A structure-based mechanism for Arf1-dependent recruitment of coatomer to membranes.

Budding of COPI-coated vesicles from Golgi membranes requires an Arf family G protein and the coatomer complex recruited from cytosol. Arf is also required with coatomer-related clathrin adaptor complexes to bud vesicles from the trans-Golgi network and endosomal compartments. To understand the structural basis for Arf-dependent recruitment of a vesicular coat to the membrane, we determined the structure of Arf1 bound to the γζ-COP subcomplex of coatomer. Structure-guided biochemical analysis reveals that a second Arf1-GTP molecule binds to βδ-COP at a site common to the &gamma.- and β-COP subunits. The Arf1-binding sites on coatomer are spatially related to PtdIns4,5P(2)-binding sites on the endocytic AP2 complex, providing evidence that the orientation of membrane binding is general for this class of vesicular coat proteins. A bivalent GTP-dependent binding mode has implications for the dynamics of coatomer interaction with the Golgi and for the selection of cargo molecules.


Identification of Arf1-binding sites on βδ/γζ-COP

(A) GTP-dependent binding of Arf1 to γζ-COP dimers. The pull-down assay was utilized to test the Arf1-binding capacity of truncated forms of γ-COP. In the experiments, γ-COP was present in a dimer with full-length ζ-COP (lanes 1.6) or truncated (1.153) ζ-COP (lanes 7.14). All of the dimeric proteins, from the longest to the shortest forms (left to right), bind to Arf1.

(B) GTP-dependent binding of Arf1 to β&delta&-COP dimers. Arf1 binds to full-length βδ-COP (lane 2) and to a βδ-COP dimer lacking the β-COP appendage domain (lane 4).

(C) Various forms of γζ-COP, including the minimal γζ-COP dimer (γ-COP 1.355 and ζ-COP 1.153) identified in (A), are fully active in the fluorescence assay.that is, all synergize with GAP to catalyze GTP hydrolysis on Arf1. For clarity, the curves have been offset incrementally along the x-axis.

(D) The βδ-COP dimer can also synergize with GAP to catalyze GTP hydrolysis on Arf1. Curves have been offset incrementally along the x-axis.

Results from:
Deperalta G, Alvarez M, Bechtel C, Dong K, McDonald R, Ling V.
MAbs. 2013 Jan 1;5(1):86-101. doi: 10.4161/mabs.22964. Epub 2012 Dec 17.
PMID: 23247543 [PubMed - in process]
PMCID: PMC3564890 [Available on 2014/1/1]