BIOGENESIS OF THE MITOCHONDRIAL INNER MEMBRANE

本申请中描述的研究计划涉及蛋白质输入线粒体的机制。实验模型是萌芽酵母酿酒酵母，这是一个理想的哺乳动物系统模型，因为蛋白质输入是高度保守的。以前的工作已经发现了线粒体内膜蛋白的一种新的输入途径，这不同于具有氨基末端靶向前序列的前体所使用的途径。这种输入途径的成分包括膜间隙的可溶性Tim8/Tim13和Tim9/Tim10复合体以及内膜的TIM22复合体(Tim12、Tim22和Tim54)。DDP1(耳聋/肌张力障碍蛋白；与Tim8同源)的突变会导致人类疾病莫尔-特兰布贾尔格综合征，这很可能是由蛋白质输入机制缺陷引起的。这项研究的目的是用生化、生物物理和遗传学相结合的方法来确定这一输入途径的分子机制。具体地说，将确定Tim8/Tim13和Tim9/Tim10络合物识别的内膜底物及其基序。此外，TIM8/TIM13和TIM9/TIM10络合物护送底物到内膜的机制将被阐明。利用温度敏感的tim12和tim22突变体，将从位置和功能方面鉴定和鉴定TIM22复合体的其他成分。拟议的项目将扩大关于蛋白质插入线粒体内膜机制的基础知识，扩展目前一般侧重于蛋白质如何到达线粒体可溶间隔的研究。此外，这些研究将有助于基本理解蛋白质如何插入膜，以及线粒体生物发生中的缺陷如何导致线粒体疾病。

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.