Hsieh, H.-H. | bioRxiv Science

bioRxiv2026

Molecular basis of ubiquitin-independent recognition and degradation of ODC/Antizyme by the 26S proteasome

The ubiquitin-proteasome system represents the main pathway for targeted protein degradation in eukaryotic cells. The majority of substrates is recruited for degradation through ubiquitin modifications, and the underlying principles are well established. However, the requirements for ubiquitin-independent substrates are still poorly understood. Here, we reveal the mechanisms for the antizyme-mediated degradation of the yeast ornithine decarboxylase (yODC), the first reported ubiquitin-independent substrate of the 26S proteasome. Using biochemical studies and cryo-EM structure determination, we show how antizyme binding makes the yODC monomer prone for degradation by exposing an interface that is normally buried in the catalytically active ODC dimer. Together with a surface on antizyme, yODC forms a two-part interface that binds the N-terminal coiled coil of two ATPase subunits, Rpt4 and Rpt5, for delivery to the 26S proteasome motor. This positions the N-terminal unstructured region of yODC for insertion into the ATPase channel to initiate degradation, which we found does not depend on a specific sequence. Interestingly, binding of the globular yODC/antizyme complex to the Rpt4/Rpt5 coiled coil allosterically stabilizes a proteasome conformation that facilitates substrate engagement by the ATPase motor and may represent a primed pre-initiation state with a general role in ubiquitin-dependent and -independent degradation.

biochemistry↗

bioRxiv2026

Chaperones shape the conformational landscape of 26S-proteasome-base assembly for allosteric ATPase motor activation

Protein homeostasis depends on the 26S proteasome, the most complex ATP-dependent protease in eukaryotic cells. The proteasome base subcomplex is responsible for mechanical substrate unfolding and translocation into an internal degradation chamber. It contains three non-ATPase subunits, Rpn1, Rpn2, and Rpn13, and a heterohexameric AAA+ motor with six distinct ATPases, Rpt1 - Rpt6. Correct base assembly requires four dedicated chaperones that initially form the Hsm3 module (Hsm3-Rpt1-Rpt2-Rpn1), the Rpn14/Nas6 module (Rpn14-Rpt6-Nas6-Rpt3-Rpn2-Rpn13), and the Nas2 module (Nas2-Rpt5-Rpt4). However, the mechanisms underlying module assembly and formation of the mature base remain unknown. Here, we in vitro reconstitute the base subcomplex of the S. cerevisiae 26S proteasome from recombinant modules. Using biochemical assays, mass photometry, single-molecule fluorescence measurements, and single-particle cryo-EM, we reveal how the chaperones direct the conformational transitions through several intermediates toward the ATP-hydrolysis-active base. The Nas2 and Rpn14/Nas6 modules associate first, and binding of the Hsm3 module creates a state in which the chaperones stabilize an open ATPase ring that lacks hydrolysis activity. Sequential chaperone release then leads to a gradual ATPase-ring closure, whereby Hsm3s unstructured C-terminal tail mimics a substrate polypeptide in the central channel and induces a processing motor state with a spiral-staircase arrangement of Rpt subunits and a closed ATPase site at Rpt4. Inaugural ATP hydrolysis in Rpt4 is subsequently required to eject Hsm3 and transition to the Nas6-bound base that is ATPase active and competent for 26S-proteasome incorporation. Our studies thus provide exciting insights into how chaperones assure correct assembly, guide the complex through an intricate conformational landscape, and thereby prevent premature ATP-hydrolysis activation or incorporation of faulty assemblies into holoenzymes.

biochemistry↗