The bacterial protein YidC accelerates MPIase-dependent integration of membrane proteins
The bacterial protein YidC accelerates MPIase-dependent integration of membrane proteins
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DOI：
10.1074/jbc.ra119.011248
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发表时间：
2019-12-06
影响因子：
4.8
通讯作者：
Nishiyama, Ken-ichi
中科院分区：
文献类型：
--
作者：
Sasaki, Masaru;Nishikawa, Hanako;Nishiyama, Ken-ichi
细菌膜蛋白是通过膜蛋白整合酶（MPIase）等一系列整合因子的协同作用整合到细胞膜上的。然而，在膜蛋白整合过程中，MPIase活性如何被其他整合因子补充还不完全清楚。在此，使用来自大肠杆菌细胞的倒置内膜囊泡和重构（蛋白质）脂质体制剂，沿着膜蛋白整合测定和PURE系统以产生膜蛋白，我们发现抗MPIase IgG抑制Sec非依赖性底物3L-Pf 3外壳和Sec依赖性底物MtlA整合到大肠杆菌中。大肠杆菌膜囊泡。MPIase耗尽的膜囊泡缺乏3L-Pf 3外壳和MtlA整合，表明MPIase参与两种蛋白质的整合。我们开发了一种重建系统，其中排除了无序的自发整合，这表明SecYEG，YidC或两者都不足以进行Sec-dependent和-independent整合。虽然YidC没有影响MPIase依赖的集成Sec-independent基板在传统的检测系统，YidC显着加速整合时，基板量增加，在我们的纯系统？基于分析对于MtlA整合观察到YidC的类似加速。在膜内的亲水腔中具有氨基酸取代的YidC突变体在SEC独立整合的加速中是有缺陷的。值得注意的是，MPIase在YidC耗尽后上调。这些结果表明，YidC加速了膜蛋白的MPIase依赖性整合，表明MPIase和YidC在膜蛋白整合的催化循环中顺序和协同地发挥作用。
Bacterial membrane proteins are integrated into membranes through the concerted activities of a series of integration factors, including membrane protein integrase (MPIase). However, how MPIase activity is complemented by other integration factors during membrane protein integration is incompletely understood. Here, using inverted inner-membrane vesicle and reconstituted (proteo)liposome preparations from Escherichia coli cells, along with membrane protein integration assays and the PURE system to produce membrane proteins, we found that anti-MPIase IgG inhibits the integration of both the Sec-independent substrate 3L-Pf3 coat and the Sec-dependent substrate MtlA into E. coli membrane vesicles. MPIase-depleted membrane vesicles lacked both 3L-Pf3 coat and MtlA integration, indicating that MPIase is involved in the integration of both proteins. We developed a reconstitution system in which disordered spontaneous integration was precluded, which revealed that SecYEG, YidC, or both, are not sufficient for Sec-dependent and -independent integration. Although YidC had no effect on MPIase-dependent integration of Sec-independent substrates in the conventional assay system, YidC significantly accelerated the integration when the substrate amounts were increased in our PURE system?based assay. Similar acceleration by YidC was observed for MtlA integration. YidC mutants with amino acid substitutions in the hydrophilic cavity inside the membrane were defective in the acceleration of the Sec-independent integration. Of note, MPIase was up-regulated upon YidC depletion. These results indicate that YidC accelerates the MPIase-dependent integration of membrane proteins, suggesting that MPIase and YidC function sequentially and cooperatively during the catalytic cycle of membrane protein integration.