ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles: a novel role for a GTP-binding protein.

ADP-ribosylation factor (ARF) is an abundant and highly conserved low molecular weight GTP-binding protein that was originally identified as a key element required for the action of cholera toxin in mammalian cells, but whose physiological role is unknown. We report that ARF family proteins are highly concentrated in non-clathrin-coated transport vesicles and are coat proteins. About three copies of ARF are present on the outside of coated vesicles per alpha-COP (and thus per coatomer). ARF is highly enriched in coated vesicles as compared with parental Golgi cisternae, as shown both by biochemical and morphological methods, and ARF is removed from transport vesicles through uncoating during transport. Furthermore, ARF binds to Golgi cisternae in a GTP-dependent manner independently of coated vesicle budding. These observations strongly suggest a new role for GTP-binding proteins: ARF proteins may modulate vesicle budding and uncoating through controlled GTP hydrolysis.     

beta-Arrestin-mediated ADP-ribosylation factor 6 activation and beta 2-adrenergic receptor endocytosis

beta-Arrestins are multifunctional adaptor proteins known to regulate internalization of agonist-stimulated G protein-coupled receptors by linking them to endocytic proteins such as clathrin and AP-2. Here we describe a previously unappreciated mechanism by which beta-arrestin orchestrates the process of receptor endocytosis through the activation of ADP-ribosylation factor 6 (ARF6), a small GTP-binding protein. Involvement of ARF6 in the endocytic process is demonstrated by the ability of GTP-binding defective and GTP hydrolysis-deficient mutants to inhibit internalization of the beta(2)-adrenergic receptor. The importance of regulation of ARF6 function is shown by the ability of the ARF GTPase-activating protein GIT1 to inhibit and of the ARF nucleotide exchange factor, ARNO, to enhance receptor endocytosis. Endogenous beta-arrestin is found in complex with ARNO. Upon agonist stimulation of the receptor, beta-arrestin also interacts with the GDP-liganded form of ARF6, thereby facilitating ARNO-promoted GTP loading and activation of the G protein. Thus, the agonist-driven formation of a complex including beta-arrestin, ARNO, and ARF6 provides a molecular mechanism that explains how the agonist-stimulated receptor recruits a small G protein necessary for the endocytic process and controls its activation.     

Molecular cloning, sequence analysis and mRNA expression of human ADP-ribosylation factor

The ADP-ribosylation factor (ARF) is the small (21 kb) GTP-binding protein required for the efficient cholera toxin-catalyzed ADP-ribosylation of purified Gs, the stimulating regulatory component of adenylate cyclase. Human ARF cDNA clones were obtained from a human cDNA library by cross-species hybridization with bovine ARF1, and the nucleotide and deduced amino acid sequences were determined. Comparison of the sequences of human and bovine ARF1 showed 90% identity at the nucleotide level and 100% identity at the amino acid level, demonstrating the highly conserved nature of the ARF protein. Using human ARF cDNA as the probe, we have detected ARF messenger RNA (approximately 2.2-2.3 kb) in a wide variety of human tissues and tumor cell lines.     

ADP ribosylation factor is an essential protein in Saccharomyces cerevisiae and is encoded by two genes.

ADP ribosylation factor (ARF) is a ubiquitous 21-kDa GTP-binding protein in eucaryotes. ARF was first identified in animal cells as the protein factor required for the efficient ADP-ribosylation of the mammalian G protein Gs by cholera toxin in vitro. A gene (ARF1) encoding a protein homologous to mammalian ARF was recently cloned from Saccharomyces cerevisiae (Sewell and Kahn, Proc. Natl. Acad. Sci. USA, 85:4620-4624, 1988). We have found a second gene encoding ARF in S. cerevisiae, ARF2. The two ARF genes are within 28 centimorgans of each other on chromosome IV, and the proteins encoded by them are 96% identical. Disruption of ARF1 causes slow growth, cold sensitivity, and sensitivity to normally sublethal concentrations of fluoride ion in the medium. Disruption of ARF2 causes no detectable phenotype. Disruption of both genes is lethal; thus, ARF is essential for mitotic growth. The ARF1 and ARF2 proteins are functionally homologous, and the phenotypic differences between mutations in the two genes can be accounted for by the level of expression; ARF1 produces approximately 90% of total ARF. Among revertants of the fluoride sensitivity of an arf1 null mutation were ARF1-ARF2 fusion genes created by a gene conversion event in which the deleted ARF1 sequences were repaired by recombination with ARF2.     

Corticosterone Regulates the Expression of ADP-Ribosylation Factor Messenger RNA and Protein in Rat Cerebral Cortex

ADP-ribosylation factors (ARFs) comprise a family of small GTP-binding proteins found in brain and other tissues. Recent studies have demonstrated that the expression of the larger heterotrimeric guanine nucleotide binding proteins is under control by steroid hormones. Therefore, in the present study, we examined the influence of glucocorticoids on the expression of ARF mRNA and protein. using specific cDNA probes and antisera, respectively. Chronic administration of corticosterone (7 days) significantly increased levels of mRNA for ARF1 and ARF3, two subtypes of ARF, in rat cerebral cortex. Chronic administration of corticosterone was also found to increase levels of ARF immunoreactivity in this brain region. However, 1-day administration of corticosterone did not influence levels of mRNA for either ARF1 or ARF3. In contrast to corticosterone, bilateral adrenalectomy (7 days after surgery) was found to decrease ARF1 and ARF3 message relative to sham controls; this effect of adrenalectomy was reversed by corticosterone treatment. These results demonstrate that the expression of ARF is under hormonal control and may underlie aspects of glucocorticoid action on neuronal function.     

Hydrolysis of bound GTP by ARF protein triggers uncoating of Golgi- derived COP-coated vesicles

The cycle of nucleotide exchange and hydrolysis by a small GTP-binding protein, ADP-ribosylation factor (ARF), helps to provide vectoriality to vesicle transport. Coat assembly is triggered when ARF binds GTP, initiating transport vesicle budding, and coat disassembly is triggered when ARF hydrolyzes GTP, allowing the uncoated vesicle to fuse.     

Purification of a protein cofactor required for ADP-ribosylation of the stimulatory regulatory component of adenylate cyclase by cholera toxin

A factor (ARF) that is required for the cholera toxin-dependent ADP-ribosylation of the stimulatory, GTP-binding regulatory component (Gs) of adenylate cyclase has been purified about 2000-fold from cholate extracts of rabbit liver membranes. ARF is an intrinsic membrane protein with Mr = 21,000. The final product can be resolved into two polypeptides with very similar molecular weights; each of these has ARF activity. The ADP-ribosylation of Gs can now be studied with defined components. GTP and ARF are both necessary cofactors. The data imply that the substrates for the activated toxin are NAD and a GTP X Gs X ARF complex, and the reaction proceeds in a lipid environment. The apparent ability of ARF to bind to the alpha subunit of Gs suggests that it may play another, unknown role in the regulation of adenylate cyclase activity.     

Separation of the 24 kDa substrate for botulinum C3 ADP-ribosyltransferase and the cholera toxin ADP-ribosylation factor

Botulinum C3 ADP-ribosyltransferase modifies a approximately 24 kDa membrane protein believed to bind guanine nucleotides. Cholera toxin ADP-ribosylation factors are approximately 19 kDa GTP-binding proteins that directly activate the toxin. To evaluate a possible relationship between C3 ADP-ribosyltransferase substrate and ADP-ribosylation factor, they were partially purified from bovine brain. ADP-ribosylation factor, but not C3 ADP-ribosyltransferase substrate, stimulated auto-ADP-ribosylation of the choleragen A1 subunit whereas C3 ADP-ribosyltransferase substrate, but not ADP-ribosylation factor, was ADP-ribosylated by C3 ADP-ribosyltransferase. Thus, although both may be GTP-binding proteins, no functional similarity between ADP-ribosylation factor and C3 ADP-ribosyltransferase substrate was found.     

Two distinct members of the ADP-ribosylation factor family of GTP-binding proteins regulate cell-free intra-Golgi transport.

We have used an intra-Golgi transport assay to identify GTP-binding proteins involved in regulation of protein traffic. Two soluble proteins of 20 kd were purified by their ability to mediate GTP gamma S-dependent inhibition of transport. These GTP-dependent Golgi binding factors, or GGBFs, exhibit a 3-fold difference in activity and are differentiated by their hydrophobicity, isoelectric points, and apparent size. Removal of 80% of GGBFs from cytosol abolishes GTP gamma S sensitivity but does not affect inhibition by aluminum fluoride. We demonstrate that GGBFs are members of the ADP-ribosylation factor (ARF) family. Recombinant ARF1 exhibits GGBF activity and myristoylation is required. The distinct biochemical properties of GGBFs indicate that members of the ARF family may have related but distinct functions in intracellular transport.     

Identification and isoprenylation of plant GTP-binding proteins

To identify isoprenylated plant GTP-binding proteins,Arabidopsis thaliana andNicotiana tabacum cDNA expression libraries were screened for cDNA-encoded proteins capable of binding [³²P]GTPin vitro. ATGB2, anArabidopsis homologue of the GTP-binding protein Rab2, was found to bind GTPin vitro and to be a substrate for a geranylgeranyl:protein transferase (GGTase) present in plant extracts. The carboxyl terminus of this protein contains a-GCCG sequence, which has not previously been shown to be recognized by any prenyl:protein transferase (PTase), but which most closely resembles that isoprenylated by the type II GGTase (-XXCC,-XCXC, or-CCXX).In vitro geranylgeranylation of anArabidopsis Rab1 protein containing a carboxyl-terminal-CCGQ sequence contirmed the presence of a type II GGTase-like activity in plant extracts. Several other proteins were also identified byin vitro GTP binding, includingArabidopsis and tobacco homologues of Rab11, ARF (ADP-ribosylation factor) and Sar proteins, as well as a novel 22 kDaArabidopsis protein (ATG81). This 22 kDa protein had consensus GTP-binding motifs and bound GTP with high specificity, but its structure was not closely related to that of any known GTP-binding protein (it most resembled proteins within the ARF/Sar and G protein -subunit superfamilies).     

Human ADP-ribosylation factors. A functionally conserved family of GTP-binding proteins

A new member, hARF4, of the ADP-ribosylation factor (ARF) family, a subset of the superfamily of regulatory GTP-binding proteins, has been cloned from a cDNA expression library. Two other human ARF cDNA sequences, designated human ARF1 and ARF3, have been reported previously and are 96% identical in amino acid sequence. A human ARF1 cDNA, significantly longer than previously described clones, was obtained, by cross-species hybridization using a bovine ARF1 cDNA probe. Bovine ARF1p and human ARF1p are 100% identical while each is only 80% identical to hARF4p. Thus, hARF4p is the most divergent of the mammalian ARF proteins identified. Northern blot analysis revealed the expression of at least three different ARF messages in human placenta and adrenal carcinoma cells. Both hARF1 and hARF4 encode GTP-binding proteins with predicted molecular masses of 20,000-21,000 Da. Biochemical analysis of the purified recombinant proteins revealed a high degree of conservation of nucleotide binding properties and in vitro ARF activities. ARF is an essential gene in the yeast, Saccharomyces cerevisiae, and is encoded by two genes. Expression of either hARF1p or hARF4p in yeast was found to rescue the lethal double mutant, arf1-arf2-, thus demonstrating the functional conservation of ARF functions between yeast and man. The combination of in vivo and in vitro assays for ARF function provides a specific and unambiguous means of determining bona fide ARF proteins from divergent species from among the rapidly increasing number of structurally related, small molecular weight GTP-binding proteins.     

Activation of phospholipase D1 by direct interaction with ADP-ribosylation factor 1 and RalA

Phospholipase D1 (PLD1) is known to be activated by ADP-ribosylation factor 1 (ARF1). We report here that ARF1 co-immunoprecipitates with PLD1 and that the ARF1-dependent PLD activation is induced by the direct interaction between ARF1 and PLD1. We found that RalA, another member of the small GTP-binding proteins, synergistically enhances the ARF1-dependent PLD activity with an EC₅₀ of about 30 nM. Using in vitro binding assay, we show that ARF1 and RalA directly interact with different sites of PLD1. The results suggest that the independent interactions of RalA and ARF1 with PLD1 are responsible for the synergistic activation.     

Role of ARF4L in Recycling Between Endosomes and the Plasma Membrane

The human ADP-ribosylation factor-like protein, ARF4L is a member of the ARF family, which are small GTP-binding proteins that play significant roles in vesicle transport and protein secretion. However, little is known about the physiological roles of ARF4L. In this study, to understand the biological functions of ARF4L, we carried out immunocytochemical analysis of ARF4L molecules with mutations in the functional domains. ARF4L was shown to be distributed to the plasma membrane following binding to GTP (Q80L), and into endosomes following binding to GDP (T35N). Moreover, the inactive-form of ARF4L (T35N) causes localization of transferrin receptors to the endosomal compartment, while the active form (Q80L) causes transport to the plasma membrane. These findings indicate that ARF4L drive the transport of cargo protein and subsequent fusion of recycling vesicles with the plasma membrane for maintenance of the cell surface.     

Decoupling of activation and effector binding underlies ARF6 priming of fast endocytic recycling

The small GTP-binding protein ADP-ribosylation factor 6 (ARF6) controls the endocytic recycling pathway of several plasma membrane receptors. We analyzed the localization and GDP/GTP cycle of GFP-tagged ARF6 by total internal reflection fluorescent microscopy. We found that ARF6-GFP associates with clathrin-coated pits (CCPs) at the plasma membrane in a GTP-dependent manner in a mechanism requiring the adaptor protein complex AP-2. In CCP, GTP-ARF6 mediates the recruitment of the ARF-binding domain of downstream effectors including JNK-interacting proteins 3 and 4 (JIP3 and JIP4) after the burst recruitment of the clathrin uncoating component auxilin. ARF6 does not contribute to receptor-mediated clathrin-dependent endocytosis. In contrast, we found that interaction of ARF6 and JIPs on endocytic vesicles is required for trafficking of the transferrin receptor in the fast, microtubule-dependent endocytic recycling pathway. Our findings unravel a novel mechanism of separation of ARF6 activation and effector function, ensuring that fast recycling may be determined at the level of receptor incorporation into CCPs.     

Fluoride is not an activator of the smaller (20-25 kDa) GTP-binding proteins

Effects of aluminum, magnesium, and fluoride (AMF) on members of both the trimeric G protein and smaller (20-25 kDa) monomeric GTP-binding protein families were examined. The dissociation of GDP from G proteins was blocked by AMF but was unchanged with the addition of AMF to any of six of the monomeric GTP-binding proteins. Biochemical activities and properties of one of the smaller GTP-binding proteins, ADP-ribosylation factor, were also found to be unaffected by AMF. It is concluded that the ability of AMF to activate the trimeric G proteins is not shared by the smaller GTP-binding proteins and thus should prove to be a useful discriminator between cellular activities regulated by these two families of regulatory proteins.     

Effect of protein kinase A activity on the association of ADP-ribosylation factor 1 to golgi membranes

The small GTP-binding protein ADP-ribosylation factor 1 (ARF1) is an essential component of the molecular machinery that catalyzes the formation of membrane-bound transport intermediates. By using an in vitro assay that reproduces recruitment of cytosolic proteins onto purified, high salt-washed Golgi membranes, we have analyzed the role of cAMP-dependent protein kinase A (PKA) on ARF1 incorporation. Addition to this assay of either pure catalytic subunits of PKA (C-PKA) or cAMP increased ARF1 binding. By contrast, ARF1 association was inhibited following C-PKA inactivation with either PKA inhibitory peptide or RIIalpha as well as after cytosol depletion of C-PKA. C-PKA also stimulated recruitment and activation of a recombinant form of human ARF1 in the absence of additional cytosolic components. The binding step could be dissociated from the activation reaction and found to be independent of guanine nucleotides and saturable. This step was stimulated by C-PKA in an ATP-dependent manner. Dephosphorylated Golgi membranes exhibited a decreased ability to recruit ARF1, and this effect was reverted by addition of C-PKA. Following an increase in the intracellular level of cAMP, ARF proteins redistributed from cytosol to the perinuclear Golgi region of intact cells. Collectively, the results show that PKA exerts a key regulatory role in the recruitment of ARF1 onto Golgi membranes. In contrast, PKA modulators did not affect recruitment of beta-COP onto Golgi membranes containing prebound ARF1.     

Two distinct populations of ARF bound to Golgi membranes

ADP-ribosylation factor (ARF) is a small molecular weight GTP-binding protein (20 kD) and has been implicated in vesicular protein transport. The guanine nucleotide, bound to ARF protein is believed to modulate the activity of ARF but the mechanism of action remains elusive. We have previously reported that ARF binds to Golgi membranes after Brefeldin A-sensitive nucleotide exchange of ARF-bound GDP for GTP gamma S. Here we report that treatment with phosphatidylcholine liposomes effectively removed 40-60% of ARF bound to Golgi membranes with nonhydrolyzable GTP, presumably by competing for binding of activated ARF to lipid bilayers. This revealed the presence of two different pools of ARF on Golgi membranes. Whereas total ARF binding did not appear to be saturable, the liposome-resistant pool is saturable suggesting that this pool of ARF is stabilized by interaction with a Golgi membrane-component. We propose that activation of ARF by a guanine nucleotide-exchange protein results in association of myristoylated ARF GTP with the lipid bilayer of the Golgi apparatus. Once associated with the membrane, activated ARF can diffuse freely to associate stably with a target protein or possibly can be inactivated by a GTPase activating protein (GAP) activity.     

Stimulation of choleragen enzymatic activities by GTP and two soluble proteins purified from bovine brain

Choleragen (cholera toxin) activates adenylate cyclase by catalyzing ADP-ribosylation of Gs alpha, the stimulatory guanine nucleotide-binding protein. It was recently found (Tsai, S.-C., Noda, M., Adamik, R., Moss, J., and Vaughan, M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5139-5142) that a bovine brain membrane protein known as ADP-ribosylation factor or ARF, which enhances ADP-ribosylation of Gs alpha, also increases the GTP-dependent NAD:arginine and NAD:protein ADP-ribosyltransferase, NAD glycohydrolase, and auto-ADP-ribosylation activities of choleragen. We report here the purification and characterization of two soluble proteins from bovine brain that similarly enhance the Gs alpha-dependent and independent ADP-ribose transfer reactions catalyzed by toxin. Like membrane ARF, both soluble factors are 19-kDA proteins dependent on GTP or GTP analogues for activity. Maximal ARF effects were observed at a molar ratio of less than 2:1, ARF/toxin A subunit. Dimyristoyl phosphatidylcholine was necessary for optimal ADP-ribosylation of Gs alpha but inhibited auto-ADP-ribosylation of the choleragen A1 subunit and NAD:agmatine ADP-ribosyltransferase activity. It appears that the soluble factors directly activate choleragen in a GTP-dependent fashion. The relationships of the ARF proteins to the ras oncogene products and to the family of guanine nucleotide-binding regulatory proteins that includes Gs alpha remains to be determined.     

ADP-ribosylation factors: a family of ∼20-kDa guanine nucleotide-binding proteins that activate cholera toxin

ADP-ribosylation factors (ARFs) comprise a family of 20 kDa guanine nucleotide-binding proteins that were discovered as one of several cofactors required in cholera toxin-catalyzed ADP-ribosylation of G_s, the guanine nucleotide-binding protein responsible for stimulation of adenylyl cyclase, and was subsequently found to enhance all cholera toxin-catalyzed reactions and to directly interact with, and activate the toxin. ARF is dependent on GTP or its analogues for activity, binds GTP with high affinity in the presence of dimyristoylphosphatidylcholine/cholate and contains consensus sequences for GTP-binding and hydrolysis. Six mammalian family members have been identified which have been classified into three groups (Class I, II, and III) based on size, deduced amino acid sequence identity, phylogenetic analysis and gene structure. ARFs are ubiquitous among eukaryotes, with a deduced amino acid sequence that is highly conserved across diverse species. They have recently been shown to associate with phospholipid and Golgi membranes in a GTP-dependent manner and are involved in regulating vesicular transport.Abbreviations ARF ADP-ribosylation factor - sARF I and sARF II soluble ADP-ribosylation factors purified from bovine brain - mARF purified membrane-associated ARF - hARF human ARF - bARF bovine ARF - yARF yeast ARF - ARF bacterially-expressed recombinant ARF - gARF Giardia ARF - dARF Drosophila ARF - G protein guanine nucleotide-binding protein - G_s G protein responsible for stimulation of adenylyl cyclase - GTPS guanosine-5-O-(3-thio-triphosphate) - CIAI cholera toxin A1 subunit - DMPC dimyristoylphosphatidylcholine - SDS sodium dodecyl sulfate