Figure 6. Polar cluster substitutions impair TatBC interactions.
Digitonin-solubilized cell lysates of the indicated strains were immunoprecipitated with antibodies against TatC and then immunoblotted with either a combination of TatB and TatC antibodies or with TatA antibodies (`α-TatC coIP’ panels). Where indicated a portion of the total cell lysate was also immunoblotted (‘input’ panels). (A,B) Co-immunoprecipitation analysis of strains expressing native levels of Tat proteins. Corresponding immunoblots of membranes isolated from the same strains are also shown (`membranes’). The TatCFEA variant is blocked in substrate interactions. The TatCMTQ variant carries the three polar cluster substitutions M205A, T208A, and Q215A. (C) Schematic representation of the results of polar cluster substitutions on Tat receptor complex composition. The red asterisks indicate the location of the polar cluster substitutions. (D) The indicated Tat proteins were overproduced approximately 500-fold in the ΔtatABCDΔtatE strain DADE-A from pUT2DH series plasmids and immunoprecipitated as in (A).