26S Proteasome ATPase Mutants Have Higher Unfolding Abilities

Abstract

ATP dependent proteases are protein complexes that degrade intracellular proteins involved in cell regulation and mediate protein quality control. In eukaryotes, the major ATP-dependent protease is the 26S proteasome. The 26S proteasome is a highly conserved protein degradation machine that consists of the 20S core particle and 19S regulatory particles on each end. The 20S core particle contains the proteolytic active sites that are responsible for degradation of the unfolded protein. The base of the 19S regulatory particle contains six regulatory particle (Rpt) triple-A ATPase subunits. Each Rpt subunit contains a walker B motif that is responsible for binding and hydrolyzing ATP in order to unfold substrate proteins. We mutated a glutamate to glutamine in the walker B motif of each Rpt subunit, preventing ATP hydrolysis in a single subunit without preventing ATP binding. We then characterized these mutant Rpt proteasomes using a previously established degradation assay in which the ability to unfold and degrade a multi-domain substrate is determined. Most of the time, the proteasome will fully degrade the substrate, however, if a domain of the substrate is stabilized making unfolding more difficult, it will result in fragment formation. Using the processivity assay, we see consistent fragment formation in wild type proteasome. The mutant proteasomes are still able to degrade the substrate (despite previous reports that some were completely degradation deficient), and surprisingly, we observed little to no fragment formation using the mutant proteasome. Our results suggest that these mutant proteasomes have an increased unfolding ability.