur when vesicles had been prepared with GTPS or with a mutant ARF that does not hydrolyze GTP (35) , consistent with an earlier conclusion that uncoating of the vesicle, following conversion of ARFGTP to ARFGDP and its release from the membrane, is a prerequisite for fusion.

The demonstration that coatomer (44) , like analogous protein components of clathrin-coated vesicles (45) , can function as a K channel (whatever its relationship to ARF) is consistent with the notion that this activity has a physiological role in vesicle formation. Together with the observation that the channel activity can be blocked specifically by certain inositol polyphosphates (44, 45) , this suggests a possible mechanism for the inhibition of vesicular transport by fluoride plus AlCl. This effect has often been interpreted as evidence of the participation of an AlF-activated heterotrimeric G protein in the transport process, which may, in fact, be correct (46) . The effect of fluoride could, however, result from its demonstrated inhibition of the turnover of inositol polyphosphates with consequent accumulation of pyrophosphate derivatives of inositol pentakisphosphate and inositol hexakisphosphate, as suggested by Menniti et al.(47) . Inhibition of conversion of the pyrophosphates back to the parent inositol polyphosphates, respectively, permitted recognition of the rapid cycling of these parent compounds, previously judged to be rather inert metabolically (47). Fluoride, by causing accumulation of inositol polyphosphate pyrophosphates, which at submicromolar concentrations can inhibit coatomer channel activity, could interfere with bud and/or vesicle formation and thus inhibit vesicular transport, if operation of the ion channel is involved in these processes (44) .

Vesicle Fusion

Fusion with the target membrane requires vesicle uncoating, which means release of coatomers, after release of ARFGDP (Fig. 2). Other 20-kDa GTP-binding proteins that have low GTPase activity interact with specific GAPs that accelerate hydrolysis of the protein-bound GTP. ARF apparently lacks GTPase activity (23) , and it is presumed that conversion of ARFGTP to ARFGDP occurs when ARF interacts with a GAP in the target membrane. Description of a long sought ARF-GAP, present in both soluble and particulate fractions of brain homogenate, was published during the past year (23) , and the complete purification was reported more recently (48) . Phosphatidylinositol 4,5-bisphosphate (PIP) appeared to be a specific activator, and phosphatidic acid markedly enhanced sensitivity of the impure GAP to PIP.

ARF activation of phospholipase D (26, 27) provided the basis for a new way of thinking about the function of ARF in vesicular transport. Phospholipase D, of which there are several isoforms in animal tissues, catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline. It also catalyzes a transphosphatidylation reaction, e.g. with ethanol replacing water to yield phosphatidylethanol instead of phosphatidic acid. Stutchfield and Cockcroft (49) had postulated a role for phosphatidic acid in receptor-stimulated secretion based on inhibition of that process by ethanol. Inhibition of intra-Golgi vesicular transport by ethanol has also been reported (50) , consistent with the notion that phosphatidic acid has an important role in these processes, whether through action in a signaling pathway or via its well documented effects as a fusogenic lipid in model membranes. Proteins required for GTPS stimulation of membrane phospholipase D activity were purified from brain cytosol and identified as ARF1 and/or ARF3 (26, 27) . Brown et al.(26) partially purified ARF-stimulated phospholipase D from HL-60 cell membranes and described the critical role for PIP in its stimulation by ARF. Subsequently, ARF-stimulated phospholipase D was separated from oleate-stimulated phospholipase D after solubilization from brain membranes, and its activation by class I, II, and III mammalian ARFs was demonstrated (51).

Vesicular transport (Fig. 2) appears to involve two seemingly very different actions of ARF. First, ARFGTP, after activation by its GEP, associates with the membrane, which is a prerequisite for binding of coatomer and subsequent vesicle budding. Then, the action of ARF on phospholipase D presumably takes place at the target membrane with which the vesicle will fuse, although activation of phospholipase D might occur at the donor membranes and be involved in vesicle release. Based on the abilities of PIP to activate phospholipase D and of phosphatidic acid to activate phosphatidylinositol 4-phosphate 5-kinase, Liscovitch et al.(52) suggested that these components could operate in a positive feedback loop to promote fusion of vesicle and target membranes. Their hypothesis, shown partially in Fig. 2 , is attractive in many ways. It includes a role for ARFGTP to initiate the loop by activating phospholipase D, resulting in changes in membrane lipid composition with increased phosphatidic acid that facilitates interaction of ARFGTP with GAP and activates phosphatidylinositol 4-phosphate 5-kinase in the target membrane. With the generation and release of ARFGDP followed by coatomer, increased vesicle PIP and phosphatidic acid could also promote fusion with the target membrane. Based on experiments with model membranes, it had earlier been suggested that fusion might be regulated by ``local enzymatic generation of fusogenic lipids such as diglycerides or phosphatidic acid'' (53) .

It seems not unlikely that the two different postulated functions of ARF in vesicular transport, i.e. the initiation of vesicle formation via coatomer binding and the initiation or facilitation of vesicle fusion, are properties of two different parts of the ARF structure. This is consistent with the recent demonstration of different structural requirements for ARF activation of cholera toxin and phospholipase D but remains to be established.


Afterword

The contributions of cholera toxin to biomedical research (as well as to disease) are well established. In addition to its use for labeling and perturbing the activity of the G component of the adenylyl cyclase system, it led to the recognition of numerous other bacterial ADP-ribosyltransferase toxins that, for reasons still not clear, tend to use GTP-binding proteins as substrates and sparked the discovery of analogous enzymes in animal cells (54) . Cholera toxin further led to the discovery of ARF, another GTP-binding protein that can also be a substrate for the toxin. The physiological function of ARF was a puzzle until the demonstration that it is an essential protein in yeast (9) . Since that time, information about the structure and function of multiple ARFs has been accumulated at an accelerating pace. We have tried to summarize some parts of it and, we hope, to convey a sense of the many questions that remain to be answered.


FOOTNOTES

*
This minireview will be reprinted in the 1995 Minireview Compendium, which will be available in December, 1995. We regret that because of the limited number of references permitted, citation of all original sources was not possible. Ref. 4 is a recent, more complete review.

The abbreviations used are: ARF, ADP-ribosylation factor; rARF, recombinant ARF; ARL, ARF-like protein; GEP, guanine nucleotide-exchange protein; GAP, GTPase-activating protein; PIP, phosphatidylinositol 4,5-bis-phosphate; GTPS, guanosine 5`-O-(thiotriphosphate).

The ARL1 sequence is at the top of Fig. 1 to facilitate visualization of the ARF-ARL chimeric proteins, discussed in a later section.


ACKNOWLEDGEMENTS

We thank Dr. Vincent C. Manganiello for critical review of this manuscript, Barbara Mihalko for expert secretarial assistance, and the many past members of the Laboratory of Cellular Metabolism as well as present members of the Pulmonary-Critical Care Medicine Branch, including especially Dr. Su-Chen Tsai, who are responsible for our ARF studies.


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