The purified and functionally reconstituted multidrug transporter LmrA of Lactococcus lactis mediates the transbilayer movement of specific fluorescent phospholipids
The purified and functionally reconstituted multidrug transporter LmrA of Lactococcus lactis mediates the transbilayer movement of specific fluorescent phospholipids
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DOI：
10.1021/bi990855s
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发表时间：
1999-12-07
期刊：
影响因子：
2.9
通讯作者：
Konings, WN
中科院分区：
文献类型：
--
作者：
Margolles, A;Putman, M;Konings, WN
乳酸乳球菌具有ATP结合盒转运蛋白LmrA，其是哺乳动物多药耐药（MDR）P-糖蛋白的同源物，并且能够转运广泛的结构上不相关的两亲性药物。在LmrA的N-末端引入组氨酸标签以促进通过镍亲和层析的纯化。组氨酸标签蛋白在L.乳酸链球菌使用一种新的蛋白表达系统的细胞毒性蛋白的基础上严格调控，乳酸链球菌素诱导的nisA启动子，该系统允许我们得到功能性过表达LmrA的水平高达30%的总膜蛋白。对于重构，用十二烷基麦芽糖苷溶解LmrA，通过镍螯合物亲和色谱法纯化，并在从乳酸乳球菌磷脂制备的十二烷基麦芽糖苷去稳定的预成型脂质体中重构。通过吸附到聚苯乙烯珠上来去除洗涤剂。LmrA蛋白以功能形式重构，并介导荧光底物Hoechst-33342的ATP依赖性转运进入脂蛋白体。有趣的是，重组LmrA也催化荧光磷脂酰乙醇胺的ATP依赖性运输，但不是荧光磷脂酰胆碱。这些数据表明，LmrA活性是独立的辅助蛋白，并支持的概念，LmrA可能参与运输特定的脂质或脂质连接的前体在L。乳酸菌。
Lactococcus lactis possesses an ATP-binding cassette transporter, LmrA, which is a homolog of the mammalian multidrug resistance (MDR) P-glycoprotein, and is able to transport a broad range of structurally unrelated amphiphilic drugs. A histidine tag was introduced at the N-terminus of LmrA to facilitate purification by nickel affinity chromatography. The histidine-tagged protein was overexpressed in L. lactis using a novel protein expression system for cytotoxic proteins based on the tightly regulated, nisin-inducible nisA promoter, This system allowed us to get functional overexpression of LmrA up to a level of 30% of total membrane protein. For reconstitution, LmrA was solubilized with dodecylmaltoside, purified by nickel-chelate affinity chromatography, and reconstituted in dodecylmaltoside-destabilized, preformed liposomes prepared from L, lactis phospholipids. The detergent was removed by adsorption onto polystyrene beads. The LmrA protein was reconstituted in a functional form, and mediated the ATP-dependent transport of the fluorescent substrate Hoechst-33342 into the proteoliposomes. Interestingly, reconstituted LmrA also catalyzed the ATP-dependent transport of fluorescent phosphatidylethanolamine, but not of fluorescent phosphatidylcholine. These data demonstrate that LmrA activity is independent of accessory proteins and support the notion that LmrA may be involved in the transport of specific lipids or lipid-linked precursors in L. lactis.