Structural and Functional Studies for Mitochondrial Protein Translocations

描述(申请人提供)：蛋白质跨线粒体膜转位在线粒体生物发生中起关键作用。蛋白质从细胞质到线粒体基质的转运是通过外膜(TOM)复合体的转位酶和内膜(TIM)复合体的转位酶来实现的。这项提议的长期目标是对酵母TOM和TIM复合体进行X射线晶体对数RAP HIE研究，以揭示这些转座子促进前体跨越线粒体外膜和内膜的基本机制。在TOM易位子中，Tom70p作为线粒体前体的受体，具有内部靶向信号。TimSOp、Tim21p和Tim44p是TIM23易位子中的重要成员。在膜间隙(IMS)中，TimSOp作为具有N端线粒体靶向序列的前体的受体。Tim21p可以与TOM复合体成员Tom22p相互作用，促进TOM转位蛋白前体的释放。Tim44p是一种外周膜蛋白，与线粒体基质侧的内膜稳定结合。我们测定了酵母菌Tom/Op和Tim44P的晶体结构，分辨率分别为3.0A和3.2A。我们已经结晶了酵母菌TimSOp和Tim21p，并对TimSOp晶体进行了2.7A的X射线衍射。通过噬菌体展示文库筛选和等温滴定量热法(ITC)相结合的方法，我们鉴定了Tom70p的多肽底物。我们构建了Tom70p与其多肽底物的蛋白质复合体。我们还构建了TimSOp与线粒体靶向多肽Cox4N的复合体。已构建了Tim21p和Tom22p C-末端结构域的蛋白质复合体，用于结晶实验。我们建议确定Tom70P-肽底物络合物的晶体结构。我们打算结晶并确定TimSOp靶向多肽复合体和Tim21p-Tom22p蛋白复合体的晶体结构。我们还计划解决Tim44P和洗涤剂FOS-胆碱复合体的晶体结构/最后，我们将进行基于结构的突变研究，以测试我们提出的TOM和TIM转座子模型。诱变研究将采用体外和体内两种检测方法。总而言之，这项建议的目的是进行全面的研究，试图了解TOM和TIM复合体在蛋白质从细胞质到线粒体的易位中发挥作用的基本机制。

DESCRIPTION (provided by applicant): Protein translocations across mitochondria membranes play critical roles in mitochondria biogenesis. The protein transports from the cell cytosol to the mitochondria matrix are carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complex. The long-term goal of this proposal is to carry out X-ray crystal log rap hie studies on yeast TOM and TIM complexes to uncover the basic mechanisms by which these translocons facilitate the precursors across the outer and inner mitochondria membranes. In the TOM translocon, Tom70p functions as the receptor for mitochondria precursors with internal targeting signals. TimSOp, Tim21p and Tim44p are important members in TIM23 translocon. In the intermembrane space (IMS), TimSOp functions as the receptor for the precursor with the N-terminal mitochondrion targeting sequence. Tim21p can interact with TOM complex member Tom22p to facilitate the release of the precursor from the TOM translocon. Tim44p is a peripheral membrane protein and is stably associated with the mitochondria inner membrane at the matrix side. We have determined the crystal structure of yeast Tom/Op and Tim44p to 3.0A and 3.2A resolution, respectively. We have crystallized yeast TimSOp and Tim21p and the TimSOp crystals diffracted X-ray to 2.7A. By use of the combination of phage peptide display library screening and Isothermal Titration Calorimetry (ITC) technique, we have identified a peptide substrate for Tom70p. We have constituted the protein complex of Tom70p and its peptide substrate. We have also constituted the complex of TimSOp and the mitochondrion targeting peptide Cox4N. The protein complex of Tim21p and Tom22p C-terminal domain has been constituted for crystallization trials. We propose to determine the crystal structures of the Tom70p-peptide substrate complex. We intend to crystallize and determine the crystal structures of the TimSOp-targeting peptide complex and Tim21p-Tom22p protein complex. We also plan to solve the crystal structure of Tim44p and detergent FOS-CHOLINE complex/finally, we will conduct the structure-based mutagenesis studies to test our proposed models for TOM and TIM translocons. Both in vitro and in vivo assays will be utilized in the mutagenesis studies. Collectively, the aims of this proposal constitute the comprehensive studies that seek to understand the basic mechanisms via which the TOM and TIM complexes function in protein translocations from cell cytosol to mitochondrion.