Characterization of YvcC (BmrA), a multidrug ABC transporter constitutively expressed in Bacillus subtilis

被引:108
作者
Steinfels, E
Orelle, C
Fantino, JR
Dalmas, O
Rigaud, JL
Denizot, F
Di Pietro, A
Jault, JM
机构
[1] Univ Grenoble 1, CEA, CNRS, Lab Biophys Mol & Cellulaire,DRDC,UMR 5090, F-38054 Grenoble 9, France
[2] Inst Curie, CNRS, UMR 168, F-75231 Paris 05, France
[3] CEA, LRC, F-75231 Paris, France
[4] CNRS, Inst Biol Struct & Microbiol, Chim Bacterienne Lab, UPR 9043, F-13402 Marseille 20, France
[5] Univ Lyon 1, CNRS, Inst Biol & Chim Prot, UMR 5086, F-69367 Lyon 07, France
[6] Univ Lyon 1, CNRS, Inst Biol & Chim Prot, IFR 128, F-69367 Lyon 07, France
关键词
D O I
10.1021/bi0362018
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The involvement of transporters in multidrug resistance of bacteria is an increasingly challenging problem, and most of the pumps identified so far use the protonmotive gradient as the energy source. A new member of the ATP-binding cassette (ABC) family, known in Bacillus subtilis as YvcC and homologous to each half of mammalian P-glycoprotein and to LmrA of Lactococcus lactis, has been studied here. The yvcC gene was constitutively expressed in B. subtilis throughout its growth, and a knockout mutant showed a lower rate of ethidium efflux than the wild-type strain. Overexpression of yvcC in Escherichia coli allowed the preparation of highly enriched inverted-membrane vesicles that exhibited high transport activities of three fluorescent drugs, namely, Hoechst 33342, doxorubicin, and 7-aminoactinomycin D. After solubilization with n-dodecyl beta-D-maltoside, the hexahistidine-tagged YvcC was purified by a one-step affinity chromatography, and its ability to bind many P-glycoprotein effectors was evidenced by fluorescence spectroscopy experiments. Collectively, these results showed that YvcC is a multidrug ABC transporter functionally active in wild-type B. subtilis, and YvcC was therefore renamed BmrA for Bacillus multidrug resistance ATP. Besides, reconstitution of YvcC into liposomes led to the highest, vanadate-sensitive, ATPase activity reported so far for an ABC transporter. Interestingly, such a high ATP hydrolysis proceeds with a positive cooperativity mechanism, a property only found so far with ABC importers.
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页码:7491 / 7502
页数:12
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