PERTUSSIS TOXIN-MEDIATED ADP-RIBOSYLATION OF TARGET PROTEINS IN CHINESE-HAMSTER OVARY CELLS INVOLVES A VESICLE TRAFFICKING MECHANISM

Pertussis toxin (PT)-catalyzed ADP-ribosylation of target proteins in intact Chinese hamster ovary (CHO) cells was evaluated with an in vitr o ADP-ribosylation assay. In this assay, a postnuclear supernatant was prepared from CHO cells and used as a source of PT-sensitive target p roteins for in vitro [P-32]ADP-ribosylation. The postnuclear supernata nt contained three proteins that were ADP-ribosylated in vitro, with a pparent molecular masses of 50, 45 and 42 kDa. The 42- and 45-kDa prot eins were membrane associated, while the 50-kDa protein was soluble. F ollowing PT treatment of CHO cells, the 42- and 45-kDa proteins were n ot available for in vitro ADP-ribosylation, while the soluble 50-kDa p rotein remained available for in vitro ADP-ribosylation. The decrease in the availability of the 42- and 45-kDa proteins to in vitro ADP-rib osylation was proportional to the PT concentration and time of incubat ion with CHO cells. Western immunoblot analysis showed that extracts f rom PT-treated CHO cells and control CHO cells possessed equivalent am ounts of two proteins that were recognized by anti-G(i) protein antise rum. The two proteins recognized by anti-G(i) protein antiserum from P T-treated cells migrated with higher apparent molecular weights than t he two proteins from control cells. This was consistent with the in vi vo ADP-ribosylation of the two proteins by PT. NH4Cl, inhibitors of in tracellular trafficking, reduced temperature, and brefeldin A inhibite d the ability of PT to ADP-ribosylate the 42- and 45-kDa proteins in v ivo. These data implicate a pH-sensitive step and intracellular traffi cking system in the in vivo ADP-ribosylation of G(i) proteins by PT.