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

A site-directed photocross-linking approach was employed to determine compo nents that act downstream of ADP-ribosylation factor (ARF). To this end, a photolabile phenylalanine analog was incorporated at various positions of t he putative effector region of the ARF molecule. Depending on the position of incorporation, we find specific and GTP-dependent interactions of ARF wi th two subunits of the coatomer complex, beta-COP and gamma-COP, as well as an interaction with a cytosolic protein (similar to 185 kDa). In addition, we observe homodimer formation of ARF molecules at the Golgi membrane. The se data suggest that the binding site of ARF to coatomer is at the interfac e of its beta- and gamma-subunits, and this is in close proximity to the se cond site of interaction of coatomer with the Golgi membrane, the binding s ite within gamma-COP for cytosolic dibasic/diphenylalanine motifs.