SMART MODE:

NORMAL GENOMIC

• Mayer G et al.
• Controlling small guanine-nucleotide-exchange factor function through cytoplasmic RNA intramers.
• Proc Natl Acad Sci U S A. 2001; 98: 4961-5
• Display abstract

• ADP-ribosylation factor (ARF) GTPases and their regulatory proteins have been implicated in the control of diverse biological functions. Two main classes of positive regulatory elements for ARF have been discovered so far: the large Sec7/Gea and the small cytohesin/ARNO families, respectively. These proteins harbor guanine-nucleotide-exchange factor (GEF) activity exerted by the common Sec7 domain. The availability of a specific inhibitor, the fungal metabolite brefeldin A, has enabled documentation of the involvement of the large GEFs in vesicle transport. However, because of the lack of such tools, the biological roles of the small GEFs have remained controversial. Here, we have selected a series of RNA aptamers that specifically recognize the Sec7 domain of cytohesin 1. Some aptamers inhibit guanine-nucleotide exchange on ARF1, thereby preventing ARF activation in vitro. Among them, aptamer M69 exhibited unexpected specificity for the small GEFs, because it does not interact with or inhibit the GEF activity of the related Gea2-Sec7 domain, a member of the class of large GEFs. The inhibitory effect demonstrated in vitro clearly is observed as well in vivo, based on the finding that M69 produces similar results as a dominant-negative, GEF-deficient mutant of cytohesin 1: when expressed in the cytoplasm of T-cells, M69 reduces stimulated adhesion to intercellular adhesion molecule-1 and results in a dramatic reorganization of F-actin distribution. These highly specific cellular effects suggest that the ARF-GEF activity of cytohesin 1 plays an important role in cytoskeletal remodeling events of lymphoid cells.

• Baumgartner F, Wiek S, Paprotka K, Zauner S, Lingelbach K
• A point mutation in an unusual Sec7 domain is linked to brefeldin A resistance in a Plasmodium falciparum line generated by drug selection.
• Mol Microbiol. 2001; 41: 1151-8
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• The malaria parasite Plasmodium falciparum has an unusual organization of its secretory compartments. As an approach to a functional identification of auxiliary proteins involved in secretion, a parasite line was generated by drug selection that is resistant to brefeldin A, an inhibitor of the secretory pathway. In the resistant line, neither protein secretion nor parasite viability were affected by the drug. The analysis of a sec7 domain, a conserved structure of guanine nucleotide exchange factors (ARF-GEF) required for the activation of ADP-ribosylation factors, revealed a single methionine-isoleucine substitution in the resistant parasite line. ARF-GEFs are key molecules in the formation of transport vesicles and the main targets of brefeldin A. The methionine residue in this position of sec7 domains is highly conserved and confers brefeldin A sensitivity. Unlike other eukaryotes that have multiple ARF-GEFs, the plasmodial genome encodes a single sec7 domain. This domain shows a distinct structural difference to all sec7 domains analysed so far; two conserved subdomains that are essential for protein function are separated in the plasmodial protein by an insertion of 146 amino acids.

• Kliche S et al.
• Signaling by human herpesvirus 8 kaposin A through direct membrane recruitment of cytohesin-1.
• Mol Cell. 2001; 7: 833-43
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• The induction of a transformed cellular phenotype by viruses requires the modulation of signaling pathways through viral proteins. We show here that the phenotypic changes induced by the kaposin A protein of human herpesvirus 8 are mediated through its direct interaction with cytohesin-1, a guanine nucleotide exchange factor for ARF GTPases and regulator of integrin-mediated cell adhesion. Focus formation, stress fiber dissolution, and activation of the ERK-1/2 MAP kinase signal cascade were reverted by the cytohesin-1 E157K mutant, which is deficient in catalyzing guanine nucleotide exchange. Furthermore, liposome-embedded kaposin A specifically stimulates cytohesin-1 dependent GTP binding of myristoylated ARF1 in vitro. These results suggest a previously unknown involvement of ARF GTPases in the control of cellular functions by herpesviruses.

• Someya A et al.
• ARF-GEP(100), a guanine nucleotide-exchange protein for ADP-ribosylation factor 6.
• Proc Natl Acad Sci U S A. 2001; 98: 2413-8
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• A human cDNA encoding an 841-aa guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARFs), named ARF-GEP(100), which contains a Sec7 domain, a pleckstrin homology (PH)-like domain, and an incomplete IQ-motif, was identified. On Northern blot analysis of human tissues, a approximately 8-kb mRNA that hybridized with an ARF-GEP(100) cDNA was abundant in peripheral blood leukocytes, brain, and spleen. ARF-GEP(100) accelerated [(35)S]GTPgammaS binding to ARF1 (class I) and ARF5 (class II) 2- to 3-fold, and to ARF6 (class III) ca. 12-fold. The ARF-GEP(100) Sec7 domain contains Asp(543) and Met(555), corresponding to residues associated with sensitivity to the inhibitory effect of the fungal metabolite brefeldin A (BFA) in yeast Sec7, but also Phe(535) and Ala(536), associated with BFA-insensitivity. The PH-like domain differs greatly from those of other ARF GEPs in regions involved in phospholipid binding. Consistent with its structure, ARF-GEP(100) activity was not affected by BFA or phospholipids. After subcellular fractionation of cultured T98G human glioblastoma cells, ARF6 was almost entirely in the crude membrane fraction, whereas ARF-GEP(100), a 100-kDa protein detected with antipeptide antibodies, was cytosolic. On immunofluorescence microscopy, both proteins had a punctate pattern of distribution throughout the cells, with apparent colocalization only in peripheral areas. The coarse punctate distribution of EEA-1 in regions nearer the nucleus appeared to coincide with that of ARF-GEP(100) in those areas. No similar coincidence of ARF-GEP(100) with AP-1, AP-2, catenin, LAMP-1, or 58K was observed. The new human BFA-insensitive GEP may function with ARF6 in specific endocytic processes.

• Beraud-Dufour S, Robineau S
• Expression, purification, and measurements of activity of ARNO1, a guanine nucleotide exchange factor for ADP-ribosylation factor 1 (ARF1).
• Methods Enzymol. 2001; 329: 264-71

• Klarlund JK, Czech MP
• Isolation and properties of GRP1, an ADP-ribosylation factor (ARF)-guanine nucleotide exchange protein regulated by phosphatidylinositol 3,4,5-trisphosphate.
• Methods Enzymol. 2001; 329: 279-89

• Macia E, Chabre M, Franco M
• Specificities for the small G proteins ARF1 and ARF6 of the guanine nucleotide exchange factors ARNO and EFA6.
• J Biol Chem. 2001; 276: 24925-30
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• ARF1 and ARF6 are distant members of the ADP-ribosylation factor (ARF) small G-protein subfamily. Their distinct cellular functions must result from specificity of interaction with different effectors and regulators, including guanine nucleotide exchange factors (GEFs). ARF nucleotide-binding site opener (ARNO), and EFA6 are analogous ARF-GEFs, both comprising a catalytic "Sec7" domain and a pleckstrin homology domain. In vivo ARNO, like ARF1, is mostly cytosolic, with minor localizations at the Golgi and plasma membrane; EFA6, like ARF6, is restricted to the plasma membrane. However, depending on conditions, ARNO appears active on ARF6 as well as on ARF1. Here we analyze the origin of these ARF-GEF selectivities. In vitro, in the presence of phospholipid membranes, ARNO activates ARF1 preferentially and ARF6 slightly, whereas EFA6 activates ARF6 exclusively; the stimulation efficiency of EFA6 on ARF6 is comparable with that of ARNO on ARF1. These selectivities are determined by the GEFs Sec7 domains alone, without the pleckstrin homology and N-terminal domains, and by the ARF core domains, without the myristoylated N-terminal helix; they are not modified upon permutation between ARF1 and ARF6 of the few amino acids that differ within the switch regions. Thus selectivity for ARF1 or ARF6 must depend on subtle folding differences between the ARFs switch regions that interact with the Sec7 domains.

• Grebe M et al.
• A conserved domain of the arabidopsis GNOM protein mediates subunit interaction and cyclophilin 5 binding.
• Plant Cell. 2000; 12: 343-56
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• The Arabidopsis GNOM protein, a guanine nucleotide exchange factor (GEF) that acts on ADP ribosylation factor (ARF)-type G proteins, is required for coordination of cell polarity along the apical-basal embryo axis. Interallelic complementation of gnom mutants suggested that dimerization is involved in GNOM function. Here, direct interaction between GNOM molecules is demonstrated in vitro and by using a yeast two-hybrid system. Interaction was confined to an N-terminal domain conserved within a subgroup of large ARF GEFs. The same domain mediated in vitro binding to cyclophilin 5 (Cyp5), which was identified as a GNOM interactor in two-hybrid screening. Cyp5 displayed peptidylprolyl cis/trans-isomerase and protein refolding activities that were sensitive to cyclosporin A. Cyp5 protein accumulated in several plant organs and, like GNOM, was partitioned between cytosolic and membrane fractions. Cyp5 protein was also expressed in the developing embryo. Our results suggest that Cyp5 may regulate the ARF GEF function of the GNOM protein during embryogenesis.

• Shimizu T, Ihara K, Maesaki R, Kuroda S, Kaibuchi K, Hakoshima T
• An open conformation of switch I revealed by the crystal structure of a Mg2+-free form of RHOA complexed with GDP. Implications for the GDP/GTP exchange mechanism.
• J Biol Chem. 2000; 275: 18311-7
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• Mg(2+) ions are essential for guanosine triphosphatase (GTPase) activity and play key roles in guanine nucleotide binding and preserving the structural integrity of GTP-binding proteins. We determined the crystal structure of a small GTPase RHOA complexed with GDP in the absence of Mg(2+) at 2.0-A resolution. Elimination of a Mg(2+) ion induces significant conformational changes in the switch I region that opens up the nucleotide-binding site. Similar structural changes have been observed in the switch regions of Ha-Ras bound to its guanine nucleotide exchange factor, Sos. This RHOA-GDP structure reveals an important regulatory role for Mg(2+) and suggests that guanine nucleotide exchange factor may utilize this feature of switch I to produce an open conformation in GDP/GTP exchange.

• Jackson CL, Casanova JE
• Turning on ARF: the Sec7 family of guanine-nucleotide-exchange factors.
• Trends Cell Biol. 2000; 10: 60-7
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• ARF proteins are important regulators of membrane dynamics and protein transport within the eukaryotic cell. The Sec7 domain is approximately 200 amino acids in size and stimulates guanine-nucleotide exchange on members of the ARF class of small GTPases. The members of one subclass of Sec7-domain proteins are direct targets of the secretion-inhibiting drug brefeldin A, which blocks the exchange reaction by trapping a reaction intermediate in an inactive, abortive complex. A separate subclass of Sec7-domain proteins is involved in signal transduction and possess a domain that mediates membrane binding in response to extracellular signals.

• Robineau S, Chabre M, Antonny B
• Binding site of brefeldin A at the interface between the small G protein ADP-ribosylation factor 1 (ARF1) and the nucleotide-exchange factor Sec7 domain.
• Proc Natl Acad Sci U S A. 2000; 97: 9913-8
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• Sec7 domains (Sec7d) catalyze the exchange of guanine nucleotide on ARFs. Recent studies indicated that brefeldin A (BFA) inhibits Sec7d-catalyzed nucleotide exchange on ARF1 in an uncompetitive manner by trapping an early intermediate of the reaction: a complex between GDP-bound ARF1 and Sec7d. Using (3)H-labeled BFA, we show that BFA binds to neither isolated Sec7d nor isolated ARF1-GDP, but binds to the transitory Sec7d-ARF1-GDP complex and stabilizes it. Two pairs of residues at positions 190-191 and 198-208 (Arno numbering) in Sec7d contribute equally to the stability of BFA binding, which is also sensitive to mutation of H80 in ARF1. The catalytic glutamic (E156) residue of Sec7d is not necessary for BFA binding. In contrast, BFA does not bind to the intermediate catalytic complex between nucleotide-free ARF1 and Sec7d. These results suggest that, on initial docking steps between ARF1-GDP and Sec7d, BFA inserts like a wedge between the switch II region of ARF1-GDP and a surface encompassing residues 190-208, at the border of the characteristic hydrophobic groove of Sec7d. Bound BFA would prevent the switch regions of ARF1-GDP from reorganizing and forming tighter contacts with Sec7d and thereby would maintain the bound GDP of ARF1 at a distance from the catalytic glutamic finger of Sec7d.

• Kuai J, Kahn RA
• Residues forming a hydrophobic pocket in ARF3 are determinants of GDP dissociation and effector interactions.
• FEBS Lett. 2000; 487: 252-6
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• Three residues of human ADP-ribosylation factor 3 (ARF3) (F51, W66 and Y81) cluster into a hydrophobic pocket in the inactive, GDP-bound protein. Disruption of the hydrophobic pocket with mutations at these residues increased the rate of GDP dissociation and association, but not always that of GTPgammaS. Several of the same mutants were found to be defective, often selectively, in binding different ARF effectors in two-hybrid assays. These results highlight three features of these hydrophobic residues in regulating (1) the rate of GDP dissociation, (2) the conformational changes that promote GTP binding and (3) their role in binding target proteins.

• Knorr T, Nagel W, Kolanus W
• Phosphoinositides determine specificity of the guanine-nucleotide exchange activity of cytohesin-1 for ADP-ribosylation factors derived from a mammalian expression system.
• Eur J Biochem. 2000; 267: 3784-91
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• ADP-ribosylation factors (ARFs) are small Ras-like GTPases which play important roles in intracellular vesicle transport and in the remodeling of the actin cytoskeleton. Guanine nucleotide exchange factors (GEFs) for ARFs have recently been identified. One of them, cytohesin-1, a 47-kDa cytoplasmic protein acts as an inside-out signaling molecule and regulates binding of the beta2 integrin leukocyte function antigen 1 (LFA-1) to its ligand intercellular adhesion molecule 1 (ICAM-1). In this study, we address the regulation of the GEF activity of cytohesin-1 by phosphoinositides, using mammalian expression of functional ARF-Ig chimeras. The fusion proteins, which can be quantitatively immunoprecipitated on protein A-Sepharose, target to the expected intracellular compartments, and they are readily induced to bind GTP in vitro. We show that both ARF1-Ig and ARF6-Ig chimeras are activated in vitro by cytohesin-1. However, GEF activity towards ARF6 is strongly suppressed by phosphatidylinositol-(3,4,5)-trisphosphate (PtdInsP3). In contrast, cytohesin-1-dependent GTP binding of ARF1 is significantly enhanced by PtdInsP3. We conclude that the membrane phospholipid PtdInsP3 determines the specificity of the GEF activity of cytohesin-1.

• Vitale N, Pacheco-Rodriguez G, Ferrans VJ, Riemenschneider W, Moss J, Vaughan M
• Specific functional interaction of human cytohesin-1 and ADP-ribosylation factor domain protein (ARD1).
• J Biol Chem. 2000; 275: 21331-9
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• Activation of ADP-ribosylation factors (ARFs) is mediated by guanine nucleotide-exchange proteins, which accelerate conversion of inactive ARF-GDP to active ARF-GTP. ARF domain protein (ARD1), a 64-kDa GTPase with a C-terminal ADP-ribosylation factor domain, is localized to lysosomes and the Golgi apparatus. When ARD1 was used as bait to screen a human liver cDNA library using the yeast two-hybrid system, a cDNA for cytohesin-1, a approximately 50-kDa protein with ARF guanine nucleotide-exchange protein activity, was isolated. In this system, ARD1-GDP interacted well with cytohesin-1 but very poorly with cytohesin-2. In agreement, cytohesin-1, but not cytohesin-2, markedly accelerated [(35)S]guanosine 5'-3-O-(thio)triphosphate binding to ARD1. The effector region of the ARF domain of ARD1 appeared to be critical for the specific interaction with cytohesin-1. Replacement of single amino acids in the Sec7 domains of cytohesin-1 and -2 showed that residue 30 is critical for specificity. In transfected COS-7 cells, overexpressed ARD1 and cytohesin-1 were partially colocalized, as determined by confocal fluorescence microscopy. It was concluded that cytohesin-1 is likely to be involved in ARD1 activation, consistent with a role for ARD1 in the regulation of vesicular trafficking.

• Ogasawara M et al.
• Similarities in function and gene structure of cytohesin-4 and cytohesin-1, guanine nucleotide-exchange proteins for ADP-ribosylation factors.
• J Biol Chem. 2000; 275: 3221-30
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• Activation of ADP-ribosylation factors (ARFs), approximately 20-kDa GTPases that are inactive in the GDP-bound form, depends on guanine nucleotide-exchange proteins (GEPs) to accelerate GTP binding. A novel ARF GEP, designated cytohesin-4, was cloned from a human brain cDNA library. Deduced amino acid sequence of the 47-kDa protein contains the same structural components present in cytohesin -1, -2, and -3, including an approximately 200-amino acid Sec7 domain with an approximately 100-residue pleckstrin homology domain near the C terminus. The Sec7 domain sequence is 77% identical to those of other cytohesins. Structures of the cytohesin-4 and cytohesin-1 genes were remarkably similar, except for an extra 3-base pair (GAG) exon present in cytohesin-1. Two mRNAs with and without the 3-base pair sequence were found in brain in different ratios for cytohesin-1, -2, and -3 but not cytohesin-4. Recombinant cytohesin-4 stimulated guanosine 5'-3-O-(thio)triphosphate binding by human ARF1 and ARF5 but not ARF6. Like other cytohesins and unlike the approximately 200-kDa ARF GEPs, it was not inhibited by brefeldin A. A cytohesin-4 mRNA of approximately 3.7 kilobases, abundant in leukocytes, was not detected in most tissues. Among separated populations of blood cells, approximately 90% of CD33(+) (monocytes), 80% of CD2(+) (NK/T), and 10-20% of CD19(+) (B) cells contained cytohesin-4 mRNA by in situ hybridization. Thus, in gene structure and brefeldin A-insensitive GEP activity, cytohesin-4 resembles other cytohesins, but its tissue distribution differs considerably, consistent with a different specific function.

• Schurmann A et al.
• The ADP-ribosylation factor (ARF)-related GTPase ARF-related protein binds to the ARF-specific guanine nucleotide exchange factor cytohesin and inhibits the ARF-dependent activation of phospholipase D.
• J Biol Chem. 1999; 274: 9744-51
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• ADP-ribosylation factor-related protein (ARP) is a membrane-associated GTPase with remote similarity to the family of ADP-ribosylation factors (ARF). In a yeast two-hybrid screen designed to identify proteins interacting with ARP, we isolated a partial cDNA of the ARF-specific guanine nucleotide exchange factor mSec7-1/cytohesin encoding its N terminus and most of the Sec7 domain (codons 1-200). ARP and ARP-Q79L (GTPase-negative ARP) exhibited a higher affinity to mSec7-1-(1-200) than ARP-T31N (nucleotide exchange-defective ARP) in the two-hybrid assay. Similarly, full-length [35S]mSec7-1/cytohesin was specifically adsorbed to glutathione-Sepharose loaded with glutathione S-transferase (GST)-ARP-Q79L, GST-ARP, or GST-ARP-T31N, the latter exhibiting the lowest binding affinity. Overexpression of ARP-Q79L, but not of ARP-T31N, in COS-7 cells reduced the fluorescence from co-expressed green fluorescent protein fused with mSec7-1/cytohesin or mSec7-2/ARNO in plasma membranes as detected by deconvolution microscopy. Recombinant ARP and ARP-Q79L, but not ARP-T31N, inhibited the phospholipase D (PLD) activity stimulated by mSec7-2/ARNO and ARF in a system of isolated membranes. Furthermore, transfection of HEK-293 cells with ARP or ARP-Q79L, but not ARP-T31N, inhibited the muscarinic acetylcholine receptor-3 induced PLD stimulation and translocation of ARF from cytosol to membranes. These data suggest that the GTP-bound form of ARP specifically binds mSec7-1/cytohesin, and that ARP may be involved in a pathway inhibiting the ARF-controlled activity of PLD.

• Beraud-Dufour S, Paris S, Chabre M, Antonny B
• Dual interaction of ADP ribosylation factor 1 with Sec7 domain and with lipid membranes during catalysis of guanine nucleotide exchange.
• J Biol Chem. 1999; 274: 37629-36
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• Sec7 domains catalyze the replacement of GDP by GTP on the G protein ADP-ribosylation factor 1 (myrARF1) by interacting with its switch I and II regions and by destabilizing, through a glutamic finger, the beta-phosphate of the bound GDP. The myristoylated N-terminal helix that allows myrARF1 to interact with membrane lipids in a GTP-dependent manner is located some distance from the Sec7 domain-binding region. However, these two regions are connected. Measuring the binding to liposomes of functional or abortive complexes between myrARF1 and the Sec7 domain of ARNO demonstrates that myrARF1, in complex with the Sec7 domain, adopts a high affinity state for membrane lipids, similar to that of the free GTP-bound form. This tight membrane attachment does not depend on the release of GDP induced by the Sec7 domain but is partially inhibited by the uncompetitive inhibitor brefeldin A. These results suggest that the conformational switch of the N-terminal helix of myrARF1 to the membrane-bound form is an early event in the nucleotide exchange pathway and is a prerequisite for a structural rearrangement at the myrARF1-GDP/Sec7 domain interface that allows the glutamic finger to expel GDP from myrARF1.

• Peyroche A, Antonny B, Robineau S, Acker J, Cherfils J, Jackson CL
• Brefeldin A acts to stabilize an abortive ARF-GDP-Sec7 domain protein complex: involvement of specific residues of the Sec7 domain.
• Mol Cell. 1999; 3: 275-85
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• We demonstrate that the major in vivo targets of brefeldin A (BFA) in the secretory pathway of budding yeast are the three members of the Sec7 domain family of ARF exchange factors: Gea1p and Gea2p (functionally interchangeable) and Sec7p. Specific residues within the Sec7 domain are important for BFA inhibition of ARF exchange activity, since mutations in these residues of Gea1p (sensitive to BFA) and of ARNO (resistant to BFA) reverse the sensitivity of each to BFA in vivo and in vitro. We show that the target of BFA inhibition of ARF exchange activity is an ARF-GDP-Sec7 domain protein complex, and that BFA acts to stabilize this complex to a greater extent for a BFA-sensitive Sec7 domain than for a resistant one.

• Moss J, Vaughan M
• Activation of toxin ADP-ribosyltransferases by eukaryotic ADP-ribosylation factors.
• Mol Cell Biochem. 1999; 193: 153-7
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• ADP-ribosylation factors (ARFs) are members of a multigene family of 20-kDa guanine nucleotide-binding proteins that are regulatory components in several pathways of intracellular vesicular trafficking. The relatively small (approximately 180-amino acids) ARF proteins interact with a variety of molecules (in addition to GTP/GDP, of course). Cholera toxin was the first to be recognized, hence the name. Later it was shown that ARF also activates phospholipase D. Different parts of the molecule are responsible for activation of the two enzymes. In vesicular trafficking, ARF must interact with coatomer to recruit it to a membrane and thereby initiate vesicle budding. ARF function requires that it alternate between GTP- and GDP-bound forms, which involves interaction with regulatory proteins. Inactivation of ARF-GTP depends on a GTPase-activating protein or GAP. A guanine nucleotide-exchange protein or GEP accelerates release of bound GDP from inactive ARF-GDP to permit GTP binding. Inhibition of GEP by brefeldin A (BFA) blocks ARF activation and thereby vesicular transport. In cells, it causes apparent disintegration of Golgi structure. Both BFA-sensitive and insensitive GEPs are known. Sequences of peptides from a BFA-sensitive GEP purified in our laboratory revealed the presence of a Sec7 domain, a sequence of approximately 200 amino acids that resembles a region in the yeast Sec7 gene product, which is involved in Golgi vesicular transport. Other proteins of unknown function also contain Sec7 domains, among them a lymphocyte protein called cytohesin-1. To determine whether it had GEP activity, recombinant cytohesin-1 was synthesized in E. coli. It preferentially activated class I ARFs 1 and 3 and was not inhibited by BFA but failed to activate ARF5 (class II). There are now five Sec7 domain proteins known to have GEP activity toward class I ARFs. It remains to be determined whether there are other Sec7 domain proteins that are GEPs for ARFs 4, 5, or 6.

• Claude A et al.
• GBF1: A novel Golgi-associated BFA-resistant guanine nucleotide exchange factor that displays specificity for ADP-ribosylation factor 5.
• J Cell Biol. 1999; 146: 71-84
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• Expression cloning from a cDNA library prepared from a mutant CHO cell line with Golgi-specific resistance to Brefeldin A (BFA) identified a novel 206-kD protein with a Sec7 domain termed GBF1 for Golgi BFA resistance factor 1. Overexpression of GBF1 allowed transfected cells to maintain normal Golgi morphology and grow in the presence of BFA. Golgi- enriched membrane fractions from such transfected cells displayed normal levels of ADP ribosylation factors (ARFs) activation and coat protein recruitment that were, however, BFA resistant. Hexahistidine-tagged-GBF1 exhibited BFA-resistant guanine nucleotide exchange activity that appears specific towards ARF5 at physiological Mg2+concentration. Characterization of cDNAs recovered from the mutant and wild-type parental lines established that transcripts in these cells had identical sequence and, therefore, that GBF1 was naturally BFA resistant. GBF1 was primarily cytosolic but a significant pool colocalized to a perinuclear structure with the beta-subunit of COPI. Immunogold labeling showed highest density of GBF1 over Golgi cisternae and significant labeling over pleiomorphic smooth vesiculotubular structures. The BFA-resistant nature of GBF1 suggests involvement in retrograde traffic.

• Langille SE et al.
• ADP-ribosylation factor 6 as a target of guanine nucleotide exchange factor GRP1.
• J Biol Chem. 1999; 274: 27099-104
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• The GRP1 protein contains a Sec7 homology domain that catalyzes guanine nucleotide exchange on ADP-ribosylation factors (ARF) 1 and 5 as well as a pleckstrin homology domain that binds phosphatidylinositol(3,4,5)P(3), an intermediate in cell signaling by insulin and other extracellular stimuli (Klarlund, J. K., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and Czech, M. P. (1997) Science 275, 1927-1930). Here we show that both endogenous GRP1 and ARF6 rapidly co-localize in plasma membrane ruffles in Chinese hamster ovary (CHO-T) cells expressing human insulin receptors and COS-1 cells in response to insulin and epidermal growth factor, respectively. The pleckstrin homology domain of GRP1 appears to be sufficient for regulated membrane localization. Using a novel method to estimate GTP loading of expressed HA epitope-tagged ARF proteins in intact cells, levels of biologically active, GTP-bound ARF6 as well as GTP-bound ARF1 were elevated when these ARF proteins were co-expressed with GRP1 or the related protein cytohesin-1. GTP loading of ARF6 in both control cells and in response to GRP1 or cytohesin-1 was insensitive to brefeldin A, consistent with previous data on endogenous ARF6 exchange activity. The ability of GRP1 to catalyze GTP/GDP exchange on ARF6 was confirmed using recombinant proteins in a cell-free system. Taken together, these results suggest that phosphatidylinositol(3,4,5)P(3) may be generated in cell membrane ruffles where receptor tyrosine kinases are concentrated in response to growth factors, causing recruitment of endogenous GRP1. Further, co-localization of GRP1 with ARF6, combined with its demonstrated ability to activate ARF6, suggests a physiological role for GRP1 in regulating ARF6 functions.

• Pacheco-Rodriguez G et al.
• Structural elements of ADP-ribosylation factor 1 required for functional interaction with cytohesin-1.
• J Biol Chem. 1999; 274: 12438-44
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• ADP-ribosylation factor 1 (ARF1) is a 20-kDa guanine nucleotide-binding protein involved in vesicular trafficking. Conversion of inactive ARF-GDP to active ARF-GTP is catalyzed by guanine nucleotide exchange proteins such as cytohesin-1. Cytohesin-1 and its Sec7 domain (C-1Sec7) exhibit guanine nucleotide exchange protein activity with ARF1 but not ARF-like protein 1 (ARL1), which is 57% identical in amino acid sequence. With chimeric proteins composed of ARF1 (F) and ARL1 (L) sequences we identified three structural elements responsible for this specificity. Cytohesin-1 increased [35S]guanosine 5'-(gamma-thio)triphosphate binding to L28/F (first 28 residues of L, remainder F) and to a much lesser extent F139/L, and mut13F139/L (F139/L with random sequence in the first 13 positions) but not Delta13ARF1 that lacks the first 13 amino acids; therefore, a nonspecific ARF N terminus was required for cytohesin-1 action. The N terminus was not, however, required for that of C-1Sec7. Both C-1Sec7 and cytohesin-1 effectively released guanosine 5'-(gamma-thio)triphosphate from ARF1, but only C-1Sec7 displaced the nonhydrolyzable GTP analog bound to mut13F139/L, again indicating that structure in addition to the Sec7 domain is involved in cytohesin-1 interaction. Some element(s) of the C-terminal region is also involved, because replacement of the last 42 amino acids with ARL sequence in F139L decreased markedly the interaction with cytohesin-1. Participation of both termini is consistent with the crystallographic structure of ARF in which the two terminal alpha-helices are in close proximity. ARF1 residues 28-50 are also important in the interaction with cytohesin-1; replacement of Lys-38 with Gln, the corresponding residue in ARL1, abolished the ability to serve as substrate for cytohesin-1 or C-1Sec7. These studies have defined multiple structural elements in ARF1, including switch 1 and the N and C termini, that participate in functional interactions with cytohesin-1 (or its catalytic domain C-1Sec7), which were not apparent from crystallographic analysis.

• Sata M, Moss J, Vaughan M
• Structural basis for the inhibitory effect of brefeldin A on guanine nucleotide-exchange proteins for ADP-ribosylation factors.
• Proc Natl Acad Sci U S A. 1999; 96: 2752-7
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• Protein secretion through the endoplasmic reticulum and Golgi vesicular trafficking system is initiated by the binding of ADP-ribosylation factors (ARFs) to donor membranes, leading to recruitment of coatomer, bud formation, and eventual vesicle release. ARFs are approximately 20-kDa GTPases that are active with bound GTP and inactive with GDP bound. Conversion of ARF-GDP to ARF-GTP is regulated by guanine nucleotide-exchange proteins. All known ARF guanine nucleotide-exchange proteins contain a Sec7 domain of approximately 200 amino acids that includes the active site and fall into two classes that differ in molecular size and susceptibility to inhibition by the fungal metabolite brefeldin A (BFA). To determine the structural basis of BFA sensitivity, chimeric molecules were constructed by using sequences from the Sec7 domains of BFA-sensitive yeast Sec7 protein (ySec7d) and the insensitive human cytohesin-1 (C-1Sec7). Based on BFA inhibition of the activities of these molecules with recombinant yeast ARF2 as substrate, the Asp965-Met975 sequence in ySec7d was shown to be responsible for BFA sensitivity. A C-1Sec7 mutant in which Ser199, Asn204, and Pro209 were replaced with the corresponding ySec7d amino acids, Asp965, Gln970, and Met975, exhibited BFA sensitivity similar to that of recombinant ySec7d (rySec7d). Single replacement in C-1Sec7 of Ser199 or Pro209 resulted in partial inhibition by BFA, whereas replacement of Gln970 in ySec7d with Asn (as found in C-1Sec7) had no effect. As predicted, the double C-1Sec7 mutant with S199D and P209M was BFA-sensitive, demonstrating that Asp965 and Met975 in ySec7d are major molecular determinants of BFA sensitivity.

• Klarlund JK et al.
• Regulation of GRP1-catalyzed ADP ribosylation factor guanine nucleotide exchange by phosphatidylinositol 3,4,5-trisphosphate.
• J Biol Chem. 1998; 273: 1859-62
• Display abstract

• Cellular levels of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) are rapidly elevated in response to activation of growth factor receptor tyrosine kinases. This polyphosphoinositide binds the pleckstrin homology (PH) domain of GRP1, a protein that also contains 200 residues with high sequence similarity to a segment of the yeast Sec7 protein that functions as an ADP ribosylation exchange factor (ARF) (Klarlund, J., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and Czech, M. P. (1997) Science 275, 1927-1930). Here we show that dioctanoyl PtdIns(3,4,5)P3 binds the PH domain of GRP1 with a Kd = 0.5 microM, an affinity 2 orders of magnitude greater than dioctanoyl-PtdIns(4,5)P2. Further, the Sec7 domain of GRP1 is found to catalyze guanine nucleotide exchange of ARF1 and -5 but not ARF6. Importantly, PtdIns(3,4,5)P3, but not PtdIns(4,5)P2, markedly enhances the ARF exchange activity of GRP1 in a reaction mixture containing dimyristoylphosphatidylcholine micelles, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and a low concentration of sodium cholate. PtdIns(3,4,5)P3-mediated ARF nucleotide exchange through GRP1 is selectively blocked by 100 microM inositol 1,3,4,5-tetrakisphosphate, which also binds the PH domain of GRP1. Taken together, these data are consistent with the hypothesis that selective recruitment of GRP1 to PtdIns(3,4,5)P3 in membranes activates ARF1 and -5, known regulators of intracellular membrane trafficking.

• Losonczi JA, Prestegard JH
• Nuclear magnetic resonance characterization of the myristoylated, N-terminal fragment of ADP-ribosylation factor 1 in a magnetically oriented membrane array.
• Biochemistry. 1998; 37: 706-16
• Display abstract

• The behavior of the N-terminal fragment of human ADP-ribosylation factor 1 (ARF1) in a membranelike environment is described. This is accomplished using heteronuclear liquid crystal NMR techniques in a magnetically oriented membrane array on a selectively 13C- and 15N-labeled peptide. After full assignment of the labeled sites, residual dipolar couplings (13C-13C, 15N-1H and, 13C-15N) and chemical shift anisotropy effects (amide 13C and 15N) were measured. The experimental data were interpreted using order matrix calculations to yield orientational and dynamic information for four separate, rigid amide planes. The experimental data obtained proves that the amphipathic peptide interacts with the bilayer in a mode that is consistent with an alpha-helix having its axis parallel to the membrane surface. Possibilities of extending the employed techniques to larger and uniformly labeled systems are discussed.

• Morinaga N, Noda M
• [Guanine nucleotide-exchange proteins (GEP) for ADP-ribosylation factors (ARF)]
• Nippon Saikingaku Zasshi. 1998; 53: 587-98

• Pacheco-Rodriguez G, Meacci E, Vitale N, Moss J, Vaughan M
• Guanine nucleotide exchange on ADP-ribosylation factors catalyzed by cytohesin-1 and its Sec7 domain.
• J Biol Chem. 1998; 273: 26543-8
• Display abstract

• ADP-ribosylation factors (ARFs) are 20-kDa guanine nucleotide-binding proteins that require specific guanine nucleotide-exchange proteins (GEPs) to accelerate the conversion of inactive ARF-GDP to active ARF-GTP. Cytohesin-1, a 46-kDa ARF GEP, contains a central Sec7 domain of 188 amino acids similar in sequence to a region of the yeast Sec7 protein. Cytohesin-1 and its 22-kDa Sec7 domain (C-1 Sec7), synthesized in Escherichia coli, were assayed with recombinant non-myristoylated ARFs and related proteins to compare their GEP activities. Both were effective with native mammalian ARFs 1 and 3. Cytohesin-1 accelerated GTPgammaS (guanosine 5'-3-O-(thio)triphosphate) binding to recombinant human ARF1 (rARF1), yeast ARF3, and ARD1 (a 64-kDa guanine nucleotide-binding protein containing a C-terminal ARF domain). In contrast, C-1 Sec7 enhanced GTPgammaS binding to recombinant human ARFs 1, 5, and 6; yeast ARFs 1, 2, and 3; ARD1; two ARD1 mutants that contain the ARF domain; and Delta13ARF1, which lacks the N-terminal alpha-helix. Neither C-1 Sec7 nor cytohesin-1 increased GTPgammaS binding to human ARF-like ARL proteins 1, 2, and 3. Thus, ARLs, initially differentiated from ARFs because of their inability to activate cholera toxin, differ also in their failure to interact functionally with C-1 Sec7 or cytohesin-1. As C-1 Sec7 was much less substrate-specific than cytohesin-1, it appears that structure outside of the Sec7 domain is important for ARF specificity. Data obtained with mutant ARF constructs are all consistent with the conclusion that the ARF N terminus is an important determinant of cytohesin-1 specificity.

• Frank SR, Hatfield JC, Casanova JE
• Remodeling of the actin cytoskeleton is coordinately regulated by protein kinase C and the ADP-ribosylation factor nucleotide exchange factor ARNO.
• Mol Biol Cell. 1998; 9: 3133-46
• Display abstract

• ARNO is a member of a family of guanine-nucleotide exchange factors with specificity for the ADP-ribosylation factor (ARF) GTPases. ARNO possesses a central catalytic domain with homology to yeast Sec7p and an adjacent C-terminal pleckstrin homology (PH) domain. We have previously shown that ARNO localizes to the plasma membrane in vivo and efficiently catalyzes ARF6 nucleotide exchange in vitro. In addition to a role in endocytosis, ARF6 has also been shown to regulate assembly of the actin cytoskeleton. To determine whether ARNO is an upstream regulator of ARF6 in vivo, we examined the distribution of actin in HeLa cells overexpressing ARNO. We found that, while expression of ARNO leads to disassembly of actin stress fibers, it does not result in obvious changes in cell morphology. However, treatment of ARNO transfectants with the PKC agonist phorbol 12-myristate 13-acetate results in the dramatic redistribution of ARNO, ARF6, and actin into membrane protrusions resembling lamellipodia. This process requires ARF activation, as actin rearrangement does not occur in cells expressing a catalytically inactive ARNO mutant. PKC phosphorylates ARNO at a site immediately C-terminal to its PH domain. However, mutation of this site had no effect on the ability of ARNO to regulate actin rearrangement, suggesting that phosphorylation of ARNO by PKC does not positively regulate its activity. Finally, we demonstrate that an ARNO mutant lacking the C-terminal PH domain no longer mediates cytoskeletal reorganization, indicating a role for this domain in appropriate membrane localization. Taken together, these data suggest that ARNO represents an important link between cell surface receptors, ARF6, and the actin cytoskeleton.

• Loizos N, Darst SA
• Mapping protein-ligand interactions by footprinting, a radical idea.
• Structure. 1998; 6: 691-5

• Tsai SC et al.
• Effects of arfaptin 1 on guanine nucleotide-dependent activation of phospholipase D and cholera toxin by ADP-ribosylation factor.
• J Biol Chem. 1998; 273: 20697-701
• Display abstract

• Arfaptin 1, a approximately 39-kDa protein based on the deduced amino acid sequence, had been initially identified in a yeast two-hybrid screen using dominant active ARF3 (Q71L) as bait with an HL-60 cDNA library. It was suggested that arfaptin 1 may be involved in Golgi functions, since the FLAG-tagged protein was associated with Golgi membranes when expressed in COS-7 cells and could be bound to Golgi in vitro in an ADP-ribosylation factor (ARF)- and GTPgammaS-dependent, brefeldin A-inhibited fashion. Arfaptin 2, found in the same two-hybrid screen as arfaptin 1, is 60% identical in amino acid sequence and may or may not have an analogous function. We now report some effects of arfaptin 1 on ARF activation of phospholipase D and cholera toxin ADP-ribosyltransferase. Arfaptin 1 inhibited activation of both enzymes in a concentration-dependent manner and was without effect in the absence of ARF. Two ARF1 mutants that activated the toxin, one lacking 13 N-terminal amino acids and the other, in which 73 residues at the N terminus were replaced with the analogous sequence from ARL1, were not inhibited by arfaptin, consistent with the conclusion that arfaptin interaction requires the N terminus of ARF. This region has also been implicated in phospholipase D activation, but whether the two proteins interact with the same structural elements in ARF remains to be determined. Arfaptin inhibition of the action of ARF5 and ARF6 was less than that of ARF1 and ARF3; its effects were less on nonmyristoylated than myristoylated ARFs. Arfaptin effects on guanine nucleotide binding by ARFs were minimal whether or not a purified ARF guanine nucleotide-exchange protein was present. These findings indicate that arfaptin acts as an inhibitor of ARF actions in vitro, raising the possibility that it has a similar role in vivo.

• Faundez V, Horng JT, Kelly RB
• ADP ribosylation factor 1 is required for synaptic vesicle budding in PC12 cells.
• J Cell Biol. 1997; 138: 505-15
• Display abstract

• Carrier vesicle generation from donor membranes typically progresses through a GTP-dependent recruitment of coats to membranes. Here we explore the role of ADP ribosylation factor (ARF) 1, one of the GTP-binding proteins that recruit coats, in the production of neuroendocrine synaptic vesicles (SVs) from PC12 cell membranes. Brefeldin A (BFA) strongly and reversibly inhibited SV formation in vivo in three different PC12 cell lines expressing vesicle-associated membrane protein-T Antigen derivatives. Other membrane traffic events remained unaffected by the drug, and the BFA effects were not mimicked by drugs known to interfere with formation of other classes of vesicles. The involvement of ARF proteins in the budding of SVs was addressed in a cell-free reconstitution system (Desnos, C., L. Clift-O'Grady, and R.B. Kelly. 1995. J. Cell Biol. 130:1041-1049). A peptide spanning the effector domain of human ARF1 (2-17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size. During in vitro incubation in the presence of the mutant ARFs, the labeled precursor membranes acquired different densities, suggesting that the two ARF mutations block at different biosynthetic steps. Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1. Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.

• Loirand G, Cario-Toumaniantz C, Chardin P, Pacaud P
• ARF-independent inhibition of the carbachol-induced contractions by brefeldin A in intestinal smooth muscle.
• Am J Physiol. 1997; 273: 81621-81621
• Display abstract

• The aim of this study was to determine whether an ADP ribosylation factor (ARF)-regulated pathway is involved in the carbachol-induced contraction in rat intestinal smooth muscle. Brefeldin A, a known inhibitor of the guanine nucleotide exchange activity on ARF, reversibly inhibited the carbachol-induced contraction in intact ileal muscle strips, whereas the carbachol- and guanosine 5'-O-(3-thiotriphosphate)-induced increases in the Ca2+ sensitivity of myofilaments in beta-escin-permeabilized strips were not affected. The high-K(+)-induced contraction in intact strips was also inhibited by brefeldin A. In isolated ileal myocytes, brefeldin A inhibited the Ca2+ channel current, indicating that the inhibitory effect of brefeldin A in intact cells is related to an inhibition of voltage-dependent Ca2+ channels. Furthermore, the loading of permeabilized strips with the combination of the recombinant fully myristoylated ARF1, the guanine nucleotide exchange factor ARNO, and guanosine 5'-triphosphate did not change the tone at constant pCa (6.45) and did not modify the carbachol- and guanosine 5'-O-(3-thiotriphosphate)-induced Ca2+ sensitization. Taken together, these findings suggest that an ARF-dependent pathway is not involved in the carbachol-induced contraction.

• Vaughan M, Moss J
• Activation of toxin ADP-ribosyltransferases by the family of ADP-ribosylation factors.
• Adv Exp Med Biol. 1997; 419: 315-20
• Display abstract

• ADP-ribosylation factors or ARFs are 20-kDa guanine nucleotide-binding proteins, initially identified as stimulators of cholera toxin-catalyzed ADP-ribosylation of Gs alpha. We now know that ARFs play a critical role in many vesicular trafficking events and ARF activation of a membrane-associated phospholipase D (PLD) has been recognized. ARF is active and associates with membranes when GTP is bound. The active state is terminated by hydrolysis of bound GTP, producing inactive ARF-GDP. The nucleotide effect on ARF association with membranes is related to alteration in orientation of the N-terminal myristoyl moiety that is important for ARF function. Cycling of ARF between active and inactive states involves guanine nucleotide-exchange proteins (GEPs) that accelerate replacement of bound GDP with GTP and GTPase-activating proteins (GAPS) that are responsible for ARF inactivation. Six mammalian ARFs have been identified by cDNA cloning. Class I ARFs 1 and 3 have been studied most extensively. Their activation (GTP binding) is catalyzed by a GEP now purified from spleen cytosol. In crude preparations, GEP was inhibited by brefeldin A (BFA), which in cells causes apparent disintegration of Golgi. Demonstration that the approximately 60 kDa purified GEP was not inhibited by BFA means that contrary to earlier belief, there must be another protein to mediate BFA inhibition. GEP activity was greatly enhanced by phosphatidyl serine. The purified GEP, equally active with ARFs 1 and 3, was inactive with ARFs 5 and 6 (Classes II and III); myristoylated ARFs were better substrates than were their non-myristoylated counterparts. ARF GAP purified from bovine spleen cytosol in our laboratory had much broader substrate specificity than the GEP. It used both ARFs 5 and 6 at least as well as ARFs 1 and 3; myristoylation was without effect. It also accelerated GTP hydrolysis by certain ARF mutants and an ARF-like protein (ARL1) that does not have ARF activity. The purified GAP also differed from the GEP in its rather specific requirement for phosphatidylinositol bisphosphate. This was also observed with a seemingly different ARF GAP that was purified and subsequently cloned in Cassel's laboratory. Activation and inactivation of ARFs present many potential sites for physiological regulation and, therefore, for pathological disruption of ARF function.

• Rosa JL, Barbacid M
• A giant protein that stimulates guanine nucleotide exchange on ARF1 and Rab proteins forms a cytosolic ternary complex with clathrin and Hsp70.
• Oncogene. 1997; 15: 1-6
• Display abstract

• We have recently identified an unusually large human protein that stimulates guanine nucleotide exchange on ARF1 and Rab proteins (EMBO J 15: 4262-4273, 1996). This protein, designated p532 based on its predicted molecular weight (EMBO J 15: 5738, 1996), contains multiple structural domains including two regions of seven internal repeats highly related to the cell cycle regulator RCC1, a guanine nucleotide exchange factor for the small GTP-binding protein Ran, seven beta-repeat domains characteristic of the beta subunit of heterotrimeric G proteins, three putative SH3 binding sites, a putative leucine-zipper and a carboxy-terminal HECT domain characteristic of E3 ubiquitin-protein ligases. Some of these domains are known to be involved in protein-protein interactions, suggesting the existence of p532-interacting proteins. To identify some of these proteins, we used the carboxy-terminal RCC1-like domain (RLD) of p532 in the yeast two-hybrid system. We report here the isolation of a clone that encodes the last 654 amino acid residues of the clathrin heavy chain. This interaction involves amino acid residues 1315-1557 of the clathrin heavy chain and the carboxy, but not the amino-terminal RLD of p532. p532 has been located in the cytosolic fraction as well as in the Golgi apparatus. The interaction between p532 and clathrin only occurs in the cytosol and is mediated by the formation of an ATP-dependent ternary complex with the heat shock protein, Hsp70. These observations suggest that p532 is involved in membrane transport processes.

• Liang JO, Kornfeld S
• Comparative activity of ADP-ribosylation factor family members in the early steps of coated vesicle formation on rat liver Golgi membranes.
• J Biol Chem. 1997; 272: 4141-8
• Display abstract

• We have compared the abilities of mammalian ADP-ribosylation factors (ARFs) 1, 5, and 6 and Saccharomyces cerevisiae ARF2 to serve as substrates for the rat liver Golgi membrane guanine nucleotide exchange factor and to initiate the formation of clathrin- and coatomer protein (COP) I-coated vesicles on these membranes. While Golgi membranes stimulated the exchange of GTPgammaS for GDP on all of the ARFs tested, mammalian ARF1 was the best substrate, with an apparent Km of 5 &mgr;M. In all cases myristoylation of ARF was required for stimulation. Agents that inhibit the Golgi membrane guanine nucleotide exchange factor (the fungal metabolite brefeldin A and trypsin treatment) selectively inhibited the guanine nucleotide exchange on mammalian ARF1. Taken together, these data indicate that of the ARFs tested, only mammalian ARF1 is activated efficiently by the Golgi guanine nucleotide exchange factor. The other ARFs are activated mainly by another mechanism, possibly phospholipid-mediated. Once activated, all of the membrane-associated, myristoylated ARFs promoted the recruitment of coatomer to about the same extent. Mammalian ARFs 1 and 5 were the most effective in promoting the recruitment of the AP-1 adaptor complex, whereas yeast ARF2 was the least active. These data indicate that the specificity for ARF action on the Golgi membranes is primarily determined by the Golgi guanine nucleotide exchange factor, which has a strong preference for myristoylated mammalian ARF1.

• Marshansky V, Bourgoin S, Londono I, Bendayan M, Vinay P
• Identification of ADP-ribosylation factor-6 in brush-border membrane and early endosomes of human kidney proximal tubules.
• Electrophoresis. 1997; 18: 538-47
• Display abstract

• The expression and distribution of ADP-ribosylation factor (ARF) small GTP-binding proteins in kidney tissue was examined. Various anti-ARF antibodies were raised against purified rec-ARF 1 and rec-ARF 6 and their specificity was determined. Using indirect immunofluorescence analysis of intact kidney, ARF proteins were found to be predominantly expressed in kidney tubules as compared to glomeruli. This result was further supported by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of purified human kidney glomeruli and proximal tubules. Both ARF 1 and ARF 6 were detected in purified human glomeruli and proximal tubules; however, ARF 1 was more abundant than ARF 6 in these kidney structures. Brush-border membrane vesicles (BBMV) and early endosomes (EE) derived from the receptor-mediated endocytosis pathway were isolated from purified proximal tubules of rat, dog and human kidney using a combination of magnesium precipitation and wheat-germ agglutinin negative selection techniques. We demonstrated that ARF 6 is associated with BBMV and with EE derived from receptor-mediated endocytosis pathway of human kidney proximal tubules. Using a combination of SDS-PAGE and quantitative enhanced chemiluminescence Western blot analysis, the quantification of the ARF 6 distribution in membrane and cytoplasmic fractions of proximal tubules was made and its predominance in membrane fractions was demonstrated. By analogy with the functional role of ARF 1 in Golgi protein transport, we suggest that ARF 6 may play an important role in the regulation of receptor-mediated endocytosis and protein reabsorption by kidney proximal tubules.

• Paris S et al.
• Role of protein-phospholipid interactions in the activation of ARF1 by the guanine nucleotide exchange factor Arno.
• J Biol Chem. 1997; 272: 22221-6
• Display abstract

• Arno is a 47-kDa human protein recently identified as a guanine nucleotide exchange factor for ADP ribosylation factor 1 (ARF1) with a central Sec7 domain responsible for the exchange activity and a carboxyl-terminal pleckstrin homology (PH) domain (Chardin, P., Paris, S., Antonny, B., Robineau, S., Beraud-Dufour, S., Jackson, C. L., and Chabre, M. (1996) Nature 384, 481-484). Binding of the PH domain to phosphatidylinositol 4,5-bisphosphate (PIP2) greatly enhances Arno-mediated activation of myristoylated ARF1. We show here that in the absence of phospholipids, Arno promotes nucleotide exchange on [Delta17]ARF1, a soluble mutant of ARF1 lacking the first 17 amino acids. This reaction is unaffected by PIP2, which suggests that the PIP2-PH domain interaction does not directly regulate the catalytic activity of Arno but rather serves to recruit Arno to membranes. Arno catalyzes the release of GDP more efficiently than that of GTP from [Delta17]ARF1, and a stable complex between Arno Sec7 domain and nucleotide-free [Delta17]ARF1 can be isolated. In contrast to [Delta17]ARF1, full-length unmyristoylated ARF1 is not readily activated by Arno in solution. Its activation requires the presence of phospholipids and a reduction of ionic strength and Mg2+ concentration. PIP2 is strongly stimulatory, indicating that binding of Arno to phospholipids is involved, but in addition, electrostatic interactions between phospholipids and the amino-terminal portion of unmyristoylated ARF1GDP seem to be important. We conclude that efficient activation of full-length ARF1 by Arno requires a membrane surface and two distinct protein-phospholipid interactions: one between the PH domain of Arno and PIP2, and the other between amino-terminal cationic residues of ARF1 and anionic phospholipids. The latter interaction is normally induced by insertion of the amino-terminal myristate into the bilayer but can also be artificially facilitated by decreasing Mg2+ and salt concentrations.

• Franco M, Chardin P, Chabre M, Paris S
• Myristoylation-facilitated binding of the G protein ARF1GDP to membrane phospholipids is required for its activation by a soluble nucleotide exchange factor.
• J Biol Chem. 1996; 271: 1573-8
• Display abstract

• We have investigated the role of N-myristoylation in the activation of bovine ADP-ribosylation factor 1 (ARF1). We previously showed that myristoylation allows some spontaneous GDP-to-GTP exchange to occur on ARF1 at physiological Mg2+ levels in the presence of phospholipid vesicles (Franco, M., Chardin, P., Chabre, M., and Paris, S. (1995) J. Biol. Chem. 270, 1337-1341). Here, we report that this basal nucleotide exchange can be accelerated (by up to 5-fold) by addition of a soluble fraction obtained from bovine retinas. This acceleration is totally abolished by brefeldin A (IC50 = 2 microM) and by trypsin treatment of the retinal extract, as expected for an ARF-specific guanine nucleotide exchange factor. To accelerate GDP release from ARF1, this soluble exchange factor absolutely requires myristoylation of ARF1 and the presence of phospholipid vesicles. The retinal extract also stimulates guanosine 5'-3-O-(thio)-triphosphate (GTP gamma S) release from ARF1 in the presence of phospholipids, but in this case myristoylation of ARF is not required. These observations, together with our previous findings that both myristoylated and non-myristoylated forms of ARF GTP-gamma S but only the myristoylated form of ARFGDP bind to membrane phospholipids, suggest that (i) the retinal exchange factor acts only on membrane-bound ARF, (ii) the myristate is not involved in the protein-protein interaction between ARF1 and the exchange factor, and (iii) N-myristoylation facilitates both spontaneous and catalyzed GDP-to-GTP exchange on ARF1 simply by facilitating the binding of ARFGDP to membrane phospholipids.

• Boman AL, Taylor TC, Berger SJ, Melancon P, Wilson KL
• Purification and mass spectrometric analysis of ADP-ribosylation factor proteins from Xenopus egg cytosol.
• Biochemistry. 1996; 35: 8244-51
• Display abstract

• The GTP analog GTP gamma S potently inhibits nuclear envelope assembly in cell-free Xenopus egg extracts. GTP gamma S does not affect vesicle binding to chromatin but blocks vesicle fusion. Fusion inhibition by GTP gamma S is mediated by a soluble factor, initially named GSF (GTP gamma S-dependent soluble factor). We previously showed that vesicles pretreated with GTP gamma S plus recombinant mammalian ARF1 were inhibited for fusion, suggesting that "GSF activity" was due to the ARF (ADP-ribosylation factor) family of small GTP-binding proteins. To ask if any soluble proteins other than ARF also inhibited vesicle fusion in the pretreatment assay, we purified GSF activity from Xenopus egg cytosol. At all steps in the purification, fractions containing ARF, but no other fractions, showed GSF activity. The purified GSF was identified as Xenopus ARF by immunoblotting and peptide sequence analysis. Reverse phase HPLC and mass spectrometry revealed that GSF contained at least three distinct ARF proteins, all of which copurified through three chromatography steps. The most abundant isoform was identified as ARF1 (62% of the total GSF), because its experimentally determined mass of 20 791 Da matched within experimental error that predicted by the sequence of the Xenopus ARF1 cDNA, which is reported here. The second-most abundant isoform (25% of GSF activity) was identified as ARF3. We concluded that ARF is most likely the only soluble protein that inhibits nuclear vesicle fusion after pretreatment with GTP gamma S.

• Lee CM et al.
• Expression in human endothelial cells of ADP-ribosylation factors, 20-kDa guanine nucleotide-binding proteins involved in the initiation of vesicular transport.
• J Mol Cell Cardiol. 1996; 28: 1911-20
• Display abstract

• ADP-ribosylation factors (ARFs) are approximately 20-kDa, guanine nucleotide-binding proteins, initially discovered as stimulators of cholera toxin ADP-ribosyltransferase activity and subsequently shown to participate in vesicular trafficking. Five of the six mammalian ARFs have been identified in human tissues by molecular cloning. They fall into three classes (class I: ARFs 1-3; class II: ARFs 4, 5; class III: ARF 6) based on deduced amino acid sequence, size, phylogenetic analysis, and gene structure. Similar to the rab family of approximately 20 kDa guanine nucleotide-binding proteins, the ARFs appear to function in specific trafficking pathways. The presence of a specific ARF might serve as a marker for that pathway. To verify expression of ARF mRNA and protein in human umbilical vein endothelial cells, immunoreactivity using antibodies specific for each ARF class, quantitative polymerase chain reaction (PCR) using ARF-specific, internal cRNA standards containing unique restriction enzyme cleavage sites introduced by point mutations, and Northern analysis with probes specific for ARFs 1, and 3-6, were utilized. PCR and Northern analysis were in agreement in showing that amounts of mRNA for ARF 1 and ARF 4 were similar and higher than those of ARF 3 and ARF 5 which were greater than ARF 6. Primarily, Class 1 ARF proteins were detected by immunoreactivity, with the majority in the supernatant fraction. The relative expression of ARFs in endothelial cells thus differs from that in neuronal tissues where it had been found that ARF3 is the predominant species.

• Price SR, Nightingale MS, Tsuchiya M, Moss J, Vaughan M
• Interspecies relationships among ADP-ribosylation factors (ARFs): evidence of evolutionary pressure to maintain individual identities.
• Mol Cell Biochem. 1996; 159: 15-23
• Display abstract

• ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that are allosteric activators of the NAD:arginine ADP-ribosyltransferase activity of cholera toxin and appear to play a role in intracellular vesicular trafficking. Although the physiological roles of these proteins have not been defined, it has been presumed that each has a specific intracellular function. To obtain genetic evidence that each ARF is under evolutionary pressure to maintain its structure, and presumably function, rat ARF cDNA clones were isolated and their nucleotide and deduced amino acid sequences were compared to those of other mammalian ARFs. Deduced amino acid sequences for rat ARFs 1, 2, 3, 5 and 6 were identical to those of the known cognate human and bovine ARFs; rat ARF4 was 96% identical to human ARF4. Nucleotide sequences of both the untranslated as well as the coding regions were highly conserved. These results indicate that the ARF proteins are, as a family, extraordinarily well conserved across mammalian species. The unusually high degree of conservation of the untranslated regions is consistent with these regions having important regulatory roles and that individual ARFs contain structurally unique elements required for specific functions.

• Tsai SC, Adamik R, Moss J, Vaughan M
• Purification and characterization of a guanine nucleotide-exchange protein for ADP-ribosylation factor from spleen cytosol.
• Proc Natl Acad Sci U S A. 1996; 93: 305-9
• Display abstract

• ADP-ribosylation factors (ARFs) are 20-kDa guanine nucleotide-binding proteins and are active in the GTP-bound state and inactive with GDP bound. ARF-GTP has a critical role in vesicular transport in several cellular compartments. Conversion of ARF-GDP to ARF-GTP is promoted by a guanine nucleotide-exchange protein (GEP). We earlier reported the isolation from bovine brain cytosol of a 700-kDa protein complex containing GEP activity that was inhibited by brefeldin A (BFA). Partial purification yielded an approximately 60-kDa BFA-insensitive GEP that enhanced binding of ARF1 and ARF3 to Golgi membranes. GEP has now been purified extensively from rat spleen cytosol in a BFA-insensitive, approximately 55-kDa form. It activated class I ARFs (ARFs 1 and 3) that were N-terminally myristoylated, but not nonmyristoylated ARFs from class-I, II, or III. GEP activity required MgCl2. In the presence of 0.6-0.8 mM MgCl2 and 1 mM EDTA, binding of guanosine 5'-[gamma[35S]thio]triphosphate ([35S]GTP gamma S) by ARF1 and ARF3 was equally high without and with GEP. At higher Mg2+ concentrations, binding without GEP was much lower; with 2-5 mM MgCl2, GEP-stimulated binding was maximal. The rate of GDP binding was much less than that of GTP gamma S with and without GEP. Phospholipids were necessary for GEP activity; phosphatidylinositol was more effective than phosphatidylserine, and phosphatidic acid was less so. Other phospholipids tested were ineffective. Maximal effects required approximately 200 microM phospholipid, with half-maximal activation at 15-20 microM. Release of bound [35S]GTP gamma S from ARF3 required the presence of both GEP and unlabeled GTP or GTP gamma S; GDP was much less effective. This characterization of the striking effects of Mg2+ concentration and specific phospholipids on the purified BFA-insensitive ARF GEP should facilitate experiments to define its function in vesicular transport.

• Morinaga N, Tsai SC, Moss J, Vaughan M
• Isolation of a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP ribosylation factor (ARF) 1 and ARF3 that contains a Sec7-like domain.
• Proc Natl Acad Sci U S A. 1996; 93: 12856-60
• Display abstract

• Brefeldin A (BFA) inhibited the exchange of ADP ribosylation factor (ARF)-bound GDP for GTP by a Golgi-associated guanine nucleotide-exchange protein (GEP) [Helms, J.B. & Rothman, J.E. (1992) Nature (London) 360, 352-354; Donaldson, J.G., Finazzi, D. & Klausner, R.D. (1992) Nature (London) 360, 350-352]. Cytosolic ARF GEP was also inhibited by BFA, but after purification from bovine brain and rat spleen, it was no longer BFA-sensitive [Tsai, S.-C., Adamik, R., Moss, J. & Vaughan, M. (1996) Proc. Natl. Acad. Sci. USA 93, 305-309]. We describe here purification from bovine brain cytosol of a BFA-inhibited GEP. After chromatography on DEAE-Sephacel, hydroxylapatite, and Mono Q and precipitation at pH 5.8, GEP was eluted from Superose 6 as a large molecular weight complex at the position of thyroglobulin (approximately 670 kDa). After SDS/PAGE of samples from column fractions, silver-stained protein bands of approximately 190 and 200 kDa correlated with activity. BFA-inhibited GEP activity of the 200-kDa protein was demonstrated following electroelution from the gel and renaturation by dialysis. Four tryptic peptides from the 200-kDa protein had amino acid sequences that were 47% identical to sequences in Sec7 from Saccharomyces cerevisiae (total of 51 amino acids), consistent with the view that the BFA-sensitive 200-kDa protein may be a mammalian counterpart of Sec7 that plays a similar role in cellular vesicular transport and Sec7 may be a GEP for one or more yeast ARFs.

• Franco M, Chardin P, Chabre M, Paris S
• Myristoylation of ADP-ribosylation factor 1 facilitates nucleotide exchange at physiological Mg2+ levels.
• J Biol Chem. 1995; 270: 1337-41
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• Recombinant N-myristoylated bovine ADP-ribosylation factor 1 (myr-rARF1) has been expressed in bacteria and purified to near homogeneity with a high (85%) myristoylation efficiency. Myr-rARF1 and nonmyristoylated rARF1 have been compared with respect to their kinetics of guanine nucleotide exchange and their interactions with phospholipids. Myristoylation is shown to allow the release of bound GDP at physiological (mM) concentrations of Mg2+. GDP dissociation is slow in the absence of phospholipids but is accelerated 2-fold in the presence of phospholipid vesicles. On the contrary, myristoylation decreases 10-fold the rate of dissociation of GTP or guanosine 5'-O-(thiotriphosphate) (GTP gamma S) in the presence of phospholipids. As a result, myr-ARF1 can be spontaneously activated by GTP or GTP gamma S (t1/2 approximately 30 min at 37 degrees C) at 1 mM Mg2+, in the sole presence of phospholipid membranes without the need for a nucleotide exchange factor. In contrast to the nonacylated protein, the GDP-bound form of myr-ARF1 interacts with phospholipids, as demonstrated by its cosedimentation with phospholipid vesicles and its comigration with phospholipid/cholate micelles on gel filtration. The interaction is, however, weaker than for the GTP-bound form, suggesting that only the myristate in myr-ARF1GDP interacts with phospholipids, whereas both the myristate and the amino-terminal hydrophobic residues in myr-ARF1GTP bind to phospholipids.

• Teal SB, Hsu VW, Peters PJ, Klausner RD, Donaldson JG
• An activating mutation in ARF1 stabilizes coatomer binding to Golgi membranes.
• J Biol Chem. 1994; 269: 3135-8
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• The Ras-related protein ADP-ribosylation factor 1 (ARF1) is a low molecular weight GTP binding protein, which in its GTP state supports the binding of coatomer, a cytosolic coat protein complex, to Golgi membranes. To create an "active" ARF, we constructed a point mutation in ARF1, Q71I, which was predicted to slow the rate of GTP hydrolysis. We demonstrate that Q71I, in contrast to wild type ARF1, exhibits a 2-3-fold increase in the half-life of ARF-GTP and is able to promote stable coatomer binding to Golgi membranes in the presence of GTP in vitro. Additionally, Q71I is able to support the binding of a significant amount of coatomer to membranes in the absence of added nucleotides, effectively bypassing the brefeldin A (BFA)-sensitive exchange activity. Furthermore, transfection of cells with Q71I, but not ARF1, renders the Golgi association of coatomer resistant to the effects of BFA in vivo. These observations provide compelling evidence that ARF1 is a necessary GTP binding protein that regulates the reversible binding of coat proteins to Golgi membranes and that the effects of BFA on this process in living cells must be a consequence of BFA's inhibition of guanine nucleotide exchange onto ARF1.

• Terui T, Kahn RA, Randazzo PA
• Effects of acid phospholipids on nucleotide exchange properties of ADP-ribosylation factor 1. Evidence for specific interaction with phosphatidylinositol 4,5-bisphosphate.
• J Biol Chem. 1994; 269: 28130-5
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• ADP-ribosylation factors (ARFs) have been implicated as ubiquitous regulators of multiple steps in both exocytic and endocytic membrane traffic in yeast and mammalian cells. More specific interactions have also been described for ARF proteins with an ARF-specific GTP-ase-activating protein and as activators of phospholipase D activity. These protein interactions have defined requirements for phosphatidylinositol 4,5-bisphosphate (PIP2). Direct interactions between ARF1 and PIP2 or other phospholipids were tested by examining effects on guanine nucleotide binding kinetics. PIP2 (400 microM) increased the rate of GDP dissociation > 100-fold. Several other acid phospholipids had more modest effects (4-7-fold) on GDP dissociation rates, while other phospholipids had no effect. PIP2 also had the greatest effect on the rate of binding of guanosine 5'-(gamma-thio)triphosphate (GTP gamma S), increasing it almost 100-fold at early time points. However, at later times (> 5 min), PIP2 caused a paradoxical loss of nucleotide binding to ARF1. PIP2 was found to stabilize the nucleotide-free form of ARF1 as subsequent dilution of PIP2 allowed ARF1 to bind GTP gamma S to high stoichiometry. The demonstration of direct interaction between ARF1 and PIP2 provides the basis for a model in which PIP2 acts as a cofactor in some of the interactions between ARF1 and other proteins.

• Erickson FL, Hannig EM, Krasinskas A, Kahn RA
• Cloning and sequence of ADP-ribosylation factor 1 (ARF1) from Schizosaccharomyces pombe.
• Yeast. 1993; 9: 923-7
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• A gene encoding a homologue of the ADP-ribosylation factor (ARF) family of small GTP binding proteins was cloned from a Schizosaccharomyces pombe cDNA library by a functional screen of suppressors of sensitivity to 3-aminotriazole in a gcn3 null strain of Saccharomyces cerevisiae. Two independent isolates each contained the full coding region of the ARF1 gene. The encoded SpARF1 protein has a predicted molecular weight of 20,618 and is 88% and 79% identical to human and S. cerevisiae ARF1 proteins, respectively. As independent isolates were obtained, this effect of the SpARF1 appears to be a real phenomenon, but cannot currently be easily understood within the context of the evidence for a role(s) for ARF proteins in the protein secretory pathway.

• Serventi IM, Cavanaugh E, Moss J, Vaughan M
• Characterization of the gene for ADP-ribosylation factor (ARF) 2, a developmentally regulated, selectively expressed member of the ARF family of approximately 20-kDa guanine nucleotide-binding proteins.
• J Biol Chem. 1993; 268: 4863-72
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• ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins that stimulate the ADP-ribosyltransferase activities of cholera toxin in vitro and function in protein trafficking in vivo. The six cloned mammalian ARFs can be grouped into three classes based on size and sequence identity. ARF 2 is a class I ARF, whose approximately 2.6-kilobase mRNA exhibits species and tissue selective expression and is developmentally regulated in rat brain. Here we report the sequence, structure, and functional promoter region of the bovine ARF 2 gene, which was facilitated by constructing a composite cDNA. The ARF 2 cDNA, constructed from a partial cDNA clone and polymerase chain reaction-amplified fragments from reverse-transcribed poly(A)+ RNA, was approximately 2270 base pairs (bp) (minus the poly(A) tail). In the 3'-untranslated region, there are two potential polyadenylation signals, ATTAAA and AATAAA, at positions 1064 and 2232, respectively, and two ATTTA motifs, believed to signal mRNA degradation, at positions 2115 and 2165. The ARF 2 gene, represented in three overlapping genomic clones, spans approximately 20 kilobase pairs with five exons and four introns. Consensus sequences for guanine nucleotide-binding and GTP hydrolysis are in separate exons, except for the NKXD sequence, which is divided by intron 4. There are multiple transcriptional initiation sites. Transient transfection of embryonic trachea cells with deletion constructs defined the functional promoter region to be within 400 bp upstream of the most 5' site of transcription initiation. This 400-bp region lacks a TATA-like sequence but contains six inverted CCAAT boxes, four potential Sp1-binding sites, and a potential AP-2-binding site. Although the pattern of expression of ARF 2 is unique among the ARFs, the structures of the class I ARF genes are conserved among its members and across species.

• Clark J et al.
• Selective amplification of additional members of the ADP-ribosylation factor (ARF) family: cloning of additional human and Drosophila ARF-like genes.
• Proc Natl Acad Sci U S A. 1993; 90: 8952-6
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• The ADP-ribosylation factor (ARF) family is one of four subfamilies of the RAS superfamily of low molecular weight GTP-binding proteins (G proteins). Highly degenerate oligonucleotides encoding two conserved regions were used in a PCR reaction to amplify cDNAs encoding each of the known ARF proteins and eight additional cDNA fragments encoding previously unreported human members of the ARF family. Additional sequences were obtained from yeast or fly libraries by using this technique. These oligonucleotides specifically amplify members of the ARF family but not the structurally related G protein alpha subunits or members of the other three subfamilies of the RAS superfamily. Fragments obtained by PCR were used to obtain full-length sequences encoding highly homologous ARF-like (ARL) gene products from human and Drosophila melanogaster libraries, termed ARL2 and Ar184F, respectively. The encoded proteins are each 184 amino acids long and are 76% identical, with 40-45% identity to human ARF1 and Drosophila arf-like (arl) proteins. These genes appear to be generally expressed in human tissues and during Drosophila development. The purified human ARL2 protein differed in several biochemical properties from human ARF proteins, including the complete absence of ARF activity. Thus, the ARF family of low molecular weight GTP-binding proteins includes at least 15 distinct but structurally conserved members, including both the functionally conserved ARF proteins and the functionally disparate ARL proteins. The latter proteins currently comprise two distinct gene products in Drosophila (arl and ARL84F) and one in man (ARL2).

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