The importance of proteasome grip depends on substrate stability

The 26S proteasome is responsible for the unfolding and degradation of intracellular proteins in eukaryotes. A hexameric ring of ATPases (Rpt1-Rpt6) grabs onto substrates and unfolds them by pulling them through a central pore and translocating them into the 20S degradation chamber. A set of pore loops containing a so-called aromatic paddle motif in each Rpt subunit is believed to be important for the proteasome's ability to unfold and translocate substrates. Based on structural and mechanistic experiments, paddles from adjacent Rpt subunits, which are arrayed in a spiral staircase conformation, grip and pull on the substrate in a hand-over-hand type mechanism, disengaging at the bottom of the staircase and re-engaging at the top. We tested the contribution of the aromatic paddles to unfolding substrates of differing stabilities by mutating the paddles singly or in combination. For an easy-to-unfold substrate (a circular permutant of green fluorescent protein; GFP), mutations had little effect on degradation rates. For a substrate with moderate stability (enhanced GFP), there were modest effects of individual mutations on GFP unfolding rates, and alternating aromatic paddle mutants had a larger detrimental effect on unfolding than sequential mutants. For a more stable substrate (superfolder GFP), unfolding is overall slower, and multiple simultaneous mutations essentially prevent unfolding. Our results highlight the context-dependent need for grip during unfolding, support the hand-over-hand model for substrate unfolding and translocation, and suggest that for hard-to-unfold substrates, it is important to have simultaneous strong contacts to the substrate for unfolding to occur. The results also suggest a kinetic proofreading model, where substrates that cannot be easily unfolded are instead clipped, removing the initiation region and preventing futile unfolding attempts.