title: Membrane topology of multidrug resistance protein expressed in Escherichia coli. N-terminal domain.
abstract: Expression of eukaryotic polytopic membrane proteins in Escherichia coli could provide an invaluable system for structure-function studies. Recently, the functional expression of a mouse multidrug resistance protein (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 present study, the phoA gene fusion approach has been utilized to determine the membrane topology of the N-terminal domain of Mdr. The results support the idea that the N-terminal half of Mdr contains six transmembrane helices (TMs). However, our observations suggest that the previously proposed TM4 (amino acid residues Thr214-Ala232) is located at the periplasmic face of the membrane. Alternatively, we propose that another stretch of amino acid residues (Leu243 (out) to Ile260 (in)) traverses the membrane. This assignment is indicated also by the following observations: 1) deletion of a segment containing the originally predicted TM4 (delta T214-K241) does not reverse the orientation of the Mdr-alkaline phosphatase hybrid that is located in the following cytoplasmic loop; 2) a "sandwich" chimera, in which alkaline phosphatase is inserted in-frame between amino acid residues Leu226 and Ser227, exhibits high alkaline phosphatase activity. The existence of an externally exposed hydrophobic domain in Mdr may have special structural and functional implications, and these may also be relevant to other members of the ABC superfamily.
authors.affiliation: Department of Biochemistry, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: E
authors.initials: E
authors.lastname: Bibi
authors.forename: O
authors.initials: O
authors.lastname: Béjà
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D004351:Drug Resistance; D004926:Escherichia coli; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase