Mechanism of organelle targeting and topogenesis of membrane proteins

本研究以膜拓扑发生和分选为研究背景，对几种典型的膜蛋白进入内质网膜的拓扑发生过程进行了深入的研究，包括所有跨膜片段的机制。I型信号锚定序列，其易位的N-末端部分，介导的易位的N-结构域远长于已经假定。对于长N-结构域的易位，既不需要NTP也不需要管腔hsp 70同源物BiP。然而，核糖体在N结构域移位中起着关键作用。建议将易位链保持在易位子的适当位置。我们还提供了新的证据支持我们的新的拓扑发生模式，通过非疏水性的部分被迫形成跨膜处置的内部I型信号锚定序列。此外，我们广泛地研究了介导高度疏水性膜蛋白ABC转运蛋白的线粒体输入的线粒体靶向前序列，并证明ABC（B10）亚型的N-末端135个残基的亲水片段是抑制SRP介导的ER靶向并介导线粒体输入的膜蛋白特异性信号序列。通过该序列，膜蛋白的靶向模式从共翻译的ER转运转变为翻译后的线粒体输入。我们还对线粒体外膜蛋白Tom 22的拓扑结构基因进行了测序，并建立了Tom 40的实验体系，在大肠杆菌中高效表达Tom 40，变性剂溶解，纯化，体外复性。

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.