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首页> 外文期刊>The FEBS journal >Complete reconstitution of an ATP-binding cassette transporter LolCDE complex from separately isolated subunits
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Complete reconstitution of an ATP-binding cassette transporter LolCDE complex from separately isolated subunits

机译：从单独分离的亚基完全重建ATP结合盒转运蛋白LolCDE复合物

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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.
机译：大肠杆菌的LolCDE复合物属于ATP结合盒转运蛋白超家族，介导脂蛋白从内膜的分离，从而启动脂蛋白分选到外膜的过程。该复合物由每个膜亚基LolC和LolE的一个拷贝，和两个ATPase亚基LolD的拷贝组成。为了建立从单独分离的亚基重建LolCDE复合物的条件，在各种条件下检查LolD和LolCDE的ATPase活性。我们发现LolD和LolCDE均在去污剂溶液中于30摄氏度孵育时被灭活。 ATP和磷脂保护LolCDE，但不保护LolD。此外，即使在LolCDE几乎完全失活之后，磷脂也能使其重新激活。当膜亚基和磷脂同时存在时，LolD也被保护免受灭活，表明LolCDE亚基的磷脂依赖性重组。实际上，将功能性脂蛋白释放机构重建为具有大肠杆菌磷脂并分别纯化的LolC，LolD和LolE的蛋白脂质体。在30°C下用磷脂预孵育对于从亚基重建功能机械至关重要。令人惊讶的是，脂蛋白的释放活性也可以由不含LolC的LolE和LolD重建，这暗示了由LolD和LolE形成最小脂蛋白释放机制的可能性。我们在这里报告从单独纯化的亚基的功能性ATP结合盒式转运蛋白的完整重建。

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