Understanding how the mitochondrial phosphatase PGAM5 regulates mitochondrial dynamics

背景：线粒体是细胞的动力源，是细胞生死的重要介质。许多信号传导和代谢途径依赖于线粒体活性和影响功能。线粒体生物学的一个方面仍然是谜，引人注目的膜动力学，发生在线粒体内部和之间。单个单位不断发生膜融合和裂变反应，这种活动的总体原因尚不清楚。我的实验室对线粒体动力学有着长期的兴趣，我们已经发现了作为调节者的蛋白质和机制。一种特别有趣的蛋白质叫做PGAM5。PGAM5是一种磷酸酶，存在于两种异构体中，一个长异构体（l-PGAM5）具有线粒体靶向和膜结合基序，一个短异构体（s-PGAM5）其n端已被去除。目前尚不清楚这些异构体是否具有不同的功能，但我们最近获得的初步数据表明，它们在调节线粒体膜动力学方面具有非常不同的作用。假设：l-PGAM5和s-PGAM5在线粒体内有不同的定位，调节线粒体融合和裂变的不同方面。具体目标：1)使用多种细胞生物学和生化方法，我们将确定PGAM5的两种亚型在细胞内稳态和细胞生存需要线粒体膜动力学时的细胞器间定位。2)我们将使用BioID方法表征和比较两种PGAM5亚型的相互作用。潜在交互作用者列表将通过包括IP和PLA分析在内的辅助方法进行合理化和验证。我们的研究将揭示PGAM5在线粒体动力学中的重要功能机制。表征不同的定位和蛋白质相互作用组应该突出PGAM5如何促进线粒体生物学的分子细节。总之，这些研究应该帮助我们理解线粒体动力学作为细胞生物学的一个基本方面的原因，在所有真核生物中都是保守的。

Background: Mitochondria are the powerhouse of the cell and are critical mediators of cellular life and death. Many signalling and metabolic pathways are dependent on mitochondrial activity and impact function. One aspect of mitochondrial biology that remains enigmatic is the compelling membrane dynamics that occur within and between mitochondria. Individual units are constantly undergoing membrane fusion and fission reactions, the overall reason for this activity remains unknown. My lab has had a long-standing interest in mitochondrial dynamics, and we have been uncovering proteins and mechanisms that act as regulators. One particularly interesting protein is called PGAM5. PGAM5 is a phosphatase that exists in two isoforms, a long isoform (l-PGAM5) with a mitochondrial targeting and membrane binding motif and a short isoform (s-PGAM5) in which the N-terminus has been removed. It is not currently understood if the isoforms have different functions, but we have recently obtained preliminary data to suggest they have very different roles in regulating mitochondrial membrane dynamics. Hypothesis: l-PGAM5 and s-PGAM5 have both different localizations within mitochondria and regulate different aspects of mitochondrial fusion and fission. Specific aims: 1) Using a variety of cell biological and biochemical approaches, we will determine the inter-organelle localization of the two isoforms of PGAM5, both during cellular homeostasis, and during time of stress when mitochondrial membrane dynamics are required for cellular survival. 2) We will characterize and compare the interactome of the two PGAM5 isoforms using the BioID approach. Lists of potential interactors will be rationalized and validated with secondary approaches including IP and PLA analyses. Our studies should reveal important mechanistic insights into the function of PGAM5 in mitochondrial dynamics. Characterizing differing localizations and protein interactomes should highlight the molecular details of how PGAM5 contributes to mitochondrial biology. In sum these studies should help us to understand the reasons for mitochondrial dynamics as a fundamental aspect of cell biology that is conserved across all eukaryotes.