Development of an in vitro mitochondrial fusion assay.

描述(申请人提供)：线粒体形态和拷贝数的变化与多种人类疾病有关，包括神经障碍和某些类型的癌症。虽然线粒体的代谢功能已经得到了广泛的研究，但调控线粒体膜动力学的分子机制尚不清楚。在过去的几年里，我们鉴定了酿酒酵母中两种不同的GTP酶，它们相互对立地调节线粒体的形态和拷贝数。Dnm1p是一种与动力蛋白相关的GTP酶，作用于线粒体外膜，调节分裂。Fzo1p(模糊洋葱)是一种跨膜GTP酶，调节线粒体停靠和/或融合。这两种GTP酶都有人类同源物，当突变时，会导致线粒体形态的缺陷。这些发现说明了酵母如何有效地被用作研究控制人类细胞线粒体动力学的分子机制的工具。 到目前为止，大多数异型膜融合事件都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)，以便在融合之前正确识别和对接两个隔室。我们的假设是，Fzo1蛋白定义了一种新型的SNARE，它利用GTP水解来调节线粒体-线粒体膜对接和/或同型融合反应。尽管我们目前正在活生生的酵母细胞中测试这一假设，但要完全理解Fzo1p的生化活性，将需要重建线粒体融合的体外测试。本申请中描述的实验的目标是开发一种使用从酿酒酵母细胞中分离出来的成分进行线粒体融合的体外检测方法。然后将使用本实验研究酿酒酵母Fzo1p在融合过程中的作用。我们提出的研究最终将提供关于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.