BIOGENESIS OF THE MITOCHONDRIAL INNER MEMBRANE

本申请中描述的研究程序涉及蛋白质输入到细胞中的机制。 实验模型是芽殖酵母酿酒酵母，这是哺乳动物系统的理想模型，因为蛋白质输入高度保守。 先前的工作已经确定了线粒体内膜蛋白质的新输入途径，这与具有氨基末端靶向前序列的前体所使用的途径不同。 该输入途径的组分包括膜间隙的可溶性Tim 8/Tim 13和Tim 9/Tim 10复合物以及内膜的TIM 22复合物（Tim 12、Tim 22和Tim 54）。 DDP 1（耳聋/肌张力障碍蛋白;与Tim 8同源）的突变导致人类疾病Mohr-Tranebjaerg综合征，这很可能是由蛋白质输入机制缺陷引起的。 本文提出的研究目的是结合生物化学、生物物理学和遗传学方法来确定这一输入途径的分子机制。 具体地，将确定由Tim 8/Tim 13和Tim 9/Tim 10复合物识别的内膜底物及其基序。 此外，将阐明Tim 8/Tim 13和Tim 9/Tim 10复合物将底物护送到内膜的机制。 使用温度敏感的tim 12和tim 22突变体，TIM 22复合物的其他成分将被确定和表征的位置和功能。 拟议的项目将扩大有关蛋白质插入线粒体内膜机制的基础知识，扩展目前的研究，这些研究通常集中在蛋白质如何到达线粒体的可溶性隔室。 此外，这些研究将有助于对蛋白质如何插入膜以及线粒体生物合成缺陷如何导致线粒体疾病的基本理解。

The research program described in this application deals with the mechanism of protein import into the mitochondrion. The experimental model is the budding yeast Saccharomyces cerevisiae, which is an ideal model for mammalian systems because protein import is highly conserved. Previous work has identified a new import pathway for proteins of the mitochondrial inner membrane, which is distinct from the pathway used by precursors with an amino-terminal targeting presequence. Components of this import pathway include the soluble Tim8/Tim13 and Tim9/Tim10 complexes of the intermembrane space and the TIM22 Complex (Tim12, Tim22, and Tim54) of the inner membrane. Mutations in DDP1 (deafness/dystonia protein; homologous to Tim8) cause the human disease Mohr-Tranebjaerg Syndrome, which is most likely caused by a defective protein import machinery. The objective of the research proposed here is to define the molecular mechanisms of this import pathway with a combined biochemical, biophysical and genetic approach. Specifically, the inner membrane substrates and their motifs, which are recognized by the Tim8/Tim13 and Tim9/Tim10 complexes, will be determined. Moreover, the mechanism by which the Tim8/Tim13 and Tim9/Tim10 complexes escort the substrates to the inner membrane will be elucidated. Using temperature-sensitive tim12 and tim22 mutants, additional components of the TIM22 complex will be identified and characterized with respect to location and function. The proposed project will expand fundamental knowledge about the mechanism of protein insertion into the mitochondrial inner membrane, extending present studies that have focused generally on how proteins reach the soluble compartments of the mitochondria. Also, these studies will contribute to the basic understanding of how proteins insert into membranes and how defects in mitochondrial biogenesis can contribute to mitochondrial diseases.