Radioisotope Center, University of Tokyo, Tokyo, Japan.
J Bacteriol. 2012 Jul;194(14):3643-50. doi: 10.1128/JB.00553-12. Epub 2012 May 4.
The Escherichia coli LolA protein is a lipoprotein-specific chaperone that carries lipoproteins from the inner to the outer membrane. A dominant negative LolA mutant, LolA(I93C/F140C), in which both (93)Ile and (140)Phe are replaced by Cys, binds tightly to the lipoprotein-dedicated ABC transporter LolCDE complex on the inner membrane and therefore inhibits the detachment of outer membrane-specific lipoproteins from the inner membrane. We found that the expression of this mutant strongly induced lolA gene transcription. The depletion of the LolA or LolB protein also triggered lolA gene transcription, indicating that the inhibition of outer membrane lipoprotein transport triggers lolA transcription. To elucidate the mechanism, we isolated mutants that are unable to induce lolA transcription using the lacZ gene fused to the lolA promoter as a reporter and found that the Rcs phosphorelay system directly regulates lolA transcription. An outer membrane lipoprotein, RcsF, was essential for this activation, while the coactivator RcsA was dispensable. Taking the observation that an RcsF mutant localized in the inner membrane constitutively activated the Rcs phosphorelay system into consideration, the results shown here strongly suggest that correct lipoprotein sorting to the outer membrane is monitored by RcsF, which plays a key role in the Rcs stress response system.
大肠杆菌 LolA 蛋白是一种脂蛋白特异性伴侣，可将脂蛋白从内膜转运到外膜。一种具有显性负效应的 LolA 突变体 LolA(I93C/F140C)，其中 93 位的异亮氨酸和 140 位的苯丙氨酸均被替换为半胱氨酸，与内膜上的脂蛋白专用 ABC 转运蛋白 LolCDE 复合物紧密结合，从而抑制外膜特异性脂蛋白从内膜上脱离。我们发现，该突变体的表达强烈诱导了 lolA 基因的转录。LolA 或 LolB 蛋白的耗竭也触发了 lolA 基因的转录，表明外膜脂蛋白转运的抑制会触发 lolA 转录。为了阐明机制，我们分离了无法使用融合到 lolA 启动子的 lacZ 基因作为报告基因诱导 lolA 转录的突变体，发现 Rcs 磷酸传递系统直接调节 lolA 转录。一种外膜脂蛋白 RcsF 对于这种激活是必需的，而共激活因子 RcsA 则是可有可无的。考虑到 RcsF 突变体在膜内的定位会导致 Rcs 磷酸传递系统持续激活这一观察结果，这些结果强烈表明，正确的脂蛋白分拣到外膜是由 RcsF 监测的，它在 Rcs 应激反应系统中起着关键作用。
