The 26S proteasome is an ~70 subunit ATP-dependent chambered protease that destroys proteins via multiple highly coordinated processing steps. The smallest and only intrinsically disordered proteasome subunit, Sem1 (DSS1 in metazoans), is critical for efficient substrate degradation despite lacking obvious enzymatic activities and being located far away from the proteasome's catalytic centers. Dissecting its role in proteolysis using cell-based approaches has been challenging because Sem1 also controls proteasome function indirectly via its role in proteasome biogenesis. To circumvent this challenge, we reconstituted Sem1-deficient proteasomes in vitro from purified components and systematically dissected its impact on distinct processing steps. Whereas most substrate processing steps are independent of Sem1, ATP-dependent unfolding is stimulated several-fold. Using structure-guided mutagenesis and engineered protein crosslinking, we demonstrate that Sem1 allosterically regulates ATP-dependent substrate unfolding via a distal conformation-dependent intersubunit contact. Together, this work reveals how a small, unstructured subunit comprising < 0.4% the total size of the proteasome can augment substrate processing from afar, and reveals a new allosteric pathway in controlling proteolysis.

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The 26S proteasome is an ~70 subunit ATP‐dependent chambered protease responsible for most proteolysis in eukaryotes. Deciphering how its smallest subunit, Sem1 (DSS1 in metazoans), contributes to proteolysis has been challenging because Sem1 moonlights in other multisubunit complexes and is required for efficient proteasome biogenesis in vivo. To circumvent these limitations, we established a system to reconstitute Sem1‐deficient proteasomes in vitro from purified components. We found that proteasomes lacking Sem1 displayed both compromised ATPase activity and substrate unfolding efficiency, whereas proteasomal substrate capture, conformational switching of the proteasome between inactive and activated states, removal of the substrate's ubiquitin targeting signal, and proteolysis were largely unchanged. Using targeted mutagenesis and protein engineering approaches, we found that Sem1 acts as a molecular brace between a key structural subunit and the ATPase ring. This work reveals how a small, unstructured subunit less than 1% the total size of the proteasome can control the efficiency of substrate degradation, and suggests that the function of the proteasomal ATPase ring is tightly regulated via exogenous intersubunit contacts.