Figure 6. Energy-dependent cytosolic activities are required to discharge CTA1 and TCRα into the cytosol.

(A) P1 from intoxicated cells was incubated with buffer, CE, or CE pretreated with the indicated ATP-γ-S or GTP-γ-S concentrations. The resulting S2′ were analyzed by immunoblotting with the indicated antibodies. (B) P1 from CT-intoxicated cells was incubated with buffer, yeast CE, or yeast CE pretreated with 1 mM GTP-γ-S or ATP-γ-S. The resulting S2′ were analyzed by immunoblotting with the indicated antibodies. (C) P1 from epoxomicin-treated cells expressing TCRα-HA was incubated with CE, or CE pretreated with 10 mM ATP-γ-S, 10 mM GTP-γ-S, or apyrase. The resulting S2′ were analyzed by immunoblotting with the indicated antibodies. Identical findings were obtained when 1 mM ATP-γ-S or GTP-γ-S was used (data not shown). (D) CT-intoxicated cells were permeabilized with digitonin buffer with or without 1 mM GTP-γ-S. P1 was washed and incubated with buffer, CE, or CE pretreated with 1 mM GTP-γ-S. The resulting S2′ were analyzed by immunoblotting with the indicated antibodies. (E) As in (D) except with ATP-γ-S. (F) Buffer, CE and CE pretreated with ATP-γ-S or GTP-γ-S were dialyzed 400-fold to remove free nucleotides. Intoxicated P1 were then incubated with undialyzed or dialyzed extracts, as indicated. The resulting S2′ were analyzed by immunoblotting with the indicated antibodies.