SMART MODE:

NORMAL GENOMIC

• Tarricone C et al.
• The structural basis of Arfaptin-mediated cross-talk between Rac and Arf signalling pathways.
• Nature. 2001; 411: 215-9
• Display abstract

• Small G proteins are GTP-dependent molecular switches that regulate numerous cellular functions. They can be classified into homologous subfamilies that are broadly associated with specific biological processes. Cross-talk between small G-protein families has an important role in signalling, but the mechanism by which it occurs is poorly understood. The coordinated action of Arf and Rho family GTPases is required to regulate many cellular processes including lipid signalling, cell motility and Golgi function. Arfaptin is a ubiquitously expressed protein implicated in mediating cross-talk between Rac (a member of the Rho family) and Arf small GTPases. Here we show that Arfaptin binds specifically to GTP-bound Arf1 and Arf6, but binds to Rac.GTP and Rac.GDP with similar affinities. The X-ray structure of Arfaptin reveals an elongated, crescent-shaped dimer of three-helix coiled-coils. Structures of Arfaptin with Rac bound to either GDP or the slowly hydrolysable analogue GMPPNP show that the switch regions adopt similar conformations in both complexes. Our data highlight fundamental differences between the molecular mechanisms of Rho and Ras family signalling, and suggest a model of Arfaptin-mediated synergy between the Arf and Rho family signalling pathways.

• Pasqualato S, Menetrey J, Franco M, Cherfils J
• The structural GDP/GTP cycle of human Arf6.
• EMBO Rep. 2001; 2: 234-8
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• The small GTP-binding protein Arf6 coordinates membrane traffic at the plasma membrane with aspects of cytoskeleton organization. This function does not overlap with that of other members of the ADP-ribosylation factor (Arf) family, although their switch regions, which are their major sites of interaction with regulators and effectors, have virtually identical sequences. Here we report the crystal structure of full-length, non-myristoylated human Arf6 bound to GTPgammaS. Unlike their GDP-bound forms, the active forms of Arf6 and Arf1 are very similar. Thus, the switch regions are discriminatory elements between Arf isoforms in their inactive but not in their active forms, a property that may generalize to other families of small G proteins. This suggests that GTP-bound Arfs may establish specific interactions outside the switch regions and/or be recognized in their cellular context rather than as isolated proteins. The structure also allows further insight into the lack of spontaneous GTPase activity of Arf proteins.

• Hillig RC, Hanzal-Bayer M, Linari M, Becker J, Wittinghofer A, Renault L
• Structural and biochemical properties show ARL3-GDP as a distinct GTP binding protein.
• Structure Fold Des. 2000; 8: 1239-45
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• BACKGROUND: Based on sequence similarities, Arf-like (ARL) proteins have been assigned to the Arf subfamily of the superfamily of Ras-related GTP binding proteins. They have been identified in several isoforms in a wide variety of species. Their cellular function is unclear, but they are proposed to regulate intracellular transport. RESULTS: The 1.7 A crystal structure of murine ARL3-GDP provides a first insight into the structural features of this subgroup of Ar proteins. The N-terminal extension of ARL3 folds into an elongated loop region that is hydrophobically anchored onto the surface by burying 1440 A2. The features observed suggest that ARL3 releases its N terminus and undergoes a beta sheet register shift upon the binding of GTP. The structure and kinetic experiments with fluorescent mGDP demonstrate that tight GDP (but not GTP) binding is achieved in the absence of a magnesium ion. This is due to a lysine residue in the active site, close to the canonical Mg2+ site found in other GTP binding proteins. This is a distinct feature separating ARL2 and ARL3 from Arf proteins. CONCLUSION: The disturbed magnesium binding site and the independence of GDP coordination from the presence of Mg2+ separate ARL2 and ARL3 from Arf proteins. The D sheet register shift, which is similar to that of Arf, that is observed in the present structure, along with the postulated release of the N-terminal extension and the concomitant exposure of a patch of conserved hydrophobic residues in this region suggest that ARL proteins might be localized to target membranes upon exchange of GDP to GTP. Contrary to the situation in Arf, however, the conformational change to ARL-GTP does not require the presence of membranes and might thus be energetically unfavored. Together with the very low affinity described for the interaction of ARL3 with Mg-GTP, this suggests that ARL protein activation requires the presence of effectors stabilizing the GTP coordination rather than guanine nucleotide exchange factors (GEFs).

• Menetrey J, Macia E, Pasqualato S, Franco M, Cherfils J
• Structure of Arf6-GDP suggests a basis for guanine nucleotide exchange factors specificity.
• Nat Struct Biol. 2000; 7: 466-9
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• Arf6 is an isoform of Arf that localizes at the periphery of the cell where it has an essential role in endocytotic pathways. Its function does not overlap with that of Arf1, although the two proteins share approximately 70% sequence identity and they have switch regions, whose conformation depends on the nature of the guanine nucleotide, with almost identical sequences. The crystal structure of Arf6-GDP at 2.3 A shows that it has a conformation similar to that of Arf1-GDP, which cannot bind membranes with high affinity. Significantly, the switch regions of Arf6 deviate by 2-5 A from those of Arf1. These differences are a consequence of the shorter N-terminal linker of Arf6 and of discrete sequence changes between Arf6 and Arf1. Mutational analysis shows that one of the positions which differs between Arf1 and Arf6 affects the configuration of the nucleotide binding site and thus the nucleotide binding properties of the Arf variant. Altogether, our results provide a structural basis for understanding how Arf1 and Arf6 can be distinguished by their guanine nucleotide exchange factors and suggest a model for the nucleotide/membrane cycle of Arf6.

• Jones D, Morgan C, Cockcroft S
• Phospholipase D and membrane traffic. Potential roles in regulated exocytosis, membrane delivery and vesicle budding.
• Biochim Biophys Acta. 1999; 1439: 229-44
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• It is now well-established that phospholipase D is transiently stimulated upon activation by G-protein-coupled and receptor tyrosine kinase cell surface receptors in mammalian cells. Over the last 5 years, a tremendous effort has gone to identify the major intracellular regulators of mammalian phospholipase D and to the cloning of two mammalian phospholipase D enzymes (phospholipase D1 and D2). In this chapter, we review the physiological function of mammalian phospholipase D1 that is synergistically stimulated by ADP ribosylation factor, Rho and protein kinase Calpha. We discuss the function of this enzyme in membrane traffic, emphasising the possible integrated relationships between consumption of vesicles in regulated exocytosis, membrane delivery and constitutive membrane traffic.

• Roth MG, Bi K, Ktistakis NT, Yu S
• Phospholipase D as an effector for ADP-ribosylation factor in the regulation of vesicular traffic.
• Chem Phys Lipids. 1999; 98: 141-52
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• A mammalian phospholipase D (PLD) activity that is stimulated by ADP-ribosylation factor (ARF) has been identified in Golgi-enriched membrane fractions. This activity is due to the PLD1 isoform and evidence from several laboratories indicates that PLD1 is important for the polymerization of vesicle coat proteins on membranes. When expressed in Chinese hamster ovary cells, PLD1 localized to dispersed small vesicles that overlapped with the location of the ERGIC53 protein, a marker for the endoplasmic reticulum (ER)-Golgi intermediate compartment. Cells having increased PLD1 expression had accelerated anterograde and retrograde transport between the ER and Golgi. Membranes from cells having elevated PLD1 activity bound more COPI, ARF, and ARF-GTPase activating protein. These membranes also produced more COPI vesicles than did membranes from control cells. It is likely that PLD1 participates in both positive and negative feedback regulation of the formation of COPI vesicles and is important for controlling the rate of this process.

• Chardin P, McCormick F
• Brefeldin A: the advantage of being uncompetitive.
• Cell. 1999; 97: 153-5

• Iyer SS, Kusner DJ
• Association of phospholipase D activity with the detergent-insoluble cytoskeleton of U937 promonocytic leukocytes.
• J Biol Chem. 1999; 274: 2350-9
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• Phospholipase D (PLD) regulates cytoskeletal-dependent antimicrobial responses of myeloid leukocytes, including phagocytosis and oxidant generation. However, the mechanisms responsible for this association between PLD activity and the actin cytoskeleton are unknown. We utilized a cell-free system from U937 promonocytes to test the hypothesis that stimulation of PLD results in stable association of the activated lipase with the detergent-insoluble membrane skeleton. Plasma membrane and cytosol were incubated +/- guanosine 5'-3-O-(thio)triphosphate (GTPgammaS), followed by re-isolation and extraction of the washed membranes with octyl glucoside. The detergent-insoluble fraction derived from membranes incubated with GTPgammaS (DIFGTPgammaS) exhibited 22-fold greater PLD activity than that derived from control membranes (DIF0), when both were assayed in the presence of GTPgammaS. The DIF contained PLD1, RhoA, and ARF, and the level of each was increased by GTPgammaS in a dose-dependent manner. The DIF also contained F-actin, vinculin, talin, paxillin, and alpha-actinin, consistent with its identification as the membrane skeleton. The physiologic relevance of these findings was demonstrated by a similar increase in DIF-associated PLD activity after stimulation of intact U937 cells with opsonized zymosan. These results indicate that stimulation of PLD1 is accompanied by stable association of the activated lipase, RhoA, and ADP-ribosylation factor with the actin-based membrane skeleton.

• Toda K, Nogami M, Murakami K, Kanaho Y, Nakayama K
• Colocalization of phospholipase D1 and GTP-binding-defective mutant of ADP-ribosylation factor 6 to endosomes and lysosomes.
• FEBS Lett. 1999; 442: 221-5
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• Phospholipase D (PLD) is involved in various aspects of cellular function. Two isoforms, PLD1 and PLD2, have been identified. PLD1, which has two splicing variants, is regulated by various factors, including ADP-ribosylation factor (ARF). We here show that both variants of PLD1 are predominantly localized to late endosomes and lysosomes, but not to the Golgi apparatus or endoplasmic reticulum in contrast to earlier studies. Furthermore, PLD1s show significant colocalization with an ARF6 mutant defective in GTP binding. The data suggest that PLD1, under the regulation of ARF6, plays a role in the function of endosomes and lysosomes.

• Arai K, Matsuda T, Sai Y, Ohkuma S
• ARF-induced lysosomal lysis in vitro.
• J Biochem (Tokyo). 1998; 123: 637-43
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• Cytosol treated with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) disintegrated lysosomes in a dose-dependent manner, as detected as the release of preloaded fluorescein isothiocyanate-dextran. The effect of GTPgammaS was suppressed by GTP or GDP, indicating a role of a GTP binding protein (G-protein) in the lysis [Sai, Y. et al. (1994) Biochem. Biophys. Res. Commun. 198, 869-877]. Gel filtration of cytosol and GTP-ligand blotting showed that a small GTP-binding protein participated in the lysosomal lysis. We partially purified the G-protein from rat liver cytosol and identified it as ARF1. GTPgammaS-stimulated lysis was reconstituted with ARF1 purified from bovine brain cytosol or recombinant ARF1. ARF bound to lysosomal membranes depending upon GTPgammaS in a dose-dependent manner. These results suggest that the transfer of ARF from the cytosol to the lysosomal membrane is necessary for GTPgammaS-stimulated lysis of lysosomes.

• Mitchell R et al.
• Rhodopsin-family receptors associate with small G proteins to activate phospholipase D.
• Nature. 1998; 392: 411-4
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• G-protein-coupled receptors of the rhodopsin family transduce many important neural and endocrine signals. These receptors activate heterotrimeric G proteins and in many cases also cause activation of phospholipase D, an enzyme that can be controlled by the small G proteins ARF and RhoA. Here we show that the activation of phospholipase D that is induced by many, but not all, Ca2+-mobilizing G-protein-coupled receptors is sensitive to inhibitors of ARF and of RhoA. Receptors of this type were co-immunoprecipitated with ARF or RhoA on exposure to agonists, and the effects of GTP analogues on ligand binding to the receptor changed to a profile that is characteristic of small G proteins. These receptors contain the amino-acid sequence AsnProXXTyr in their seventh transmembrane domain, whereas receptors capable of activating phospholipase D without involving ARF contain the sequence AspProXXTyr. Mutation of this latter sequence to AsnProXXTyr in the gonadotropin-releasing hormone receptor conferred sensitivity to an inhibitor of ARF, and the reciprocal mutation in the 5-HT2A receptor for 5-hydroxy-tryptamine reduced its sensitivity to the inhibitor. Receptors carrying the AsnProXXTyr motif thus seem to form functional complexes with ARF and RhoA.

• Akisue T, Jinnai H, Hitomi T, Miwa N, Yoshida K, Nakamura S
• Purification of a heat-stable activator protein for ADP-ribosylation factor-dependent phospholipase D.
• FEBS Lett. 1998; 422: 108-12
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• A heat-stable activator for ADP-ribosylation factor (ARF)-dependent phospholipase D (PLD) was purified to near homogeneity from rat kidney cytosol by a sequential column chromatography. The purified activator has a molecular mass of 23 kDa on SDS-PAGE. Using a partially purified ARF-dependent PLD from rat kidney, the activator synergistically stimulates PLD with ARF in time- and dose-dependent manner. In the absence of ARF, the activator has little or no effect. The purified activator also stimulates PLD under several conditions including permeabilized cell system, suggesting that the activator is a physiologically relevant regulator of PLD.

• Rudge SA et al.
• ADP-Ribosylation factors do not activate yeast phospholipase Ds but are required for sporulation.
• Mol Biol Cell. 1998; 9: 2025-36
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• ADP-ribosylation factor (ARF) proteins in Saccharomyces cerevisiae are encoded by two genes, ARF1 and ARF2. The addition of the c-myc epitope at the C terminus of Arf1 resulted in a mutant (arf1-myc arf2) that supported vegetative growth and rescued cells from supersensitivity to fluoride, but homozygous diploids failed to sporulate. arf1-myc arf2 mutants completed both meiotic divisions but were unable to form spores. The SPO14 gene encodes a phospholipase D (PLD), whose activity is essential for mediating the formation of the prospore membrane, a prerequisite event for spore formation. Spo14 localized normally to the developing prospore membrane in arf1-myc arf2 mutants; however, the synthesis of the membrane was attenuated. This was not a consequence of reduced PLD catalytic activity, because the enzymatic activity of Spo14 was unaffected in meiotic arf1-myc arf2 mutants. Although potent activators of mammalian PLD1, Arf1 proteins did not influence the catalytic activities of either Spo14 or ScPld2, a second yeast PLD. These results demonstrate that ARF1 is required for sporulation, and the mitotic and meiotic functions of Arf proteins are not mediated by the activation of any known yeast PLD activities. The implications of these results are discussed with respect to current models of Arf signaling.

• Moss J, Vaughan M
• Molecules in the ARF orbit.
• J Biol Chem. 1998; 273: 21431-4

• Martin A et al.
• Role and regulation of phospholipase D signalling.
• Biochem Soc Trans. 1997; 25: 1157-60

• Clark JM, Hodgkin MN, Wakelam MJ
• HL60 nuclei lacking the nuclear double membrane contain a PLD activity which is insensitive to the ADP-ribosylation factor.
• Biochem Soc Trans. 1997; 25: 590-590

• Wakelam MJ et al.
• Role and regulation of phospholipase D activity in normal and cancer cells.
• Adv Enzyme Regul. 1997; 37: 29-34
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• PLD is regulated by the small GTP binding proteins Rho and Arf, though predominantly by the latter. The PA product of PLD activation is an activator of Rho-regulated actin stress fibre formation and in invasive cells of MMP-9 synthesis and activation. Together this may explain the increased invasion of cells in response to PA.

• Roth MG, Sternweis PC
• The role of lipid signaling in constitutive membrane traffic.
• Curr Opin Cell Biol. 1997; 9: 519-26
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• Several lines of evidence indicate that enzymes that modify membrane lipids function in the regulation of constitutive membrane traffic. Recent evidence suggests that specific phosphatidylinositides may regulate the activity of proteins with diverse functions in membrane transport, such as dynamin, the clathrin-associated AP-2 complex, and proteins that stimulate guanine nucleotide exchange on ADP-ribosylation factors (ARFs). ARF proteins activate a phospholipase D that produces phosphatidic acid from phosphatidylcholine, and this may be essential for the formation of certain types of transport vesicles or may be constitutive vesicular transport to signal transduction pathways.

• Randazzo PA
• Functional interaction of ADP-ribosylation factor 1 with phosphatidylinositol 4,5-bisphosphate.
• J Biol Chem. 1997; 272: 7688-92
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• The relationship between ADP-ribosylation factor (Arf) 1 and phosphoinositides, which have been independently implicated as regulators of membrane traffic, was examined. Because both Arf-dependent phospholipase D and Arf1 GTPase-activating protein (GAP) require phosphatidylinositol 4,5-bisphosphate (PIP2), Arf1 complexed with PIP2 has been proposed to interact with target proteins. This hypothesis was tested using Arf1 GAP as a model system. Arf1 was shown to bind to PIP2 in Triton X-100 micelles with a Kd of 45 +/- 13 &mgr;M. Arf1 also bound phosphatidic acid but with 10-fold lower affinity. PIP2 binding was specifically disrupted by mutating lysines 15, 16, and 181 and arginine 178 to leucines. Decreased PIP2 binding resulted in an increased EC50 of PIP2 for activation of Arf GAP. None of the mutations that decreased PIP2 binding affected binding to or activation of GAP by phosphatidic acid, which acts at a functionally distinct site. These data support the hypothesis that PIP2 binding to Arf1 promotes interaction with Arf GAP. The implications of lipid-directed protein-protein interactions for membrane traffic are discussed.

• Saitoh O, Oshima T, Agata K, Watanabe K, Nakata H
• Corrigendum to "Molecular cloning of a novel ADP-ribosylation factor (ARF) expressed in planarians" [Biochim. Biophys. Acta 1309 (1996) 205-210]
• Biochim Biophys Acta. 1997; 1352: 231-231

• Antonny B, Beraud-Dufour S, Chardin P, Chabre M
• N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipids upon GDP to GTP exchange.
• Biochemistry. 1997; 36: 4675-84
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• GDP/GTP exchange modulates the interaction of the small G-protein ADP-ribosylation factor-1 with membrane lipids: if ARF(GDP) is mostly soluble, ARF(GTP) binds tightly to lipid vesicles. Previous studies have shown that this GTP-dependent binding persists upon removal of the N-terminal myristate but is abolished following further deletion of the 17 N-terminal residues. This suggests a role for this amphipathic peptide in lipid membrane binding. In the ARF(GDP) crystal structure, the 2-13 peptide is helical, with its hydrophobic residues buried in the protein core. When ARF switches to the GTP state, these residues may insert into membrane lipids. We have studied the binding of ARF to model unilamellar vesicles of defined composition. ARF(GDP) binds weakly to vesicles through hydrophobic interaction of the myristate and electrostatic interaction of cationic residues with anionic lipids. Phosphatidylinositol 4,5-bis(phosphate) shows no specific effects other than strictly electrostatic. By using fluorescence energy transfer, the strength of the ARF(GTP)-lipid interaction is assessed via the dissociation rate of ARF(GTPgammaS) from labeled lipid vesicles. ARF(GTPgammaS) dissociates slowly (tau(off) approximately 75 s) from neutral PC vesicles. Including 30% anionic phospholipids increases tau(off) by only 3-fold. Reducing the N-terminal peptide hydrophobicity by point mutations had larger effects: F9A and L8A-F9A substitutions accelerate the dissociation of ARF(GTPgammaS) from vesicles by factors of 7 and 100, respectively. This strongly suggests that, upon GDP/GTP exchange, the N-terminal helix is released from the protein core so its hydrophobic residues can interact with membrane phospholipids.

• Kahn RA, Terui T, Randazzo PA
• Effects of acid phospholipids on ARF activities: potential roles in membrane traffic.
• J Lipid Mediat Cell Signal. 1996; 14: 209-14
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• ADP-ribosylation factors are a family of approximately 21 kDa GTP binding proteins which have been implicated as ubiquitous regulators of multiple steps in both exocytic and endocytic membrane traffic in mammals and yeast. Reversible membrane associations are thought to be an essential component in the physiological actions of ARF and are regulated by GTP binding. ARFs are unique among the superfamily of GTP binding proteins in having a strict dependence on phospholipids for nucleotide exchange. In addition, ARF proteins were found to bind phospatidylinositol 4,5-bisphosphate (PIP2) specifically. PIP2 was found to increase the rate of GDP dissociation and stabilize the nucleotide-free form of the protein. The previously described requirements for PIP2 in the ARF stimulated phospholipase D (PLD) activity and ARF GTPase activating protein (ARF GAP) assays provide the basis for a model in which PIP2 acts as a cofactor in one or more ARF pathways. There are potentially two distinct phospholipid binding sites each of which are coupled to the nucleotide binding site of ARFs.

• Saitoh O, Oshima T, Agata K, Watanabe K, Nakata H
• Molecular cloning of a novel ADP-ribosylation factor (ARF) expressed in planarians.
• Biochim Biophys Acta. 1996; 1309: 205-10

• Hosaka M, Toda K, Takatsu H, Torii S, Murakami K, Nakayama K
• Structure and intracellular localization of mouse ADP-ribosylation factors type 1 to type 6 (ARF1-ARF6).
• J Biochem (Tokyo). 1996; 120: 813-9
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• ADP-ribosylation factors (ARFs) are a family of small GTP-binding proteins that are proposed to be involved in the formation of coated transport vesicles. Although six ARF sequences have been reported in mammals to date, it has been unclear how many ARF members are present in a single organism. In this study, we provide the first direct evidence by cDNA cloning for the presence of all six ARF members in mouse. These proteins are highly conserved across mammalian species and Northern blot analysis revealed that mRNAs for all the members were expressed ubiquitously. Transfection of cells with epitope-tagged ARFs revealed that ARFs 1-3 displayed a perinuclear Golgi localization, while ARFs 4-6 appeared to be widely dispersed throughout the cytoplasm. These results suggest that although all the ARF proteins play fundamental and critical roles in cellular function, they are involved in different vesicular transport processes.

• Frohman MA, Morris AJ
• Rho is only ARF the story. Phospholipid signalling.
• Curr Biol. 1996; 6: 945-7
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• The GTP-binding proteins ARF and Rho control a number of important cellular processes, such as protein traffic and cell morphology; there is increasing evidence that phospholipase D is a key mediator of these ARF/Rho-regulated events.

• Shimooku K et al.
• Reconstitution of GTP-gamma-S-dependent phospholipase D activity with ARF, RhoA, and a soluble 36-kDa protein.
• FEBS Lett. 1996; 387: 141-4
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• For activation of kidney membrane phospholipase D (PLD), cytosol is absolutely needed in addition to GTP-gamma-S. The active component of cytosol consists of three protein factors: ADP-ribosylation factor, RhoA, and a soluble 36-kDa protein. Any combination of these two factors synergistically activates PLD to some extent, but the presence of the three factors causes full activation. The 36-kDa protein is stable at 60 degrees C but inactivated at 80 degrees C for 10 min. Tissue distribution of the 36-kDa protein roughly coincides with that of PLD, suggesting physiological relevance of the protein in the regulation of PLD.

• Cockcroft S
• ARF-regulated phospholipase D: a potential role in membrane traffic.
• Chem Phys Lipids. 1996; 80: 59-80
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• Phospholipase D activity is stimulated rapidly upon occupation of cell-surface receptors. One of the intracellular regulators of phospholipase D activity has been identified as ADP ribosylation factor (ARF). ARF is a small GTP binding protein whose function has been elucidated in vesicular traffic. This review puts into context the connection between the two fields of signal transduction and vesicular transport.

• Smith SA, Holik PR, Stevens J, Melis R, White R, Albertsen H
• Isolation and mapping of a gene encoding a novel human ADP-ribosylation factor on chromosome 17q12-q21.
• Genomics. 1995; 28: 113-5
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• A gene encoding a low-molecular-weight GTP-binding protein was isolated from a retinal cDNA library and mapped to human chromosome 17q12-q21. Comparison of the predicted protein with the protein databases revealed striking homology to the family of ADP-ribosylation factors (ARFs), which are thought to be involved in membrane trafficking and protein secretion. The greatest homology observed was with the rat ARF-like 4 protein (ARL4), with which it shared 58% identity, while the more highly conserved human ARF1 and ARF3 proteins each shared 46% identity. Inspection of the predicted new protein showed that it contained each of the six conserved motifs that are required for guanine nucleotide binding and hydrolysis, and thus it is probably a novel ARF isoform. We have designated the new protein and its corresponding gene ARF4L.

• Randazzo PA, Terui T, Sturch S, Fales HM, Ferrige AG, Kahn RA
• The myristoylated amino terminus of ADP-ribosylation factor 1 is a phospholipid- and GTP-sensitive switch.
• J Biol Chem. 1995; 270: 14809-15
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• ADP-ribosylation factor 1 (Arf1) is an essential N-myristoylated 21-kDa GTP-binding protein with activities that include the regulation of membrane traffic and phospholipase D activity. Both the N terminus of the protein and the N-myristate bound to glycine 2 have previously been shown to be essential to the function of Arf in cells. We show that the bound nucleotide affects the conformation of either the N terminus or residues of Arf1 that are in direct contact with the N terminus. This was demonstrated by examining the effects of mutations in this N-terminal domain on guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) and GDP binding and dissociation kinetics. Arf1 mutants, lacking 13 or 17 residues from the N terminus or mutated at residues 3-7, had a greater affinity for GTP gamma S and a lower affinity for GDP than did the wild-type protein. As the N terminus is required for interactions with target proteins, we conclude that the N terminus of Arf1 is a GTP-sensitive effector domain. When Arf1 was acylated, the GTP-dependent conformational changes were codependent on added phospholipids. In the absence of phospholipids, myristoylated Arf1 has a lower affinity for GTP gamma S than for GDP, and in the presence of phospholipids, the myristoylated protein has a greater affinity for GTP gamma S than for GDP. Thus, N-myristoylation is a critical component in the construction of this phospholipid- and GTP-dependent switch.

• Peters PJ et al.
• Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments.
• J Cell Biol. 1995; 128: 1003-17
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• The ARF GTP binding proteins are believed to function as regulators of membrane traffic in the secretory pathway. While the ARF1 protein has been shown in vitro to mediate the membrane interaction of the cytosolic coat proteins coatomer (COP1) and gamma-adaptin with the Golgi complex, the functions of the other ARF proteins have not been defined. Here, we show by transient transfection with epitope-tagged ARFs, that whereas ARF1 is localized to the Golgi complex and can be shown to affect predictably the assembly of COP1 and gamma-adaptin with Golgi membranes in cells, ARF6 is localized to the endosomal/plasma membrane system and has no effect on these Golgi-associated coat proteins. By immuno-electron microscopy, the wild-type ARF6 protein is observed along the plasma membrane and associated with endosomes, and overexpression of ARF6 does not appear to alter the morphology of the peripheral membrane system. In contrast, overexpression of ARF6 mutants predicted either to hydrolyze or bind GTP poorly shifts the distribution of ARF6 and affects the structure of the endocytic pathway. The GTP hydrolysis-defective mutant is localized to the plasma membrane and its overexpression results in a profound induction of extensive plasma membrane vaginations and a depletion of endosomes. Conversely, the GTP binding-defective ARF6 mutant is present exclusively in endosomal structures, and its overexpression results in a massive accumulation of coated endocytic structures.

• Zhang GF et al.
• Different ARF domains are required for the activation of cholera toxin and phospholipase D.
• J Biol Chem. 1995; 270: 21-4
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• ADP-ribosylation factors (ARFs), initially described as activators of cholera toxin ADP-ribosyltransferase activity, regulate intracellular vesicular membrane trafficking and stimulate a phospholipase D (PLD) isoform. ARF-like (ARL) proteins are structurally related to ARFs but do not activate cholera toxin and have relatively little effect on PLD. A new human ARL gene termed hARL1, which shares 57% amino acid identity with hARF1, was identified using a polymerase chain reaction-based cloning method. To determine whether different structural elements are responsible for the activation structural elements are responsible for the activation of the A subunit of cholera toxin and PLD, chimeric proteins were constructed by switching the amino-terminal 73 amino acids of ARF1 and ARL1. The recombinant rL73/F protein, in which the amino-terminal 73 amino acids of ARL1 replaced those of ARF1, activated the A subunit of cholera toxin, whereas the rF73/L protein, in which the NH2-terminal 73 amino acids of ARF1 replaced those of ARL1, was inactive. The two chimeric proteins had quite opposite effects on PLD activity. rF73/L activated PLD as effectively as rARF1, whereas rL73/F protein activated PLD only slightly. It appears that the amino-terminal region of ARF1 is not critical for its action as a GTP-dependent activator of cholera toxin, whereas it is necessary for activation of the putative effector enzyme, PLD.

• Hong JX, Zhang X, Moss J, Vaughan M
• Isolation of an amino-terminal deleted recombinant ADP-ribosylation factor 1 in an activated nucleotide-free state.
• Proc Natl Acad Sci U S A. 1995; 92: 3056-9
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• ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that activate cholera toxin ADP-ribosyltransferase in vitro and participate in intracellular vesicular membrane trafficking. ARFs are activated when bound GDP is replaced by GTP and inactivated by hydrolysis of bound GTP to yield ARF-GDP. Usually, ARFs are isolated in an inactive GDP-bound state and require addition of GTP along with detergent or phospholipid for activity. Purified mutant recombinant ARF1 lacking the first 13 amino acids (r delta 13ARF1-P) stimulated cholera toxin activity essentially equally with or without added GTP (and phospholipid or detergent), at least in part due to the presence of bound nucleotides, which later were identified as GTP and GDP. Nucleotide-free r delta 13ARF1 (r delta 13ARF1-F), prepared by dialysis against 7 M urea, was active without added GTP in the absence of SDS but inactive without added GTP in its presence. Renaturation of r delta 13ARF1-F in the presence of GTP, ITP, or GDP yielded, respectively, r delta 13ARF1-GTP and r delta 13ARF1-ITP, which were active, and r delta 13ARF1-GDP, which was inactive. Effects of phospholipids and detergents on nucleotide exchangeability evaluated as effects on activity of rARF1 and r delta 13ARF1-F differed. With r delta 13ARF1-F, 100 microM ITP and 100 microM GTP were essentially equally effective in the presence of cardiolipin or SDS. The finding that r delta 13ARF1 differs from rARF1 in the effects of phospholipids and detergents on nucleotide binding is consistent with the conclusion that the ARF amino terminus plays an important role in nucleotide binding and its specificity as well as the molecular conformation and associated activity.

• Randazzo PA, Weiss O, Kahn RA
• Preparation of recombinant ADP-ribosylation factor.
• Methods Enzymol. 1995; 257: 128-35

• Cockcroft S et al.
• Phospholipase D: a downstream effector of ARF in granulocytes.
• Science. 1994; 263: 523-6
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• Activation of the phospholipase D (PLD) pathway is a widespread response when cells are activated by agonists that bind receptors on the cell surface. A 16-kD cytosolic component can reconstitute guanosine triphosphate (GTP)-mediated activation of phospholipase D in HL60 cells depleted of their cytosol by permeabilization. This factor was purified and identified as two small GTP-binding proteins, ARF1 and ARF3. Recombinant ARF1 substituted for purified ARF proteins in the reconstitution assay. These results indicate that phospholipase D is a downstream effector of ARF1 and ARF3. The well-established role of ARF in vesicular traffic would suggest that alterations in lipid content by PLD are an important determinant in vesicular dynamics.

• Amor JC, Harrison DH, Kahn RA, Ringe D
• Structure of the human ADP-ribosylation factor 1 complexed with GDP.
• Nature. 1994; 372: 704-8
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• ADP-ribosylation factors (ARFs) are essential and ubiquitous in eukaryotes, being involved in vesicular transport and functioning as an activator of phospholipase D (refs 1, 2) and cholera toxin. The functions of ARF proteins in membrane traffic and organelle integrity are intimately tied to its reversible association with membranes and specific interactions with membrane phospholipids. One common feature of these functions is their regulation by the binding and hydrolysis of GTP. Here we report the three-dimensional structure of full-length human ARF1 (M(r) 21,000) in its GDP-bound non-myristoylated form. The presence of a unique amino-terminal alpha-helix and loop, together with differences in Mg2+ ligation and the existence of a non-crystallographic dimer, set this structure apart from other GTP-binding proteins. These features provide a structural basis for the GTP-dependent modulation of membrane affinity, the lack of intrinsic GTPase activity, and the nature of effector binding surfaces.

• Cavenagh MM et al.
• ADP-ribosylation factor (ARF)-like 3, a new member of the ARF family of GTP-binding proteins cloned from human and rat tissues.
• J Biol Chem. 1994; 269: 18937-42
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• The human and rat homologues of a new member of the ADP-ribosylation factor (ARF) family of 21-kDa GTP-binding proteins, termed Arl3, were identified as an expressed sequence tag (human) and as a product of polymerase chain reaction amplification using degenerate probes derived from conserved sequences in members of the ARF family (rat). Alignments of the full-length open reading frames of the human and rat homologues revealed the encoded proteins to be over 97% identical to each other and 43% identical to human ARF1. Northern blots of mRNA from seven human tissues and four rat tissues revealed the presence of a ubiquitous band of about 1 kilobase in length that hybridized with the corresponding Arl3 probes. A number of human tumor cell lines expressed Arl3, as determined by immunoblotting with an Arl-specific antibody, raised against a peptide derived from the human Arl3 sequence. The level of Arl3 expressed in these cell lines was on the order of 0.01% of total cell protein. Purified recombinant human Arl3 was shown to bind guanine nucleotides but lacks ARF activity and intrinsic or ARF GTPase-activating protein-stimulated GTPase activity. In contrast to ARF proteins, the Arl3 protein has reduced dependence on phospholipids and magnesium for guanine nucleotide exchange. Thus, Arl3 is a ubiquitously expressed GTP-binding protein in the ARF family with distinctive biochemical properties consistent with its having unique, but unknown, role(s) in cell physiology.

• Regad F, Bardet C, Tremousaygue D, Moisan A, Lescure B, Axelos M
• cDNA cloning and expression of an Arabidopsis GTP-binding protein of the ARF family.
• FEBS Lett. 1993; 316: 133-6
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• A cDNA clone encoding a small GTP-binding protein, the ADP-ribosylation factor (ARF) was isolated from a cDNA library of Arabidopsis thaliana cultured cells. The predicted amino acid sequence was highly homologous to the known yeast, bovine and human ARF sequences. Southern analysis of Arabidopsis genomic DNA suggested the existence of at least two copies of ARF genes. The level of ARF mRNA was found to be nearly constant during all cell growth stages in suspension cultures.

• Mishima K, Tsuchiya M, Nightingale MS, Moss J, Vaughan M
• ARD 1, a 64-kDa guanine nucleotide-binding protein with a carboxyl-terminal ADP-ribosylation factor domain.
• J Biol Chem. 1993; 268: 8801-7
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• Clones referred to as ARD 1 were isolated from human and rat cDNA libraries. ARD 1 genes encode a putative 64-kDa protein that contains an 18-kDa ADP-ribosylation factor (ARF) domain at the carboxyl terminus and is much larger than the other monomeric approximately 20-kDa guanine nucleotide-binding ARF proteins thus far identified. ARD 1 mRNAs of 3.7 and 4.1 kilobases were detected in all rat tissues as well as in mouse and rabbit brain, human fibroblasts, and human neuroblastoma cells but not in HL-60 cells. Based on sequence identities, ARD 1 is highly conserved between rat and human. The ARF domain of ARD 1 contains the consensus sequences believed to be involved in guanine nucleotide binding, which are conserved in the ARFs and other GTP-binding proteins. Recombinant ARD 1 or the ARF domain of ARD 1, which lacks the 15 amino acids corresponding to the amino-terminal regions of ARFs stimulated, in a GTP-dependent manner, cholera toxin ADP-ribosyltransferase activity in the presence of 0.3% Tween 20. It had no effect in the presence of SDS, dimyristoylphosphatidylcholine/cholate, or cardiolipin. These observations are consistent with the conclusion that the amino-terminal region of ARF proteins is not required for activation of cholera toxin. In addition, the characteristic features of ARF proteins may be found as domains of larger mammalian proteins.

• Brown HA, Gutowski S, Moomaw CR, Slaughter C, Sternweis PC
• ADP-ribosylation factor, a small GTP-dependent regulatory protein, stimulates phospholipase D activity.
• Cell. 1993; 75: 1137-44
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• The hydrolysis of phosphatidylcholine by phospholipase D (PLD) results in the production of phosphatidic acid and choline. An assay that uses an exogenous substrate was developed to measure this activity in membranes and solubilized preparations from HL60 cells. A cytosolic factor markedly enhanced PLD activity in membranes and was essential for GTP gamma S-dependent stimulation of an enriched preparation of PLD. The factor was purified to homogeneity from bovine brain cytosol and identified as a member of the ADP-Ribosylation Factor (ARF) subfamily of small G proteins. Subsequently, recombinant myristoylated ARF1 was found to be a better activator of PLD activity than was the nonmyristoylated form. ARF proteins have been implicated recently as factors for regulation of intracellular vesicle traffic. The current finding suggests that PLD activity plays a prominent role in the action of ARF and that ARF may be a key component in the generation of second messengers via phospholipase D.

• Kahn RA, Yucel JK, Malhotra V
• ARF signaling: a potential role for phospholipase D in membrane traffic.
• Cell. 1993; 75: 1045-8

• Kahn RA et al.
• The amino terminus of ADP-ribosylation factor (ARF) is a critical determinant of ARF activities and is a potent and specific inhibitor of protein transport.
• J Biol Chem. 1992; 267: 13039-46
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• Deletion of the amino-terminal 17 residues from human ADP-ribosylation factor (ARF) resulted in a protein ([delta 1-17]mARF1p) devoid of ARF activity but which retained the ability to bind guanine nucleotides with high affinity. Unlike the wild type, the binding of guanine nucleotides to this deletion mutant was found to be independent of added phospholipids. A chimeric protein was produced, consisting of 10% (the amino-terminal 17 amino acids) human ARF1p and 90% ARL1p, an ARF-like protein (55% identical protein sequence) from Drosophila. This chimera was found to have ARF activity, lacking in the parental ARL1 protein. Thus, the amino terminus of ARF1p was shown to be a critical component of ARF activity. A synthetic peptide, derived from the amino terminus of ARF1p, has no ARF activity. Rather, the peptide was found to be a specific inhibitor of ARF activities. This peptide was also found to be a potent and specific inhibitor of both an in vitro intra-Golgi transport assay and the guanosine 5'-3-O-(thio)triphosphate-stimulated accumulation of coated vesicles and buds from Golgi preparations. We conclude that ARF is required for the budding of coated vesicles from the Golgi stacks and serves a regulatory role in protein secretion through the Golgi in eukaryotic cells.

• Graves RA, Tontonoz P, Spiegelman BM
• Analysis of a tissue-specific enhancer: ARF6 regulates adipogenic gene expression.
• Mol Cell Biol. 1992; 12: 3313-3313

• Tsai SC, Haun RS, Tsuchiya M, Moss J, Vaughan M
• Isolation and characterization of the human gene for ADP-ribosylation factor 3, a 20-kDa guanine nucleotide-binding protein activator of cholera toxin.
• J Biol Chem. 1991; 266: 23053-9
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• ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that stimulate the ADP-ribosyltransferase activity of cholera toxin in vitro. Five different human ARFs have been identified by cDNA cloning. Northern analysis using ARF 3-specific oligonucleotides identified two mRNAs of 3.7 and 1.2 kilobases (kb). We report here the complete nucleotide sequence of the 3.7-kb ARF 3 mRNA derived from three overlapping cDNAs isolated from human hippocampus and fetal brain cDNA libraries, as well as the structure of human ARF 3 gene. Sequences of two overlapping genomic clones indicated that the ARF 3 gene spans approximately 18.3 kb and contains five exons and four introns. The conserved amino acid sequences involved in guanine nucleotide binding by ARF 3 are distributed among separate exons, as found in other GTP-binding protein genes. Translation initiates in exon 2 which includes the sequence GXXXXGK that probably participates in phosphate binding and GTP hydrolysis. The sequence DVGG in exon 3 coordinates binding of Mg2+ and the beta-phosphate of GDP. In the ARF 3 gene in contrast to those of other GTP-binding proteins, the sequence NKXD (which is thought to contribute to the specificity of interaction with the guanine ring) is divided between exons 4 and 5. The latter encodes the COOH-terminal 53 amino acids of ARF 3 and contains greater than 2500 base pairs of untranslated DNA. The sequence AATTAA is 19 bases 5' to the polyadenylation addition site of the 3.7-kb mRNA. Multiple transcription start sites were identified by primer extension and S1 and mung bean nuclease analyses. The 5'-flanking region of exon 1 contains neither a TATA nor a CAAT box, but is high in GC content (greater than 70%) and includes three potential Sp1-binding sites (GC box), consistent with the promoters described for several housekeeping genes. The 1.2-kb ARF 3 mRNA is shown to arise by use of an alternative polyadenylation signal (AACAAA) at nucleotide 1091 within the ARF 3 cDNA.

• Moss J et al.
• Structural and functional characterization of ADP-ribosylation factors, 20 kDa guanine nucleotide-binding proteins that activate cholera toxin.
• Adv Second Messenger Phosphoprotein Res. 1990; 24: 83-8

• Tsuchiya M, Price SR, Nightingale MS, Moss J, Vaughan M
• Tissue and species distribution of mRNA encoding two ADP-ribosylation factors, 20-kDa guanine nucleotide binding proteins.
• Biochemistry. 1989; 28: 9668-73
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• Cholera toxin catalyzed ADP-ribosylation of Gs alpha, the stimulatory guanine nucleotide binding protein of the adenylyl cyclase system, is enhanced by approximately 20-kDa guanine nucleotide binding proteins, termed ADP-ribosylation factors or ARFs. ARF is an allosteric activator of the A1 catalytic protein of the toxin. Bovine ARF cDNA clones, ARF-1 isolated from adrenal (Sewell & Kahn, 1988) and ARF-2B from retina (Price et al., 1988), exhibit nucleotide and deduced amino acid sequences that are 80% and 96% identical, respectively, in the coding region. To determine tissue and species distribution of ARF-like mRNAs, bovine ARF-2B and human ARF-1 cDNAs and 30- or 48-base oligonucleotide probes that distinguish between ARF-1 and ARF-2B cDNAs in coding and 3'-untranslated regions were used for Northern analysis of poly(A+) RNA from different tissues and species. On the basis of hybridization with specific oligonucleotide probes, all bovine tissues contained mRNAs of 1.7 and 2.1 kb that were related to ARF-1 and ARF-2B, respectively. Northern analysis of brain poly(A+) RNA from different species with ARF-2B and ARF-1 cDNAs at low stringency demonstrated several bands varying in size from 0.9 to 3.7 kb. A 1.7-kb band consistently hybridized with an ARF-1 30-base coding-region probe but not with a probe for the 3'-untranslated region. Similar ARF-2B oligonucleotide probes did not hybridize with rat, mouse, rabbit, or human brain mRNA. Cleavage of ARF-2B cDNA with PvuII generated two fragments, one containing coding and the other 3'-noncoding region.(ABSTRACT TRUNCATED AT 250 WORDS)

• Sewell JL, Kahn RA
• Sequences of the bovine and yeast ADP-ribosylation factor and comparison to other GTP-binding proteins.
• Proc Natl Acad Sci U S A. 1988; 85: 4620-4
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• The ADP-ribosylation factor (ARF) is a 21-kDa GTP-binding protein that serves as the cofactor in the cholera toxin-catalyzed activation of the stimulatory guanine nucleotide-binding protein of adenylate cyclase (Gs). An oligonucleotide probe based on the partial amino acid sequence was used to clone ARF from a bovine adrenal chromaffin cDNA library. The yeast (Saccharomyces cerevisiae) ARF gene was then cloned from a YCp50 genomic library by cross-species hybridization by using the coding region of the bovine gene. RNA gel blots of poly(A)+ RNA indicate that only one ARF message size (900 and 2000 base pairs) is present in yeast and cows, respectively. Comparison of the cDNA-derived amino acid sequences of ARF to other GTP-binding proteins reveals a structural relationship between ARF and the ras family of proteins. A slightly better structural relationship is detected when ARF is compared to the alpha subunits of the trimeric GTP-binding proteins, including Gs alpha. All of the biochemical characteristics of the purified ARF, including the lack of GTPase activity and the posttranslational myristoylation, are consistent with the derived sequences. Comparison of the ARF sequences to that of the chicken processed pseudogene (CPS-1), previously reported as a ras homologue, reveals that CPS-1 is actually an ARF-derived gene. These results demonstrate that ARF is a GTP-binding protein with structural features of both the ras and the trimeric GTP-binding protein families.

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