Dominant Negative lptE Mutation That Supports a Role for LptE as a Plug in the LptD Barrel
Dominant Negative lptE Mutation That Supports a Role for LptE as a Plug in the LptD Barrel
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DOI：
10.1128/jb.02142-12
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发表时间：
2013-03-01
影响因子：
3.2
通讯作者：
Silhavy, Thomas J.
中科院分区：
文献类型：
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作者：
Grabowicz, Marcin;Yeh, Jennifer;Silhavy, Thomas J.
脂多糖（LPS）是革兰氏阴性外膜（OM）双层的主要外叶成分。 LptD 和 LptE 的二分蛋白复合物将 LPS 组装到 OM 中。已经确定 LptE 有助于其 β 桶伙伴 LptD 的折叠和组装，但报道的生化证据表明 LptE 还具有其他功能。在这里，我们分离出显性失活 lptE 突变，试图了解这些功能。即使存在野生型 lptE 基因，lptE14 突变也会增加 OM 对红霉素的渗透性。我们发现 lptE14 突变不会导致 LptD 组装或 LPS 输出缺陷。 secA 中自发的 IS1 插入通过去除 SecA 的 C 端 SecB 结合域来抑制 lptE14 红霉素敏感性。虽然这种抑制突变广泛阻碍了前蛋白的 SecB 依赖性分泌，但我们表明抑制是 OM 中 LptD 水平降低的直接且特定的结果。我们认为，lptE14 会导致 LptD beta 桶的堵塞不良，并且 OM 中无效堵塞的 LptD-LptE14 复合物的减少会降低红霉素的渗透性。因此，lptE14 支持所提出的插塞式 LptE-LptD 布置。
Lipopolysaccharide (LPS) is the major outer leaflet constituent of the Gram-negative outer membrane (OM) bilayer. A bipartite protein complex of LptD and LptE assembles LPS into the OM. It has been established that LptE assists folding and assembly of its beta-barrel partner LptD, yet reported biochemical evidence suggested additional LptE functions. Here, we isolated dominant negative lptE mutations, seeking to inform these functions. The lptE14 mutation increased OM permeability to erythromycin, even when the wild-type lptE gene was present. We show that the lptE14 mutation does not cause a defect in either LptD assembly or LPS export. A spontaneous IS1 insertion in secA suppressed lptE14 erythromycin sensitivity by removing the C-terminal SecB-binding domain of SecA. While this suppressor mutation broadly impeded SecB-dependent secretion of preproteins, we show that suppression was a direct and specific consequence of reduced LptD levels in the OM. We suggest that lptE14 causes poor plugging of the LptD beta barrel and that a reduction of ineffectively plugged LptD-LptE14 complexes in the OM decreases permeability to erythromycin. Hence, lptE14 supports a proposed plug-and-barrel LptE-LptD arrangement.