MECHANISMS OF MITOCHONDRIAL FISSION

描述（由申请人提供）：控制线粒体分裂的基本分子机制从酵母到人类都是保守的。我们正在使用遗传学、生物化学和结构方法来了解芽殖酵母线粒体裂变的机制。我们之前发现了一种与动力蛋白相关的GTPase，称为Dnm1p。Dnm1p从细胞质循环到线粒体外膜，在那里形成线粒体裂变复合体（mfc），将线粒体室分裂成更小的单位。第二种蛋白Mdv1p与Dnm1p结合，是mfc激活所必需的。我们最近在线粒体外膜上发现了第三种裂变蛋白，叫做Fis1p。Fis1p独立地将Dnm1p和Mdv1p招募到线粒体膜上，并在功能性mfc的组装中发挥关键作用。另外Dnm1p-Dnm1p、Dnm1p- Mdv1p和Mdv1p-Mdv1p相互作用在膜募集和MFC组装中发挥作用。本文提出的研究旨在了解Mdv1p和Dnm1p膜募集和MFC组装背后的时间和物理相互作用。具体而言，我们提出：1)验证Mdv1p的NTE （N-Terminal Extension）与Fis1p TPR折叠结合袋相互作用的假设；2)绘制Dnm1p膜募集和MFC组装所需的Fis1p残基；3)求解酵母Fis1p和Fis1p-Mdv1p结合位点复合物的溶液结构；4)确定不同的Dnm1p分子相互作用在MFC前后组装事件中的作用。我们将确定和描述的一些相互作用是阻断线粒体裂变的抑制剂的潜在目标。在临床环境中，此类抑制剂可用于减少或预防由药物治疗、创伤或疾病引起的线粒体介导的细胞损伤或死亡。

DESCRIPTION (provided by applicant): The basic molecular machinery controlling mitochondrial fission is conserved from yeast to humans. We are using genetic, biochemical and structural approaches to understand the mechanism of mitochondrial fission in budding yeast. We previously identified a dynamin-related GTPase called Dnm1p. Dnm1p cycles from the cytoplasm onto the outer mitochondrial membrane where it forms Mitochondrial Fission Complexes (MFCs) that divide mitochondrial compartments into smaller units. A second protein, Mdv1p associates with Dnm1p and is required for activation of MFCs. We recently identified a third fission protein on the outer mitochondrial membrane called Fis1p. Fis1p independently recruits Dnm1p and Mdv1p to the mitochondrial membrane and plays a critical role in assembly of functional MFCs. Additional Dnm1p-Dnm1p, Dnm1p- Mdv1p and Mdv1p-Mdv1p interactions play a role in membrane recruitment and MFC assembly. The studies proposed here are aimed at understanding the temporal and physical interactions that underlie Mdv1p and Dnm1p membrane recruitment and MFC assembly. Specifically, we propose to: 1) test the hypothesis that the NTE (N-Terminal Extension) of Mdv1p interacts with the binding pocket of the Fis1p TPR fold, 2) map residues in Fis1p required for Dnm1p membrane recruitment and MFC assembly, 3) solve the solution structure of yeast Fis1p and the Fis1p-Mdv1p binding site complex, and 4) define the role of different Dnm1p molecular interactions in pre-and post-MFC assembly events. Some of the interactions we will identify and characterize are potential targets for inhibitors that block mitochondrial fission. In a clinical setting, such inhibitors could be used to reduce or prevent mitochondrial-mediated cell damage or death resulting from drug treatment, trauma or disease.