Interactions of Arf1 with coatomer probed by site-directed photo–cross-linking. Arf1 variants with a photolabile amino acid residue in either position 49 or 167 were prepared as described in Materials and methods and used for coatomer recruitment to Golgi membranes. Membranes were separated by centrifugation and UV irradiated followed by SDS-PAGE and Western blotting. (A) Analysis of photo–cross-link products with the photolabile amino acid derivative in position 49. Lanes 1 and 2 show Cys-wt and Cys-Y35A, respectively, decorated with anti-Arf1 antibodies. Lanes 3 and 4 show samples as in lanes 1 and 2 decorated with anti–γ-COP and, as a control, with anti–α-COP antibodies. (B) Analysis of photo–cross-link products with the photolabile amino acid derivative in position 167. Lanes 1 and 2 are decorated with anti-Arf1 antibodies, and lanes 3 and 4 are decorated with anti–δ-COP and anti–α-COP antibodies. For details of preparation of site-directed photolabile Arf1 derivatives see Materials and methods. Molecular masses are given in kilodaltons.