MEMBRANE TOPOLOGY OF MULTIDRUG-RESISTANCE PROTEIN EXPRESSED IN ESCHERICHIA-COLI - N-TERMINAL DOMAIN

Expression of eukaryotic polytopic membrane proteins in Escherichia co li could provide an invaluable system for structure-function studies. Recently, the functional expression of a mouse multidrug resistance pr otein (Mdr1) in E. coli was described (Bibi, E., Gros, P., and Kaback, H. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9209-9213). In the p resent study, the phoA gene fusion approach has been utilized to deter mine the membrane topology of the N-terminal domain of Mdr. The result s support the idea that the N-terminal half of Mdr contains six transm embrane helices (TMs). However, our observations suggest that the prev iously proposed TM4 (amino acid residues Thr(214)-Ala(232)) is located at the periplasmic face of the membrane. Alternatively, we propose th at another stretch of amino acid residues (Leu(243) (out) to Ile(260) (in)) traverses the membrane. This assignment is indicated also by the following observations: 1) deletion of a segment containing the origi nally predicted TM4 (Delta T214-K241) does not reverse the orientation of the Mdr-alkaline phosphatase hybrid that is located in the followi ng cytoplasmic loop; 2) a ''sandwich'' chimera, in which alkaline phos phatase is inserted in-frame between amino acid residues Leu(226) and Ser(227), exhibits high alkaline phosphatase activity. The existence o f an externally exposed hydrophobic domain in Mdr may have special str uctural and functional implications, and these may also be relevant to other members of the ABC superfamily.