Biogenesis of multispan proteins of the mitochondrial outer membrane

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他部分之间的大量相互作用。这种膜的生物发生需要将新合成的蛋白质定向到细胞器，并将它们整合到脂质双层中。一组重要的这种MOM蛋白是那些跨越膜的具有多个α-螺旋片段的蛋白。尽管越来越多的证据表明这些蛋白质具有生物学意义，但我们对它们的生物发生过程的了解还很少。这项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多跨蛋白和内膜载体蛋白？3)真菌MIM复合体在哺乳动物中的功能同源物是什么？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操纵菌株的细胞器的情况。为了解决目标1，将通过从无细胞系统中纯化Mim1和Mim2来研究多跨区蛋白的膜整合。洗涤剂增溶的Mim蛋白识别MOM底物的能力将被研究。此外，这两种蛋白质将结合在一起形成MIM复合体，然后该复合体将被重组为人造脂泡。为了测试MIM复合体是否建立了膜整合的最小机制并发挥整合酶的功能，将通过测试它们介导底物蛋白质膜整合的能力来监测它在这类蛋白脂质体中的功能。AIM 2将通过光诱导的位点特异性交叉连接外膜或内膜底物以及Tom70本身中参与受体-底物相互作用的区域来解决。交联物的鉴定将通过蛋白质印迹和质谱学进行。此外，各种底物蛋白与Tom70的结合对该受体与TOM复合体或MIM复合体的其余部分的结合的影响将通过交联和下拉分析来研究。最后，为了解决目标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。我们将寻找那些其蛋白产物能够挽救同时缺失MIM1和MIM2(MIM1ΔMIM2Δ)的酵母细胞的生长缺陷的基因。潜在候选者的功能将在酵母和哺乳动物细胞中得到验证。综上所述，这项拟议的研究将为线粒体外膜蛋白生物发生中的关键过程提供新的线索。

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.