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.