Anti-parallel membrane topology of a homo-dimeric multidrug transporter, EmrE.
EmrE in Escherichia coli belongs to the small multidrug resistance (SMR) transporter family. It functions as a homo-dimer, but the orientation of the two monomers in the membrane (membrane topology) is under debate. We expressed various single-cysteine EmrE mutants in E. coli cells lacking a major efflux transporter. Efflux from cells expressing the P55C or T56C mutant was blocked by the external application of membrane-impermeable SH-reagents. This is difficult to explain by the parallel topology configuration, because Pro55 and Thr56 are considered to be located in the cytoplasm. From both the periplasm and the cytoplasm, biotin-PE-maleimide, a bulky membrane-impermeable SH-reagent, could access the cysteine residue at the 25th position in the presence of transport substrates and at the 108th position. These observations support the anti-parallel topology in the membrane.
Nara
Toshifumi
T
Laboratory of Biophysical Chemistry, College of Pharmaceutical Sciences, Matsuyama University, Matsuyama 790-8578, Japan. tnara@cc.matsuyama-u.ac.jp
Kouyama
Tomoko
T
Kurata
Yuko
Y
Kikukawa
Takashi
T
Miyauchi
Seiji
S
Kamo
Naoki
N
D017920:Antiporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D001710:Biotin / Q000494:pharmacology; D002463:Cell Membrane Permeability; D003545:Cysteine / Q000235:genetics; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D019897:Periplasm / Q000737:chemistry / Q000378:metabolism*; D020959:Polyethylene / Q000494:pharmacology; D011392:Proline / Q000235:genetics; D017433:Protein Structure, Secondary; D013912:Threonine / Q000235:genetics
D017920:Antiporters; D001710:Biotin; D003545:Cysteine; D029968:Escherichia coli Proteins; D008301:Maleimides; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D020959:Polyethylene; D011392:Proline; D013912:Threonine; C075436:EmrE protein, E coli; C043592:maleimide