The Tol/Pal system function requires an interaction between the C-terminal domain of TolA and the N-terminal domain of TolB

The Tol/Pal system of Escherichia coli is composed of the YbgC, ToIQ, ToIA, ToIR, ToIB, Pal and YbgF proteins. It is involved in maintaining the integrity of the outer membrane, and is required for the uptake of group A colicins and DNA of filamentous bacteriophages. To identify new interactions between the components of the Tol/Pal system and gain insight into the mechanism of colicin import, we performed a yeast two-hybrid screen using the different components of the Tol/Pal system and colicin A. Using this system, we confirmed the already known interactions and identified several new interactions. ToIB dimerizes and the periplasmic domain of ToIA interacts with YbgF and ToIB. Our results indicate that the central domain of ToIA (TolAll) is sufficient to interact with YbgF, that the C-terminal domain of ToIA (ToIAIII) is sufficient to interact with ToIB, and that the amino terminal domain of ToIB (D1) is sufficient to bind TolAlll. The TolA/ToIB interaction was confirmed by crosslinking experiments on purified proteins. Moreover, we show that the interaction between ToIA and ToIB is required for the uptake of colicin A and for the membrane integrity. These results demonstrate that the TolA/ToIB interaction allows the formation of a trans-envelope complex that brings the inner and outer membranes in close proximity.