Development of an in vitro mitochondrial fusion assay.

体外线粒体融合测定的开发。

• 批准号: 6560524
• 负责人: Janet M. Shaw
• 金额: $ 11.22万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-07 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6560524
• 关键词: SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; bioassay; cell free system; fluorescence resonance energy transfer; fluorimetry; green fluorescent proteins; guanosinetriphosphatases; hydrolysis; membrane fusion; membrane proteins; membrane reconstitution /synthesis; method development; mitochondrial membrane; protein structure function; western blottings; SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; bioassay; cell free system; fluorescence resonance energy transfer; fluorimetry; green fluorescent proteins; guanosinetriphosphatases; hydrolysis; membrane fusion; membrane proteins; membrane reconstitution /synthesis; method development; mitochondrial membrane; protein structure function; western blottings

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.

描述（由申请人提供）：线粒体形态和拷贝数的变化与包括神经系统疾病和某些类型的癌症在内的多种人类疾病有关。尽管已经对线粒体的代谢功能进行了广泛的研究，但不了解调节线粒体膜动力学的分子机制。在过去的几年中，我们在酿酒酵母中表征了两种不同的GTPases，这些GTPase反对调节线粒体形态和拷贝数。 DNM1P是一种与动力学相关的GTPase，可作用于外部线粒体膜以调节裂变。 FZO1P（模糊洋葱）是一种跨膜GTPase，可调节线粒体对接和/或融合。两种GTP酶都有人类的同源物，当突变时会导致线粒体形态的缺陷。这些发现说明了如何有效地将酵母用作研究人类细胞中线粒体动力学的分子机制的工具。 迄今为止特征的大多数异型膜融合事件都需要在供体膜上（称为V-SNARES）上的蛋白质和靶膜上的蛋白质（称为T-SNARES），该蛋白在融合之前可以正确识别和对接这两个腔室。我们的假设是，FZO1蛋白定义了一种使用GTP水解来调节线粒体线粒体膜对接和/或同质型融合反应的新型圈套。尽管我们目前正在活酵母细胞中检验这一假设，但对FZO1P的生化活性的完全了解将需要一个体外测定，以重构线粒体融合。本应用中描述的实验的目的是使用从酿酒酵母细胞中分离出的成分来开发用于线粒体融合的体外测定。然后将使用此测定法研究酿酒酵母在融合过程中的作用。我们提出的研究最终将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。