FIGURE 4.
Mutation of lysines in the native A1-chain rescues HA-CT from the proteasome. A, cells were pretreated with lactacystin or vehicle alone before retrotranslocation experiments. WT toxin (lanes 6 and 7) and carbamylated WT CT with Lys-4 and Lys-17 mutations (WT K4RK17R*) (lanes 8 and 9) are shown. HA-CT (lanes 10 and 11), HA-CT K4RK17R (lanes 12 and 13), or the carbamylated HA-CT K4RK17R mutant (HA-CT K4RK17R*) (lanes 14 and 15) are shown. Lanes 1–5 are controls. All samples were analyzed by SDS-PAGE and immunoblotted (IB) using antibodies recognizing the A-subunit (CTA) and B-subunit (CTB) or HA tag. B, in a time course of CT-induced Cl− secretion (Isc), T84 cells were either treated with HA-CT K4RK17R* (filled circle) or pretreated with lactacystin followed by HA-CT K4RK17R* (open circle). C, this was performed as in panel B, but HA-CT K4RK17R (filled diamond) or pretreated HA-CT K4RK17R (open diamond) was tested. D, summarized data from three independent experiments, each done in triplicate, of rescue by lactacystin is shown. The percentage of increase in maximal Isc for lactacystin-pretreated T84 cells versus untreated cells was plotted on the y axis for both HA-CT K4RK17R and HA-CT K4RK17R*. Error bars in indicate S.D.