title: Topology of amphipathic motifs mediating Golgi localization in ArfGAP1 and its splice isoforms.
abstract: The interaction of the Arf1-directed GTPase-activating protein ArfGAP1 with the Golgi apparatus depends on motifs in its noncatalytic part that are unstructured in solution but are capable of folding into amphipathic helices in vitro upon interaction with poorly packed lipids. In previous studies a few hydrophobic residues that are critical for lipid binding and Golgi localization were identified, but the precise topology of the amphipathic motifs has not been determined. Here we present a detailed analysis of the Golgi targeting and in vitro folding features of the region encompassing the amphipathic motifs (residues 199-294). Point mutation analysis revealed that most hydrophobic residues within this region contribute to Golgi localization, whereas analysis by proline replacements and alanine insertions revealed that Golgi interaction depends on folding into two amphipathic helices with a short interrupting sequence. Analysis of splice isoforms containing 10-residue in-frame insertions within their first amphipathic motifs revealed that the insertion causes a truncation of the amphipathic helix that does not extend beyond the insertion sequence. Lastly, a lysine replacement mutant recently reported to bind to negatively charged liposomes in a curvature-independent manner showed normal cellular distribution, suggesting that Golgi targeting of Arf-GAP1 may involve factors other than sensing lipid packing.
authors.affiliation: Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Sharon
authors.initials: S
authors.lastname: Levi
authors.forename: Moran
authors.initials: M
authors.lastname: Rawet
authors.forename: Lena
authors.initials: L
authors.lastname: Kliouchnikov
authors.forename: Anna
authors.initials: A
authors.lastname: Parnis
authors.forename: Dan
authors.initials: D
authors.lastname: Cassel
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003600:Cytosol / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008081:Liposomes; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D055672:Static Electricity
chemical_list: D020690:GTPase-Activating Proteins; D008081:Liposomes; D020033:Protein Isoforms