Functional analysis of mitochondrial ribosomes: Biogenesis and Function

线粒体包含一个微型基因组，编码少数疏水蛋白，这些蛋白由膜相关的线粒体核糖体(有丝分裂糖体)合成。线粒体翻译机制的生物发生或功能缺陷损害呼吸，是人类严重疾病的原因。有丝分裂体与细菌或真核细胞胞浆的核糖体有惊人的不同。有丝分裂糖体含有70-80个不同的蛋白质亚基(在面包酵母中为73个)，因此与胞质对应的蛋白质亚基相似。在酵母的遗传筛选中发现了线粒体中的许多核糖体组装因子、解旋酶、修饰酶和调节成分。尽管如此，有丝分裂体是如何组装的在很大程度上仍然是未知的，这也是这项提议的主题。在这里，我们申请了该项目的第二个资助期，其中将解决两个目标。目标1将专注于有丝分裂体蛋白的进口。与其他基质蛋白相比，许多有丝分裂核糖体蛋白缺乏N端基质靶向信号。我们最近在模型蛋白mrp17中发现了靶向信息，这与前序列中发现的特征惊人地不同。我们的初步数据表明，MRp17走上了TOM-TIM23的导入途径，该途径也被其他基质蛋白使用，但它不利用依赖于Hsp70的导入马达。因此，MRP17导入是不依赖于ATP的，但需要内膜极高的膜电位。在这个项目中，我们将阐明MRp17和其他有丝分裂体蛋白非传统进口过程的分子机制。一方面，我们想要描述这种不依赖于ATP的蛋白质输入途径的机制细节。另一方面，我们计划揭开这一非常规进口过程的生理后果。为此，我们将开发新的竞争性进口试验，使其能够直接在体外和体内比较不同类型线粒体的进口效率。目的2重点研究线粒体核糖体的组装过程。我们开发了一种系统，在该系统中，线粒体核糖体可以通过下调线粒体RNA聚合酶暂时从线粒体中耗尽。在重新启动转录时，线粒体核糖体被重建。我们将使用这个同步系统来监测有丝分裂体大小亚基的组装，并使用复杂组学分析将组装过程分成单独的反应。初步研究已经揭示了部分稳定的组装中间产物的积累，我们现在将用蛋白质组学进一步表征这些中间产物。此外，我们将确定组装因子和修饰酶在核糖体生物发生过程中的相关性。因此，我们期望对线粒体蛋白质翻译机制的生物合成和功能的生物学过程有基本的了解。

Mitochondria contain a miniature genome that codes for a handful of hydrophobic proteins which are synthesized by membrane-associated mitochondrial ribosomes (mitoribosomes). Defects in the biogenesis or function of the mitochondrial translation machinery compromise respiration and are cause of severe diseases in humans.Mitoribosomes are surprisingly different to ribosomes of the bacterial or eukaryotic cytosol. Mitoribosomes contain 70-80 different protein subunits (73 in baker’s yeast) and thus are similarly complex than their cytosolic counterparts. Genetic screens in yeast identified many ribosome assembly factors, helicases, modifying enzymes and regulatory components in the mitochondria. Nonetheless, how mitoribosomes are assembled is still largely unknown and the topic of this proposal. Here, we apply for the second funding period of the project in which two aims will be addressed.Aim 1 will focus on the import of mitoribosomal proteins. In contrast to other matrix proteins, many mitoribosomal proteins lack N-terminal matrix targeting signals. We recently identified the targeting information in the model protein Mrp17 which is surprisingly different to the features found in presequences. Our preliminary data indicate that Mrp17 embarks on the TOM-TIM23 import pathway that is also used by other matrix proteins, however, it does not make use of the Hsp70-dependent import motor. Hence, Mrp17 import is ATP-independent but requires an extremely high membrane potential of the inner membrane. In this project we will elucidate the molecular mechanisms of the unconventional import process of Mrp17 and other mitoribosomal proteins. On the one hand, we want to characterize the mechanistic details of this ATP-independent protein import pathway. On the other hand, we plan to unravel the physiological consequences of this unconventional import process. To this end, we will develop novel competitive import assays which allow it to compare the import efficiency of different types of mitochondria directly in vitro as well as in vivo. Aim 2 focuses on the processes by which mitochondrial ribosomes are assembled. We developed a system in which mitochondrial ribosomes can be temporarily depleted from mitochondria by down-regulation of the mitochondrial RNA polymerase. Upon re-initiation of transcription, mitochondrial ribosomes are re-built. We will use this synchronized system to monitor the assembly of the large and small subunit of the mitoribosome and stage the assembly process into individual reactions using complexome analysis. Preliminary studies already revealed the accumulation of partially stable assembly intermediates which we will now further characterize by proteomics. Furthermore, we will identify the relevance of assembly factors and modifying enzymes during ribosome biogenesis. Thereby, we expect fundamental insights into the biological processes underlying the biosynthesis and function of mitochondrial protein translation machinery.