Structural and Functional Studies for Mitochondrial Protein Translocations

描述（由申请人提供）：蛋白质跨线粒体膜易位在线粒体生物发生中起关键作用。蛋白质从细胞质转运到线粒体基质是通过外膜复合物转位酶（translocase of outer membrane，TOM）和内膜复合物转位酶（translocase of inner membrane，TIM）完成的。该提案的长期目标是对酵母TOM和TIM复合物进行X射线晶体记录研究，以揭示这些translocon促进前体穿过线粒体外膜和内膜的基本机制。在TOM易位子中，Tom 70 p作为线粒体前体的受体发挥作用，具有内部靶向信号。TimSOp、Tim 21 p和Tim 44 p是TIM 23易位子的重要成员。在膜间隙（IMS）中，TimSOp作为具有N-末端双链体靶向序列的前体的受体发挥作用。Tim 21 p可以与TOM复合体成员Tom 22 p相互作用以促进前体从TOM易位子的释放。Tim 44 p是一种外周膜蛋白，在基质侧与线粒体内膜稳定结合。我们测定了酵母Tom/Op和Tim 44 p的晶体结构，分辨率分别为3.0和3.2。我们对酵母TimSOp和Tim 21 p进行了结晶，晶体的X射线衍射强度达到2.7A。利用噬菌体肽库筛选和等温滴定量热法（ITC）相结合的方法，我们鉴定了Tom 70 p的一个肽底物。我们构建了Tom 70 p与其肽底物的蛋白复合物。我们还构建了TimSOp与靶向肽Cox 4 N的复合物。Tim 21 p和Tom 22 p C-末端结构域的蛋白质复合物已被构建用于结晶试验。我们建议确定Tom 70 p-肽底物复合物的晶体结构。我们打算结晶并确定TimSOp靶向肽复合物和Tim 21 p-Tom 22 p蛋白质复合物的晶体结构。我们还计划解决Tim 44 p和洗涤剂FOS-CHOLINE复合物的晶体结构，最后，我们将进行基于结构的诱变研究，以测试我们提出的TOM和TIM转座子模型。体外和体内试验均将用于诱变研究。总的来说，这项建议的目的构成了全面的研究，试图了解的基本机制，通过该TOM和TIM复合物的功能，从细胞胞质溶胶的蛋白质易位到细胞外基质。

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.