PERTUSSIS TOXIN-CATALYZED ADP-RIBOSYLATION OF G(I-2), AND G(I-3) IN CHO CELLS IS MODULATED BY INHIBITORS OF INTRACELLULAR TRAFFICKING

In previous studies, an in vitro ADP-ribosylation assay was developed to quantitatively evaluate the in vivo ADP-ribosylation of eukaryotic target proteins in intact Chinese hamster ovary (CHO) cells by pertuss is toxin (PT). Immunoblot analysis identified the two PT-sensitive tar get proteins in CHO cells as G(i-2) and G(i-3). In this in vitro ADP-r ibosylation assay, the ability of PT to ADP-ribosylate G(i-2), and G(i -3) in intact CHO cells was not inhibited by cytochalasin D but was in hibited by chloroquine, monensin, and nocodazole. These data implicate d the involvement of a cytochalasin D-independent endocytic mechanism, a pa-sensitive step, and microtubules in the ADP-ribosylation of G(i- 2) and G(i-3) by PT in intact CHO cells. Preincubation of CHO cells wi th cycloheximide, at concentrations that reduced protein synthesis by >95%, did not inhibit the ability of PT to ADP-ribosylate G(i-2), and G(i-3). Control experiments showed that these agents did not affect ei ther the ability of PT to directly ADP-ribosylate the heterotrimeric G protein, G(t), or the binding of PT to CHO cells, except that monensi n slightly inhibited the binding of PT to CHO cells. These results are consistent with a model in which PT is internalized by receptor-media ted endocytosis, probably via a cytochalasin D-independent pathway, wh ich involves intracellular trafficking through late endosomes and the Golgi apparatus, An alternative model predicts the presence of a eukar yotic factor that traffics within cells via this pathway and is requir ed by PT to ADP-ribosylate G(i) proteins.