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Martha Vaughan - One of the best experts on this subject based on the ideXlab platform.

  • the brefeldin a inhibited guanine nucleotide exchange protein big2 regulates the constitutive release of tnfr1 exosome like vesicles
    Journal of Biological Chemistry, 2007
    Co-Authors: Aminul Islam, Joel Moss, Martha Vaughan, Xiaoyan Shen, Toyoko Hiroi, Stewart J Levine
    Abstract:

    Abstract The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released from cells to the extracellular space where it can bind and modulate TNF bioactivity. Extracellular TNFR1 release occurs by two distinct pathways: the inducible proteolytic cleavage of TNFR1 ectodomains and the constitutive release of full-length TNFR1 in exosome-like vesicles. Regulation of both TNFR1 release pathways appears to involve the trafficking of cytoplasmic TNFR1 vesicles. Vesicular trafficking is controlled by ADP-ribosylation factors (ARFs), which are active in the GTP-bound state and inactive when bound to GDP. ARF activation is enhanced by guanine nucleotide-exchange factors that catalyze replacement of GDP by GTP. We investigated whether the brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins, BIG1 and/or BIG2, are required for TNFR1 release from human umbilical vein endothelial cells. Effects of specific RNA interference (RNAi) showed that BIG2, but not BIG1, regulated the release of TNFR1 exosome-like vesicles, whereas neither BIG2 nor BIG1 was required for the IL-1β-induced proteolytic cleavage of TNFR1 ectodomains. BIG2 co-localized with TNFR1 in diffusely distributed cytoplasmic vesicles, and the association between BIG2 and TNFR1 was disrupted by BFA. Consistent with the preferential activation of class I ARFs by BIG2, ARF1 and ARF3 participated in the extracellular release of TNFR1 exosome-like vesicles in a nonredundant and additive fashion. We conclude that the association between BIG2 and TNFR1 selectively regulates the extracellular release of TNFR1 exosome-like vesicles from human vascular endothelial cells via an ARF1- and ARF3-dependent mechanism.

  • arf gep100 a guanine nucleotide exchange protein for adp ribosylation factor 6
    Proceedings of the National Academy of Sciences of the United States of America, 2001
    Co-Authors: Akimasa Someya, Joel Moss, Makoto Sata, Kazuyo Takeda, Gustavo Pachecorodriguez, Victor J Ferrans, Martha Vaughan
    Abstract:

    A human cDNA encoding an 841-aa guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARFs), named ARF-GEP100, 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 ≈8-kb mRNA that hybridized with an ARF-GEP100 cDNA was abundant in peripheral blood leukocytes, brain, and spleen. ARF-GEP100 accelerated [35S]GTPγS binding to ARF1 (class I) and ARF5 (class II) 2- to 3-fold, and to ARF6 (class III) ca. 12-fold. The ARF-GEP100 Sec7 domain contains Asp543 and Met555, corresponding to residues associated with sensitivity to the inhibitory effect of the fungal metabolite brefeldin A (BFA) in yeast Sec7, but also Phe535 and Ala536, 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-GEP100 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-GEP100, 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-GEP100 in those areas. No similar coincidence of ARF-GEP100 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.

  • brefeldin a inhibited activity of the sec7 domain of p200 a mammalian guanine nucleotide exchange protein for adp ribosylation factors
    Journal of Biological Chemistry, 1999
    Co-Authors: Naoko Morinaga, Joel Moss, Ronald Adamik, Martha Vaughan
    Abstract:

    Abstract 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 ∼50% inhibition at ≤50 μm. The inactive BFA analogue B36 did not inhibit the Sec7 domain or p200. Thus, the Sec7 domain of p200, like that of Sec7 itself (Sata, M., Donaldson, J. G., Moss, J., and Vaughan, M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4204–4208), plays a role in BFA inhibition as well as in GEP activity, although the latter is markedly modified by other structural elements.

  • purification and cloning of a brefeldin a inhibited guanine nucleotide exchange protein for adp ribosylation factors
    Journal of Biological Chemistry, 1999
    Co-Authors: Akira Togawa, Naoko Morinaga, Masahito Ogasawara, Joel Moss, Martha Vaughan
    Abstract:

    Abstract Activation of ADP-ribosylation factors (ARFs), ∼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: ∼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 ∼50-kDa GEPs that are insensitive to BFA. We describe here two human brain cDNAs that encode BFA-inhibited GEPs. One is a ∼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 ∼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 ∼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 andBIG2 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 ∼50-kDa BFA-insensitive ARF GEPs are known, these new clones for the ∼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.

  • brefeldin a inhibited guanine nucleotide exchange activity of sec7 domain from yeast sec7 with yeast and mammalian adp ribosylation factors
    Proceedings of the National Academy of Sciences of the United States of America, 1998
    Co-Authors: Makoto Sata, Julie G. Donaldson, Joel Moss, Martha Vaughan
    Abstract:

    The Saccharomyces cerevisiae Sec7 protein (ySec7p), which is an important component of the yeast secretory pathway, contains a sequence of ≈200 amino acids referred to as a Sec7 domain. Similar Sec7 domain sequences have been recognized in several guanine nucleotide-exchange proteins (GEPs) for ADP ribosylation factors (ARFs). ARFs are ≈20-kDa GTPases that regulate intracellular vesicular membrane trafficking and activate phospholipase D. GEPs activate ARFs by catalyzing the replacement of bound GDP with GTP. We, therefore, undertook to determine whether a Sec7 domain itself could catalyze nucleotide exchange on ARF and found that it exhibited brefeldin A (BFA)-inhibitable ARF GEP activity. BFA is known to inhibit ARF GEP activity in Golgi membranes, thereby causing reversible apparent dissolution of the Golgi complex in many cells. The His6-tagged Sec7 domain from ySec7p (rySec7d) synthesized in Escherichia coli enhanced binding of guanosine 5′-[γ-[35S]thio]triphosphate by recombinant yeast ARF1 (ryARF1) and ryARF2 but not by ryARF3. The effects of rySec7d on ryARF2 were inhibited by BFA in a concentration-dependent manner but not by inactive analogues of BFA (B-17, B-27, and B-36). rySec7d also promoted BFA-sensitive guanosine 5′-[γ-thio]triphosphate binding by nonmyristoylated recombinant human ARF1 (rhARF1), rhARF5, and rhARF6, although the effect on rhARF6 was very small. These results are consistent with the conclusion that the yeast Sec7 domain itself contains the elements necessary for ARF GEP activity and its inhibition by BFA.

Crislyn Dsouzaschorey - One of the best experts on this subject based on the ideXlab platform.

  • adp ribosylation factor 6 regulates tumor cell invasion through the activation of the mek erk signaling pathway
    Proceedings of the National Academy of Sciences of the United States of America, 2004
    Co-Authors: Sarah Tague, Vandhana Muralidharan, Crislyn Dsouzaschorey
    Abstract:

    Tumor cell invasion through the extracellular matrix is accompanied by the formation of invadopodia, which are actin-rich protrusions at the adherent surface of cells at sites of extracellular matrix degradation. Using the invasive human melanoma cell line LOX as a model system, we demonstrate that the ADP-ribosylation factor 6 (ARF6) GTPase is an important regulator of invadopodia formation and cell invasion. We show that ARF6 localizes to invadopodia of LOX cells. Sustained activation of ARF6 significantly enhances the invasive capacity of melanoma as well as breast tumor cell lines, whereas dominant negative ARF6 abolishes basal cell invasive capacity as well as invasion induced by growth factors. Furthermore, using biochemical assays, we show that enhanced invasive capacity is accompanied by the activation of endogenous ARF6. Finally, we provide evidence that ARF6-enhanced melanoma cell invasion depends on the activation of the extracellular signal-regulated kinase (ERK), and that the ARF6 GTPase cycle regulates ERK activation. This study describes a vital role for ARF6 in melanoma cell invasion and documents a link between ARF6-mediated signaling and ERK activation.

  • localization and activation of the ARF6 gtpase during cleavage furrow ingression and cytokinesis
    Journal of Biological Chemistry, 2002
    Co-Authors: Jill Kuglin Schweitzer, Crislyn Dsouzaschorey
    Abstract:

    The ARF6 GTPase mediates cell shape changes in interphase cells through its effects on membrane cycling and actin remodeling. In this study, we focus our attention on the dynamics of cell division and present evidence supporting a novel role for ARF6 during cleavage furrow ingression and cytokinesis. We demonstrate that endogenous ARF6 redistributes during mitosis and concentrates near the cleavage furrow during telophase. Constitutively activated ARF6 localizes to the plasma membrane at the site of cleavage furrow ingression and midbody formation, and dominant negative ARF6 remains cytoplasmic. By using a novel pull-down assay for ARF6-GTP, we find an abrupt, but transient, increase in ARF6-GTP levels as cells progress through cytokinesis. Whereas high levels of expression of a GTPase-defective ARF6 mutant induce aberrant phenotypes in cells at cytokinesis, cells expressing low levels of ARF6 mutants do not display a significant mitotic delay or cytokinesis defect, presumably due to compensatory or redundant mechanisms that allow cytokinesis to proceed when the ARF6 GTPase cycle is disrupted. Finally, actin accumulation and phospholipid metabolism at the cleavage furrow are unchanged in cells expressing ARF6 mutants, suggesting that ARF6 may be involved in membrane remodeling during cytokinesis via effector pathways that are distinct from those operative in interphase cells.

  • an essential role for ARF6 regulated membrane traffic in adherens junction turnover and epithelial cell migration
    The EMBO Journal, 2001
    Co-Authors: Felipe Palacios, Leo Price, Jill Kuglin Schweitzer, John G Collard, Crislyn Dsouzaschorey
    Abstract:

    We describe a novel role for the ARF6 GTPase in the regulation of adherens junction (AJ) turnover in MDCK epithelial cells. Expression of a GTPase-defective ARF6 mutant, ARF6(Q67L), led to a loss of AJs and ruffling of the lateral plasma membrane via mechanisms that were mutually exclusive. ARF6-GTP-induced AJ disassembly did not require actin remodeling, but was dependent on the internalization of E-cadherin into the cytoplasm via vesicle transport. ARF6 activation was accompanied by increased migratory potential, and treatment of cells with hepatocyte growth factor (HGF) induced the activation of endogenous ARF6. The effect of ARF6(Q67L) on AJs was specific since ARF6 activation did not perturb tight junction assembly or cell polarity. In contrast, dominant-negative ARF6, ARF6(T27N), localized to AJs and its expression blocked cell migration and HGF-induced internalization of cadherin-based junctional components into the cytoplasm. Finally, we show that ARF6 exerts its role downstream of v-Src activation during the disassembly of AJs. These findings document an essential role for ARF6- regulated membrane traffic in AJ disassembly and epithelial cell migration.

  • adp ribosylation factor 6 regulates actin cytoskeleton remodeling in coordination with rac1 and rhoa
    Molecular and Cellular Biology, 2000
    Co-Authors: Rita L Boshans, Stacey Szanto, Linda Van Aelst, Crislyn Dsouzaschorey
    Abstract:

    In this study, we have documented an essential role for ADP-ribosylation factor 6 (ARF6) in cell surface remodeling in response to physiological stimulus and in the down regulation of stress fiber formation. We demonstrate that the G-protein-coupled receptor agonist bombesin triggers the redistribution of ARF6- and Rac1-containing endosomal vesicles to the cell surface. This membrane redistribution was accompanied by cortical actin rearrangements and was inhibited by dominant negative ARF6, implying that bombesin is a physiological trigger of ARF6 activation. Furthermore, these studies provide a new model for bombesin-induced Rac1 activation that involves ARF6-regulated endosomal recycling. The bombesin-elicited translocation of vesicular ARF6 was mimicked by activated Galphaq and was partially inhibited by expression of RGS2, which down regulates Gq function. This suggests that Gq functions as an upstream regulator of ARF6 activation. The ARF6-induced peripheral cytoskeletal rearrangements were accompanied by a depletion of stress fibers. Moreover, cells expressing activated ARF6 resisted the formation of stress fibers induced by lysophosphatidic acid. We show that the ARF6-dependent inhibition of stress fiber formation was due to an inhibition of RhoA activation and was overcome by expression of a constitutively active RhoA mutant. The latter observations demonstrate that activation of ARF6 down regulates Rho signaling. Our findings underscore the potential roles of ARF6, Rac1, and RhoA in the coordinated regulation of cytoskeletal remodeling.

  • efa6 a sec7 domain containing exchange factor for ARF6 coordinates membrane recycling and actin cytoskeleton organization
    The EMBO Journal, 1999
    Co-Authors: Michel Franco, Crislyn Dsouzaschorey, Peter J Peters, Joelle Boretto, Elly Van Donselaar, Antonino Neri, Philippe Chavrier
    Abstract:

    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.

Richard A Kahn - One of the best experts on this subject based on the ideXlab platform.

  • several adp ribosylation factor arf isoforms support copi vesicle formation
    Journal of Biological Chemistry, 2011
    Co-Authors: Vincent Popoff, Julian D Langer, Ingeborg Reckmann, Andrea Hellwig, Richard A Kahn, Britta Brugger, Felix T Wieland
    Abstract:

    Newly synthesized proteins and lipids are transported in vesicular carriers along the secretory pathway. Arfs (ADP-ribosylation factors), a family of highly conserved GTPases within the Ras superfamily, control recruitment of molecular coats to membranes, the initial step of coated vesicle biogenesis. Arf1 and coatomer constitute the minimal cytosolic machinery leading to COPI vesicle formation from Golgi membranes. Although some functional redundancies have been suggested, other Arf isoforms have been poorly analyzed in this context. In this study, we found that Arf1, Arf4, and Arf5, but not Arf3 and ARF6, associate with COPI vesicles generated in vitro from Golgi membranes and purified cytosol. Using recombinant myristoylated proteins, we show that Arf1, Arf4, and Arf5 each support COPI vesicle formation individually. Unexpectedly, we found that Arf3 could also mediate vesicle biogenesis. However, Arf3 was excluded from the vesicle fraction in the presence of the other isoforms, highlighting a functional competition between the different Arf members.

  • Dynamic structure of membrane-anchored Arf•GTP
    Nature Structural & Molecular Biology, 2010
    Co-Authors: Richard A Kahn, James H Prestegard
    Abstract:

    ADP ribosylation factors (Arfs) are N-myristoylated GTP/GDP switch proteins that have key regulatory roles in vesicle transport in eukaryotic cells. ARFs execute their roles by anchoring to membrane surfaces, where they interact with other proteins to initiate budding and maturation of transport vesicles. However, existing structures of Arf•GTP are limited to nonmyristoylated and truncated forms with impaired membrane binding. We report a high-resolution NMR structure for full-length myristoylated yeast ( Saccharomyces cerevisiae ) Arf1 in complex with a membrane mimic. The two-domain structure, in which the myristoylated N-terminal helix is separated from the C-terminal domain by a flexible linker, suggests a level of adaptability in binding modes for the myriad of proteins with which Arf interacts and allows predictions of specific lipid binding sites on some of these proteins. ARF proteins play key roles in the budding and maturation of eukaryotic transport vesicles. The high-resolution NMR structure of full-length yeast Arf1-GTP anchored to a membrane mimic reveals a flexible linker between the myristoylated N-terminal helix and the GTPase domain, which would facilitate interaction with multiple partners.

  • isoform selective effects of the depletion of adp ribosylation factors 1 5 on membrane traffic
    Molecular Biology of the Cell, 2005
    Co-Authors: Laura A Volpicellidaley, Chunjiang Zhang, Richard A Kahn
    Abstract:

    The ADP-ribosylation factors (Arfs) are six proteins within the larger Arf family and Ras superfamily that regulate membrane traffic. Arfs all share numerous biochemical activities and have very similar specific activities. The use of dominant mutants and brefeldin A has been important to the discovery of the cellular functions of Arfs but lack specificity between Arf isoforms. We developed small interference RNA constructs capable of specific depletion of each of the cytoplasmic human Arfs to examine the specificity of Arfs in live cells. No single Arf was required for any step of membrane traffic examined in HeLa cells. However, every combination of the double knockdowns of Arf1, Arf3, Arf4, and Arf5 yielded a distinct pattern of defects in secretory and endocytic traffic, demonstrating clear specificity for Arfs at multiple steps. These results suggest that the cooperation of two Arfs at the same site may be a general feature of Arf signaling and provide candidates at several cellular locations that when paired with data on the localization of the many different Arf guanine nucleotide exchange factors, Arf GTPase activating proteins, and effectors will aid in the description of the mechanisms of specificity in this highly conserved and primordial family of regulatory GTPases.

  • adp ribosylation factors arfs and arf like 1 arl1 have both specific and shared effectors characterizing arl1 binding proteins
    Journal of Biological Chemistry, 2001
    Co-Authors: Hillary Van Valkenburgh, Jack F Shern, Daniel J Sharer, Xinjun Zhu, Richard A Kahn
    Abstract:

    Abstract Despite the 40–60% identity between ADP-ribosylation factors (ARFs) and ARF-like (ARL) proteins, distinct functional roles have been inferred from findings that ARLs lack the biochemical or genetic activities characteristic of ARFs. The potential for functional overlap between ARFs and ARLs was examined by comparing effects of expression on intact cells and the ability to bind effectors. Expression of [Q71L]ARL1 in mammalian cells led to altered Golgi structure similar to, but less dramatic than, that reported previously for [Q71L]ARF1 (1). Two previously identified partners of ARFs, MKLP1 and Arfaptin2/POR1, also bind ARL1 but not ARL2 or ARL3. Two-hybrid screens of human cDNA libraries with dominant active mutants of human ARL1, ARL2, and ARL3 identified eight different but overlapping sets of binding partners. Specific interactions between ARL1 and two binding proteins, SCOCO and Golgin-245, are defined and characterized in more detail. Like ARFs and ARL1, the binding of SCOCO to Golgi membranes is rapidly reversed by brefeldin A, suggesting the presence of a brefeldin A-sensitive ARL1 exchange factor. These data reveal a complex network of interactions between GTPases in the ARF family and their effectors and reveal a potential for cross-talk not demonstrated previously.

Julie G. Donaldson - One of the best experts on this subject based on the ideXlab platform.

  • The small GTPase ARF6 regulates sea urchin morphogenesis
    Differentiation, 2017
    Co-Authors: Nadezda A. Stepicheva, Megan Elissa Dumas, Priscilla Kobi, Julie G. Donaldson, Jia L. Song
    Abstract:

    Abstract The small GTPase ARF6 is a conserved protein that is expressed in all metazoans. ARF6 remodels cytoskeletal actin and mediates membrane protein trafficking between the plasma membrane in its active form and endosomal compartments in its inactive form. While a rich knowledge exists for the cellular functions of ARF6, relatively little is known about its physiological role in development. This study examines the function of ARF6 in mediating cellular morphogenesis in early development. We dissect the function of ARF6 with a loss-of-function morpholino and constitutively active ARF6-Q67L construct. We focus on the two cell types that undergo active directed migration: the primary mesenchyme cells (PMCs) that give rise to the sea urchin skeleton and endodermal cells that form the gut. Our results indicate that ARF6 plays an important role in skeleton formation and PMC migration, in part due to its ability to remodel actin. We also found that embryos injected with ARF6 morpholino have gastrulation defects and embryos injected with constitutively active ARF6 have endodermal cells detached from the gut epithelium with decreased junctional cadherin staining, indicating that ARF6 may mediate the recycling of cadherin. Thus, ARF6 impacts cells that undergo coordinated movement to form embryonic structures in the developing embryo.

  • active ARF6 recruits arno cytohesin gefs to the pm by binding their ph domains
    Molecular Biology of the Cell, 2007
    Co-Authors: Lee Ann Cohen, Akira Honda, Fraser D Brown, Peter Varnai, Tamas Balla, Julie G. Donaldson
    Abstract:

    ARNO is a soluble guanine nucleotide exchange factor (GEF) for the Arf family of GTPases. Although in biochemical assays ARNO prefers Arf1 over ARF6 as a substrate, its localization in cells at the plasma membrane (PM) suggests an interaction with ARF6. In this study, we found that ARNO activated Arf1 in HeLa and COS-7 cells resulting in the recruitment of Arf1 on to dynamic PM ruffles. By contrast, ARF6 was activated less by ARNO than EFA6, a canonical ARF6 GEF. Remarkably, ARF6 in its GTP-bound form recruited ARNO to the PM and the two proteins could be immunoprecipitated. ARNO binding to ARF6 was not mediated through the catalytic Sec7 domain, but via the pleckstrin homology (PH) domain. Active ARF6 also bound the PH domain of Grp1, another ARNO family member. This interaction was direct and required both inositol phospholipids and GTP. We propose a model of sequential Arf activation at the PM whereby ARF6-GTP recruits ARNO family GEFs for further activation of other Arf isoforms.

  • recycling of raft associated prohormone sorting receptor carboxypeptidase e requires interaction with ARF6
    Molecular Biology of the Cell, 2003
    Co-Authors: Irina Arnaoutova, Julie G. Donaldson, Catherine L Jackson, Omayma S Alawar, Peng Y Loh
    Abstract:

    Little is known about the molecular mechanism of recycling of intracellular receptors and lipid raft-associated proteins. Here, we have investigated the recycling pathway and internalization mechanism of a transmembrane, lipid raft-associated intracellular prohormone sorting receptor, carboxypeptidase E (CPE). CPE is found in the trans-Golgi network (TGN) and secretory granules of (neuro)endocrine cells. An extracellular domain of the IL2 receptor α-subunit (Tac) fused to the transmembrane domain and cytoplasmic tail of CPE (Tac-CPE 2 5 ) was used as a marker to track recycling of CPE. We show in (neuro)endocrine cells, that upon stimulated secretory granule exocytosis, raft-associated Tac-CPE 2 5 was rapidly internalized from the plasma membrane in a clathrin-independent manner into early endosomes and then transported through the endocytic recycling compartment to the TGN. A yeast two-hybrid screen and in vitro binding assay identified the CPE cytoplasmic tail sequence S 4 7 2 ETLNF 4 7 7 as an interactor with active small GTPase ADP-ribosylation factor (ARF) 6, but not ARF1. Expression of a dominant negative, inactive ARF6 mutant blocked this recycling. Mutation of residues S 4 7 2 or E 4 7 3 to A in the cytoplasmic tail of CPE obliterated its binding to ARF6, and internalization from the plasma membrane of Tac-CPE 2 5 mutated at S 4 7 2 or E 4 7 3 was significantly reduced. Thus, CPE recycles back to the TGN by a novel mechanism requiring ARF6 interaction and activity.

  • acaps are ARF6 gtpase activating proteins that function in the cell periphery
    Journal of Cell Biology, 2000
    Co-Authors: Trevor R Jackson, Julie G. Donaldson, Victor W Hsu, Fraser D Brown, Zhongzhen Nie, Koichi Miura, Letizia Foroni, Jianlan Sun, Paul A Randazzo
    Abstract:

    The GTP-binding protein ADP-ribosylation factor 6 (ARF6) regulates endosomal membrane trafficking and the actin cytoskeleton in the cell periphery. GTPase-activating proteins (GAPs) are critical regulators of Arf function, controlling the return of Arf to the inactive GDP-bound state. Here, we report the identification and characterization of two ARF6 GAPs, ACAP1 and ACAP2. Together with two previously described Arf GAPs, ASAP1 and PAP, they can be grouped into a protein family defined by several common structural motifs including coiled coil, pleckstrin homology, Arf GAP, and three complete ankyrin-repeat domains. All contain phosphoinositide-dependent GAP activity. ACAP1 and ACAP2 are widely expressed and occur together in the various cultured cell lines we examined. Similar to ASAP1, ACAP1 and ACAP2 were recruited to and, when overexpressed, inhibited the formation of platelet-derived growth factor (PDGF)–induced dorsal membrane ruffles in NIH 3T3 fibroblasts. However, in contrast with ASAP1, ACAP1 and ACAP2 functioned as ARF6 GAPs. In vitro, ACAP1 and ACAP2 preferred ARF6 as a substrate, rather than Arf1 and Arf5, more so than did ASAP1. In HeLa cells, overexpression of either ACAP blocked the formation of ARF6-dependent protrusions. In addition, ACAP1 and ACAP2 were recruited to peripheral, tubular membranes, where activation of ARF6 occurs to allow membrane recycling back to the plasma membrane. ASAP1 did not inhibit ARF6-dependent protrusions and was not recruited by ARF6 to tubular membranes. The additional effects of ASAP1 on PDGF-induced ruffling in fibroblasts suggest that multiple Arf GAPs function coordinately in the cell periphery.

  • a requirement for ARF6 in fcγ receptor mediated phagocytosis in macrophages
    Journal of Biological Chemistry, 1998
    Co-Authors: Qing Zhang, Julie G. Donaldson, Dianne Cox, Ching Chun Tseng, Steven Greenberg
    Abstract:

    Abstract Phagocytosis requires extension of F-actin-rich pseudopods and is accompanied by membrane fusion events. Members of the ARF family of GTPases are essential for many aspects of membrane trafficking. To test a role for this family of proteins in Fcγ receptor-mediated phagocytosis, we utilized the fungal metabolite brefeldin A (BFA). The addition of 100 μm BFA to a subclone of RAW 264.7 macrophages disrupted the appearance and function of the Golgi apparatus as indicated by altered immunofluorescent distribution of β-COP and reduced efflux of BODIPY C5-ceramide, a phospholipid that normally accumulates in the Golgi apparatus. In contrast, BFA had no effect on phagocytosis of IgG-coated erythrocytes. These results suggested that activation of BFA-sensitive ARFs is not required for phagocytosis. ARF6 is unique among members of the ARF family in that its membrane association is unaffected by BFA. Expression of ARF6 mutants defective in either GTP hydrolysis (Q67L) or binding (T27N) inhibited phagocytosis of IgG-coated erythrocytes and attenuated the focal accumulation of F-actin beneath the test particles. These results indicate a requirement for ARF6 in Fcγ receptor-mediated phagocytosis and suggest that ARF6 is an important mediator of cytoskeletal alterations after Fcγ receptor activation.

Joel Moss - One of the best experts on this subject based on the ideXlab platform.

  • the brefeldin a inhibited guanine nucleotide exchange protein big2 regulates the constitutive release of tnfr1 exosome like vesicles
    Journal of Biological Chemistry, 2007
    Co-Authors: Aminul Islam, Joel Moss, Martha Vaughan, Xiaoyan Shen, Toyoko Hiroi, Stewart J Levine
    Abstract:

    Abstract The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released from cells to the extracellular space where it can bind and modulate TNF bioactivity. Extracellular TNFR1 release occurs by two distinct pathways: the inducible proteolytic cleavage of TNFR1 ectodomains and the constitutive release of full-length TNFR1 in exosome-like vesicles. Regulation of both TNFR1 release pathways appears to involve the trafficking of cytoplasmic TNFR1 vesicles. Vesicular trafficking is controlled by ADP-ribosylation factors (ARFs), which are active in the GTP-bound state and inactive when bound to GDP. ARF activation is enhanced by guanine nucleotide-exchange factors that catalyze replacement of GDP by GTP. We investigated whether the brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins, BIG1 and/or BIG2, are required for TNFR1 release from human umbilical vein endothelial cells. Effects of specific RNA interference (RNAi) showed that BIG2, but not BIG1, regulated the release of TNFR1 exosome-like vesicles, whereas neither BIG2 nor BIG1 was required for the IL-1β-induced proteolytic cleavage of TNFR1 ectodomains. BIG2 co-localized with TNFR1 in diffusely distributed cytoplasmic vesicles, and the association between BIG2 and TNFR1 was disrupted by BFA. Consistent with the preferential activation of class I ARFs by BIG2, ARF1 and ARF3 participated in the extracellular release of TNFR1 exosome-like vesicles in a nonredundant and additive fashion. We conclude that the association between BIG2 and TNFR1 selectively regulates the extracellular release of TNFR1 exosome-like vesicles from human vascular endothelial cells via an ARF1- and ARF3-dependent mechanism.

  • arf gep100 a guanine nucleotide exchange protein for adp ribosylation factor 6
    Proceedings of the National Academy of Sciences of the United States of America, 2001
    Co-Authors: Akimasa Someya, Joel Moss, Makoto Sata, Kazuyo Takeda, Gustavo Pachecorodriguez, Victor J Ferrans, Martha Vaughan
    Abstract:

    A human cDNA encoding an 841-aa guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARFs), named ARF-GEP100, 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 ≈8-kb mRNA that hybridized with an ARF-GEP100 cDNA was abundant in peripheral blood leukocytes, brain, and spleen. ARF-GEP100 accelerated [35S]GTPγS binding to ARF1 (class I) and ARF5 (class II) 2- to 3-fold, and to ARF6 (class III) ca. 12-fold. The ARF-GEP100 Sec7 domain contains Asp543 and Met555, corresponding to residues associated with sensitivity to the inhibitory effect of the fungal metabolite brefeldin A (BFA) in yeast Sec7, but also Phe535 and Ala536, 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-GEP100 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-GEP100, 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-GEP100 in those areas. No similar coincidence of ARF-GEP100 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.

  • brefeldin a inhibited activity of the sec7 domain of p200 a mammalian guanine nucleotide exchange protein for adp ribosylation factors
    Journal of Biological Chemistry, 1999
    Co-Authors: Naoko Morinaga, Joel Moss, Ronald Adamik, Martha Vaughan
    Abstract:

    Abstract 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 ∼50% inhibition at ≤50 μm. The inactive BFA analogue B36 did not inhibit the Sec7 domain or p200. Thus, the Sec7 domain of p200, like that of Sec7 itself (Sata, M., Donaldson, J. G., Moss, J., and Vaughan, M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4204–4208), plays a role in BFA inhibition as well as in GEP activity, although the latter is markedly modified by other structural elements.

  • purification and cloning of a brefeldin a inhibited guanine nucleotide exchange protein for adp ribosylation factors
    Journal of Biological Chemistry, 1999
    Co-Authors: Akira Togawa, Naoko Morinaga, Masahito Ogasawara, Joel Moss, Martha Vaughan
    Abstract:

    Abstract Activation of ADP-ribosylation factors (ARFs), ∼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: ∼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 ∼50-kDa GEPs that are insensitive to BFA. We describe here two human brain cDNAs that encode BFA-inhibited GEPs. One is a ∼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 ∼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 ∼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 andBIG2 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 ∼50-kDa BFA-insensitive ARF GEPs are known, these new clones for the ∼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.

  • brefeldin a inhibited guanine nucleotide exchange activity of sec7 domain from yeast sec7 with yeast and mammalian adp ribosylation factors
    Proceedings of the National Academy of Sciences of the United States of America, 1998
    Co-Authors: Makoto Sata, Julie G. Donaldson, Joel Moss, Martha Vaughan
    Abstract:

    The Saccharomyces cerevisiae Sec7 protein (ySec7p), which is an important component of the yeast secretory pathway, contains a sequence of ≈200 amino acids referred to as a Sec7 domain. Similar Sec7 domain sequences have been recognized in several guanine nucleotide-exchange proteins (GEPs) for ADP ribosylation factors (ARFs). ARFs are ≈20-kDa GTPases that regulate intracellular vesicular membrane trafficking and activate phospholipase D. GEPs activate ARFs by catalyzing the replacement of bound GDP with GTP. We, therefore, undertook to determine whether a Sec7 domain itself could catalyze nucleotide exchange on ARF and found that it exhibited brefeldin A (BFA)-inhibitable ARF GEP activity. BFA is known to inhibit ARF GEP activity in Golgi membranes, thereby causing reversible apparent dissolution of the Golgi complex in many cells. The His6-tagged Sec7 domain from ySec7p (rySec7d) synthesized in Escherichia coli enhanced binding of guanosine 5′-[γ-[35S]thio]triphosphate by recombinant yeast ARF1 (ryARF1) and ryARF2 but not by ryARF3. The effects of rySec7d on ryARF2 were inhibited by BFA in a concentration-dependent manner but not by inactive analogues of BFA (B-17, B-27, and B-36). rySec7d also promoted BFA-sensitive guanosine 5′-[γ-thio]triphosphate binding by nonmyristoylated recombinant human ARF1 (rhARF1), rhARF5, and rhARF6, although the effect on rhARF6 was very small. These results are consistent with the conclusion that the yeast Sec7 domain itself contains the elements necessary for ARF GEP activity and its inhibition by BFA.