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Mechanism of organelle targeting and topogenesis of membrane proteins

膜蛋白的细胞器靶向和拓扑发生机制

基本信息

批准号：
14380294
负责人：
SAKAGUCHI Masao
金额：
$ 9.47万
依托单位：
University of Hyogo (2004)Kyushu University (2002-2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

In this research, based on our research background of membrane topogenesis and sorting, we have extensively examined whole topogenesis process of several typical membrane proteins into the endoplasmic reticulum membranes, including mechanism for all the transmembrane segments. The type I signal-anchor sequence, which translocates the N-terminal portion, mediates the translocation of N-domain much longer than had been assumed. For the translocation of the long N-domain, neither NTP's nor luminal hsp70 homologue, BiP, is required. Ribosome, however, plays critical function for the N-domain translocation. It is suggested to maintain the translocating chain at an appropriate position of the translocon. We also provided new evidences supporting our novel topogenic mode via which non-hydrophobic segment is forced to form transmembrane disposition by internal type I signal-anchor sequence. Furthermore, we extensively examined the mitochondrial targeting presequence which mediate the mitochondrial import of highly hydrophobic membrane proteins, ABC transporters and demonstrated the N-terminal 135 residues hydrophilic segment of ABC(B10) isoform is a membrane protein specific signal sequence that suppresses SRP-mediated ER targeting and mediate mitochondrial import. By the sequence the targeting mode of membrane proteins switches from co-translational ER translocation to the post-translational mitochondrial import. We also clarified topogeinc sequences of Tom22, mitochondrial outer membrane protein, and established experimental system for Tom40, by which Tom40 is over-expressed in E.coli, solubilized by denaturing reagent, purified, and renatured in vitro.

在本研究中，我们基于膜拓扑学和膜分选的研究背景，对几种典型的膜蛋白进入内质网膜的整个拓扑化过程进行了广泛的研究，包括所有跨膜片段的发生机制。I类信号-锚定序列移位N-末端部分，介导N-结构域移位的时间比之前假设的要长得多。对于长N-结构域的易位，既不需要NTP的，也不需要管腔HSP70的同源物Bip。然而，核糖体在N结构域易位中起着关键作用。建议将转运链维持在转运子的适当位置。我们还提供了新的证据支持我们的新的拓扑发生模式，通过内部I型信号锚定序列迫使非疏水片段形成跨膜处置。此外，我们广泛研究了线粒体靶向前序列，它介导了高度疏水的膜蛋白ABC转运体的输入，并证明ABC(B10)亚型的N-末端135个残基亲水片段是一个膜蛋白特异的信号序列，它抑制SRP介导的ER靶向并介导线粒体的输入。通过该序列，膜蛋白的靶向模式从共翻译的内质网易位转变为翻译后的线粒体输入。我们还明确了线粒体外膜蛋白Tom22的拓扑基因序列，并建立了Tom40在大肠杆菌中高效表达、变性试剂溶解、纯化和体外复性的实验体系。