Characterization of YvcC (BmrA), a Multidrug ABC Transporter Constitutively Expressed in Bacillus subtilis
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- 作者：Emmanuelle Steinfels ; Cé ; dric Orelle ; Jean-Raphaë ; l Fantino ; Olivier Dalmas ; Jean-Louis Rigaud ; Franç ; ois Denizot ; Attilio Di Pietro ; and Jean-Michel Jault
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：June 15, 2004
- 年：2004
- 卷：43
- 期：23
- 页码：7491 - 7502
- 全文大小：871K
- 年卷期：v.43,no.23(June 15, 2004)
- ISSN：1520-4995
文摘
The involvement of transporters in multidrug resistance of bacteria is an increasingly challengingproblem, and most of the pumps identified so far use the protonmotive gradient as the energy source. Anew member of the ATP-binding cassette (ABC) family, known in Bacillus subtilis as YvcC andhomologous to each half of mammalian P-glycoprotein and to LmrA of Lactococcus lactis, has beenstudied here. The yvcC gene was constitutively expressed in B. subtilis throughout its growth, and aknockout mutant showed a lower rate of ethidium efflux than the wild-type strain. Overexpression ofyvcC in Escherichia coli allowed the preparation of highly enriched inverted-membrane vesicles thatexhibited high transport activities of three fluorescent drugs, namely, Hoechst 33342, doxorubicin, and7-aminoactinomycin D. After solubilization with n-dodecyl 
-D-maltoside, the hexahistidine-tagged YvcCwas purified by a one-step affinity chromatography, and its ability to bind many P-glycoprotein effectorswas evidenced by fluorescence spectroscopy experiments. Collectively, these results showed that YvcCis a multidrug ABC transporter functionally active in wild-type B. subtilis, and YvcC was therefore renamedBmrA for Bacillus multidrug resistance ATP. Besides, reconstitution of YvcC into liposomes led to thehighest, vanadate-sensitive, ATPase activity reported so far for an ABC transporter. Interestingly, such ahigh ATP hydrolysis proceeds with a positive cooperativity mechanism, a property only found so farwith ABC importers.
-D-maltoside, the hexahistidine-tagged YvcCwas purified by a one-step affinity chromatography, and its ability to bind many P-glycoprotein effectorswas evidenced by fluorescence spectroscopy experiments. Collectively, these results showed that YvcCis a multidrug ABC transporter functionally active in wild-type B. subtilis, and YvcC was therefore renamedBmrA for Bacillus multidrug resistance ATP. Besides, reconstitution of YvcC into liposomes led to thehighest, vanadate-sensitive, ATPase activity reported so far for an ABC transporter. Interestingly, such ahigh ATP hydrolysis proceeds with a positive cooperativity mechanism, a property only found so farwith ABC importers.