Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan.
Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
J Biol Chem. 2014 Apr 11;289(15):10530-10539. doi: 10.1074/jbc.M113.539270. Epub 2014 Feb 25.
The Lol system comprising five Lol proteins, LolA through LolE, sorts Escherichia coli lipoproteins to outer membranes. The LolCDE complex, an ATP binding cassette transporter in inner membranes, releases outer membrane-specific lipoproteins in an ATP-dependent manner, causing formation of the LolA-lipoprotein complex in the periplasm. LolA transports lipoproteins through the periplasm to LolB on outer membranes. LolB is itself a lipoprotein anchored to outer membranes, although the membrane anchor is functionally dispensable. LolB then localizes lipoproteins to outer membranes through largely unknown mechanisms. The crystal structure of LolB is similar to that of LolA, and it possesses a hydrophobic cavity that accommodates acyl chains of lipoproteins. To elucidate the molecular function of LolB, a periplasmic version of LolB, mLolB, was mutagenized at various conserved residues. Despite the lack of acyl chains, most defective mutants were insoluble. However, a derivative with glutamate in place of leucine 68 was soluble and unable to localize lipoproteins to outer membranes. This leucine is present in a loop protruding from mLolB into an aqueous environment, and no analogous loop is present in LolA. Thus, leucine 68 was replaced with other residues. Replacement by acidic, but not hydrophobic, residues generated for the first time mLolB derivatives that can accept but cannot localize lipoproteins to outer membranes. Moreover, deletion of the leucine with neighboring residues impaired the lipoprotein receptor activity. Based on these observations, the roles of the protruding loop of LolB in the last step of lipoprotein sorting are discussed.
Lol 系统由五个 Lol 蛋白（LolA 至 LolE）组成，将大肠杆菌脂蛋白分拣到外膜。LolCDE 复合物是内膜中的一种 ATP 结合盒转运蛋白，以 ATP 依赖的方式释放外膜特异性脂蛋白，导致 LolA-脂蛋白复合物在外周质中形成。LolA 将脂蛋白通过周质转运到外膜上的 LolB。LolB 本身是一种脂蛋白，锚定在外膜上，尽管膜锚定在功能上是可有可无的。LolB 然后通过大致未知的机制将脂蛋白定位到外膜上。LolB 的晶体结构与 LolA 相似，它具有一个容纳脂蛋白酰链的疏水性腔。为了阐明 LolB 的分子功能，对各种保守残基进行了突变的periplasmic 版本的 LolB，mLolB。尽管缺乏酰基链，大多数有缺陷的突变体都是不可溶的。然而，用谷氨酸取代亮氨酸 68 的衍生物是可溶的，并且无法将脂蛋白定位到外膜上。该亮氨酸存在于从 mLolB 突入水溶液的环中，而 LolA 中不存在类似的环。因此，用其他残基取代了亮氨酸 68。用酸性而不是疏水性残基取代，首次生成了可以接受但不能将脂蛋白定位到外膜的 mLolB 衍生物。此外，缺失带有相邻残基的亮氨酸会损害脂蛋白受体活性。基于这些观察结果，讨论了 LolB 突出环在脂蛋白分拣的最后一步中的作用。
