E. Bibi et O. Beja, MEMBRANE TOPOLOGY OF MULTIDRUG-RESISTANCE PROTEIN EXPRESSED IN ESCHERICHIA-COLI - N-TERMINAL DOMAIN, The Journal of biological chemistry, 269(31), 1994, pp. 19910-19915
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.