Cooperativity and communication in archaeal Cdc48·20S, an ancient proteolytic machine

Abstract

ATP dependent proteolysis is a process essential for life and is carried out by AAA+ proteases. AAA+ unfoldases use the energy of ATP hydrolysis to power the unfolding and translocation of protein substrates into compartmentalized peptidases for regulated proteolysis. Cdc48 is a highly conserved AAA+ homohexameric unfoldase which is made up of two AAA+ rings. Each ring can, in principle, bind and hydrolyzing ATP, but it is unclear what roles are played by each ring and how they coordinate their activities. A regulatory N domain functions to control the activity of the enzyme and binding to its partner peptidase, the 20S proteasome. In this thesis I present experiments which investigate the role of inter-ring communication in ATP hydrolysis, protein unfolding, and allosteric interactions with the 20S and show how these features affect enzyme function. Experiments also show how the N domain controls D1-D2 interactions that govern ATP hydrolysis and substrate unfolding. Finally, I present experiments that take steps toward developing a system for screening protein substrates of Cdc48-20S and identify several substrates from E. coli lysates.