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

大肠杆菌细胞表面脂蛋白膜定位和质量控制的分子机制

基本信息

批准号：
14037212
负责人：
TOKUDA Hajime
金额：
$ 82.69万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2006
项目状态：
已结题

项目摘要

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.

大肠大肠杆菌的突变体，该突变体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Asp至Pro突变。发现内膜特异性分选信号是Lo1CDE回避信号，从而导致脂蛋白在内膜中的保留。进一步研究了磷脂组成对Lo1CDE回避功能的影响，并解析了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有一个疏水腔，这很可能是脂蛋白的结合位点。在Lo1B的功能重要的色氨酸残基进行了鉴定。Lo1A的43位Arg突变为其他残基。这些突变体的活动表明，在Lo1A和Lo1B之间的脂蛋白的疏水相互作用的强度的差异是至关重要的脂蛋白从Lo1A到Lo1B的有效转移。Lo1A突变体的分离表明，疏水腔经历打开和关闭后的结合和释放的脂蛋白。Lo1CDE用紧密结合的脂蛋白纯化。这是第一个可以与其底物分离的ABC转运蛋白的例子。研究发现，铜绿假单胞菌还具有5个Lo1蛋白，它们在脂蛋白的外膜分选中起重要作用。然而，铜绿假单胞菌中的分选信号被发现与E.杆菌