Regulation of ESCRT-III activity in yeast

酵母中 ESCRT-III 活性的调节

基本信息

批准号：
10386800
负责人：
CHARLES G ODORIZZI
金额：
$ 31.96万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10386800
关键词：
Affect Binding Biochemical Genetics Biological Assay Biological Models Biological Process Cell Nucleus Cell membrane Cell physiology Cells Communicable Diseases Complex Data Deubiquitination Disease Electron Microscopy Electron Transport Complex III Endosomes Foundations Functional disorder Genetic Diseases Goals HIV-1 Homeostasis Human Hydrolase In Vitro Integral Membrane Protein Knowledge Link Lysosomes Measures Mediating Membrane Methodology Microscopic Mission National Institute of General Medical Sciences Neck Neurodegenerative Disorders Outcome Pathway interactions Pharmacology Physiological Prevention Process Property Protein Biochemistry Public Health Publishing Regulation Research Retroviridae Role Saccharomyces cerevisiae Saccharomycetales Side Site Testing Ubiquitin Vesicle Work Yeasts cofactor disease diagnosis endosome membrane in vivo innovation insight light microscopy preservation prevent protein complex protein degradation reconstitution trafficking

项目摘要

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.