The yeast ADP-ribosylation factor GAP, Gcs1p, is involved in maintenance of mitochondrial morphology

2002 ◽  
Vol 115 (2) ◽  
pp. 275-282 ◽  
Author(s):  
Chun-Fang Huang ◽  
Chien-Cheng Chen ◽  
Luh Tung ◽  
Leh-Miauh Buu ◽  
Fang-Jen S. Lee
Keyword(s):  
Membrane Trafficking ◽  
Minimal Medium ◽  
Retrograde Transport ◽  
Carboxypeptidase Y ◽  
Mitochondrial Morphology ◽  
Ph Domain ◽  
Rich Medium ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

Membrane trafficking is regulated, in part, by small GTP-binding proteins of the ADP-ribosylation factor (ARF) family. ARF function depends on the controlled binding and hydrolysis of GTP. In vitro, the GTPase activity of yeast ARF proteins can be stimulated by Gcs1p. Although Gcs1p was implicated in the regulation of retrograde transport from the Golgi to the ER and in actin cytoskeletal organization, its intracellular functions and distribution remain to be established. Following subcellular fractionation of yeast grown in rich medium, Gcs1p was localized in denser fractions than it was in cells grown in minimal medium. In yeast grown in rich or minimal medium, Gcs1p was distributed over the cytoplasm in a fine punctate pattern with more concentrated staining in the perinuclear regions. Overexpressed Gcs1p in yeast was localized partially with mitochondria and partially in perinuclear structures close to mitochondria. The Gcs1p PH-domain was required for localization in mitochondria but not for the perinuclear region. Transport of carboxypeptidase Y and invertase was not significantly altered by disruption of the gcs1 gene. This mutation did, however, reduce mitochondrial lateral distribution and branching when yeast were grown in rich medium. In yeast overexpressing Gcs1p, mitochondrial morphology was aberrant, with unbranched tubules and large spherical structures. We suggest that Gcs1p may be involved in the maintenance of mitochondrial morphology, possibly through organizing the actin cytoskeleton in Saccharomyces.

scholarly journals ADP-Ribosylation Factor (ARF) Interaction Is Not Sufficient for Yeast GGA Protein Function or Localization

2002 ◽  
Vol 13 (9) ◽  
pp. 3078-3095 ◽  
Author(s):  
Annette L. Boman ◽  
Paul D. Salo ◽  
Melissa J. Hauglund ◽  
Nicole L. Strand ◽  
Shelly J. Rensink ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Carboxypeptidase Y ◽  
Minor Role ◽  
Temperature Sensitive ◽  
Adp Ribosylation ◽  
And Function ◽  
Adp Ribosylation Factor

Golgi-localized γ-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only ∼25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs andVPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function.

scholarly journals ADP-Ribosylation Factor 1 (ARF1) Regulates Recruitment of the AP-3 Adaptor Complex to Membranes

1998 ◽  
Vol 142 (2) ◽  
pp. 391-402 ◽  
Author(s):  
Chean Eng Ooi ◽  
Esteban C. Dell'Angelica ◽  
Juan S. Bonifacino
Keyword(s):  
Membrane Trafficking ◽  
Brefeldin A ◽  
Membrane Association ◽  
Coat Proteins ◽  
Nucleotide Exchange ◽  
Membrane Recruitment ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

Small GTP-binding proteins such as ADP- ribosylation factor 1 (ARF1) and Sar1p regulate the membrane association of coat proteins involved in intracellular membrane trafficking. ARF1 controls the clathrin coat adaptor AP-1 and the nonclathrin coat COPI, whereas Sar1p controls the nonclathrin coat COPII. In this study, we demonstrate that membrane association of the recently described AP-3 adaptor is regulated by ARF1. Association of AP-3 with membranes in vitro was enhanced by GTPγS and inhibited by brefeldin A (BFA), an inhibitor of ARF1 guanine nucleotide exchange. In addition, recombinant myristoylated ARF1 promoted association of AP-3 with membranes. The role of ARF1 in vivo was examined by assessing AP-3 subcellular localization when the intracellular level of ARF1-GTP was altered through overexpression of dominant ARF1 mutants or ARF1- GTPase-activating protein (GAP). Lowering ARF1-GTP levels resulted in redistribution of AP-3 from punctate membrane-bound structures to the cytosol as seen by immunofluorescence microscopy. In contrast, increasing ARF1-GTP levels prevented redistribution of AP-3 to the cytosol induced by BFA or energy depletion. Similar experiments with mutants of ARF5 and ARF6 showed that these other ARF family members had little or no effect on AP-3. Taken together, our results indicate that membrane recruitment of AP-3 is promoted by ARF1-GTP. This finding suggests that ARF1 is not a regulator of specific coat proteins, but rather is a ubiquitous molecular switch that acts as a transducer of diverse signals influencing coat assembly.

scholarly journals Remodeling of the Actin Cytoskeleton Is Coordinately Regulated by Protein Kinase C and the ADP-Ribosylation Factor Nucleotide Exchange Factor ARNO

1998 ◽  
Vol 9 (11) ◽  
pp. 3133-3146 ◽  
Author(s):  
Scott R. Frank ◽  
Jessica C. Hatfield ◽  
James E. Casanova
Keyword(s):  
Actin Cytoskeleton ◽  
Catalytic Domain ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
Surface Receptors ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor ◽  
Actin Rearrangement

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

scholarly journals ASAP1, a Phospholipid-Dependent Arf GTPase-Activating Protein That Associates with and Is Phosphorylated by Src

1998 ◽  
Vol 18 (12) ◽  
pp. 7038-7051 ◽  
Author(s):  
Megan T. Brown ◽  
Josefa Andrade ◽  
Harish Radhakrishna ◽  
Julie G. Donaldson ◽  
Jonathan A. Cooper ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Membrane Trafficking ◽  
Tyrosine Kinases ◽  
Sh3 Domain ◽  
Ph Domain ◽  
Gtpase Activating Protein ◽  
Binding Motifs ◽  
Hydrolysis Of ◽  
Adp Ribosylation Factor

ABSTRACT 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.

scholarly journals Poliovirus Proteins Induce Membrane Association of GTPase ADP-Ribosylation Factor

2005 ◽  
Vol 79 (11) ◽  
pp. 7207-7216 ◽  
Author(s):  
George A. Belov ◽  
Mark H. Fogg ◽  
Ellie Ehrenfeld
Keyword(s):  
Membrane Trafficking ◽  
Fluorescent Protein ◽  
Rna Replication ◽  
Small Gtpases ◽  
Coat Proteins ◽  
Protein Fusion ◽  
Adp Ribosylation ◽  
Cell Extracts ◽  
Adp Ribosylation Factor

ABSTRACT Poliovirus infection results in the disintegration of intracellular membrane structures and formation of specific vesicles that serve as sites for replication of viral RNA. The mechanism of membrane rearrangement has not been clearly defined. Replication of poliovirus is sensitive to brefeldin A (BFA), a fungal metabolite known to prevent normal function of the ADP-ribosylation factor (ARF) family of small GTPases. During normal membrane trafficking in uninfected cells, ARFs are involved in vesicle formation from different intracellular sites through interaction with numerous regulatory and coat proteins as well as in regulation of phospholipase D activity and cytoskeleton modifications. We demonstrate here that ARFs 3 and 5, but not ARF6, are translocated to membranes in HeLa cell extracts that are engaged in translation of poliovirus RNA. The accumulation of ARFs on membranes correlates with active replication of poliovirus RNA in vitro, whereas ARF translocation to membranes does not occur in the presence of BFA. ARF translocation can be induced independently by synthesis of poliovirus 3A or 3CD proteins, and we describe mutations that abolished this activity. In infected HeLa cells, an ARF1-enhanced green fluorescent protein fusion redistributes from Golgi stacks to the perinuclear region, where poliovirus RNA replication occurs. Taken together, the data suggest an involvement of ARF in poliovirus RNA replication.

scholarly journals Involvement of a novel ADP-ribosylation factor GTPase-activating protein, SMAP, in membrane trafficking: Implications in cancer cell biology

2006 ◽  
Vol 97 (9) ◽  
pp. 801-806 ◽  
Author(s):  
Kenji Tanabe ◽  
Shunsuke Kon ◽  
Waka Natsume ◽  
Tetsuo Torii ◽  
Toshio Watanabe ◽  
...  
Keyword(s):  
Cancer Cell ◽  
Membrane Trafficking ◽  
Cell Biology ◽  
Gtpase Activating Protein ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

scholarly journals Recombinant protein of Haemonchus contortus small GTPase ADP-ribosylation factor 1 (HcARF1) modulate the cell mediated immune response in vitro

Oncotarget ◽  
2017 ◽  
Vol 8 (68) ◽  
pp. 112211-112221 ◽  
Author(s):  
Javaid Ali Gadahi ◽  
Muhammad Ehsan ◽  
Shuai Wang ◽  
Zhenchao Zhang ◽  
Ruofeng Yan ◽  
...  
Keyword(s):  
Immune Response ◽  
Recombinant Protein ◽  
Haemonchus Contortus ◽  
Small Gtpase ◽  
Adp Ribosylation ◽  
Cell Mediated Immune Response ◽  
Adp Ribosylation Factor ◽  
Immune Response In Vitro

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

1990 ◽  
Vol 10 (12) ◽  
pp. 6690-6699
Author(s):  
T Stearns ◽  
R A Kahn ◽  
D Botstein ◽  
M A Hoyt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cold Sensitivity ◽  
Animal Cells ◽  
Gene Encoding ◽  
Phenotypic Differences ◽  
Adp Ribosylation ◽  
Protein Factor ◽  
Adp Ribosylation Factor ◽  
Sublethal Concentrations

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

scholarly journals Dual role for phosphoinositides in regulation of yeast and mammalian phospholipase D enzymes

2002 ◽  
Vol 159 (6) ◽  
pp. 1039-1049 ◽  
Author(s):  
Vicki A. Sciorra ◽  
Simon A. Rudge ◽  
Jiyao Wang ◽  
Stuart McLaughlin ◽  
JoAnne Engebrecht ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Membrane Trafficking ◽  
Dual Role ◽  
Biological Functions ◽  
Ph Domain ◽  
Catalytically Active ◽  
Acidic Phospholipids ◽  
Stimulation Of

Phospholipase D (PLD) generates lipid signals that coordinate membrane trafficking with cellular signaling. PLD activity in vitro and in vivo is dependent on phosphoinositides with a vicinal 4,5-phosphate pair. Yeast and mammalian PLDs contain an NH2-terminal pleckstrin homology (PH) domain that has been speculated to specify both subcellular localization and regulation of PLD activity through interaction with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2). We report that mutation of the PH domains of yeast and mammalian PLD enzymes generates catalytically active PI(4,5)P2-regulated enzymes with impaired biological functions. Disruption of the PH domain of mammalian PLD2 results in relocalization of the protein from the PI(4,5)P2-containing plasma membrane to endosomes. As a result of this mislocalization, mutations within the PH domain render the protein unresponsive to activation in vivo. Furthermore, the integrity of the PH domain is vital for yeast PLD function in both meiosis and secretion. Binding of PLD2 to model membranes is enhanced by acidic phospholipids. Studies with PLD2-derived peptides suggest that this binding involves a previously identified polybasic motif that mediates activation of the enzyme by PI(4,5)P2. By comparison, the PLD2 PH domain binds PI(4,5)P2 with lower affinity but sufficient selectivity to function in concert with the polybasic motif to target the protein to PI(4,5)P2-rich membranes. Phosphoinositides therefore have a dual role in PLD regulation: membrane targeting mediated by the PH domain and stimulation of catalysis mediated by the polybasic motif.

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