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Dissection of polypeptide translocator function of SecY

SecY 多肽易位子功能剖析

基本信息

批准号：
02404087
负责人：
ITO Koreaki
金额：
$ 10.11万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

项目摘要

SecY is an integral membrane protein and believed to be a central component of the protein translocator in Escherichia coli. We have identified dominant negative mutations of secY whose expression interfered with protein export. Such mutations proved to reside in the C-terminal third of SecY, mostly in the cytoplasmic domain 5 (C5). On of them (secY^d1), a three residue deletion in the C5-TM9 interface, was strongly dominant-negative. The dominant negative mutant protein will sequester the interacting components in an inactive complex, and hence compete with SecY^+ for the formtion of the functiona SecY complex. Linker insertion mutations that suppressed secY^d1 were localized within the C4-TM7 region is important for SecY's interaction with other components of the system.Consistent with our model, the secY24(Ts) mutation in the C4 domain was found to impair the SecY's interaction with SecE. Also, overproduction of either SecE or "ORF12" in the secDF operon overcame the export interference. Futhermore, we were able to identify a new gene, ydr, as another dosage-dependent overcomer of secY^d1. Although overexpressed (and presumably uncomplexed) SecY was rapidly degraded in vivo, it was significantly stabilized by simultaneous overproduction of either SecE, Orf12,or Ydr. Interestingly, overproduction of Ydr in the secY24 mutant cell severely interfered with protein export and the viability. Ydr, a hydrophilic 181-residue protein with is C-terminal region potentially forming an amphiphilic alpha-helix, is loosely associated with the membrane.Finally we addressed whether any factors are required for membrane protein integration. Our mutational analyses suggested that FtsH, a putative membrane bound ATPase with homology to a family of eukaryotic ATPases, has a role in assuring efficient anchoring of a hydrophobic stretch to the membrane as well as in the translocation process itself.

SecY是一种完整的膜蛋白，被认为是大肠杆菌中蛋白质转运体的中心组分。我们已经鉴定出secY的显性负突变，其表达干扰蛋白质输出。这种突变被证明存在于SecY的C-末端三分之一，主要是在胞质结构域5（C5）。其中一个（secY ^d1）是C5-TM 9界面上的三个残基缺失，为强显性负性。显性失活突变蛋白会将相互作用的组分隔离在一个失活的复合物中，从而与SecY^+竞争功能性SecY复合物的形成。抑制secY ^d1的连接子插入突变位于C4-TM 7区域，对于SecY与系统其他组分的相互作用是重要的。与我们的模型一致，发现C4结构域中的secY 24（Ts）突变削弱了SecY与SecE的相互作用。此外，SecDF操纵子中SecE或“ORF 12”的过量产生克服了输出干扰。因此，我们能够鉴定出一个新的基因ydr，作为secY ^d1的另一个剂量依赖性克服者。虽然过度表达（大概未复合）SecY在体内迅速降解，它是显着稳定的同时过量生产的SecE，Orf 12，或YDR。有趣的是，在secY 24突变体细胞中过量产生Ydr严重干扰蛋白质输出和活力。Ydr是一个亲水性的181个氨基酸残基的蛋白质，其C-末端区域可能形成一个两亲性的α-螺旋，与膜松散地结合。我们的突变分析表明，FtsH，一个假定的膜结合ATP酶与真核ATP酶家族的同源性，具有确保有效锚定的疏水拉伸膜，以及在易位过程本身的作用。