Folding and trimerization of signal sequence-less mature TolC in the cytoplasm of Escherichia coli.

TolC is a multifunctional outer-membrane protein (OMP) of Escherichia coli that folds into a unique alpha/beta-barrel structure. Previous studies have shown that unlike the biogenesis of beta-barrel OMPs, such as porins, TolC assembles independently from known periplasmic folding factors. Yet, the assembly of TolC, like that of beta-barrel OMPs, is dependent on BamA and BamD, two essential components of the beta-barrel OMP assembly machinery. We have investigated the folding properties and cellular trafficking of a TolC derivative that lacks the entire signal sequence (TolCDelta2-22). A significant amount of TolCDelta2-22 was found to be soluble in the cytoplasm, and a fraction of it folded and trimerized into a conformation similar to that of the normal outer membrane-localized TolC protein. Some TolCDelta2-22 was found to associate with membranes, but failed to assume a wild-type-like folded conformation. The null phenotype of TolCDelta2-22 was exploited to isolate suppressor mutations, the majority of which mapped in secY. In the secY suppressor background, TolCDelta2-22 resumed normal function and folded like wild-type TolC. Proper membrane insertion could not be achieved upon in vitro incubation of cytoplasmically folded TolCDelta2-22 with purified outer membrane vesicles, showing that even though TolC is intrinsically capable of folding and trimerization, for successful integration into the outer membrane these events need to be tightly coupled to the insertion process, which is mediated by the Bam machinery. Genetic and biochemical data attribute the unique folding and assembly pathways of TolC to its large soluble alpha-helical domain.