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The Ras superfamily is comprised of at least four large families of regulatory guanosine triphosphate-binding proteins, including the Arfs. The Arf family includes three different groups of proteins: the Arfs, Arf-like (Arls), and SARs. Several Arf family members have been very highly conserved throughout eukaryotic evolution and have orthologues in evolutionally diverse species. The different means by which Arf family members have been identified have resulted in an inconsistent and confusing array of names. This confusion is further compounded by differences in nomenclature between different species. We propose a more consistent nomenclature for the human members of the Arf family that may also serve as a guide for nomenclature in other species.

G proteins are molecular switches that control a wide variety of physiological functions, including neurotransmission, transcriptional activation, cell migration, cell growth. and proliferation. The ability of GTPases to participate in signaling events is determined by the ratio of GTP-bound to GDP-bound forms in the cell. All known GTPases exist in an inactive (GDP-bound) and an active (GTP-bound) conformation, which are catalyzed by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs), respectively. In this study, we identified and characterized a new family of bifunctional GTP-binding and GTPase-activating proteins, named GGAP. GGAPs contain an N-terminal Ras homology domain, called the G domain, followed by a pleckstrin homology (PH) domain, a C-terminal GAP domain, and a tandem ankyrin (ANK) repeat domain. Expression analysis indicates that this new family of proteins has distinct cell localization, tissue distribution, and even message sizes. GTPase assays demonstrate that GGAPs have high GTPase activity through direct intramolecular interaction of the N-terminal G domain and the C-terminal GAP domain. In the absence of the GAP domain, the N-terminal G domain has very low activity, suggesting a new model of GGAP protein regulation via intramolecular interaction like the multidomain protein kinases. Overexpression of GGAPs leads to changes in cell morphology and activation of gene transcription.

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.

ADP-ribosylation factor 6 (ARF6) appears to play an essential role in the endocytic/recycling pathway in several cell types. To determine whether ARF6 is involved in insulin-regulated exocytosis, 3T3-L1 adipocytes were infected with recombinant adenovirus expressing wild-type ARF6 or an ARF6 dominant negative mutant (D125N) that encodes a protein with nucleotide specificity modified from guanine to xanthine. Overexpression of these ARF6 proteins affected neither basal nor insulin-regulated glucose uptake in 3T3-L1 adipocytes, nor did it affect the subcellular distribution of Glut1 or Glut4. In contrast, the secretion of adipsin, a serine protease specifically expressed in adipocytes, was increased by the expression of wild-type ARF6 and was inhibited by the expression of D125N. These results indicate a requirement for ARF6 in basal and insulin-regulated adipsin secretion but not in glucose transport. Our results suggest the existence of at least two distinct pathways that undergo insulin-stimulated exocytosis in 3T3-L1 adipocytes, one for adipsin release and one for glucose transporter translocation.

The novel ARF-like GTPase ARL7 is a close relative of ARL4 and ARL6 (71% and 59%) identical amino acids). A striking characteristic of these GTPases is their basic C-terminus which, when fused to the C-terminus of green fluorescent protein (GFP), targets the constructs to the nucleus of transfected COS-7 cells. Full length ARL4 was detected in both nuclear and extranuclear compartments, whereas a construct of ARL4 lacking its C-terminus was excluded from the nucleus. Nucleotide exchange rates of recombinant ARL4, ARL6 and ARL7 were similar and appeared considerably higher than those of other members of the ARF family (ARF1, ARP). It is concluded that ARL4, ARL6 and ARL7 form a subgroup within the ARF family with similar, possibly nuclear, function.

ADP-ribosylation factor 1 (ARF1) is a key regulator of transport in the secretory system. Like all small GTPases, deactivation of ARF1 requires a GTPase-activating protein (GAP) that promotes hydrolysis of GTP to GDP on ARF1. Structure-function analysis of a GAP for ARF1 revealed that its activity in vivo requires not only a domain that catalyzes hydrolysis of GTP on ARF1 but also a non-catalytic domain. In this study, we show that the non-catalytic domain of GAP is required for its recruitment from cytosol to membranes and that this domain mediates the interaction of GAP with the transmembrane KDEL receptor. Blocking its interaction with the KDEL receptor leaves the GAP cytosolic and prevents the deactivation in vivo of Golgi-localized ARF1. Thus, these findings suggest that the KDEL receptor plays a critical role in the function of GAP by regulating its recruitment from cytosol to membranes, where it can then act on its membrane-restricted target, the GTP-bound form of ARF1.

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.

The ADP-ribosylation factor (ARF) subfamily of small GTPases regulates intracellular transport. Although much is known about how ARF1 regulates transport in the secretory pathways, regulation of the endocytic pathways by ARF6 remains less understood. In particular, whereas cycling of ARF1 between membrane and cytosol represents a major mechanism of regulating its function, this regulation has been questioned for ARF6. In this study, we found that ARF6 is distributed both on membranes and in the cytosol. Cytosolic ARF6 is recruited to membranes in a GTP-dependent manner that is fundamentally similar to ARF1. However, unlike ARF1, release of membrane-bound ARF6 to the cytosol requires hydrolysis of GTP that is sensitive to the level of magnesium. These findings suggest that the GTPase cycle of ARF6 also regulates its distribution between membrane and cytosol and that this form of regulation will also likely be important for the function of ARF6. Moreover, as ARF6 has little intrinsic ability to hydrolyze GTP, magnesium concentration most likely affects the release of membrane-bound ARF6 by altering the activity of its GTPase-activating protein.

ADP-ribosylation factors (ARFs) regulate coatomer assembly on the Golgi as well as recruitment of clathrin adapter proteins and are therefore involved in vesicle budding from the Golgi and vesicular transport. They are also regulators of phospholipase D (PLD) activity. Arfaptin 1 is an ARF binding protein that inhibits PLD activation, vesicular trafficking and secretion. In the present report, we show that arfaptin 1 interacts with 'high speed' membranes independently of ARF. However, addition of myristoylated ARF3 (myrARF3) increases the association of arfaptin 1 with the membranes, suggesting that arfaptin 1 and ARF form a complex on the Golgi. Utilizing several deletion mutants of arfaptin 1 it is shown that the association of arfaptin 1 with myrARF3 is mediated via two binding sites on arfaptin 1. These two domains are needed for arfaptin 1 inhibition of PLD activation by myrARF3 in vitro.

The small GTP-binding protein, ADP-ribosylation factor 1 (ARF1) is essential for the formation of coatomer-coated vesicles from the Golgi and is also an activator of phospholipase D (PLD). Moreover, ARF1-regulated PLD is part of the signal-transduction pathway that can lead to secretion. In this study, substitution and deletion mutants of ARF1 were tested for their ability to activate PLD. These map the PLD effector region of ARF1 to the alpha2 helix, part of the beta2-strand and the N-terminal helix and its ensuing loop. ARF mutants with an increased or decreased ability to activate PLD showed similar characteristics when tested for their ability to stimulate secretion from HL60 cells. ARF1, deleted of the N-terminal 17 amino acid residues (Ndel17), did not support PLD activity or secretion, and neither did it inhibit the activity of wild-type myristoylated ARF1 (myrARF1). In contrast, Ndel17 effectively competed with wild-type myrARF1 to prevent coatomer binding to membranes. This appears to define a structural role for Ndel17, as it can bind a high-molecular mass complex in cytosol. In addition, ethanol has no effect on recruitment of coatomer to membrane. We conclude that the function of ARF-regulated PLD is in the signal-transduction pathway leading to secretion of lysosomal granules, and not as an essential component of ARF1-mediated coatomer binding.

Phospholipase D (PLD) has been implicated as a crucial signaling enzyme in secretory pathways. Two 20-kDa guanine nucleotide-binding proteins, Rho and ADP-ribosylation factor (ARF), are involved in the regulation of secretion and can activate PLD in vitro. We investigated in intact (human adenocarcinoma A549 cells) the role of RhoA and ARF in activation of PLD by phorbol 12-myristate 13-acetate, bradykinin, and/or sphingosine 1-phosphate. To express recombinant Clostridium botulinum C3 exoenzyme (using double subgenomic recombinant Sindbis virus C3), an ADP-ribosyltransferase that inactivates Rho, or dominant-negative Rho containing asparagine at position 19 (using double subgenomic recombinant Sindbis virus Rho19N), cells were infected with Sindbis virus, a novel vector that allows rapid, high level expression of heterologous proteins. Expression of C3 toxin or Rho19N increased basal and decreased phorbol 12-myristate 13-acetate-stimulated PLD activity. Bradykinin or sphingosine 1-phosphate increased PLD activity with additive effects that were abolished in cells expressing C3 exoenzyme or Rho19N. In cells expressing C3, modification of Rho appeared to be incomplete, suggesting the existence of pools that differed in their accessibility to the enzyme. Similar results were obtained with cells scrape-loaded in the presence of C3; however, results with virus infection were more reproducible. To assess the role of ARF, cells were incubated with brefeldin A (BFA), a fungal metabolite that disrupts Golgi structure and inhibits enzymes that catalyze ARF activation by accelerating guanine nucleotide exchange. BFA disrupted Golgi structure, but did not affect basal or agonist-stimulated PLD activity, i.e. it did not alter a rate-limiting step in PLD activation. It also had no effect on Rho-stimulated PLD activity, indicating that RhoA action did not involve a BFA-sensitive pathway. A novel PLD activation mechanism, not sensitive to BFA and involving RhoA, was identified in human airway epithelial cells by use of a viral infection technique that preserves cell responsiveness.

ADP-ribosylation factors (ARFs) play important roles in both constitutive and regulated membrane trafficking to the plasma membrane in other cells. Here we have examined their role in insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. These cells express ARF5 and ARF6. ARF5 was identified in the soluble protein and intracellular membranes; in response to insulin some ARF5 was observed to re-locate to the plasma membrane. In contrast, ARF6 was predominantly localized to the plasma membrane and did not redistribute in response to insulin. We employed myristoylated peptides corresponding to the NH2 termini of ARF5 and ARF6 to investigate the function of these proteins. Myr-ARF6 peptide inhibited insulin-stimulated glucose transport and GLUT4 translocation by approximately 50% in permeabilized adipocytes. In contrast, myr-ARF1 and myr-ARF5 peptides were without effect. Myr-ARF5 peptide also inhibited the insulin stimulated increase in cell surface levels of GLUT1 and transferrin receptors. Myr-ARF6 peptide significantly decreased cell surface levels of these proteins in both basal and insulin-stimulated states, but did not inhibit the fold increase in response to insulin. These data suggest an important role for ARF6 in regulating cell surface levels of GLUT4 in adipocytes, and argue for a role for both ARF5 and ARF6 in the regulation of membrane trafficking to the plasma membrane.

A brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARF) was purified earlier from bovine brain cytosol. Cloning and expression of the cDNA confirmed that the recombinant protein (p200) is a BFA-sensitive ARF GEP. p200 contains a domain that is 50% identical in amino acid sequence to a region in yeast Sec7, termed the Sec7 domain. Sec7 domains have been identified also in other proteins with ARF GEP activity, some of which are not inhibited by BFA. To identify structural elements that influence GEP activity and its BFA sensitivity, several truncated mutants of p200 were made. Deletion of sequence C-terminal to the Sec7 domain did not affect GEP activity. A protein lacking 594 amino acids at the N terminus, as well as sequence following the Sec7 domain, also had high activity. The mutant lacking 630 N-terminal amino acids was, however, only 1% as active, as was the Sec7 domain itself (mutant lacking 697 N-terminal residues). It appears that the Sec7 domain of p200 contains the catalytic site but additional sequence (perhaps especially that between positions 595 and 630) modifies activity dramatically. Myristoylated recombinant ARFs were better than non-myristoylated as substrates; ARFs 1 and 3 were better than ARF5, and no activity was detected with ARF6. Physical interaction of the Sec7 domain with an ARF1 mutant was demonstrated, but it was much weaker than that of the cytohesin-1 Sec7 domain with the same ARF protein. Effects of BFA on p200 and all mutants with high activity were similar with approximately 50% inhibition at

Brefeldin A (BFA) is a fungal metabolite that disassembles the Golgi apparatus into tubular networks and causes the dissociation of coatomer proteins from Golgi membranes. We have previously shown that an additional effect of BFA is to stimulate the ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa (brefeldin A-ADP-riboslyated substrate (BARS)) and that this effect greatly facilitates the Golgi-disassembling activity of the toxin. In this study, BARS has been purified from rat brain cytosol and microsequenced, and the BARS cDNA has been cloned. BARS shares high homology with two known proteins, C-terminal-binding protein 1 (CtBP1) and CtBP2. It is therefore a third member of the CtBP family. The role of BARS in Golgi disassembly by BFA was verified in permeabilized cells. In the presence of dialyzed cytosol that had been previously depleted of BARS or treated with an anti-BARS antibody, BFA potently disassembled the Golgi. However, in cytosol complemented with purified BARS, or even in control cytosols containing physiological levels of BARS, the action of BFA on Golgi disassembly was strongly inhibited. These results suggest that BARS exerts a negative control on Golgi tubulation, with important consequences for the structure and function of the Golgi complex.

Hybridization of a blot containing 50 human RNAs with an ADP-ribosylation factor 5-specific (ARF5) oligonucleotide probe revealed that the ARF5 gene is expressed in all tissues; however, the level of expression varies significantly with highest levels in pancreas, pituitary gland, and placenta. The 5'-flanking region of the human ARF5 gene lacks a TATA or CAAT box and is highly GC-rich. Primer extension analysis indicates that transcription initiates at a discrete site 62 bp 5' to the start of translation; however, the sequence surrounding the transcription initiation site does not resemble the initiator elements described for other TATA-less genes. Transient transfection of ARF5/luciferase deletion constructs into human IMR-32 neuroblastoma cells revealed that sequences within 169 bp of the transcription initiation site were necessary for full expression. Two GC boxes within this region were modified by site-directed mutagenesis and found to be critical for expression of the reporter constructs. Electrophoretic mobility-shift assays demonstrated specific DNA/protein complexes could be formed with oligonucleotides containing each of the GC boxes and these complexes could be effectively competed by oligonucleotides containing either ARF5 Sp1 site or by an oligonucleotide containing a previously characterized Sp1-binding sequence. The level of ARF5 gene expression, therefore, is dependent upon Sp1 or an Sp1-like factor but does not rely upon a canonical initiator element for accurate transcription initiation.

Centaurin-alpha is a 46 kDa in vitro binding protein for the lipid second messenger PtdIns(3,4,5)P3. In this report we have addressed whether centaurin-alpha1, a human homologue of centaurin-alpha, binds PtdIns(3,4,5)P3 in vivo and furthermore, identified a potential physiological function for centaurin-alpha1. Using confocal microscopy of live PC12 cells, transiently transfected with a chimera of green fluorescent protein (GFP) fused to the N-terminus of centaurin-alpha1 (GFP-centaurin-alpha1), we demonstrated the rapid plasma membrane recruitment of cytosolic GFP-centaurin-alpha1 following stimulation with either nerve growth factor or epidermal growth factor. This recruitment was dependent on the centaurin-alpha1 pleckstrin homology domains and was blocked by the PtdIns(4,5)P2 3-kinase (PI 3-kinase) inhibitors wortmannin (100 nM) and LY294002 (50 microM), and also by co-expression with a dominant negative p85. Functionally, we demonstrated that centaurin-alpha1 could complement a yeast strain deficient in the ADP-ribosylation factor (ARF) GTPase-activating protein Gcs1; a complementation that was blocked by mutagenesis of conserved cysteine residues within the ARF GTPase-activating protein analogous domain of centaurin-alpha1. Taken together, our data demonstrated that centaurin-alpha1 could potentially function as an ARF GTPase-activating protein that, on agonist stimulation, was recruited to the plasma membrane possibly through an ability to interact with PtdIns(3,4,5)P3.

Paxillin is a focal adhesion adaptor protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Repeats of a leucine-rich sequence named paxillin LD motifs (Brown M.C., M.S. Curtis, and C.E. Turner. 1998. Nature Struct. Biol. 5:677-678) have been implicated in paxillin binding to focal adhesion kinase (FAK) and vinculin. Here we demonstrate that the individual paxillin LD motifs function as discrete and selective protein binding interfaces. A novel scaffolding function is described for paxillin LD4 in the binding of a complex of proteins containing active p21 GTPase-activated kinase (PAK), Nck, and the guanine nucleotide exchange factor, PIX. The association of this complex with paxillin is mediated by a new 95-kD protein, p95PKL (paxillin-kinase linker), which binds directly to paxillin LD4 and PIX. This protein complex also binds to Hic-5, suggesting a conservation of LD function across the paxillin superfamily. Cloning of p95PKL revealed a multidomain protein containing an NH2-terminal ARF-GAP domain, three ankyrin-like repeats, a potential calcium-binding EF hand, calmodulin-binding IQ motifs, a myosin homology domain, and two paxillin-binding subdomains (PBS). Green fluorescent protein- (GFP-) tagged p95PKL localized to focal adhesions/complexes in CHO.K1 cells. Overexpression in neuroblastoma cells of a paxillin LD4 deletion mutant inhibited lamellipodia formation in response to insulin-like growth fac- tor-1. Microinjection of GST-LD4 into NIH3T3 cells significantly decreased cell migration into a wound. These data implicate paxillin as a mediator of p21 GTPase-regulated actin cytoskeletal reorganization through the recruitment to nascent focal adhesion structures of an active PAK/PIX complex potentially via interactions with p95PKL.

A site-directed photocross-linking approach was employed to determine components that act downstream of ADP-ribosylation factor (ARF). To this end, a photolabile phenylalanine analog was incorporated at various positions of the putative effector region of the ARF molecule. Depending on the position of incorporation, we find specific and GTP-dependent interactions of ARF with two subunits of the coatomer complex, beta-COP and gamma-COP, as well as an interaction with a cytosolic protein (approximately 185 kDa). In addition, we observe homodimer formation of ARF molecules at the Golgi membrane. These data suggest that the binding site of ARF to coatomer is at the interface of its beta- and gamma-subunits, and this is in close proximity to the second site of interaction of coatomer with the Golgi membrane, the binding site within gamma-COP for cytosolic dibasic/diphenylalanine motifs.

The mammalian ARF-INK4a locus uniquely encodes two cell cycle inhibitors by using separate promoters and alternative reading frames. p16INK4a maintains the retinoblastoma protein in its growth suppressive state while ARF stabilizes p53. We report that human ARF protein predominantly localizes to the nucleolus via a sequence within the exon 2-encoded C-terminal domain and is induced to leave the nucleolus by MDM2. ARF forms nuclear bodies with MDM2 and p53 and blocks p53 and MDM2 nuclear export. Tumor-associated mutations in ARF exon 2 disrupt ARF's nucleolus localization and reduce ARF's ability to block p53 nuclear export and to stabilize p53. Our results suggest an ARF-regulated MDM2-dependent p53 stabilization and link the human tumor-associated mutations in ARF with a functional alteration.

Retrograde transport of proteins from the Golgi to the endoplasmic reticulum (ER) has been the subject of some interest in the recent past. Here a new thermosensitive yeast mutant defective in retrieval of dilysine-tagged proteins from the Golgi back to the endoplasmic reticulum was characterized. The ret4-1 mutant also exhibited a selective defect in forward ER-to-Golgi transport of some secreted proteins at the non-permissive temperature. The corresponding RET4 gene was found to encode Glo3p, a GTPase-activating protein (GAP) specific for ADP-ribosylation factor (ARF). In vitro, the Glo3 thermosensitive mutant showed a reduced ARF1-GAP activity. The Glo3 protein belongs to a family of zinc finger proteins that may include additional ARF-GAPs. Gene deletion experiments of other family members showed that only GLO3 deletion resulted in impaired retrieval of dilysine-tagged proteins back to the ER. These results demonstrate that Glo3p is the main ARF-GAP specifically involved in ER retrieval.

Activation of ADP-ribosylation factors (ARFs), approximately 20-kDa guanine nucleotide-binding proteins that play an important role in intracellular vesicular trafficking, depends on guanine nucleotide-exchange proteins (GEPs), which accelerate replacement of bound GDP with GTP. Two major families of ARF GEPs are known: approximately 200-kDa molecules that are inhibited by brefeldin A (BFA), a fungal metabolite that blocks protein secretion and causes apparent disintegration of Golgi structure, and approximately 50-kDa GEPs that are insensitive to BFA. We describe here two human brain cDNAs that encode BFA-inhibited GEPs. One is a approximately 209-kDa protein 99.5% identical in deduced amino acid sequence (1, 849 residues) to a BFA-inhibited ARF GEP (p200) from bovine brain. The other smaller protein, which is approximately 74% identical (1, 785 amino acids), represents a previously unknown gene. We propose that the former, p200, be named BIG1 for (brefeldin A-inhibited GEP1) and the second, which encodes a approximately 202-kDa protein, BIG2. A protein containing sequences found in BIG2 had been purified earlier from bovine brain. Human tissues contained a 7.5-kilobase BIG1 mRNA and a 9.4-kilobase BIG2 transcript. The BIG1 and BIG2 genes were localized, respectively, to chromosomes 8 and 20. BIG2, synthesized as a His6 fusion protein in Sf9 cells, accelerated guanosine 5'-3-O-(thio)triphosphate binding by recombinant ARF1, ARF5, and ARF6. It activated native ARF (mixture of ARF1 and ARF3) more effectively than it did any of the nonmyristoylated recombinant ARFs. BIG2 activity was inhibited by BFA in a concentration-dependent manner but not by B17, a structural analog without effects on Golgi function. Although several clones for approximately 50-kDa BFA-insensitive ARF GEPs are known, these new clones for the approximately 200-kDa BIG1 and BIG2 should facilitate characterization of this rather different family of proteins as well as the elucidation of mechanisms of regulation of BFA-sensitive ARF function in Golgi transport.

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.

The assembly of clathrin-coated vesicles on Golgi membranes is initiated by the GTP-binding protein ADP ribosylation factor (ARF), which generates high-affinity membrane-binding sites for the heterotetrameric AP-1 adaptor complex. Once bound, the AP-1 recruits clathrin triskelia, which polymerize to form the coat. We have found that ARF.GTP also recruits AP-1 and clathrin onto protein-free liposomes. The efficiency of this process is modulated by the composition of the liposomes, with phosphatidylserine being the most stimulatory phospholipid. There is also a requirement for cytosolic factor(s) other than ARF. Thin-section electron microscopy shows the presence of clathrin-coated buds and vesicles that resemble those formed in vivo. These results indicate that AP-1-containing clathrin-coated vesicles can form in the absence of integral membrane proteins. Thus, ARF.GTP, appropriate lipids, and cytosolic factor(s) are the minimal components necessary for AP-1 clathrin-coat assembly.

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.

Phospholipase D (PLD) activity has been shown to be GTP-dependent both in vivo and in vitro. One protein that confers GTP sensitivity to PLD activity in vitro is the low-molecular-mass G-protein ADP-ribosylation factor (Arf). However, members of the Rho family and protein kinase C (PKC) have also been reported to activate PLD in various cell systems. We have characterized the stimulation of PLD in HL60 cell membranes by these proteins. The results demonstrate that a considerable proportion of HL60 PLD activity is located in a detergent-insoluble fraction of the cell membrane that is unlikely to be a caveolae-like domain, but is probably cytoskeletal. This PLD activity required the presence of Arf1, a Rho-family member and PKC for efficient catalysis of the lipid substrate, suggesting that the activity represents PLD1. We show that recombinant human PLD1b is regulated in a similar manner to HL60-membrane PLD, and that PKCalpha and PKCdelta are equally effective PLD activators. Therefore maximum PLD activity requires Arf, a Rho-family member and PKC, emphasizing the high degree of regulation of this enzyme.

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.

Background: Adenosine diphosphate (ADP)-ribosylation factors (ARFs) are small guanosine triphosphatases involved in membrane traffic regulation. Aiming to explore the possible involvement of ARF1 and ARF6 in the reabsorptive properties of the nephron, we evaluated their distribution along the different renal epithelial segments., Methods: ARFs were detected by immunofluorescence and immunogold cytochemistry on renal sections, using specific anti-ARF antibodies., Results: ARF1 was detected in proximal and distal tubules, thick ascending limbs of Henle's loops, and cortical and medullary collecting ducts. By immunofluorescence, labeling was mostly localized to the cell cytoplasm, particularly in Golgi areas. By electron microscopy, the Golgi apparatus and the endosomal compartment of proximal and distal tubular cells were labeled. ARF6 immunofluorescence was observed in brush border membranes and the cytoplasm of proximal convoluted tubular cells, whereas it was restricted to the apical border of proximal straight tubules. ARF6 immunogold labeling was detected over microvilli and endocytic compartments of proximal tubular cells., Conclusions: This study demonstrates the following: (a) the heterogeneous distributions of ARF1 and ARF6 along the nephron, (b) the existence of cytosolic and membrane-bound forms for both ARFs, and (c) their association with microvilli and endocytic compartments, suggesting an active participation in renal reabsorption.

The crystal structure of the complex of ARF1 GTPase bound to GDP and the catalytic domain of ARF GTPase-activating protein (ARFGAP) has been determined at 1.95 A resolution. The ARFGAP molecule binds to switch 2 and helix alpha3 to orient ARF1 residues for catalysis, but it supplies neither arginine nor other amino acid side chains to the GTPase active site. In the complex, the effector-binding region appears to be unobstructed, suggesting that ARFGAP could stimulate GTP hydrolysis while ARF1 maintains an interaction with its effector, the coatomer complex of COPI-coated vesicles. Biochemical experiments show that coatomer directly participates in the GTPase reaction, accelerating GTP hydrolysis a further 1000-fold in an ARFGAP-dependent manner. Thus, a tripartite complex controls the GTP hydrolysis reaction triggering disassembly of COPI vesicle coats.

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.

We have identified a human cDNA encoding a novel protein, exchange factor for ARF6 (EFA6), which contains Sec7 and pleckstrin homology domains. EFA6 promotes efficient guanine nucleotide exchange on ARF6 and is distinct from the ARNO family of ARF1 exchange factors. The protein localizes to a dense matrix on the cytoplasmic face of plasma membrane invaginations, induced on its expression. We show that EFA6 regulates endosomal membrane recycling and promotes the redistribution of transferrin receptors to the cell surface. Furthermore, expression of EFA6 induces actin-based membrane ruffles that are inhibited by co-expression of dominant-inhibitory mutant forms of ARF6 or Rac1. Our results demonstrate that by catalyzing nucleotide exchange on ARF6 at the plasma membrane and by regulating Rac1 activation, EFA6 coordinates endocytosis with cytoskeletal rearrangements.

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.

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.

The ARF6 GTPase regulates a novel endosomal-plasma membrane recycling pathway and influences cortical actin remodeling. Here we examined the relationship between ARF6 and Rac1, a Rho family GTPase, implicated in cortical actin rearrangements. Endogenous Rac1 colocalized with ARF6 at the plasma membrane and on the ARF6 recycling endosome in untransfected HeLa and primary human fibroblast cells. In transfected HeLa cells Rac1 and ARF6 also colocalized. Cells expressing wild-type ARF6 or Rac1 formed actin-containing surface protrusions and membrane ruffles, respectively, upon treatment with the G protein activator aluminum fluoride. Aluminum fluoride-treatment of cells transfected with equivalent amounts of plasmid resulted in enhanced membrane ruffling, with protrusions appearing as Rac expression was lowered. Co-expression of the dominant negative, GTP binding-defective ARF6 T27N mutant inhibited the aluminum fluoride-induced ruffling observed in cells expressing Rac1, and the constitutive ruffling observed in cells expressing the activated Rac1 Q61L mutant. In contrast, co-expression of the GTP-binding-defective, T17N mutant of either Rac1 or Cdc42 with ARF6 did not inhibit the aluminum fluoride-induced surface protrusions, nor did inactivation of Rho with C3-transferase. These observations suggest that ARF6, a non-Rho family GTPase, can, by itself, alter cortical actin and can influence the ability of Rac1 to form lamellipodia, in part, by regulating its trafficking to the plasma membrane.

Recent cloning of a rat brain phosphatidylinositol 3,4, 5-trisphosphate binding protein, centaurin alpha, identified a novel gene family based on homology to an amino-terminal zinc-binding domain. In Saccharomyces cerevisiae, the protein with the highest homology to centaurin alpha is Gcs1p, the product of the GCS1 gene. GCS1 was originally identified as a gene conditionally required for the reentry of cells into the cell cycle after stationary phase growth. Gcs1p was previously characterized as a guanosine triphosphatase-activating protein for the small guanosine triphosphatase Arf1, and gcs1 mutants displayed vesicle-trafficking defects. Here, we have shown that similar to centaurin alpha, recombinant Gcs1p bound phosphoinositide-based affinity resins with high affinity and specificity. A novel GCS1 disruption strain (gcs1Delta) exhibited morphological defects, as well as mislocalization of cortical actin patches. gcs1Delta was hypersensitive to the actin monomer-sequestering drug, latrunculin-B. Synthetic lethality was observed between null alleles of GCS1 and SLA2, the gene encoding a protein involved in stabilization of the actin cytoskeleton. In addition, synthetic growth defects were observed between null alleles of GCS1 and SAC6, the gene encoding the yeast fimbrin homologue. Recombinant Gcs1p bound to actin filaments, stimulated actin polymerization, and inhibited actin depolymerization in vitro. These data provide in vivo and in vitro evidence that Gcs1p interacts directly with the actin cytoskeleton in S. cerevisiae.

The effect of ligating the alpha2-macroglobulin signaling receptor (alpha2MSR) with receptor-recognized forms of alpha2M (alpha2M*) was studied with respect to phospholipase D (PLD) activity in murine macrophages, their plasma membranes, and nuclei. PLD activity in plasma membranes and nuclei increased linearly up to a ligand concentration of about 100 pM of either alpha2M* or a cloned and expressed receptor binding fragment (RBF). The RBF binding site mutant K1370A, which binds with high affinity to alpha2MSR, also increased nuclear PLD activity comparable to RBF and alpha2M*. Phorbol dibutyrate caused a two- to threefold stimulation of membrane and nuclear PLD activity, whereas PLD activity was nearly abolished by downregulation of protein kinase C; prior treatment with staurosporin, genestein, cyclosporin A, actinomycin D; or chelation of intracellular Ca2+. In permeabilized macrophages, isolated plasma membranes, and nuclei, GTP-gamma-S increased alpha2M*-stimulated PLD activity via a pertussis toxin-insensitive G protein and this effect was abolished on preincubation with GDP-beta-S. Incubation of plasma membranes with polyclonal antibody against sARFII, or the addition of cytosol which was immunoprecipitated with antibody against sARFII, greatly reduced alpha2M*-stimulated PLD activity in the presence of GTP-gamma-S. Preincubation of plasma membranes with GDP-beta-S prior to the addition of GTP-gamma-S and recombinant ARF1 significantly inhibited alpha2M*-stimulation of PLD activity. Nuclear PLD activity was maximally stimulated in the presence of both GTP-gamma-S and rARF1, whereas plasma membrane PLD activity was maximally stimulated in the presence of rARF1, GTP-gamma-S, RhoA, and ATP. In contrast, nuclear PLD activity was not affected by RhoA either alone or in combination with GTP-gamma-S or ATP., (Copyright 1999 Academic Press.)

Protein tyrosine kinase Pyk2 is activated by a variety of G-protein-coupled receptors and by extracellular signals that elevate intracellular Ca2+ concentration. We have identified a new Pyk2 binding protein designated Pap. Pap is a multidomain protein composed of an N-terminal alpha-helical region with a coiled-coil motif, followed by a pleckstrin homology domain, an Arf-GAP domain, an ankyrin homology region, a proline-rich region, and a C-terminal SH3 domain. We demonstrate that Pap forms a stable complex with Pyk2 and that activation of Pyk2 leads to tyrosine phosphorylation of Pap in living cells. Immunofluorescence experiments demonstrate that Pap is localized in the Golgi apparatus and at the plasma membrane, where it is colocalized with Pyk2. In addition, in vitro recombinant Pap exhibits strong GTPase-activating protein (GAP) activity towards the small GTPases Arf1 and Arf5 and weak activity towards Arf6. Addition of recombinant Pap protein to Golgi preparations prevented Arf-dependent generation of post-Golgi vesicles in vitro. Moreover, overexpression of Pap in cultured cells reduced the constitutive secretion of a marker protein. We propose that Pap functions as a GAP for Arf and that Pyk2 may be involved in regulation of vesicular transport through its interaction with Pap.

ADP-ribosylation factors (ARFs) are highly conserved, approximately 20-kDa guanine nucleotide-binding proteins that enhance the ADP-ribosyltransferase activity of cholera toxin and have an important role in vesicular transport. Several cDNAs for ARF-like proteins (ARLs) have been cloned from human, Drosophila, rat, and yeast, although the biological function(s) of ARLs is unknown. We have identified a yeast gene (yARL3) encoding a protein that is structurally related (>43% identical) to the mammalian ARF-like protein ARP. Biochemical studies of purified recombinant yARL3 protein revealed properties similar to those of ARF and ARL proteins, including the ability to bind and hydrolyze GTP. Like other ARLs, recombinant yARL3 did not stimulate cholera toxin-catalyzed auto-ADP-ribosylation. Anti-yARL3 antibodies did not cross-react with yARFs or yARL1. yARL3 was not essential for cell viability, but disruption of yARL3 resulted in cold-sensitive cell growth. At the nonpermissive temperature, processing of alkaline phosphatase and carboxypeptidase Y in arl3 mutant was slowed. yARL3 might be required for protein transport from endoplasmic reticulum to Golgi or from Golgi to vacuole at nonpermissive temperatures. On subcellular fractionation, unlike its mammalian homologue ARP, yARL3 was detected in the soluble fraction but not in the plasma membrane. Indirect immunofluorescence analysis revealed that yARL3 when overexpressed was associated in part with the endoplasmic reticulum-nuclear envelope. Thus, the structural and functional characteristics of yARL3 indicate that it may have a unique role(s) in vesicular trafficking.

ADP ribosylation factors (ARFs) represent a family of small monomeric G proteins that switch from an inactive, GDP-bound state to an active, GTP-bound state. One member of this family, ARF6, translocates on activation from intracellular compartments to the plasma membrane and has been implicated in regulated exocytosis in neuroendocrine cells. Because GDP release in vivo is rather slow, ARF activation is facilitated by specific guanine nucleotide exchange factors like cytohesin-1 or ARNO. Here we show that msec7-1, a rat homologue of cytohesin-1, translocates ARF6 to the plasma membrane in living cells. Overexpression of msec7-1 leads to an increase in basal synaptic transmission at the Xenopus neuromuscular junction. msec7-1-containing synapses have a 5-fold higher frequency of spontaneous synaptic currents than control synapses. On stimulation, the amplitudes of the resulting evoked postsynaptic currents of msec7-1-overexpressing neurons are increased as well. However, further stimulation leads to a decline in amplitudes approaching the values of control synapses. This transient effect on amplitude is strongly reduced on overexpression of msec7-1E157K, a mutant incapable of translocating ARFs. Our results provide evidence that small G proteins of the ARF family and activating factors like msec7-1 play an important role in synaptic transmission, most likely by making more vesicles available for fusion at the plasma membrane.

ARF proteins, which mediate vesicular transport, have little or no intrinsic GTPase activity. They rely on the actions of GTPase-activating proteins (GAPs) for their function. The in vitro GTPase activity of the Saccharomyces cerevisiae ARF proteins Arf1 and Arf2 is stimulated by the yeast Gcs1 protein, and in vivo genetic interactions between arf and gcs1 mutations implicate Gcs1 in vesicular transport. However, the Gcs1 protein is dispensable, indicating that additional ARF GAP proteins exist. We show that the structurally related protein Glo3, which is also dispensable, also exhibits ARF GAP activity. Genetic and in vitro approaches reveal that Glo3 and Gcs1 have an overlapping essential function at the endoplasmic reticulum (ER)-Golgi stage of vesicular transport. Mutant cells deficient for both ARF GAPs cannot proliferate, undergo a dramatic accumulation of ER and are defective for protein transport between ER and Golgi. The glo3Delta and gcs1Delta single mutations each interact with a sec21 mutation that affects a component of COPI, which mediates vesicular transport within the ER-Golgi shuttle, while increased dosage of the BET1, BOS1 and SEC22 genes encoding members of a v-SNARE family that functions within the ER-Golgi alleviates the effects of a glo3Delta mutation. An in vitro assay indicates that efficient retrieval from the Golgi to the ER requires these two proteins. These findings suggest that Glo3 and Gcs1 ARF GAPs mediate retrograde vesicular transport from the Golgi to the ER.

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.

Escherichia coli heat-labile enterotoxin (LT), an oligomeric protein with one A subunit (LTA) and five B subunits, exerts its effects via the ADP-ribosylation of Gsalpha, a guanine nucleotide-binding (G) protein that activates adenylyl cyclase. LTA also ADP-ribosylates simple guanidino compounds (e.g., arginine) and catalyzes its own auto-ADP-ribosylation. All LTA-catalyzed reactions are enhanced by ADP-ribosylation factors (ARFs), 20-kDa guanine nucleotide-binding proteins. Replacement of arginine-7 (R7K), valine-53 (V53D), serine-63 (S63K), valine 97 (V97K), or tyrosine-104 (Y104K) in LTA resulted in fully assembled but nontoxic proteins. S63K, V53D, and R7K are catalytic-site mutations, whereas V97K and Y104K are amino acid replacements adjacent to and outside of the catalytic site, respectively. The effects of mutagenesis were quantified by measuring ADP-ribosyltransferase activity (i.e., auto-ADP-ribosylation and ADP-ribosylagmatine synthesis) and interaction with ARF (i.e., inhibition of ARF-stimulated cholera toxin ADP-ribosyltransferase activity and effects of ARF on mutant auto-ADP-ribosylation). All mutants were inactive in the ADP-ribosyltransferase assay; however, auto-ADP-ribosylation in the presence of recombinant human ARF6 was detected, albeit much less than that of native LT (Y104K > V53D > V97K > R7K, S63K). Based on the lack of inhibition by free ADP-ribose, the observed auto-ADP-ribosylation activity was enzymatic and not due to the nonenzymatic addition of free ADP-ribose. V53D, S63K, and R7K were more effective than Y104K or V97K in blocking ARF stimulation of cholera toxin ADP-ribosyltransferase. Based on these data, it appears that ARF-binding and catalytic sites are not identical and that a region outside the NAD cleft may participate in the LTA-ARF interaction.

Members of the ADP-ribosylation factor (ARF) family of small guanosine triphosphate-binding proteins play an essential role in membrane trafficking which subserves constitutive protein transport along exocytic and endocytic pathways within eukaryotic cell bodies. In growing neurons, membrane trafficking within motile growth cones distant from the cell body underlies the rapid plasmalemmal expansion which subserves axon elongation. We report here that ARF is a constituent of axonal growth cones, and that application of brefeldin A to neurons in culture produces a rapid arrest of axon extension that can be ascribed to inhibition of ARF function in growth cones. Our findings demonstrate a role for ARF in growth cones that is coupled tightly to the rapid growth of neuronal processes characteristic of developmental and regenerative axon elongation, and indicate that ARF participates not only in constitutive membrane traffic within the cell body, but also in membrane dynamics within growing axon endings.

Focal adhesion assembly and actin stress fiber formation were studied in serum-starved Swiss 3T3 fibroblasts permeabilized with streptolysin-O. Permeabilization in the presence of GTPgammaS stimulated rho-dependent formation of stress fibers, and the redistribution of vinculin and paxillin from a perinuclear location to focal adhesions. Addition of GTPgammaS at 8 min after permeabilization still induced paxillin recruitment to focal adhesion-like structures at the ends of stress fibers, but vinculin remained in the perinuclear region, indicating that the distributions of these two proteins are regulated by different mechanisms. Paxillin recruitment was largely rho-independent, but could be evoked using constitutively active Q71L ADP-ribosylation factor (ARF1), and blocked by NH2-terminally truncated Delta17ARF1. Moreover, leakage of endogenous ARF from cells was coincident with loss of GTPgammaS- induced redistribution of paxillin to focal adhesions, and the response was recovered by addition of ARF1. The ability of ARF1 to regulate paxillin recruitment to focal adhesions was confirmed by microinjection of Q71LARF1 and Delta17ARF1 into intact cells. Interestingly, these experiments showed that V14RhoA- induced assembly of actin stress fibers was potentiated by Q71LARF1. We conclude that rho and ARF1 activate complimentary pathways that together lead to the formation of paxillin-rich focal adhesions at the ends of prominent actin stress fibers.

The treatment of normal rat kidney cells with N-ethylmaleimide caused the release of beta-COP, a component of coatomer, from the Golgi apparatus without causing disassembly of the organelle. The release of beta-COP, which was not due to depolymerization of microtubules, was markedly blocked by the activation of GTP-binding proteins by aluminum fluoride or a nonhydrolyzable analogue of GTP. To determine which component is N-ethylmaleimide-sensitive, we reconstituted the recruitment of coatomer from the bovine brain cytosol onto the Golgi apparatus in digitonin-permeabilized cells. In cells treated with N-ethylmaleimide before permeabilization, beta-COP was still recruited onto the Golgi apparatus. In contrast, beta-COP was not recruited when N-ethylmaleimide-treated bovine brain cytosol was used. These results suggest that the N-ethylmaleimide-sensitive factor(s) are present in the cytosol. It is known that coatomer and ADP-ribosylation factor-1 (ARF1) are the only cytoplasmic proteins needed for the assembly of Golgi-derived coated vesicles. N-Ethylmaleimide treatment of a coatomer-rich fraction did not affect the binding of beta-COP to the Golgi apparatus, whereas the same treatment of an ARF-rich fraction abolished beta-COP binding. Similar results were obtained using purified recombinant ARF1. Concomitant with inactivation, 0.85 mol of N-ethylmaleimide was incorporated into 1 mol of ARF1. ARF1 contains only one cysteine residue (Cys-159), which is located near the base moiety of the bound guanine nucleotide.

Membrane trafficking is regulated in part by small GTP-binding proteins of the ADP-ribosylation factor (Arf) family. Arf function depends on the controlled exchange and hydrolysis of GTP. We have purified and cloned two variants of a 130-kDa phosphatidylinositol 4, 5-biphosphate (PIP2)-dependent Arf1 GTPase-activating protein (GAP), which we call ASAP1a and ASAP1b. Both contain a pleckstrin homology (PH) domain, a zinc finger similar to that found in another Arf GAP, three ankyrin (ANK) repeats, a proline-rich region with alternative splicing and SH3 binding motifs, eight repeats of the sequence E/DLPPKP, and an SH3 domain. Together, the PH, zinc finger, and ANK repeat regions possess PIP2-dependent GAP activity on Arf1 and Arf5, less activity on Arf6, and no detectable activity on Arl2 in vitro. The cDNA for ASAP1 was independently identified in a screen for proteins that interact with the SH3 domain of the tyrosine kinase Src. ASAP1 associates in vitro with the SH3 domains of Src family members and with the Crk adapter protein. ASAP1 coprecipitates with Src from cell lysates and is phosphorylated on tyrosine residues in cells expressing activated Src. Both coimmunoprecipitation and tyrosine phosphorylation depend on the same proline-rich class II Src SH3 binding site required for in vitro association. By directly interacting with both Arfs and tyrosine kinases involved in regulating cell growth and cytoskeletal organization, ASAP1 could coordinate membrane remodeling events with these processes.

The role of small G proteins of the ADP-ribosylation factor (ARF) and Rho families on the activation of phospholipase D (PLD) by platelet-derived growth factor (PDGF) and phorbol esters (PMA) has been investigated. The activation of PLD by PDGF and PMA was blocked by brefeldin A (BFA), an inhibitor of ARF activation, but not by Clostridium botulinum C3 exotoxin, an inhibitor of the activity of Rho. PDGF and PMA, in the presence of GTPgammaS, promoted the association of ARF and RhoA with cell membranes. Cells depleted of ARF and Rho by digitonin permeabilization showed a significant reduction of the activity of phospholipase D. Recombinant ARF was sufficient to restore agonist-induced PLD activity to digitonin-permeabilized, cytoplasm-depleted cells. In contrast, isoprenylated recombinant RhoA had no effects in this reconstitution assay. HIRcB cells were transiently transfected with wild-type and dominant-negative mutants of ARF1 and ARF6. Neither wt-ARF1 nor wt-ARF6 had any effects on agonist-dependent PLD activity. However, dominant-negative ARF1 and ARF6 mutants blocked the stimulation of PLD by PDGF but only partially inhibited the effects of PMA. These results demonstrate that ARF rather than Rho proteins mediate the activation of PLD by PDGF and phorbol esters in HIRcB fibroblasts.

The finding that ADP-ribosylation factor (ARF) can activate phospholipase D has led to debate as to whether ARF recruits coat proteins through direct binding or indirectly by catalytically increasing phosphatidic acid production. Here we test critical aspects of these hypotheses. We find that Golgi membrane phosphatidic acid levels do not rise-in fact they decline-during cell-free budding reactions. We confirm that the level of membrane-bound ARF can be substantially reduced without compromising coat assembly [Ktistakis, N. T., Brown, H. A., Waters, M. G., Sternweis, P. C. & Roth, M. G. (1996) J. Cell Biol. 134, 295-306], but find that under all conditions, ARF is present on the Golgi membrane in molar excess over bound coatomer. These results do not support the possibility that the activation of coat assembly by ARF is purely catalytic, and they are consistent with ARF forming direct interactions with coatomer. We suggest that ARF, like many other G proteins, is a multifunctional protein with roles in trafficking and phospholipid signaling.