A mutation in the membrane subunit of an ABC transporter LolCDE complex causing outer membrane localization of lipoproteins against their inner membrane‐specific signals
A mutation in the membrane subunit of an ABC transporter LolCDE complex causing outer membrane localization of lipoproteins against their inner membrane‐specific signals
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DOI：
10.1046/j.1365-2958.2003.03569.x
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发表时间：
2003-07
影响因子：
3.6
通讯作者：
Shin-ichiro Narita;K. Kanamaru;S. Matsuyama;H. Tokuda
中科院分区：
文献类型：
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作者：
Shin-ichiro Narita;K. Kanamaru;S. Matsuyama;H. Tokuda
革兰氏阴性细菌中的脂蛋白通过连接到 N 末端半胱氨酸的脂肪酸锚定在内膜或外膜上。第 2 位的残基决定了膜的特异性。 ATP结合盒转运蛋白LolCDE复合物从内膜释放第2位具有天冬氨酸以外残基的脂蛋白，而第2位具有天冬氨酸的脂蛋白被LolCDE排斥，因此保留在内膜中。为了进一步了解这种排斥机制，我们开发了一种新的策略来选择突变体，其中释放2位带有天冬氨酸的脂蛋白。分离的突变体在 LolC（LolCDE 复合物的膜亚基）的 40 位处携带丙氨酸至脯氨酸突变。当 LolC 突变体表达时，内膜脂蛋白 L10P(DQ) 的很大一部分定位于外膜。周质伴侣 LolA 与释放的 L10P(DQ) 形成复合物，随后以 LolB 依赖性方式掺入外膜，表明 LolA 和 LolB 都不排斥 2 位具有天冬氨酸的脂蛋白。膜溶解后与 LolD 和 LolE 共纯化的 LolC 突变体的量显着减少。综上所述，这些结果表明突变导致 LolCDE 复合物不稳定，并随之阻碍脂蛋白分选信号的准确识别。
Lipoproteins in Gram‐negative bacteria are anchored to the inner or outer membrane via fatty acids attached to the N‐terminal cysteine. The residue at position 2 determines the membrane specificity. An ATP binding cassette transporter LolCDE complex releases lipoproteins with residues other than aspartate at position 2 from the inner membrane, whereas those with aspartate at position 2 are rejected by LolCDE and therefore remain in the inner membrane. For further understanding of this rejection mechanism, a novel strategy was developed to select mutants in which lipoproteins with aspartate at position 2 are released. The isolated mutants carried an alanine to proline mutation at position 40 of LolC, a membrane subunit of the LolCDE complex. A significant portion of an inner membrane lipoprotein, L10P(DQ), was localized to the outer membrane when the LolC mutant was expressed. Periplasmic chaperone LolA formed a complex with the released L10P(DQ), which was subsequently incorporated into the outer membrane in a LolB‐dependent manner, indicating that neither LolA nor LolB rejects lipoproteins with aspartate at position 2. The amount of the LolC mutant co‐purified with LolD and LolE after membrane solubilization was reduced significantly. Taken together, these results indicate that the mutation causes destabilization of the LolCDE complex and concomitantly prevents the accurate recognition of lipoprotein‐sorting signals.