LolA-dependent release of a lipid-modified protein from the inner membrane of Escherichia coli requires nucleoside triphosphate
LolA-dependent release of a lipid-modified protein from the inner membrane of Escherichia coli requires nucleoside triphosphate
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DOI：
10.1074/jbc.273.49.32576
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发表时间：
1998-12-04
影响因子：
4.8
通讯作者：
Tokuda, H
中科院分区：
文献类型：
--
作者：
Yakushi, T;Yokota, N;Tokuda, H
外膜导向的脂蛋白以与周质伴侣蛋白LOLA的复合体形式从大肠杆菌的内膜释放出来。依赖于LOLA的脂蛋白释放对脂蛋白的分选至关重要，因为它依赖于外膜特异性的分选信号。为了阐明LOLA依赖的脂蛋白释放所涉及的分子事件，我们试图建立一个体外检测系统。主要的外膜脂蛋白(LPP)在内膜加工成成熟的LPP后不久就失去了释放能力，因此不能作为体外系统的底物。构建了一种LPP衍生物L10P，并发现其在成熟后长期保持释放能力。L10P是在没有LOLA的情况下合成的，并在球体中进行放射性标记，因此它仍然锚定在球体的内膜上。然后制备含有L10P的右侧外膜微囊，并用于考察L10P的释放。除了LOLA，L10P的释放绝对需要核苷三磷酸(NTP)。非水解性NTP类似物强烈抑制NTP依赖的释放。外膜特异性分选信号对于L10P的体外释放是必不可少的，而且体外释放的L10P被特异性地掺入到外膜中，这些结果表明L10P的体外释放是体内反应，需要能量。
The outer membrane-directed lipoproteins are released from the inner membrane of Escherichia coli as a complex with LolA, a periplasmic chaperone. The LolA-dependent release of lipoproteins is critical for lipoprotein sorting as it depends on the outer membrane-specific sorting signal. To clarify molecular events involved in the LolA-dependent lipoprotein release, we attempted to establish an in vitro assay system. The major outer membrane lipoprotein (Lpp) was found to lose its release competence soon after being processed to mature Lpp in the inner membrane and therefore could not be used as a substrate for an in vitro system. An Lpp derivative, L10P, was constructed and found to retain the release competence long after its maturation. L10P was synthesized and radiolabeled in spheroplasts in the absence of LolA; therefore, it remained anchored to the inner membrane of spheroplasts. Right-side out membrane vesicles containing L10P were then prepared and used to examine the release of L10P. In addition to LolA, L10P release absolutely required nucleoside triphosphate (NTP). A non-hydrolyzable NTP analogue strongly inhibited the NTP-dependent release. The outer membrane-specific sorting signal was essential for the in vitro release of L10P, Furthermore, L10P released in vitro was specifically incorporated into the outer membrane, These results indicate that the in vitro release of L10P represents an in vivo reaction and requires energy.