Mechanistic Insights into Potentiation of the AAA+ Disaggregase Hsp104

Abstract

Ubiquitination is a common posttranslational modification in which the protein ubiquitin is appended to substrate proteins to target them to the proteasome for degradation among a myriad of other cellular functions. In addition to recognition of ubiquitin, successful proteasomal degradation relies upon engagement at an unstructured region within the substrate protein. However, many proteasome clients intrinsically lack these requisite unstructured regions. Previous studies have suggested that ubiquitin modification may destabilize the target protein. However, experimental characterization of the energetics of ubiquitin-conjugated substrates with native isopeptide bonds has been limited by both purification constraints and complication due to the signal from ubiquitin itself. We have developed a generalizable protocol for purification of milligram quantities of homogenously monoubiquitinated single-lysine proteins for experimental measurement of ubiquitininduced energetic effects. These effects span from no observable changes to dramatic destabilization and depend on the exact site of modification. Further, there exists a trend between ubiquitin-induced substrate destabilization and faster proteasomal degradation rates. We also find that polyubiquitination at destabilizing sites is sufficient to induce the requisite unstructured region required for successful proteasomal degradation. Taken together, these data establish a connection between ubiquitin-induced changes in substrate energetics and proteasomal processing. We propose that ubiquitin's varied and site-specific modulation of substrate energy landscapes plays a consequential role for substrate engagement and successful degradation at the proteasome.