Membrane topology of the xenobiotic-exporting subunit, MexB, of the MexA,B-OprM extrusion pump in Pseudomonas aeruginosa

The MexA,B-OprM efflux pump assembly of Pseudomonas aeruginosa consists of two inner membrane proteins and one outer membrane protein. The cytoplasmic membrane protein, MexB, appears to function as the xenobiotic-exporting su bunit, whereas the MexA and OprM proteins are supposed to function as the m embrane fusion protein and the outer membrane channel protein, respectively . Computer-aided hydropathy analyses of MexB predicted the presence of up t o 17 potential transmembrane segments. To verify the prediction, we analyze d the membrane topology of MexB using the alkaline phosphatase gene fusion method. We obtained the following unique characteristics. MexB bears 12 mem brane spanning segments leaving both the amino and carboxyl termini in the cytoplasmic side of the inner membrane. Both the first and fourth periplasm ic loops had very long hydrophilic domains containing 311 and 314 amino aci d residues, respectively. This fact suggests that these loops may interact with other pump subunits, such as the membrane fusion protein MexA and the outer membrane protein OprM, Alignment of the amino- and the carboxyl-termi nal halves of MexB showed a 30% homology and transmembrane segments 1, 2, 3 , 4, 5, and 6 could be overlaid with the segments 7, 8, 9, 10, 11, and 12, respectively. This result suggested that the MexB has a a-fold repeat that strengthen the experimentally determined topology model. This paper reports the structure of the pump subunit, MexB, of the MexA,B-OprM efflux pump as sembly. This is the first time to verify the topology of the resistant-nodu lation-division efflux pump protein.