The ubiquitin-selective ATPase p97 (also known as Cdc48 and VCP) is an important motor and regulator of cellular protein turnover. It uses energy from ATP hydrolysis to extract or segregate ubiquitylated target proteins from stable protein assemblies, membranes and chromatin. The molecular mechanism underlying this "segregase" activity of p97 is still unknown. In this project, we will develop a quantitative segregase assay using purified components. We will use this assay to perform a comprehensive analysis of structure-guided p97 mutations in order to identify residues and structural elements required for segregase activity. Moreover, we will use the segregase assay to characterize functional defects of different p97 mutants that cause neurodegenerative diseases.