Pulling and gripping: Proteasomal translocation pathway elements differentially contribute to substrate unfolding

Eukaryotic proteomes are regulated by the ubiquitin-proteasome system. Unwanted proteins are tagged with ubiquitin, which is recognized by the 26S proteasome for degradation. To enter the proteasome's 20S core particle and be hydrolyzed into peptides, the substrate must be unfolded and threaded through the 19S regulatory particle. Tyrosine "aromatic paddles" of the regulatory particle's Rpt motor subunits unfold and translocate the substrate by pulling it toward the core particle. We previously showed that for substrates degraded from their N-termini, polyglycine tracts inserted before a stable domain impair the proteasome's ability to grip or unfold the substrate at multiple points along the translocation pathway. Herein we compare unfolding from the N- and C-termini of model substrates. We find that a combination of local substrate structural elements, grip sequence, and possibly fundamental asymmetry of the proteasomal unfolding machinery that favors N-terminal unfolding affect the proteasome's ability to unfold substrates. Although the basic unfolding and translocation mechanism is conserved regardless of substrate orientation, we find differences in the ability of polyglycine tracts to disrupt unfolding from the N versus the C terminus. Finally, comparison of the rates of unfolding versus substrate release shows that different regions along the translocation pathway employ different mechanisms to facilitate unfolding and translocation. Interactions with aromatic paddles primarily increase the rate of substrate unfolding, while interactions predicted to take place with the core particle α-ring N-termini primarily decrease the rate of premature release of partially degraded substrates.