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Studies on Escherichia coli YidC insertase during membrane protein insertion.

机译：膜蛋白插入过程中大肠杆菌YidC插入酶的研究。

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In bacteria, it has been widely recognized that SecYEG translocon is the major translocase used to insert proteins into or export proteins across the membrane bilayer. It has also been found that SecYEG translocase helps proteins adopt their membrane topology and proper conformation. Recent studies have added another pathway into the paradigm. The Oxa1/Alb3/YidC family is a group of evolutionarily conserved proteins existing in mitochondria, chloroplasts, and bacteria. The Oxa1/Alb3/YidC pathway is believed to independently insert proteins into membrane bilayer and facilitate their subunits assembly in the membrane. In addition, the Oxa1/Alb3/YidC proteins can assist the Sec pathway by helping release the membrane proteins from the Sec translocase channel and moving them laterally into the lipid bilayer. In this research, there are two objectives. First, the interaction of YidC with the SecDF components of the SecYEG translocase was studied in details. The specific region responsible for the interaction was evaluated and the importance of YidC-SecDF binding in membrane protein insertion was investigated. Second, the features of the YidC substrates that promote membrane protein insertion were studied. In particular, the hydrophobicity threshold converting a YidC substrate to Sec pathway and the individual contribution of the twenty amino acids for transmembrane insertion was measured.; In chapter 2, we investigated the interaction of YidC with SecDF of the SecYEG translocase. YidC was previously discovered to interact with Sec translocase using SecDF as a bridge. SecD and SecF are accessory proteins in the SecYEG complex that facilitate membrane insertion but their exact function is not known. To address the importance of the YidC-SecDF interaction, we constructed a series of deletion mutants and tested their binding efficiency. The co-purification experiments showed that the loss of amino acids 24 to 264 causes a defect in YidC binding to SecDF. Then we cloned different sequences within this region of YidC and tested the ability of those sequences to bind to SecDF in vitro. In this study we found that the amino acid segment 215 to 265 is able to interact with SecDF. In addition, we investigated the membrane insertion of some "YidC only" substrates and YidC/SecYEG substrates under these deletion mutants. To our surprise, the YidC mutant lacking residues 24 to 264 can insert both substrates into the membrane very well. This result suggests that the interacting with SecDF is not critical to the function of YidC in the SecYEG pathway. Another possibility is that the high amount of YidC compensates the low binding affinity. The natural copies of YidC and SecYEG in E. coli cells are at a ratio of about 250 to one.; Presently, only a few membrane proteins have been confirmed to be a YidC pathway substrates. The transmembrane domains of YidC substrates were shown to physically contact the YidC molecules during the membrane insertion process by crosslinking studies. We assumed that hydrophobic interaction is the force bringing the two molecules together. To quantify the hydrophobic force we set up a model system. Two endonuclease sites were engineered at both sides of the gene encoding transmembrane domain of M13 procoat, so we can replace the transmembrane domain with the artificial sequences. The theoretical apparent free energy of twenty amino acids was measured using this system. A 19 amino acid segment consisting of only alanine, which is not considered a hydrophobic amino acid, is able to insert into and anchor the protein into the membrane bilayer. The 19 amino acids stretch of tyrosine, which is more hydrophilic as shown in our hydrophobicity scale, is unable to be inserted into membrane. When the artificial segment becomes even more hydrophilic, as the 19 serine stretch, this protein domain is secreted across membrane via SecYEG pathway. We also determined that the shortest hydrophobic domain to be inserted into membrane is a
机译：在细菌中，已经广泛认识到SecYEG转座子是主要的转座酶，用于将蛋白质插入膜双层或跨膜双层输出蛋白质。还已经发现SecYEG易位酶帮助蛋白质采用其膜拓扑结构和适当的构象。最近的研究为该范式添加了另一条途径。 Oxa1 / Alb3 / YidC家族是存在于线粒体，叶绿体和细菌中的一组进化保守蛋白。人们认为Oxa1 / Alb3 / YidC途径可将蛋白质独立插入膜双层中，并促进其亚基在膜中的组装。另外，Oxa1 / Alb3 / YidC蛋白可以通过帮助从Sec转移酶通道蛋白释放膜蛋白并将其横向移动到脂质双层中来辅助Sec途径。在这项研究中，有两个目标。首先，详细研究了YidC与SecYEG易位酶的SecDF组分的相互作用。评价了负责相互作用的特定区域，并研究了YidC-SecDF结合在膜蛋白插入中的重要性。其次，研究了促进膜蛋白插入的YidC底物的特征。特别地，测量了将YidC底物转化为Sec途径的疏水性阈值以及二十种氨基酸对跨膜插入的贡献。在第二章中，我们研究了YidC与SecYEG易位酶的SecDF的相互作用。以前发现YidC使用SecDF作为桥与Sec易位酶相互作用。 SecD和SecF是SecYEG复合物中的辅助蛋白，可促进膜插入，但其确切功能尚不清楚。为了解决YidC-SecDF相互作用的重要性，我们构建了一系列缺失突变体并测试了它们的结合效率。共纯化实验表明，氨基酸24至264的缺失会导致YidC与SecDF的结合缺陷。然后，我们在YidC的这个区域内克隆了不同的序列，并测试了这些序列在体外与SecDF结合的能力。在这项研究中，我们发现第215至265位氨基酸能够与SecDF相互作用。另外，我们研究了在这些缺失突变体下某些“仅YidC”底物和YidC / SecYEG底物的膜插入。令我们惊讶的是，缺少24至264位残基的YidC突变体可以将两种底物很好地插入膜中。该结果表明，与SecDF的相互作用对于SecYEG途径中YidC的功能并不关键。另一种可能性是，大量的YidC补偿了低的结合亲和力。 YidC和SecYEG在大肠杆菌细胞中的天然拷贝比例约为250：1。目前，仅少数膜蛋白被证实是YidC途径的底物。通过交联研究显示，在膜插入过程中，YidC底物的跨膜结构域与YidC分子物理接触。我们假设疏水相互作用是将两个分子结合在一起的力。为了量化疏水力，我们建立了一个模型系统。在编码M13 procoat跨膜结构域的基因的两侧设计了两个核酸内切酶位点，因此我们可以用人工序列代替跨膜结构域。使用该系统测量了二十个氨基酸的理论表观自由能。仅由丙氨酸组成的19个氨基酸区段（不认为是疏水氨基酸）能够插入蛋白质并将其锚定在膜双层中。酪氨酸的19个氨基酸段（在我们的疏水性等级中显示为更亲水）无法插入膜中。当人造片段变得更加亲水时，随着19个丝氨酸的延伸，该蛋白结构域会通过SecYEG途径跨膜分泌。我们还确定了要插入膜中的最短疏水域是

著录项

  • 作者

    Xie, Kun.;

  • 作者单位

    The Ohio State University.;

  • 授予单位 The Ohio State University.;
  • 学科 Biology Cell.
  • 学位 Ph.D.
  • 年度 2007
  • 页码 110 p.
  • 总页数 110
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 细胞生物学;
  • 关键词

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