Development of an in vitro mitochondrial fusion assay.

描述（由申请人提供）：线粒体形态和拷贝数的变化与多种人类疾病相关，包括神经系统疾病和某些类型的癌症。虽然线粒体的代谢功能已被广泛研究，但调节线粒体膜动力学的分子机制尚不清楚。在过去的几年里，我们在S.酿酒酵母的作用相反，调节线粒体形态和拷贝数。Dnm 1 p是一种动力蛋白相关的GTP酶，作用于线粒体外膜以调节裂变。Fzo 1 p（fuzzy onions）是一种跨膜GTdR，调节线粒体对接和/或融合。这两种GTP酶都有人类同源物，当突变时，会导致线粒体形态缺陷。这些发现说明了酵母如何有效地用作研究控制人类细胞线粒体动力学的分子机制的工具。 迄今为止表征的大多数异型膜融合事件需要供体膜上的蛋白质（称为v-SNARE）和靶膜上的蛋白质（称为t-SNARE），其允许在融合之前正确识别和对接两个隔室。我们的假设是，Fzo 1蛋白定义了一种新型的SNARE，利用GTP水解来调节细胞-线粒体膜对接和/或同型融合反应。虽然我们目前正在活酵母细胞中测试这一假设，但要完全了解Fzo 1 p的生物化学活性，还需要进行重建线粒体融合的体外试验。本申请中描述的实验的目的是开发使用从S.酿酒酵母细胞S的作用。然后将使用该测定研究融合过程中酿酒酵母Fzo 1 p的表达。我们提出的研究将最终提供有关真核细胞线粒体融合的分子和生化基础的新信息。

DESCRIPTION (provided by applicant): Changes in mitochondrial morphology and copy number are associated with a variety of human diseases including neurological disorders and some types of cancer. Although the metabolic functions of mitochondria have been extensively studied, the molecular mechanisms that regulate mitochondrial membrane dynamics are not understood. In the past few years, we characterized two different GTPases in S. cerevisiae that act in opposition to regulate mitochondrial morphology and copy number. Dnm1p is a dynamin-related GTPase that acts on the outer mitochondrial membrane to regulate fission. Fzo1p (fuzzy onions) is a transmembrane GTPase that regulates mitochondrial docking and/or fusion. Both GTPases have human homologues that, when mutated, cause defects in mitochondrial morphology. These findings illustrate how effectively yeast can be used as a tool to study the molecular mechanisms that control mitochondrial dynamics in human cells. Most heterotypic membrane fusion events characterized to date require proteins on the donor membrane (called v-SNAREs) and proteins on the target membranes (called t-SNAREs) that allow proper recognition and docking of the two compartments prior to fusion. It is our hypothesis that the Fzo1 protein defines a new type of SNARE that utilizes GTP hydrolysis to regulate mitochondrial-mitochondrial membrane docking and/or homotypic fusion reactions. Although we are currently testing this hypothesis in living yeast cells, a complete understanding of the biochemical activity of Fzo1p will require an in vitro assay that reconstitutes mitochondrial fusion. The goal of the experiments described in this application is to develop an in vitro assay for mitochondrial fusion using components isolated from S. cerevisiae cells. The role of S. cerevisiae Fzo1p during fusion will then be studied using this assay. The studies we propose will ultimately provide new information about the molecular and biochemical basis of mitochondrial fusion in eukaryotic cells.