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Journal of Biochemistry 2007 142(5):621-625; doi:10.1093/jb/mvm169
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© 2007 The Japanese Biochemical Society

Anti-parallel Membrane Topology of a Homo-dimeric Multidrug Transporter, EmrE

Toshifumi Nara1,*, Tomoko Kouyama2, Yuko Kurata2, Takashi Kikukawa3, Seiji Miyauchi2 and Naoki Kamo2

1Laboratory of Biophysical Chemistry, College of Pharmaceutical Sciences, Matsuyama University, Matsuyama 790-8578; 2Laboratory of Biophysical Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812; and 3Laboratory of Biomolecular Systems, Creative Research Initiative ‘Sosei’ (CRIS), Hokkaido University, Sapporo 001-0021, Japan

*To whom correspondence should be addressed. Tel: +81-89-926-7204, Fax: +81-89-926-7162, E-mail: tnara{at}cc.matsuyama-u.ac.jp


  Abstract

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.

Key Words: multidrug resistance, SMR, membrane-impermeable SH-reagent (DTNB, AMS, biotin-PE-maleimide), ethidium efflux, dual membrane topology, EbrAB

Abbreviations: AMS, 4-acetamido-4'-maleimidylstilbene-2-2'disulfonic acid; AP, alkaline phosphatase; AS1, drug-hypersensitive E. coli; BM, biotin-PE-maleimide (N'-[2-N-maleimido)ethyl]-N–piperazinyl-D-biotinamide hydrochloride); CL, cysteine-less mutant in which all native cysteine residues are replaced with serine; DTNB, 5,5'-dithiobis-(2-nitrobenzoate); TPMP, triphenylmethylphosphonium


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