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有关，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，但调控线粒体膜动力学的分子机制尚不清楚。在过去的几年中，我们在酿酒酵母中发现了两种不同的gtpase，它们在调节线粒体形态和拷贝数方面起相反的作用。Dnm1p是一种与动力蛋白相关的GTPase，作用于线粒体外膜调节裂变。Fzo1p（模糊洋葱）是一个跨膜GTPase，调节线粒体对接和/或融合。这两种gtpase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用作研究人类细胞中控制线粒体动力学的分子机制的工具。

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.