ACTIVATION OF ADP-RIBOSYLATION FACTOR BY GOLGI MEMBRANES - EVIDENCE FOR A BREFELDIN A-SENSITIVE AND PROTEASE-SENSITIVE ACTIVATING FACTOR ONGOLGI MEMBRANES

Recent evidence has implicated ADP-ribosylation factor (ARF) proteins as critical regulators of the protein secretory pathway, particularly in the endoplasmic reticulum-Golgi pathway. We have examined whether G olgi membranes contain activators of ARF and the consequences of ARF a ctivation and acylation on its membrane association. Two means were us ed to assess ARF activation. First, guanosine 5'-3-O-(thio)triphosphat e (GTPgammaS) binding to protein was found to be greater when ARF and Golgi were incubated together than when either was incubated alone. Th ese data suggested that ARF . GTPgammaS was formed. This was confirmed by showing that the GTPgammaS-bound protein functioned as a cofactor for cholera toxin-stimulated ADP-ribosylation of G(salpha), a reaction for which activated ARF is a necessary cofactor. Trypsin treatment of Golgi, an inhibitory ARF peptide, and brefeldin A each inhibited Golg i-mediated activation by approximately 70%, demonstrating that a speci fic protein interaction is required for the majority of the ARF activa tion. This ARF-activating protein is a strong candidate for the molecu lar target for brefeldin A. The ubiquitous nature of ARF proteins and their importance in both the exocytic and endocytic pathways may expla in the effects of brefeldin A on both exocytic and endocytic membrane traffic in animal cells. A protease-insensitive activation of ARF by G olgi could also be demonstrated and was the dominant activity observed in submicromolar concentrations of magnesium. We believe this to be t he lipid-mediated process described previously for purified ARF protei ns. ARF activation resulted in tight association of ARF with phospholi pid vesicles. Vesicle association required amino-terminal myristoylati on of ARF whereas activation did not. These studies indicate that the brefeldin A-sensitive ARF-activating protein and other factors that de termine the level of activation of ARF in animal cells are fundamental regulators of membrane traffic in animal cells.