Regulation of ESCRT-III activity in yeast

项目总结 ESCRT-III复合体的膜断裂是蛋白质所需的一种高度保守的细胞机制 在溶酶体和其他细胞过程中的降解。ESCRT-III膜断裂活性最好 在体外重建试验的背景下被定义，但在体内操作的机制控制 ESCRT-III在生理条件下的活性还知之甚少。这样做的长期目标是 该项目的目的是了解内吞途径中的膜转运机制。的目标是 本应用是以发芽酵母酿酒酵母为模型系统，对其进行机械结构鉴定。 调节内体ESCRT-III的核糖体异构体该项目的中心假设是膜断裂 通过ESCRT-III，内体的去泛素化机制是负调控的。该计划的基本原理 拟议的研究是，一旦知道ESCRT-III膜断裂活动是如何调节的，这 这一过程很可能是通过药理学来操纵的，为新的和创新的方法铺平了道路 在预防和治疗与ESCRT-III有关的遗传和传染病方面。的具体目标 该项目旨在确定由DOA4调控ESCRT-III的机制，并确定机制-- 一种将Doa4从抑制中解放出来的主义。Doa4是一种泛素水解酶，可使跨膜脱泛素。 蛋白质货物被分选到ILV中，但DoA4也通过调节在ILV膜的断裂中发挥非酶作用。 晚期ESCRT-III复合体稳定性。指导该项目每个具体目标的工作假设是 DoA4抑制ESCRT-III复合体的拆解，DoA4通过以下方式解除其抑制结合 Bro1，它是Doa4的辅因子。该项目将使用的方法学包括电子显微镜、光 显微镜、蛋白质生物化学和功能分析。预计拟议研究的贡献如下： ED是控制ESCRT-III活性的调节机制的确定；鉴于高度的 在ESCRT-III功能的保护方面，预计该项目的结果也将产生洞察 ESCRT-III活性是如何在人类细胞中被控制的。这一贡献意义重大，因为定义这些注册表- 体内的调控机制对于理解ESCRT-III活性是如何在正常生理状态下被控制至关重要的。 生物条件以及在疾病状态下它是如何易受攻击的。

PROJECT SUMMARY Membrane scission by the ESCRT-III complex is a highly conserved cellular mechanism required for protein degradation in lysosomes as well as other cellular processes. ESCRT-III membrane scission activity has best been defined in the context of in vitro reconstitution assays, but the mechanisms that operate in vivo to control the activity of ESCRT-III under physiological conditions are poorly understood. The long-term goal of this project is to understand the mechanisms of membrane trafficking in the endocytic pathway. The objective of this application is to use the budding yeast Saccharomyces cerevisiae as a model system to identify mecha- nisms that regulate ESCRT-III at endosomes The central hypothesis of the project is that membrane scission by ESCRT-III is negatively regulated by the deubiquitination machinery at endosomes. The rationale for the proposed research is that, once it is known how ESCRT-III membrane scission activity is regulated, this process can likely be manipulated pharmacologically, paving the way toward new and innovative approaches in the prevention and treatment of genetic and infectious diseases linked to ESCRT-III.. The specific aims of the project are to determine the mechanism by which ESCRT-III is regulated by Doa4 and to define the mech- anism that relieves Doa4 from inhibition. Doa4 is a ubiquitin hydrolase that deubiquitinates transmembrane protein cargoes sorted into ILVs; but Doa4 also functions non-enzymatically in ILV membrane scission by regu- lating ESCRT-III complex stability. The working hypotheses that guide each specific aim of the project are that Doa4 inhibits disassembly of ESCRT-III complexes and that Doa4 is relieved from its inhibitory binding by Bro1, which is a Doa4 cofactor. The methodology to be used in the project includes electron microscopy, light microscopy, protein biochemistry, and functional assays. The contribution of the proposed research is expect- ed to be the determination of regulatory mechanisms that control ESCRT-III activity; given the high degree of conservation in ESCRT-III function, it is expected that the results from this project will also yield insight into how ESCRT-III activity is controlled in human cells. This contribution is significant because defining these reg- ulatory mechanisms in vivo is crucial for understanding how ESCRT-III activity is controlled under normal phys- iological conditions and how it is vulnerable in disease states.