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 different 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) vesicle 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 translocation across the endoplasmic reticulum during protein synthesis. In such a hydrophobic environment, crosslink yields of up to 30% have been observed. We have now applied this method to study the mechanism of vesicle budding from the cytosolic face of the Golgi apparatus, an aqueous environment. Although the crosslink yield is significantly lower under these conditions, due to predominant reaction of the photolabile probes with water, a specific interaction of Arf1 with subunits of coatomer, the major coat protein of COPI vesicles, could readily be identified.