Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan.
J Bacteriol. 2011 Sep;193(18):4832-40. doi: 10.1128/JB.05013-11. Epub 2011 Jul 8.
Bacterial lipoproteins represent a subset of membrane-associated proteins that are covalently modified with lipids at the N-terminal cysteine. The final step of lipoprotein modification, N-acylation of apolipoproteins, is mediated by apolipoprotein N-acyltransferase (Lnt). Examinations with reconstituted proteoliposomes and a conditional mutant previously indicated that N-acylation of lipoproteins is required for their efficient release from the inner membrane catalyzed by LolA and LolCDE, the lipoprotein-specific chaperone and ABC transporter, respectively. Because Lnt is essential for Escherichia coli, a mutant lacking Lnt activity has not been isolated. However, we report here that lnt-null strains can be constructed when LolCDE is overproduced in strains lacking either the major outer membrane lipoprotein Lpp or transpeptidases that cross-link Lpp with peptidoglycan. Lipoproteins purified from the lnt-null strain exhibited increased mobility on SDS-PAGE compared to those from wild-type cells and could be sequenced by Edman degradation, indicating that lipoproteins in this mutant exist as apolipoproteins that lack N-acylation. Overexpression of Lpp in the lnt-null strain resulted in the accumulation of apoLpp in the inner membrane and caused growth arrest. In contrast to the release of mature Lpp in the presence of LolA and LolCDE, that of apoLpp from the inner membrane was significantly retarded. Furthermore, the amount of lipoproteins copurified with LolCDE was significantly reduced in the lnt-null strain. These results indicate that the affinity of LolCDE for apolipoprotein is very low, and therefore, overexpression of LolCDE is required for its release and sorting to the outer membrane.
细菌脂蛋白是一类与膜相关的蛋白质，它们在 N 端半胱氨酸上通过脂酰化共价修饰。脂蛋白修饰的最后一步，即载脂蛋白的 N 酰化，由载脂蛋白 N-酰基转移酶（Lnt）介导。用重组质体和以前的条件突变体进行的研究表明，脂蛋白的 N 酰化对于它们在 LolA 和 LolCDE 催化下从内膜有效释放是必需的，LolA 和 LolCDE 分别是脂蛋白特异性伴侣和 ABC 转运蛋白。由于 Lnt 对大肠杆菌是必需的，因此尚未分离出缺乏 Lnt 活性的突变体。然而，我们在这里报告，当 LolCDE 在缺乏主要外膜脂蛋白 Lpp 或连接 Lpp 与肽聚糖的转肽酶的菌株中过表达时，可以构建缺乏 Lnt 活性的突变体。从 lnt 缺失株中纯化的脂蛋白在 SDS-PAGE 上的迁移率与野生型细胞中的脂蛋白相比有所增加，并且可以通过 Edman 降解进行测序，表明该突变体中的脂蛋白作为缺乏 N 酰化的载脂蛋白存在。在 lnt 缺失株中过表达 Lpp 导致内膜中 apoLpp 的积累，并引起生长停滞。与 LolA 和 LolCDE 存在时成熟 Lpp 的释放相比，apoLpp 从内膜的释放明显延迟。此外，在 lnt 缺失株中，与 LolCDE 共纯化的脂蛋白数量显著减少。这些结果表明，LolCDE 对载脂蛋白的亲和力非常低，因此需要过表达 LolCDE 才能将其释放并分选到外膜。
