Site-specific photocrosslinking to probe interactions of Arf1 with proteins involved in budding of COPI vesicles

ADP-ribosylation factor 1 (Arf1) plays an important role in early and intra -Golgi protein trafficking During this process, Arf1 interacts with many di fferent proteins and other molecules that regulate its state of activation or are involved in its intracellular function. To determine which of these proteins interact directly with Arf1 during coat protein type I (COPI) vesi cle biogenesis, we probed the molecular environment of Arf1 by use of site- specific photocrosslinking. This method was first used successfully in the field of protein trafficking to study the mechanisms involved in protein tr anslocation across the endoplasmic reticulum during protein synthesis. In s uch a hydrophobic environment, crosslink yields of up to 30% have been obse rved. We have now applied this method to study the mechanism of vesicle bud ding from the cytosolic face of the Golgi apparatus, an aqueous environment . Although the crosslink yield is significantly lower under these condition s, due to predominant reaction of the photolabile probes with water, a spec ific interaction of Arf1 with subunits of coatomer, the major coat protein of COPI vesicles, could readily be identified. (C) 2000 Academic Press.