Molecular construction of a multidrug exporter system, AcrAB: Molecular interaction between AcrA and AcrB, and cleavage of the N-terminal signal sequence of AcrA

The AcrAB system of Escherichia coli is an intrinsic efflux protein with a broad substrate specificity. AcrA was thought to be localized in the peripl asmic space, and to be linked to AcrB and TolC, The AcrAB-TolC system direc tly exports diverse substrates from the cell interior to the medium. In thi s study, we have determined the cellular localization of AcrA. By using the osmotic shock method, sucrose density gradient centrifugation, urea washin g and Western blotting analysis, we reveal that AcrA is a peripheral inner membrane protein. A mutant plasmid encoding both the AcrA-TetBCt fusion pro tein and the AcrB-His fusion protein was constructed. Membrane vesicles pre pared from cells expressing these fusion proteins were solubilized and AcrB -His was immunoprecipitated with an anti-polyhistidine antibody. After SDS- PAGE, Western blotting was performed with anti-TetBCt antiserum, resulting in the appearance of a 40 KDa band, indicating that AcrA co-precipitated wi th AcrB, Next we performed site-directed chemical labeling of Cys-introduce d mutants of AcrA with [C-14]N-ethylmaleimide. As judged from the labeling pattern and the molecular mass shift, the N-terminus of AcrA was removed an d the mature protein is on the periplasmic surface. On the other hand, C25A mutants retained the N-terminal signal sequence on the cytoplasmic side of the membrane. We conclude that AcrA exists as a complex with AcrB on the p eriplasmic surface of the inner membrane after removal of the signal sequen ce.