Golgi localization determinants in ArfGAP1 and in new tissue-specific ArfGAP1 isoforms.
The Arf1-directed GTPase-activating protein ArfGAP1 is a Golgi-localized protein that controls the dynamics of the COPI coat of carriers that mediate transport in the endoplasmic reticulum-Golgi shuttle. Previously the interaction of ArfGAP1 with the Golgi was allocated to a portion of the non-catalytic, carboxyl part of the protein, but the mechanism of this interaction has not been established. In this study we identify a short stretch in the non-catalytic part of ArfGAP1 (residues 204-214) in which several hydrophobic residues contribute to Golgi localization. Even single alanine replacement of two of these residues (Leu-207 and Trp-211) strongly diminished Golgi localization. Mutations in the hydrophobic residues also diminished the in vitro activity of ArfGAP1 on Arf1 bound to Golgi membranes. The stretch containing the hydrophobic residues was recently shown to mediate the binding of ArfGAP1 to loosely packed lipids of highly curved liposomes (Bigay, J., Casella, J. F., Drin, G., Mesmin, B., and Antonny, B. (2005) EMBO J. 24, 2244-2253). Whereas short fragments containing the hydrophobic stretch were not Golgi-localized, a proximal 10-residue in-frame insertion that is present in new ArfGAP1 isoforms that we identified in brain and heart tissues could confer Golgi localization on these fragments. This localization was abrogated by alanine replacement of residues Phe-240 or Trp-241 of the insertion sequence but not by their replacement with leucines. Our findings indicate that ArfGAP1 interacts with the Golgi through multiple hydrophobic motifs and that alternative modes of interaction may exist in tissue-specific ArfGAP1 isoforms.
Parnis
Anna
A
Department of Biology, Technion-Israel Institute of Technology, Haifa.
Rawet
Moran
M
Regev
Lior
L
Barkan
Batya
B
Rotman
Miriam
M
Gaitner
Michal
M
Cassel
Dan
D
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017124:Conserved Sequence; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000502:physiology; D006056:Golgi Apparatus / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009928:Organ Specificity; D020033:Protein Isoforms; D013329:Structure-Activity Relationship
C503237:ARFGAP1 protein, human; D020690:GTPase-Activating Proteins; D020033:Protein Isoforms