GTP-dependent binding of ADP-ribosylation factor to coatomer in close proximity to the binding site for dilysine retrieval motifs and p23

Abstract

A site-directed photocross-linking approach was employed to determine components that act downstream of ADP-ribosylation factor (ARF). To this end, a photolabile phenylalanine analog was incorporated at various positions of the putative effector region of the ARF molecule. Depending on the position of incorporation, we find specific and GTP-dependent interactions of ARF with two subunits of the coatomer complex, β-COP and γ-COP, as well as an interaction with a cytosolic protein (~185 kDa). In addition, we observe homodimer formation of ARF molecules at the Golgi membrane. These data suggest that the binding site of ARF to coatomer is at the interface of its β- and γ-subunits, and this is in close proximity to the second site of interaction of coatomer with the Golgi membrane, the binding site within γ- COP for cytosolic dibasic/diphenylalanine motifs.