A Unique Role for the Rpt3 Pore Loop in Proteasome Activation and Proteolysis

Abstract

The 26S proteasome is a chambered protease that uses ATP-powered movements of pore loops within a ring of AAA+ ATPase subunits to drive unfolding and translocation of substrates into the peptidase core for proteolysis. Whereas most AAA+ family proteases contain a homohexameric ATPase ring in which each ATPase pore loop is thought to function identically and cooperatively, the proteasome contains six highly similar but non-identical ATPase subunits. Some limited evidence suggests their contributions to proteolysis are non-equivalent. We hypothesized that inactivation of each pore loop may distinctly impact substrate selection and/or processing by the proteasome. Using a collection of congenic pore loop mutant cells, we discovered many unique impacts on cell health, stress response, and ubiquitin homeostasis. Mutating the pore loop of one particular ATPase, Rpt3, led to a significant decrease in overall proteolytic activity. Genetic and biochemical analyses in yeast indicate that this pore loop transmits a signal to the peptidase component of the proteasome to trigger translocation of the substrate into the peptidase core for proteolysis. Together, our findings suggest an allosteric mechanism that couples pore loop movements with access to the peptidase centers for substrate processing.