Abstract
The proteasome recognizes ubiquitinated proteins and can also edit ubiquitin marks, allowing substrates to be rejected based on ubiquitin chain topology. In yeast, editing is mediated by deubiquitinating enzyme Ubp6. The proteasome activates Ubp6, whereas Ubp6 inhibits the proteasome through deubiquitination and a noncatalytic effect. Here, we report cryo-EM structures of the proteasome bound to Ubp6, based on which we identify mutants in Ubp6 and proteasome subunit Rpt1 that abrogate Ubp6 activation. The Ubp6 mutations define a conserved region that we term the ILR element. The ILR is found within the BL1 loop, which obstructs the catalytic groove in free Ubp6. Rpt1-ILR interaction opens the groove by rearranging not only BL1 but also a previously undescribed network of three interconnected active-site-blocking loops. Ubp6 activation and noncatalytic proteasome inhibition are linked in that they are eliminated by the same mutations. Ubp6 and ubiquitin together drive proteasomes into a unique conformation associated with proteasome inhibition. Thus, a multicomponent allosteric switch exerts simultaneous control over both Ubp6 and the proteasome.
Highlights
The proteasome recognizes ubiquitinated proteins and can edit ubiquitin marks, allowing substrates to be rejected based on ubiquitin chain topology
Ubp[6] has two domains: an N-terminal ubiquitin-like (UBL) domain, which binds the proteasome via subunit Rpn[135–37], and a C-terminal catalytic domain, which contacts Rpt[1] (Supplementary Fig. 4a)
We have identified an allosteric network composed of the activation loop of Rpt[1]; its target, the ILR element of Ubp[6]; and downstream elements, the blocking loop 1 (BL1) ß-hairpin, the BL2 loop, and the switching loop (SL) loop
Summary
The proteasome recognizes ubiquitinated proteins and can edit ubiquitin marks, allowing substrates to be rejected based on ubiquitin chain topology. Ubiquitinated substrates are first recognized by the 19subunit proteasome regulatory particle (RP), translocated through a channel into the proteasome core particle (CP) to be degraded. The deubiquitinating activity of Ubp[6], in contrast to that of Rpn[11], is not ATP-dependent or linked to substrate translocation. In the absence of its catalytic activity, Ubp[6] remains capable of suppressing protein degradation by the proteasome[19,20]. This second, noncatalytic mode of inhibition is promoted by binding of ubiquitin to its active site[19,20]. The functional consequences of this interaction have not been examined
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