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 vitro ADP-ribosylation assay. In this assay, a postnuclear supernatant was prepared from CHO cells and used as a source of PT-sensitive target proteins for in vitro [32P[ADP-ribosylation. The postnuclear supernatant contained three proteins that were ADP-ribosylated in vitro, with apparent molecular masses of 50, 45, and 42 kDa. The 42- and 45-kDa proteins were membrane associated, while the 50-kDa protein was soluble. Following PT treatment of CHO cells, the 42- and 45-kDa proteins were not available for in vitro ADP-ribosylation, while the soluble 50-kDa protein remained available for in vitro ADP-ribosylation. The decrease in the availability of the 42- and 45-kDa proteins to in vitro ADP-ribosylation was proportional to the PT concentration and time of incubation with CHO cells. Western immunoblot analysis showed that extracts from PT-treated CHO cells and control CHO cells possessed equivalent amounts of two proteins that were recognized by anti-Gi protein antiserum. The two proteins recognized by anti-Gi protein antiserum from PT-treated cells migrated with higher apparent molecular weights than the two proteins from control cells. This was consistent with the in vivo ADP-ribosylation of the two proteins by PT.(ABSTRACT TRUNCATED AT 250 WORDS)
Xu
Y
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Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226.
Barbieri
J T
JT
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D020126:Brefeldin A; D016466:CHO Cells; D002451:Cell Compartmentation; D006224:Cricetinae; D003517:Cyclopentanes / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D008954:Models, Biological; D037342:Pertussis Toxin; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013347:Subcellular Fractions / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D003517:Cyclopentanes; D003902:Detergents; D011500:Protein Synthesis Inhibitors; D010566:Virulence Factors, Bordetella; D000643:Ammonium Chloride; D020126:Brefeldin A; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins