Molecules in the ARF Orbit*

ADP-ribosylation factors (ARFs) are 20-kDa guanine nucleotide-binding proteins, members of the Ras GTPase superfamily that were initially recognized and purified because of their ability to stimulate the ADP-ribosyltransferase activity of the cholera toxin A subunit (CTA). We now know that they are critical components of several different vesicular trafficking pathways in all eukaryotic cells and activators of specific phospholipase Ds (PLDs) (reviewed in Refs. 1–3). ARF interacts with many proteins and other molecules that regulate its state of activation or are involved in its intracellular function (Fig. 1). As with other members of the Ras superfamily, ARF with GDP bound is inactive. Replacement of bound GDP with GTP produces active ARF-GTP, which can associate with membranes. ARF-GTP is the form that activates CTA and PLD. Both forms are important in vesicular transport, which requires that the ARF molecule cycle between active and inactive states. Like the many other GTPbinding proteins or GTPases that are molecular switches for the selection, amplification, timing, and delivery of signals from diverse sources, ARF functions via differences in conformation that depend on whether GTP or GDP is bound. Vectorial signaling results from the necessary sequence of GTP binding, hydrolysis of bound GTP, and release of the GDP product (Fig. 2). Under physiological conditions, release of GDP from ARF, the prerequisite for GTP binding, is very slow and is markedly accelerated by guanine nucleotide-exchange proteins or GEPs, several of which are now known. Hydrolysis of bound GTP to yield ARF-GDP, i.e. the inactivation or “turn-off” reaction, is similarly very slow (undetectable) in the absence of specific GTPase-activating proteins or GAPs. During the last 3 years, a large part of the new information related to ARF structure and function concerns these two major types of ARF regulatory proteins, which are the subjects of this review. In addition, the relatively small ;180-amino acid ARF proteins interact with numerous other molecules (not all simultaneously). These include CTA, PLD, and guanylyl nucleotide, of course, as well as PIP2 (4), coatomers (5), arfaptin (6), G protein bg subunits (7, 8), and Gas (7), about which information is much more limited (Fig. 1). Self-association of ARF in functional dimers or tetramers has also been suggested (5), and the crystal structure of a dimer is published (9). Criteria for designation as an ARF have been the ability to activate cholera toxin and to rescue mutant Saccharomyces cerevisiae bearing the lethal double deletion of ARF1 and ARF2 genes. ARF-like proteins (ARLs), which are structurally very similar to ARFs, were initially believed not to possess these ARF activities. It is now known, however, that under suitable assay conditions, at least some ARLs can exhibit ARF activity (10), and there is perhaps a continuum of ARF-ARL function. Neither of the two criteria may, in fact, reflect accurately the properties that are required for physiological ARF function in vesicular trafficking or PLD activation. The mutant yeast rescue assay seems ambiguous, because diverse ARFs from many species can be effective when overexpressed, although yeast ARF3, which resembles most closely mammalian ARF6, is ineffective (11). In the assay of CTA (or PLD or guanine nucleotide binding), dramatic effects of specific phospholipids and detergents as well as concentrations of Mg and salt on the activities of individual ARFs are well known and make it difficult to draw valid inferences about the functional relevance of many in vitro observations. Mammalian ARFs are divided into three classes based on size, amino acid sequence, gene structure, and phylogenetic analysis; ARF1, ARF2, and ARF3 are in class I, ARF4 and ARF5 are in class II, and ARF6 is in class III (3). Non-mammalian class I, II, and III ARFs have also been found (3). A role for class I ARFs 1 and 3 in ER to Golgi and intra-Golgi transport is well established (1, 2). ARF6 has been implicated in a pathway involving plasma membrane and a tubulovesicular compartment that is distinct from previously characterized endosomes (12, 13). Vesicular transport has been extensively studied in the Golgi and ER to Golgi pathways (1, 2). The mechanisms, including the molecules and their functions, are likely very similar in other pathways. Formation of a transport vesicle begins when activated ARF with GTP bound associates with the cytoplasmic surface of a donor membrane. Just how the initiation site is identified remains unknown. Activated ARF interacts with a coat protein, one of seven in the coatomer complex. Recruitment of multiple ARF molecules followed by coatomers causes membrane deformation and budding. Bilayer fusion at the base of a bud induced by fatty acyl-CoA results in vesicle release. Roles for PLD in both vesicle formation (14) and fusion (15) have been suggested. Removal of the coat, which is necessary for vesicle fusion at the target membrane, requires inactivation of ARF by hydrolysis of bound GTP to GDP. This description is necessarily greatly oversimplified. There is no consideration of the many other molecules, protein and lipid, in each transport vesicle that have structural, metabolic, or signaling functions. Emr and Malhotra (57) have edited an excellent series of reviews on several aspects of vesicle-mediated transport. Some are cited individually here. * This minireview will be reprinted in the 1998 Minireview Compendium, which will be available in December, 1998. This is the third article of five in the “Small GTPases Minireview Series.” ‡ To whom correspondence should be addressed: Rm. 5N-307, Bldg. 10, 10 Center Dr. MSC 1434, National Institutes of Health, Bethesda, MD 208921434. Tel.: 301-496-4554; Fax: 301-402-1610; E-mail: vaughanm@ gwgate.nhlbi.nih.gov. 1 The abbreviations used are: ARF, ADP-ribosylation factor; ARL, ARFlike protein; CTA, cholera toxin A subunit; PLD, phospholipase D; ER, endoplasmic reticulum; PIP2, phosphatidylinositol 4,5-bisphosphate; GEP, guanine nucleotide-exchange protein; GAP, GTPase-activating protein; PH, pleckstrin homology; ARNO, ARF nucleotide-binding site opener; BFA, brefeldin A; PC, phosphatidylcholine; GST, glutathione S-transferase; GTPgS, guanosine 59-O-(3-thiotriphosphate); GDPbS, guanosine 59-O-(2thiodiphosphate); GRP1, general receptor for phosphoinositides. FIG. 1. Molecules in the ARF orbit. ARF interacts with three general types of molecules. Regulatory proteins GEP and GAP (Fig. 2) are discussed in the text. Arfaptins (6) are ;44-kDa proteins identified in a yeast twohybrid screen of a HL-60 cDNA library using dominant active ARF3 (Q71L) as bait. The recombinant GST-arfaptin fusion protein bound activated recombinant human ARFs 1, 3, 5, and 6 to an extent decreasing in that order. The possibility that the complex of activated ARF and arfaptin is a functional entity remains to be explored. Small non-protein molecules like guanylyl nucleotides and PIP2 with specific binding sites have major effects on ARF conformation and, therefore, activity. PLD and coatomer are two major effectors with which ARF interacts. The functional significance of its demonstrated interaction with G protein bg subunits (7, 8) is unknown. Minireview THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 34, Issue of August 21, pp. 21431–21434, 1998 Printed in U.S.A.