Regulation of ESCRT-III Activity in Yeast

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DESCRIPTION (provided by applicant): The endosomal sorting complexes required for transport (ESCRTs) comprise a cellular machinery conserved from yeast to man. ESCRTs function in the biogenesis of late endosomal multivesicular bodies (MVBs) by executing the formation of intralumenal vesicles (ILVs), which bud directly from the endosomal membrane toward the lumen of the compartment. ILVs perform a critical role in maintaining cellular homeostasis by carrying unneeded, damaged, or dangerous transmembrane proteins to be degraded in the hydrolytic interior of the lysosome upon endolysosomal fusion. Dysfunction of the ESCRT-III complex has been linked to neurodegenerative disease, and its exploitation is essential for the spread of HIV-1 and other retroviruses as well as certain bacterial pathogens. In vitro studies have shown that ESCRT-III catalyzes membrane scission in the ILV budding pathway at MVBs. The objective of this research is to determine the regulatory mechanisms that control ESCRT-III activity in vivo, using the budding yeast Saccharomyces cerevisiae as a model system. The central hypothesis is that ESCRT-III activity is regulated in vivo by proteins that control the assembly and disassembly of this complex. This research will study how the structure and function of ESCRT-III is impacted in yeast by proteins that regulate assembly and disassembly of the complex. The methodology to be used includes electron microscopy, light microscopy, protein biochemistry, and functional assays. The rationale for this research is that the regulation of ESCRT-III must be defined if therapeutic strategies are to be developed that will treat ESCRT-III dysfunction or combat exploitations of ESCRT-III by human pathogens. Using yeast as a model system to understand this regulation is directly relevant to human health because the mechanism of ESCRT-III function is highly conserved. With respect to expected outcomes, it is anticipated that completion of this research will reveal the core, broadly conserved mechanisms that regulate ESCRT-III activity. Such results are expected to have an important positive impact because they will yield fundamental insight into a poorly understood regulatory step in the endocytic pathway, reveal potential targets for the prevention and treatment of diseases linked to ESCRT-III, and inspire new and innovative approaches to understand the mechanisms of ESCRT-III function.