COMPARATIVE ACTIVITY OF ADP-RIBOSYLATION FACTOR FAMILY MEMBERS IN THEEARLY STEPS OF COATED VESICLE FORMATION ON RAT-LIVER GOLGI MEMBRANES

We have compared the abilities of mammalian ADP-ribosylation factors ( ARFs) 1, 5, and 6 and Saccharomyces cerevisiae ARF2 to serve as substr ates for the rat liver Golgi membrane guanine nucleotide exchange fact or and to initiate the formation of clathrin- and coatomer protein (CO P) I-coated vesicles on these membranes. While Golgi membranes stimula ted the exchange of GTP gamma S for GDP on all of the ARFs tested, mam malian ARF1 was the best substrate, with an apparent K-m of 5 mu M. In all cases myristoylation of ARF was required for stimulation. Agents that inhibit the Golgi membrane guanine nucleotide exchange factor (th e fungal metabolite brefeldin A and trypsin treatment) selectively inh ibited the guanine nucleotide exchange on mammalian ARF1. Taken togeth er, these data indicate that of the ARFs tested, only mammalian ARF1 i s activated efficiently by the Golgi guanine nucleotide exchange facto r. The other ARFs are activated mainly by another mechanism, possibly phospholipid-mediated. Once activated, all of the membrane-associated myristoylated ARPs promoted the recruitment of coatomer to about the s ame extent. Mammalian ARFs 1 and 5 were the most effective in promotin g the recruitment of the AP-1 adaptor complex, whereas yeast ARFB was the least active, These data indicate that the specificity for ARF act ion on the Golgi membranes is primarily determined by the Golgi guanin e nucleotide exchange factor, which has a strong preference for myrist oylated mammalian ARF1.