scholarly journals ADP Ribosylation Factor 1 Is Required for Synaptic Vesicle Budding in PC12 Cells

1997 ◽  
Vol 138 (3) ◽  
pp. 505-515 ◽  
Author(s):  
Victor Faúndez ◽  
Jim-Tong Horng ◽  
Regis B. Kelly
Keyword(s):  
Pc12 Cell ◽  
Cell Membranes ◽  
Brefeldin A ◽  
T Antigen ◽  
Gtpase Activity ◽  
Vesicle Budding ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

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

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 Adp Ribosylation Factor-like Protein 2 (Arl2) Regulates the Interaction of Tubulin-Folding Cofactor D with Native Tubulin

2000 ◽  
Vol 149 (5) ◽  
pp. 1087-1096 ◽  
Author(s):  
Arunashree Bhamidipati ◽  
Sally A. Lewis ◽  
Nicholas J. Cowan
Keyword(s):  
Cultured Cells ◽  
Gtpase Activating Protein ◽  
Adp Ribosylation ◽  
Chaperone Protein ◽  
Ras Family ◽  
Mutant Forms ◽  
Adp Ribosylation Factor ◽  
Β Tubulin

The ADP ribosylation factor-like proteins (Arls) are a family of small monomeric G proteins of unknown function. Here, we show that Arl2 interacts with the tubulin-specific chaperone protein known as cofactor D. Cofactors C, D, and E assemble the α/β- tubulin heterodimer and also interact with native tubulin, stimulating it to hydrolyze GTP and thus acting together as a β-tubulin GTPase activating protein (GAP). We find that Arl2 downregulates the tubulin GAP activity of C, D, and E, and inhibits the binding of D to native tubulin in vitro. We also find that overexpression of cofactors D or E in cultured cells results in the destruction of the tubulin heterodimer and of microtubules. Arl2 specifically prevents destruction of tubulin and microtubules by cofactor D, but not by cofactor E. We generated mutant forms of Arl2 based on the known properties of classical Ras-family mutations. Experiments using these altered forms of Arl2 in vitro and in vivo demonstrate that it is GDP-bound Arl2 that interacts with cofactor D, thereby averting tubulin and microtubule destruction. These data establish a role for Arl2 in modulating the interaction of tubulin-folding cofactors with native tubulin in vivo.

Exocytotic Stimulation Promotes Association of the ADP-Ribosylation Factor with PC12 Cell Membranes

1998 ◽  
Vol 354 (1) ◽  
pp. 144-150 ◽  
Author(s):  
Toshihiko Murayama ◽  
Tomoyoshi Naganuma ◽  
Haruko Oda ◽  
Yasuyuki Nomura
Keyword(s):  
Pc12 Cell ◽  
Cell Membranes ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

scholarly journals ADP-ribosylation factor and coatomer couple fusion to vesicle budding

1994 ◽  
Vol 124 (4) ◽  
pp. 415-424 ◽  
Author(s):  
Z Elazar ◽  
L Orci ◽  
J Ostermann ◽  
M Amherdt ◽  
G Tanigawa ◽  
...  
Keyword(s):  
Brefeldin A ◽  
Retrograde Transport ◽  
Coated Vesicles ◽  
Coat Proteins ◽  
Vesicle Budding ◽  
Adp Ribosylation ◽  
Golgi Membranes ◽  
Transport Vesicles ◽  
Donor And Acceptor ◽  
Adp Ribosylation Factor

The coat proteins required for budding COP-coated vesicles from Golgi membranes, coatomer and ADP-ribosylation factor (ARF) protein, are shown to be required to reconstitute the orderly process of transport between Golgi cisternae in which fusion of transport vesicles begins only after budding ends. When either coat protein is omitted, fusion is uncoupled from budding-donor and acceptor compartments pair directly without an intervening vesicle. Coupling may therefore results from the sequestration of fusogenic membrane proteins into assembling coated vesicles that are only exposed when the coat is removed after budding is complete. This mechanism of coupling explains the phenomenon of "retrograde transport" triggered by uncouplers such as the drug brefeldin A.

scholarly journals The Cholera Toxin A13 Subdomain Is Essential for Interaction with ADP-Ribosylation Factor 6 and Full Toxic Activity but Is Not Required for Translocation from the Endoplasmic Reticulum to the Cytosol

2006 ◽  
Vol 74 (4) ◽  
pp. 2259-2267 ◽  
Author(s):  
Ken Teter ◽  
Michael G. Jobling ◽  
Danielle Sentz ◽  
Randall K. Holmes
Keyword(s):  
Endoplasmic Reticulum ◽  
Cholera Toxin ◽  
Toxic Activity ◽  
Hydrophobic Domain ◽  
Vesicular Traffic ◽  
Adp Ribosylation ◽  
Quality Control Mechanism ◽  
Adp Ribosylation Factor

ABSTRACT Cholera toxin (CT) moves from the plasma membrane to the endoplasmic reticulum (ER) by retrograde vesicular traffic. In the ER, the catalytic CTA1 polypeptide dissociates from the rest of the toxin and enters the cytosol by a process that involves the quality control mechanism of ER-associated degradation (ERAD). The cytosolic CTA1 then ADP ribosylates Gsα, resulting in adenylate cyclase activation and intoxication of the target cell. It is hypothesized that the C-terminal A13 subdomain of CTA1 plays two crucial roles in the intoxication process: (i) it contains a hydrophobic domain that triggers the ERAD mechanism and (ii) it facilitates interaction with the cytosolic ADP-ribosylation factors (ARFs) that serve as allosteric activators of CTA1. In this study, we examined the role(s) of the CTA13 subdomain in CT intoxication. Full-length CTA1 constructs and truncated CTA1 constructs lacking the A13 subdomain were generated and used to conduct two-hybrid studies of interactions with ARF6, in vitro enzyme assays, in vivo toxicity assays, and in vivo processing/degradation assays. Direct, plasmid-mediated expression of CTA1 constructs in the ER or cytosol of transfected CHO cells was used to perform the in vivo assays. With these methods, we found that the A13 subdomain of CTA1 is important both for interaction with ARF6 and for full expression of enzyme activity in vivo. Surprisingly, however, the A13 subdomain was not required for ERAD-mediated passage of CTA1 from the ER to the cytosol. A possible alternative trigger for CTA1 to activate the ERAD mechanism is discussed.

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 Identification of centaurin-α1 as a potential in vivo phosphatidylinositol 3,4,5-trisphosphate-binding protein that is functionally homologous to the yeast ADP-ribosylation factor (ARF) GTPase-activating protein, Gcs1

1999 ◽  
Vol 340 (2) ◽  
pp. 359-363 ◽  
Author(s):  
Kanamarlapudi VENKATESWARLU ◽  
Paru B. OATEY ◽  
Jeremy M. TAVARÉ ◽  
Trevor R. JACKSON ◽  
Peter J. CULLEN
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Dominant Negative ◽  
Gtpase Activating Protein ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

Centaurin-α 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-α1, a human homologue of centaurin-α, binds PtdIns(3,4,5)P3in vivo and furthermore, identified a potential physiological function for centaurin-α1. Using confocal microscopy of live PC12 cells, transiently transfected with a chimera of green fluorescent protein (GFP) fused to the N-terminus of centaurin-α1 (GFP-centaurin-α1), we demonstrated the rapid plasma membrane recruitment of cytosolic GFP-centaurin-α1 following stimulation with either nerve growth factor or epidermal growth factor. This recruitment was dependent on the centaurin-α1 pleckstrin homology domains and was blocked by the PtdIns(4,5)P2 3-kinase (PI 3-kinase) inhibitors wortmannin (100 nM) and LY294002 (50 μM), and also by co-expression with a dominant negative p85. Functionally, we demonstrated that centaurin-α1 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-α1. Taken together, our data demonstrated that centaurin-α1 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.

scholarly journals ADP-ribosylation Factor 1-independent Protein Sorting and Export from thetrans-Golgi Network

2004 ◽  
Vol 279 (50) ◽  
pp. 52735-52743 ◽  
Author(s):  
Mark A. Ellis ◽  
Mark T. Miedel ◽  
Christopher J. Guerriero ◽  
Ora A. Weisz
Keyword(s):  
Small Gtpase ◽  
Proton Channel ◽  
Coat Proteins ◽  
Adp Ribosylation ◽  
Influenza Hemagglutinin ◽  
Intact Membrane ◽  
Golgi Network ◽  
Adp Ribosylation Factor

Polarized epithelial cells efficiently sort newly synthesized apical and basolateral proteins into distinct transport carriers that emerge from thetrans-Golgi network (TGN), and this sorting is recapitulated in nonpolarized cells. While the targeting signals of basolaterally destined proteins are generally cytoplasmically disposed, apical sorting signals are not typically accessible to the cytosol, and the transport machinery required for segregation and export of apical cargo remains largely unknown. Here we investigated the molecular requirements for TGN export of the apical marker influenza hemagglutinin (HA) in HeLa cells using anin vitroreconstitution assay. HA was released from the TGN in intact membrane-bound compartments, and export was dependent on addition of an ATP-regenerating system and exogenous cytosol. HA release was inhibited by guanosine 5′-O-(3-thiotriphosphate) (GTPγS) as well as under conditions known to negatively regulate apical transportin vivo, including expression of the acid-activated proton channel influenza M2. Interestingly, release of HA was unaffected by depletion of ADP-ribosylation factor 1, a small GTPase that has been implicated in the recruitment of all known adaptors and coat proteins to the Golgi complex. Furthermore, regulation of HA release by GTPγS or M2 expression was unaffected by cytosolic depletion of ADP-ribosylation factor 1, suggesting that HA sorting remains functionally intact in the absence of the small GTPase. These data suggest that TGN sorting and export of influenza HA does not require classical adaptors involved in the formation of other classes of exocytic carriers and thus appears to proceed via a novel mechanism.

scholarly journals N-terminal autoprocessing and acetylation of MARTX Makes-Caterpillars Floppy-like effector is stimulated by ADP-Ribosylation Factor 1 in advance of Golgi fragmentation

2019 ◽  
Author(s):  
Alfa Herrera ◽  
John Muroski ◽  
Ranjan Sengupta ◽  
Hong Hanh Nguyen ◽  
Shivangi Agarwal ◽  
...  
Keyword(s):  
Vibrio Vulnificus ◽  
Fluorescent Microscopy ◽  
Adp Ribosylation ◽  
Cellular Effects ◽  
Section Electron ◽  
Host Cell Factors ◽  
Golgi Fragmentation ◽  
Adp Ribosylation Factor

ABSTRACTStudies have successfully elucidated the mechanism of action of several effector domains that comprise the MARTX toxins of Vibrio vulnificus. However, the biochemical linkage between the cysteine proteolytic activity of Makes Caterpillars Floppy-like (MCF) effector and its cellular effects remains unknown. In this study, we identify the host cell factors that activate in vivo and in vitro MCF autoprocessing as ADP-Ribosylation Factor 1 (ARF1) and ADP-ribosylation Factor 3 (ARF3). Autoprocessing activity is enhanced when ARF1 is in its active (GTP-bound) form compared to the inactive (GDP-bound) form. Subsequent to auto-cleavage, MCF is acetylated on its exposed N-terminal glycine residue. Acetylation apparently does not dictate subcellular localization, as MCF is found localized throughout the cell. However, the cleaved form of MCF gains the ability to bind to the specialized lipid phosphatidylinositol 5-phosphate enriched in Golgi and other membranes necessary for endocytic trafficking, suggesting a fraction of MCF may be subcellular localized. Traditional thin-section electron microscopy, high-resolution cryoAPEX localization, and fluorescent microscopy show that MCF causes Golgi dispersal resulting in extensive vesiculation. In addition, host mitochondria are disrupted and fragmented. Surprisingly, ARF1 is not itself processed or post-translationally modified by MCF. Further, only catalytically inactive MCF stably associates with ARF1, thus serving as a substrate trap. Our data indicate that ARF1 is a cross-kingdom activator of MCF, but reveal that MCF mediates cytotoxicity likely by directly targeting another yet to be identified protein. This study begins to elucidate the biochemical activity of this important domain and gives insight into how it may promote disease progression.

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

1987 ◽  
Vol 26 (01) ◽  
pp. 1-6 ◽  
Author(s):  
S. Selvaraj ◽  
M. R. Suresh ◽  
G. McLean ◽  
D. Willans ◽  
C. Turner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
T Antigen ◽  
Human Carcinomas ◽  
Animal Tumor ◽  
Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

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