Fig. 6: Model for assembly of the Ubp6 catalytic complex.

Free Ubp6 has an inactive conformation (slashed circle), its active site (red semioval) being blocked by BL1, BL2, and SL (blue wave). Step 1, Ubp6 and the proteasome are complexed via the Ubp6^UBL-Rpn1 interaction². For clarity, only a fraction of proteasome subunits are represented. The Ubp6^UBL-Rpn1 interaction does not activate Ubp6 (ref. ³⁵) but is proposed to promote association of the Ubp6 catalytic domain to Rpt1 through avidity (step 2). Ubp6-Rpt1 interaction in this context may partially destabilize the blocking loop network to enable ubiquitin loading; this remains conjectural. Activated Ubp6 remains highly selective in that it will efficiently cleave only ubiquitin-protein conjugates that carry more than one ubiquitin modification¹⁸. Thus, a second docking event wherein substrate-bound “helper ubiquitin” (H) is docked at a ubiquitin receptor such as Rpn10, is required (step 3). With step 4, docking of the “target ubiquitin” (T), the catalytic complex is assembled: blocking loops are displaced more completely, and the proteasome assumes the si state, imposing noncatalytic proteasome inhibition. Thus, assembly of a competent catalytic complex essentially requires three docking events involving ubiquitin or ubiquitin-like protein domains: Ubp6^UBL at Rpn1, helper ubiquitin at a ubiquitin receptor, and target ubiquitin in the Ubp6 active site.