Biogenesis of alpha-helical mitochondrial outer membrane proteins in higher eukaryotes

项目摘要/摘要 线粒体是起源于内共生的重要细胞器，它已经进化成在 真核生理学。线粒体的活性取决于镶嵌在线粒体外部的蛋白质 膜(OMM)，介导线粒体-细胞质的通讯，以及细胞的关键方面 功能，如细胞凋亡和先天免疫反应。A-螺旋蛋白是一个重要的子集 OMM蛋白质。然而，在哺乳动物细胞中，它们是如何被插入OMM的脂双层中的，直到 最近并不清楚。 我与丽贝卡·沃里斯和乔纳森·韦斯曼的实验室合作的工作已经确定 OMM驻留蛋白MTCH2，一个新的插入酶家族的定义成员，作为必需的和 足以将a-螺旋蛋白插入OMM。MTCH2是溶质的发散成员 载体家族25(SLC25)，并已进化为利用典型的转运蛋白折叠进行插入。生物信息学 分析表明，MTCH2在过氧化物体中有同源基因，而在全息虫中有同源基因，表明MTCH2是 A-螺旋蛋白跨膜和真核生物插入的共同机制。 在这一发现的基础上，这项建议旨在对a螺旋如何形成一个全面的理解 蛋白质被正确地定位于OMM，并被插入真核生物的脂质双层中。这项工作将 解决细胞生物学中的一个基本问题，因为OMM蛋白质组已经进化到支持越来越多的 高等真核生物中更复杂的功能。A-螺旋蛋白是由一个或多个 跨膜结构域(TA)，尽管它们的跨膜结构域在数量和生物物理范围上可能有很大的变化 疏水性等特征。这项建议旨在首先加深对MTCH2的理解 功能。首先，体内和生化技术将结合起来绘制底物TM所走的路线 通过MTCH2进入脂质双层，直接检测MTCH2的S保守的亲水沟是否具有 在这一过程中发挥直接作用。其次，这项工作将确定MTCH2的结构同源物是否具有 在真核生物中保留了它们的插入酶能力。 通过定义MTCH2的分子基础获得的生化技能和知识将是 结合系统功能基因组学来确定a-螺旋蛋白是如何靶向OMM的， 以及是否需要除MTCH2以外的其他因素来支持这一不同类别的生物发生。 总而言之，这一提议将提供对已演变为支持 真核生物的生命。此外，对管理a-螺旋OMM蛋白质插入的机制的深入了解将 对于开发与外线粒体膜蛋白失调相关的新治疗方法至关重要， 包括帕金森氏症和阿尔茨海默氏症等神经退行性疾病。

Project Summary/Abstract Mitochondria are essential organelles of endosymbiotic origin which have evolved to play critical roles in eukaryotic physiology. Mitochondrial activity is dependent on proteins embedded in the outer mitochondrial membrane (OMM), which mediate mitochondrial-cytoplasmic communication, and critical aspects of cellular function such as apoptosis and the innate immune response. a-helical proteins are an important subset of OMM proteins. However, how they get inserted into the lipid bilayer of the OMM in mammalian cells has until recently been unclear. Work by myself, in collaboration with Rebecca Voorhees and Jonathan Weissman’s labs, has identified the OMM resident protein MTCH2, a defining member of a novel family of insertases, as both necessary and sufficient for the insertion of a-helical proteins into the OMM. MTCH2 is a diverged member of the solute carrier family 25 (SLC25), and has evolved to exploit the canonical transporter fold for insertion. Bioinformatic analysis reveals that MTCH2 has a homolog in peroxisomes, and orthologs across holozoa, suggesting a common mechanism for a-helical protein insertion across membranes and eukaryotes. Building on this finding, this proposal aims to develop a comprehensive understanding of how a-helical proteins are correctly targeted to the OMM and inserted into the lipid bilayer across eukaryotes. This work will address a fundamental question in cell biology, as the OMM proteome has evolved to support increasingly more complex functions in higher eukaryotes. a-helical proteins are defined by the presence of one or more transmembrane domains (TAs), though their TMs can vary significantly in number and a range of biophysical characteristics such as hydrophobicity. This proposal aims to first develop a deeper understanding of MTCH2 function. First, in vivo and biochemical techniques will be combined to map the route a substrate TM takes through MTCH2 into the lipid bilayer, directly testing whether MTCH2’s conserved hydrophilic groove has a direct role in this process. Second, this work will establish whether structural homologs of MTCH2 have retained their insertase ability across eukaryotes. The biochemical skills and knowledge acquired by defining the molecular basis for MTCH2 will be combined with systematic functional genomics to establish how a-helical proteins get targeted to the OMM, and whether other factors besides MTCH2 are required to support the biogenesis of this diverse class. Cumulatively, this proposal will provide important insights into the mechanisms that have evolved to support eukaryotic life. Further, intimate knowledge of the machinery that governs a-helical OMM protein insertion will be critical for developing new treatments associated with outer mitochondrial membrane protein dysregulation, including neurodegenerative diseases such as Parkinson’s and Alzheimer’s.