Regulation of ESCRT-III activity in yeast

项目摘要 ESCRT-III复合物引起的膜断裂是蛋白质降解所需的高度保守的细胞机制。 溶酶体中的降解以及其他细胞过程。ESCRT-III膜断裂活性最好， 已经在体外重建测定的背景下定义，但是在体内操作以控制 对ESCRT-III在生理条件下活性知之甚少。长期目标是 项目是了解内吞途径中的膜运输机制。的目标 本申请是使用芽殖酵母酿酒酵母（Saccharomycescerevisiae）作为模型系统来鉴定机制， 在核内体调节ESCRT-III的酶该项目的中心假设是， 由ESCRT-III负调控的内体去泛素化机制。的理由 一项拟议的研究是，一旦知道ESCRT-III膜断裂活性是如何调节的， 这一过程可能会被人为操纵，从而为新的创新方法铺平道路 预防和治疗与ESCRT-III相关的遗传和传染病。的具体目标 该项目是确定ESCRT-III受Doa4监管的机制，并定义该机制， 使Doa4从抑制中解脱出来。Doa4是一种泛素水解酶， 蛋白质货物被分选到ILV中;但是Doa4也通过调节在ILV膜断裂中非酶促地起作用。 降低ESCRT-III复合物的稳定性。指导该项目每个具体目标的工作假设是， Doa4抑制ESCRT-III复合物的分解，并且Doa4通过以下方式从其抑制性结合中释放： Bro 1，是Doa 4辅因子。在该项目中使用的方法包括电子显微镜，光 显微镜、蛋白质生物化学和功能测定。预计该研究的贡献- 艾德决定了控制ESCRT-III活性的调节机制;鉴于ESCRT-III活性的高度依赖性， 保护ESCRT-III功能，预计该项目的结果还将深入了解 ESCRT-III活性在人类细胞中是如何控制的。这一贡献是重要的，因为定义这些注册- 体内的调节机制对于理解ESCRT-III活性在正常生理条件下是如何控制的至关重要。 生物学条件以及它在疾病状态下如何脆弱。

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.