Biogenesis of multispan proteins of the mitochondrial outer membrane

线粒体外膜多跨度蛋白的生物发生

• 批准号: 234739973
• 负责人: Professor Dr. Doron Rapaport
• 金额: --
• 依托单位: Interfakultäres Institut für Biochemie (IFIB)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/234739973?language=en
• 关键词: Biogenesis; multispan; proteins; mitochondrial; outer

The mitochondrial outer membrane (MOM) facilitates numerous interactions between the mitochondrial metabolic and genetic systems and the rest of the eukaryotic cell. Biogenesis of this membrane requires targeting of newly synthesized proteins to the organelle and their integration into the lipid bilayer. An important group of such MOM proteins are those that span the membrane with multiple α-helical segments. Despite ever-increasing evidence for the biological significance of these proteins, our understanding of their biogenesis process is scarce. The general objective of this study is to define the molecular mechanisms that underlie the biogenesis of α-helical multispan MOM proteins. We plan to address the following questions: 1) How do the membrane insertion factors Mim1 and Mim2 facilitate the membrane integration of MOM multispan proteins? 2) How does the import receptor Tom70 discriminate between MOM multispan proteins and carrier proteins destined to the inner membrane? and 3) What is the mammalian functional homologue of the fungal MIM complex?To that end, we will analyze in vitro, in organello and in vivo the import of precursor proteins into wild type mitochondria and into organelles from manipulated strains. To address aim 1, the membrane integration of multispan proteins will be studied by purifying Mim1 and Mim2 from a cell-free system. The ability of the detergent-solubilized Mim proteins to recognize MOM substrates will be investigated. Furthermore, the two proteins will be combined for the formation of the MIM complex and then the complex will be reconstituted into artificial lipid vesicles. To test whether the MIM complex builds the minimal machinery for membrane integration and functions as integrase, its functionality in such proteoliposomes will be monitored by testing their ability to mediate membrane integration of substrate proteins. Aim 2 will be addressed by mapping with photo-induced site-specific cross-linking the regions in either outer or inner membrane substrates, and within Tom70 itself, that are involved in receptor-substrate interactions. The identity of the cross-linking adducts will be investigated by Western blotting and mass spectrometry. Furthermore, the influence of the binding of various substrate proteins to Tom70 on the association of this receptor with either the rest of the TOM complex or the MIM complex will be studied by cross-linking and pull-down assays. Finally, to address Aim 3, mammalian functional homologues of Mim1/2 will be searched for by using yeast expression libraries comprising mouse or rat cDNAs. We will search for those cDNAs whose protein product is able to rescue the growth defect of yeast cells deleted for both MIM1 and MIM2 (mim1Δmim2Δ). The function of potential candidates will be verified in both yeast and mammalian cells. Taken together, this proposed study will shed new light on crucial processes in the biogenesis of mitochondrial outer membrane proteins.

线粒体外膜（MOM）促进了线粒体代谢和遗传系统与真核细胞其余部分之间的许多相互作用。这种膜的生物发生需要新合成的蛋白质靶向细胞器并整合到脂质双分子层中。一类重要的MOM蛋白是那些跨越多个α-螺旋片段的膜。尽管越来越多的证据表明这些蛋白质的生物学意义，但我们对它们的生物发生过程的了解很少。本研究的总体目标是确定α-螺旋多跨MOM蛋白生物发生的分子机制。我们计划解决以下问题：1)膜插入因子Mim1和Mim2如何促进MOM多跨蛋白的膜整合？2)输入受体Tom70如何区分MOM多跨蛋白和到达内膜的载体蛋白？3)真菌MIM复合物的哺乳动物功能同源物是什么？为此，我们将在体外、细胞器和体内分析前体蛋白进入野生型线粒体和从操纵菌株进入细胞器的情况。为了解决目标1，将通过从无细胞系统中纯化Mim1和Mim2来研究多跨蛋白的膜整合。洗涤剂溶解的Mim蛋白识别MOM底物的能力将被研究。再将这两种蛋白结合形成MIM复合物，再将该复合物重组为人工脂质囊泡。为了测试MIM复合物是否构建了最小的膜整合机制并发挥整合酶的功能，将通过测试其介导底物蛋白膜整合的能力来监测其在蛋白质脂质体中的功能。目的2将通过光诱导的位点特异性交联来映射膜外或膜内底物以及Tom70本身中参与受体-底物相互作用的区域。交联加合物的身份将通过Western blotting和质谱法进行研究。此外，各种底物蛋白与Tom70结合对该受体与TOM复合物或MIM复合物其余部分结合的影响将通过交联和下拉试验进行研究。最后，为了解决Aim 3，将使用包含小鼠或大鼠cdna的酵母表达文库来搜索Mim1/2的哺乳动物功能同源物。我们将寻找那些蛋白产物能够挽救因MIM1和MIM2缺失的酵母细胞生长缺陷的cdna （mim1Δmim2Δ）。潜在候选分子的功能将在酵母和哺乳动物细胞中得到验证。综上所述，这项拟议的研究将揭示线粒体外膜蛋白生物发生的关键过程。

项目成果

The chaperone-binding activity of the mitochondrial surface receptor Tom70 protects the cytosol against mitoprotein-induced stress.

• DOI: 10.1016/j.celrep.2021.108936
• 发表时间: 2021-04-06
• 期刊: Cell reports
• 影响因子: 8.8
• 作者: Backes S;Bykov YS;Flohr T;Räschle M;Zhou J;Lenhard S;Krämer L;Mühlhaus T;Bibi C;Jann C;Smith JD;Steinmetz LM;Rapaport D;Storchová Z;Schuldiner M;Boos F;Herrmann JM
• 通讯作者: Herrmann JM