Analysis of MICOS independent Contact Sites between the inner and the outer membrane of mitochondria

线粒体在真核细胞中是必不可少的。它们以ATP的形式产生能量，并参与许多其他功能。例如，Fe-S簇、血红素、脂质和蛋白质在线粒体中合成。为了执行所有这些功能，线粒体必须产生并维持复杂的超微结构。这必须适应细胞的不同代谢状态。线粒体超微结构代表膜系统的形状。与大多数其他细胞器不同，线粒体被两层膜包围，即线粒体内膜和外膜。结构元素，接触点，连接这两个膜。这些接触位点由存在于线粒体内膜和外膜两者中的膜蛋白复合物组成。蛋白质、信号分子和代谢物必须穿过两个膜进行交换。因此，接触位点被认为对线粒体的功能以及细胞的功能非常重要。在电子显微镜分析时长时间观察接触部位。然而，接触位点的分子性质和功能在很大程度上是未知的，相比之下，介导的MICOS复合物，这是必不可少的线粒体超微结构的产生。此外，我们已经确定了一组六种线粒体蛋白，它们符合接触位点蛋白的条件。这组蛋白包括线粒体内膜的三种蛋白和线粒体外膜的三种蛋白。我们在之前的接触位点蛋白筛选中鉴定了这些候选物，这导致了MICOS的鉴定。然而，这些蛋白质不是MICOS的亚基。因此，它们很可能是新的、尚未表征的接触部位的组成部分。为了扩展候选蛋白的列表，我们将重复筛选具有显著修饰的接触位点蛋白，例如分析MICOS缺陷的线粒体。这就保证了新的候选人是除MICOS之外的联系网站的组成部分。候选者将成为通过共隔离分析识别新接触部位的基础。最后，我们将结合生物化学、细胞生物学和生物物理学方法对新的接触部位进行功能表征。迄今为止在酵母中鉴定的所有接触位点蛋白都是进化上高度保守的。因此，我们相信，在拟议的项目中获得的结果也是非常重要的高等真核生物，就像它是与MICOS复杂的情况。

Mitochondria are essential in eukaryotic cells. They produce energy in from of ATP and are involved in a multitude of additional functions. For instance, Fe-S-cluster, heme, lipids and proteins are synthesized in mitochondria. In order to perform all these functions, mitochondria have to generate and maintain a complex ultrastructure. This has to be adapted in response to the different metabolic states of the cell. Mitochondrial ultrastructure represents the shape of the membrane system. In contrast to most of the other organelles, mitochondria are surrounded by two membranes, the mitochondrial inner and outer membrane. Structural elements, the contact sites, connect these two membranes. These contact sites consist of membrane protein complexes present in both, the mitochondrial inner and outer membrane. Proteins, signal molecules and metabolites have to be exchanged across both membranes. Therefore, contact sites are supposed to be highly important for the functionality of mitochondria and thus for the cell. Contact sites are observed upon electron microscopy analysis for a long time. However, the molecular nature and the functions of contact sites are largely unknown, in contrast to those mediated by the MICOS complex, which is essential for generation of mitochondrial ultrastructure.The data we obtained so far show that in addition to MICOS further contact sites exist. Moreover, we already identified a set of six mitochondrial proteins, which qualify for contact site proteins. This set includes three proteins of the inner mitochondrial membrane and three of the outer mitochondrial membrane. We identified these candidates in our previous screen for contact site proteins, which led to the identification of MICOS. However, these proteins are not subunits of MICOS. Thus, they are in likely components of new, yet uncharacterized contact sites. To extend the list of candidate proteins, we will repeat the screen for contact site proteins with significant modifications, such as analysis of MICOS deficient mitochondria. This guarantees that the new candidates are components of contact sites, which exist in addition to MICOS. The candidates will be a basis for the identification of new contact sites by co-isolation analyses. Finally, we will functionally characterize the new contact sites with a combination of biochemical, cell-biological and biophysical methods. All contact site proteins, which were identified in yeast so far, are evolutionary highly conserved. Therefore, we are convinced that the results obtained in the proposed project are highly important also for higher eukaryotes, just as it is the case with MICOS complex.