The Arf1 GTPase-activating protein ArfGAP1 regulates vesicular traffic through the COPI system This protein consists of N-terminal catalytic domain, lipid packing sensors (the ALPS motifs) in the central region, and a carboxy part of unknown function The carboxy part contains several diaromatic sequences that are reminiscent of motifs known to interact with clathrin adaptors In pull-down experiments using GST-fused peptides from rat ArfGAP1, a peptide containing a (WETF)-W-329 sequence interacted strongly with clathrin adaptors AP1 and AP2, whereas a major coatomer-binding determinant was identified within the extreme carboxy terminal peptide ((405)AADEGWDNQNW) Mutagenesis and peptide competition experiments revealed that this determinant is required for coatomer binding to full-length ArfGAP1. and that interaction is mediated through the delta-subunit of the coatomer adaptor-like subcomplex Evidence for a role of the carboxy motif in ArfGAP1-coatomer interaction in vivo is provided by means of a reporter fusion assay. Our findings point to mechanistic differences between ArfGAP1 and the other ArfGAPs known to function in the COPI system (C) 2010 Elsevier Inc All rights reserved