Classifying β-Barrel Assembly Substrates by Manipulating Essential Bam Complex Members
Classifying β-Barrel Assembly Substrates by Manipulating Essential Bam Complex Members
复制标题
DOI：
10.1128/jb.00263-16
复制
发表时间：
2016-07-01
影响因子：
3.2
通讯作者：
Silhavy, Thomas J.
中科院分区：
文献类型：
--
作者：
Mahoney, Tara F.;Ricci, Dante P.;Silhavy, Thomas J.
大肠杆菌外膜（OM）的生物发生是一个保守而重要的过程。完整的β-桶外膜蛋白（OMPs），它代表了OM的主要组成部分，组装取决于周质伴侣和异五聚体β-桶组装机（BAM复合物）在OM。然而，并不是所有的外膜蛋白都受到相同分子伴侣或非必需Bam复合物成员中的无效突变的影响，这表明存在对有效组装具有不同要求的底物类别。我们先前已经证明了两类底物，一类包括大的、低丰度的和难以组装的底物，其严重依赖于SurA以及Skp和FkpA，另一类包括相对简单和丰富的底物，其不依赖于SurA，但强烈依赖于BamB进行组装。在这里，我们描述了bamD中的新突变，其在不改变成熟蛋白质序列的情况下使BamD水平降低10倍和>25倍。我们利用这些突变，以及先前表征的降低野生型BamA水平的突变，揭示了第三类底物。这些突变优先引起多聚体蛋白水平的显著降低。这种易感性的多聚体，以降低数量的BAM机器在细胞中可能表明，需要多个BAM复合物，以有效地组装成OM的多聚体蛋白质。重要的是外膜（OM）的革兰氏阴性菌，如大肠杆菌，作为一个选择性的渗透屏障，防止吸收有毒分子和抗生素。整合β-桶蛋白（OMP）由β-桶组装机（BAM）组装，其组分在线粒体、叶绿体和所有革兰氏阴性细菌（包括许多临床相关的致病物种）中是保守的。BAM是必不可少的OM生物合成，并容纳了各种各样的客户端蛋白质，然而，一个机械模型，占BAM的选择性和广泛的底物范围是缺乏的。在这里，我们表明，多聚体外膜蛋白的组装比单体外膜蛋白的组装受到更强烈的影响时，必不可少的BAM复合物的成分是有限的，这表明需要多个BAM复合物组装多聚体蛋白。
The biogenesis of the outer membrane (OM) of Escherichia coli is a conserved and vital process. The assembly of integral beta-barrel outer membrane proteins (OMPs), which represent a major component of the OM, depends on periplasmic chaperones and the heteropentameric beta-barrel assembly machine (Bam complex) in the OM. However, not all OMPs are affected by null mutations in the same chaperones or nonessential Bam complex members, suggesting there are categories of substrates with divergent requirements for efficient assembly. We have previously demonstrated two classes of substrates, one comprising large, low-abundance, and difficult-to-assemble substrates that are heavily dependent on SurA and also Skp and FkpA, and the other comprising relatively simple and abundant substrates that are not as dependent on SurA but are strongly dependent on BamB for assembly. Here, we describe novel mutations in bamD that lower levels of BamD 10-fold and>25-fold without altering the sequence of the mature protein. We utilized these mutations, as well as a previously characterized mutation that lowers wildtype BamA levels, to reveal a third class of substrates. These mutations preferentially cause a marked decrease in the levels of multimeric proteins. This susceptibility of multimers to lowered quantities of Bam machines in the cell may indicate that multiple Bam complexes are needed to efficiently assemble multimeric proteins into the OM.IMPORTANCEThe outer membrane (OM) of Gram-negative bacteria, such as Escherichia coli, serves as a selective permeability barrier that prevents the uptake of toxic molecules and antibiotics. Integral beta-barrel proteins (OMPs) are assembled by the beta-barrel assembly machine (Bam), components of which are conserved in mitochondria, chloroplasts, and all Gram-negative bacteria, including many clinically relevant pathogenic species. Bam is essential for OM biogenesis and accommodates a diverse array of client proteins; however, a mechanistic model that accounts for the selectivity and broad substrate range of Bam is lacking. Here, we show that the assembly of multimeric OMPs is more strongly affected than that of monomeric OMPs when essential Bam complex components are limiting, suggesting that multiple Bam complexes are needed to assemble multimeric proteins.