Complete reconstitution of an ATP-binding cassette transporter LolCDE complex from separately isolated subunits
Complete reconstitution of an ATP-binding cassette transporter LolCDE complex from separately isolated subunits
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DOI：
10.1111/j.1742-4658.2007.05832.x
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发表时间：
2007-07-01
期刊：
影响因子：
5.4
通讯作者：
Tokuda, Hajime
中科院分区：
文献类型：
--
作者：
Kanamaru, Kyoko;Taniguchi, Naohiro;Tokuda, Hajime
大肠杆菌的LolCDE复合体属于atp结合盒转运蛋白超家族，它介导脂蛋白从内膜分离，从而启动脂蛋白向外膜的分选。该复合物由膜亚基LolC和LolE各一个拷贝和atp酶亚基LolD两个拷贝组成。为了确定从分离的亚基重新构建LolCDE复合物的条件，在不同条件下检测了LolD和LolCDE的atp酶活性。我们发现LolD和LolCDE都在30℃的洗涤剂溶液中失活。ATP和磷脂对LolCDE有保护作用，而对LolD无保护作用。此外，磷脂在其几乎完全失活后重新激活了LolCDE。当膜亚基和磷脂同时存在时，LolD也不会失活，这表明LolCDE亚基的重组依赖于磷脂。事实上，用大肠杆菌磷脂将功能性脂蛋白释放机制重组为蛋白脂质体，并分别纯化了LolC、LolD和LolE。在30℃下用磷脂进行预孵育对于从亚基重建功能机制是必不可少的。引人注目的是，在没有LolC的情况下，LolE和LolD也能重建脂蛋白释放活性，这表明LolD和LolE可能形成最低限度的脂蛋白释放机制。我们在此报告了从单独纯化的亚基中完全重构功能性atp结合盒转运体。
The LolCDE complex of Escherichia coli belongs to the ATP-binding cassette transporter superfamily and mediates the detachment of lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane. The complex is composed of one copy each of membrane subunits LolC and LolE, and two copies of ATPase subunit LolD. To establish the conditions for reconstituting the LolCDE complex from separately isolated subunits, the ATPase activities of LolD and LolCDE were examined under various conditions. We found that both LolD and LolCDE were inactivated on incubation at 30 degrees C in a detergent solution. ATP and phospholipids protected LolCDE, but not LolD. Furthermore, phospholipids reactivated LolCDE even after its near complete inactivation. LolD was also protected from inactivation when membrane subunits and phospholipids were present together, suggesting the phospholipid-dependent reassembly of LolCDE subunits. Indeed, the functional lipoprotein-releasing machinery was reconstituted into proteoliposomes with E. coli phospholipids and separately purified LolC, LolD and LolE. Preincubation with phospholipids at 30 degrees C was essential for the reconstitution of the functional machinery from subunits. Strikingly, the lipoprotein-releasing activity was also reconstituted from LolE and LolD without LolC, suggesting the intriguing possibility that the minimum lipoprotein-releasing machinery can be formed from LolD and LolE. We report here the complete reconstitution of a functional ATP-binding cassette transporter from separately purified subunits.