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

被引:43
作者
XU, YR [1 ]
BARBIERI, JT [1 ]
机构
[1] MED COLL WISCONSIN,DEPT MICROBIOL,MILWAUKEE,WI 53226
关键词
D O I
10.1128/IAI.63.3.825-832.1995
中图分类号
R392 [医学免疫学]; Q939.91 [免疫学];
学科分类号
100102 ;
摘要
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 [P-32]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-G(i) protein antiserum. The two proteins recognized by anti-G(i) 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. NH4Cl, inhibitors of intracellular trafficking, reduced temperature, and brefeldin A inhibited the ability of PT to ADP-ribosylate the 42- and 45-kDa proteins in vivo. These data implicate a pH-sensitive step and intracellular trafficking system in the in vivo ADP-ribosylation of G(i) proteins by PT.
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页码:825 / 832
页数:8
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