Fig. 3.
Complementation indicates an intersubunit interaction model. (a) Planar lipid bilayer and cellular translocation assays. Both single mutants K397Q and D426K are translocation-defective. ∗, D426K translocation could not be measured, because there was no block by LFN observed. To create mixed heptamers, K397Q and D426K mutants (PA83) were combined in equal quantities, nicked with trypsin, and purified over a Q-Sepharose column (see Materials and Methods). In planar lipid bilayers, mixed heptamers were able to bind and translocate LFN with a translocation half-time of 63 ± 3 seconds (n = 3). In the LFN-DTA cellular translocation assay, both single mutants were at least six orders of magnitude defective, but the mixture was approximately one order of magnitude defective. (b) Model illustrating intersubunit interactions. In wild-type heptamers, K397 interacts with D426 from a neighboring subunit. In K/K and Q/D homoheptamers, these interactions are disrupted. In heteroheptamers, translocation K–D and Q–K interactions are restored.