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。它们以三磷酸腺苷的形式产生能量，并参与许多额外的功能。例如，铁-S-簇、血红素、脂质和蛋白质都是在线粒体中合成的。为了执行所有这些功能，线粒体必须产生并维持一个复杂的超微结构。这必须根据细胞的不同新陈代谢状态进行调整。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的是，线粒体周围有两层膜，即线粒体内膜和外膜。结构元素，接触部位，连接这两层膜。这些接触部位由存在于线粒体内膜和外膜的膜蛋白复合体组成。蛋白质、信号分子和代谢物必须通过两层膜进行交换。因此，接触部位被认为对线粒体的功能非常重要，从而对细胞也是如此。通过电子显微镜分析，长期观察到接触部位。然而，与MICOS复合体介导的分子性质和功能相比，接触位点的分子性质和功能在很大程度上是未知的。MICOS复合体是线粒体超微结构形成所必需的。我们迄今获得的数据表明，除了MICOS之外，还存在更多的接触位点。此外，我们已经鉴定了一组六种线粒体蛋白，它们符合接触位点蛋白的条件。这套蛋白包括线粒体内膜的三种蛋白质和线粒体外膜的三种蛋白质。我们在之前的接触位点蛋白筛选中确定了这些候选蛋白，这导致了MICOS的鉴定。然而，这些蛋白质不是MICOS的亚基。因此，它们可能是新的、但尚未确定特征的接触点的组成部分。为了扩展候选蛋白质的列表，我们将重复筛选具有显著修改的接触位点蛋白质，例如分析MICOS缺陷的线粒体。这保证了新的候选人是除MICOS之外还存在的联系地点的组成部分。候选人将成为通过共隔离分析确定新的接触地点的基础。最后，我们将结合生化、细胞生物学和生物物理方法对新的接触部位进行功能表征。到目前为止在酵母中发现的所有接触位点蛋白在进化上都是高度保守的。因此，我们相信，在拟议的项目中获得的结果对高等真核生物也是非常重要的，就像MICOS复合体一样。

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.