The reconstituted Escherichia coli MsbA protein displays lipid flippase activity
The reconstituted Escherichia coli MsbA protein displays lipid flippase activity
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DOI：
10.1042/bj20100144
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发表时间：
2010-07-01
影响因子：
4.1
通讯作者：
Sharom, Frances J.
中科院分区：
文献类型：
--
作者：
Eckford, Paul D. W.;Sharom, Frances J.
MsbA蛋白是革兰氏阴性菌中必不可少的ABC （atp结合盒）超家族成员。这种65 kDa的膜蛋白被认为是一种同源二聚体atp依赖的脂质转位酶或翻转酶，将脂质a从细胞质膜的内叶转运到外叶。我们之前已经证明，从大肠杆菌中纯化的MsbA具有高atp酶活性，并与脂质和类脂分子结合，包括脂质A，在低微摩尔范围内具有亲和力。从大肠杆菌中分离出的过表达His(6)标记的MsbA的细菌膜囊泡显示出几种荧光NBD（7-硝基苯-2-氧-1,3-二唑）标记的磷脂的atp依赖性易位。纯化的MsbA重组为大肠杆菌脂质蛋白脂质体，并表征了其转运nbd标记的脂质衍生物的能力。在该体系中，对于酰基链标记的PE（磷脂酰乙醇胺）衍生物，在20分钟内，蛋白质的脂质翻转酶活性最高，为每毫克蛋白质7.7 nmol脂质易位。当重组成大肠杆菌脂质混合物时，该蛋白显示出最高的翻转酶活性。大量的翻转酶活性也被观察到用于各种其他nbd标记的磷脂和糖脂，包括标记在头基或酰基链上的分子。脂质翻转酶的活性需要ATP水解，并依赖于ATP和NBD脂质的浓度。NBD PE的转位被假定的生理底物脂质a的存在所抑制。本文首次报道了在重组系统中直接测量纯化MsbA的脂质翻转酶活性。
The MsbA protein is an essential ABC (ATP-binding-cassette) superfamily member in Gram-negative bacteria. This 65 kDa membrane protein is thought to function as a homodimeric ATP-dependent lipid translocase or flippase that transports lipid A from the inner to the outer leaflet of the cytoplasmic membrane. We have previously shown that purified MsbA from Escherichia coli displays high ATPase activity, and binds to lipids and lipid-like molecules, including lipid A, with affinity in the low micromolar range. Bacterial membrane vesicles isolated from E. coli overexpressing His(6)-tagged MsbA displayed ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species. Purified MsbA was reconstituted into proteoliposomes of E. coli lipid and its ability to translocate NBD-labelled lipid derivatives was characterized. In this system, the protein displayed maximal lipid flippase activity of 7.7 nmol of lipid translocated per mg of protein over a 20 min period for an acyl chain-labelled PE (phosphatidylethanolamine) derivative. The protein showed the highest rates of flippase activity when reconstituted into an E. coli lipid mixture. Substantial flippase activity was also observed for a variety of other NBD-labelled phospholipids and glycolipids, including molecules labelled on either the headgroup or the acyl chain. Lipid flippase activity required ATP hydrolysis, and was dependent on the concentration of ATP and NBD lipid. Trans location of NBD PE was inhibited by the presence of the putative physiological substrate lipid A. The present paper represents the first report of a direct measurement of the lipid flippase activity of purified MsbA in a reconstituted system.