Abstract
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.
Highlights
Process of coat formation by the direct binding of coat subunits (4 – 6)
ArfGAP1 was originally identified as a protein that cycles between cytosol and the Golgi complex [11], and a subsequent study revealed the presence of a Golgi localization determinant in a fragment of the noncatalytic part [28]
We have identified a small determinant between residues 204 –214 in which several hydrophobic residues contribute to the Golgi localization of non-catalytic fragments as well as of the full-length ArfGAP1
Summary
Process of coat formation by the direct binding of coat subunits (4 – 6). The coat in turn recruits cargo and polymerizes causing membrane deformation to form a bud. The positive curvature in the central region of the bud would cause ArfGAP1 recruitment and facilitate GTP hydrolysis on Arf, whose presence may not be required following the polymerization of the coat, whereas the negative curvature in the bud rims would exclude ArfGAP1 from this region, allowing Arf11⁄7GTP-dependent recruitment of new coat subunits [23, 25, 26]. We report the identification of a short stretch within the noncatalytic part of ArfGAP1 containing hydrophobic residues that are necessary for Golgi localization These residues closely match those recently reported to constitute a lipid-binding motif in ArfGAP1 [24], suggesting a role of lipid binding in ArfGAP1 targeting. We describe new tissue-specific ArfGAP1 isoforms that contain a small in-frame insertion and deletion in proximity to the lipid-binding motif and provide evidence that these insertion and deletion regions function as Golgi localization determinants
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