Molecular Mechanisms underlying membrane localization and qualify control of lipoproteins in Escherichia coli cell surface

分离出一种大肠杆菌突变体，可将内膜特异性脂蛋白转运到外膜。该突变体在Lo1CDE复合物的Lo1C亚基中携带从Asp到Pro的突变。内膜特异性分选信号被发现是一个Lo1CDE回避信号，从而导致脂蛋白滞留在内膜。然后发现Lo1CDE的避免功能受到磷脂组成的影响。分析了Lo1A和Lo1B的晶体结构。尽管它们的氨基酸序列不同，但它们的结构非常相似。Lo1A和Lo1B具有疏水空腔，这很可能是脂蛋白的结合位点。鉴定出Lo1B中功能重要的Trp残基。Lo1A的43位Arg突变为其他残基。这些突变体的活性表明，Lo1A和Lo1B之间与脂蛋白疏水相互作用强度的差异对于脂蛋白从Lo1A到Lo1B的有效转移至关重要。Lo1A突变体的分离表明，疏水腔在脂蛋白的结合和释放过程中开启和关闭。用紧密相关脂蛋白纯化Lo1CDE。这是ABC转运蛋白可以与其底物分离的第一个例子。铜绿假单胞菌还具有5种Lo1蛋白，它们在脂蛋白的外膜分选中起重要作用。然而，铜绿假单胞菌的分选信号与大肠杆菌不同。

An E. coli mutant, which transports the inner membrane-specific lipoprotein to the outer membrane, was isolated. The mutant carried the Asp to Pro mutation in Lo1C subunit of the Lo1CDE complex. The inner membrane-specific sorting signal was found to be a Lo1CDE avoidance signal, thereby causing the retention of lipoproteins in the inner membrane. It was then found that the Lo1CDE avoidance function was affected by phospholipids compositions.The crystal structures of Lo1A and Lo1B were solved. Their structures were very similar despite that their amino acid sequences were dissimilar. Lo1A and Lo1B have a hydrophobic cavity, which is most likely the binding site for lipoproteins. Functionally important Trp residues in Lo1B were identified. Arg at position 43 of Lo1A was mutated to other residues. Activities of these mutants revealed that differences in the strength of hydrophobic interaction with lipoproteins between Lo1A and Lo1B are critically important for efficient transfer of lipoproteins from Lo1A to Lo1B. Isolation of Lo1A mutants suggested that the hydrophobic cavity undergoes opening and closing upon the binding and release of lipoproteins. Lo1CDE was purified with tightly associated lipoproteins. This is the first example of an ABC transporter that can be isolated with its substrate.It was found that Pseudomonas aeruginosa also possesses five Lo1 proteins, which play an important role in the outer membrane sorting of lipoproteins. However, the sorting signal in P. aeruginosa was found to be different from that in E. coli.