title: Two human ARFGAPs associated with COP-I-coated vesicles.
abstract: ADP-ribosylation factors (ARFs) are critical regulators of vesicular trafficking pathways and act at multiple intracellular sites. ADP-ribosylation factor-GTPase-activating proteins (ARFGAPs) are proposed to contribute to site-specific regulation. In yeast, two distinct proteins, Glo3p and Gcs1p, together provide overlapping, essential ARFGAP function required for coat protein (COP)-I-dependent trafficking. In mammalian cells, only the Gcs1p orthologue, named ARFGAP1, has been characterized in detail. However, Glo3p is known to make the stronger contribution to COP I traffic in yeast. Here, based on a conserved signature motif close to the carboxy terminus, we identify ARFGAP2 and ARFGAP3 as the human orthologues of yeast Glo3p. By immunofluorescence (IF), ARFGAP2 and ARFGAP3 are closely colocalized with coatomer subunits in NRK cells in the Golgi complex and peripheral punctate structures. In contrast to ARFGAP1, both ARFGAP2 and ARFGAP3 are associated with COP-I-coated vesicles generated from Golgi membranes in the presence of GTP-gamma-S in vitro. ARFGAP2 lacking its zinc finger domain directly binds to coatomer. Expression of this truncated mutant (DeltaN-ARFGAP2) inhibits COP-I-dependent Golgi-to-endoplasmic reticulum transport of cholera toxin (CTX-K63) in vivo. Silencing of ARFGAP1 or a combination of ARFGAP2 and ARFGAP3 in HeLa cells does not decrease cell viability. However, silencing all three ARFGAPs causes cell death. Our data provide strong evidence that ARFGAP2 and ARFGAP3 function in COP I traffic.
authors.affiliation: Department of Clinical Biochemistry, Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge CB2 2XY, United Kingdom.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Frigerio
authors.forename: Neil
authors.initials: N
authors.lastname: Grimsey
authors.forename: Martin
authors.initials: M
authors.lastname: Dale
authors.forename: Irina
authors.initials: I
authors.lastname: Majoul
authors.forename: Rainer
authors.initials: R
authors.lastname: Duden
mesh_terms: D020727:ADP-Ribosylation Factors / Q000737:chemistry / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D022181:COP-Coated Vesicles / Q000378:metabolism*; D002522:Chlorocebus aethiops; D020755:Coat Protein Complex I / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D014709:Vero Cells
chemical_list: C503240:ARFGAP3 protein, human; D020755:Coat Protein Complex I; D020690:GTPase-Activating Proteins; C514182:Glo3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D020727:ADP-Ribosylation Factors; C525860:ARFGAP2 protein, human