Determining the mechanism of mitochondrial outer membrane fusion

项目摘要 线粒体是无数细胞功能所必需的。细胞内的线粒体网络已经建立 并由运动、融合和分裂的协调活动维持。线粒体融合 特别是有益的，因为它增加细胞能量，防止细胞死亡。分子机器 介导线粒体外膜和内膜融合的分子是动力蛋白超家族的成员。AS 这样，它们是大的自组装的GTP酶，利用核苷酸水解的能量来重塑 膜。这些蛋白质如何将自组装和催化循环与膜系留和脂质相结合 混合情况尚不清楚。本工作的重点是研究线粒体外区的分子机制。 膜融合。尽管线粒体外区都需要Mitofusin1(Mfn1)和Mitofusin2(Mfn2) 膜融合，它们在功能上是不同的。我们发现线粒体融合是最有效的 当Mfn1和Mfn2在相反的膜上时。这表明它们有独特的分子 特点。为了解决这个问题，我们将在整个分析过程中比较和对比Mfn1和Mfn2 确定它们独特的生化特性。我们对GTPase结构域的评估将揭示 核苷酸结合、水解和释放的机制。我们将重建对Mitofusin的依赖 膜系留。这将使我们能够确定催化循环在膜系留和 脂质成分对丝裂原蛋白系留活性的影响。利用与疾病相关的错义突变，我们 将确定GTPase结构域的独特特征。我们将确定Mitofusin的分子决定因素 复杂的装配。我们将结合细胞研究和强大的生化分析，包括 脂类的重组和与蛋白脂质体的含量混合。这些研究将为我们提供对 线粒体外膜融合的独特机制。

Project Summary Mitochondria are required for a myriad of cellular functions. The mitochondrial network in cells is established and maintained by the coordinate activities of movement, fusion and division. Mitochondrial fusion in particular is beneficial as it increases cellular energy and protects against cell death. The molecular machines that mediate mitochondrial outer and inner membrane fusion are members of the dynamin superfamily. As such, they are large self-assembling GTPases that harness the energy from nucleotide hydrolysis to remodel membranes. How these proteins couple self-assembly and the catalytic cycle to membrane tethering and lipid mixing is not known. The focus of this work is to examine the molecular mechanism of mitochondrial outer membrane fusion. Although Mitofusin1 (Mfn1) and Mitofusin2 (Mfn2) are both required for mitochondrial outer membrane fusion, they are functionally distinct. We discovered that mitochondrial fusion is most efficient when Mfn1 and Mfn2 are on opposite membranes. This suggests that they have unique molecular characteristics. To address this, we will compare and contrast Mfn1 and Mfn2 throughout our analyses and determine their unique biochemical properties. Our evaluation of the GTPase domain will reveal the mechanism of nucleotide binding, hydrolysis and release. We will reconstitute Mitofusin-dependent membrane tethering. This will allow us to determine the role of the catalytic cycle in membrane tethering and the role of lipid composition on Mitofusin tethering activity. Using disease associated missense mutations, we will identify unique features of the GTPase domain. We will identify the molecular determinants of Mitofusin complex assembly. We will combine cellular studies with powerful biochemical analyses, including reconstitution of lipid and content mixing with proteoliposomes. These studies will provide new insight into the unique mechanism of mitochondrial outer membrane fusion.