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  • Another important question concerns the mechanism by

    2022-12-06

    Another important question concerns the mechanism by which a polyubiquitinated substrate is released from the Cdc48 complex and passed on to downstream components, such as the proteasome. There are at least three proposed models. In the first, a ubiquitinated substrate is transferred by ubiquitin-binding shuttling factors to the proteasome (Richly et al., 2005). In the second, the substrate is completely deubiquitinated, processed by Cdc48, and then re-ubiquitinated to enable shuttling factor and proteasome binding (for review, see Liu and Ye, 2012). In the third, the substrate is only partially deubiquitinated, leaving sufficient ubiquitin moieties for interaction with downstream components. A candidate for a deubiquitinase involved in Cdc48-dependent reactions is Otu1 (called YOD1 in mammals). Otu1 binds via its UBX-like Triflurdine mg domain to the N domain of Cdc48, an interaction that stimulates its deubiquitination activity (Ernst et al., 2009, Stein et al., 2014). Expression of an enzymatically inactive Otu1 mutant blocks Cdc48 function in vivo and leads to the accumulation of polyubiquitinated proteins on the Cdc48/p97 complex. However, the precise role of Otu1 has yet to be clarified. Specifically, it is unclear whether it deubiquitinates substrates before they are processed by Cdc48, thus acting as a negative regulator (Rumpf and Jentsch, 2006), or synergistically cooperates with Cdc48. Here, we have elucidated the mechanism of Cdc48 from S. cerevisiae with purified components that include Cdc48 itself, its UN cofactor, and model substrates bearing K48-linked polyubiquitin chains. We demonstrate that polyubiquitinated substrate first binds to UN. Following ATP binding to the D1 ring, Cdc48 unfolds the substrate by passing it through the central pore, starting from the D1 side and moving it all the way through the D2 ring. This translocation process relies on ATP hydrolysis by the D2 domain. We show that subsequent substrate release from Cdc48 requires hydrolysis of ATP in D1 and partial trimming of the ubiquitin chain by the deubiquitinase Otu1. The remaining ubiquitin Triflurdine mg follow the substrate through the central pore and are released on the D2 side of the pore. Our results establish a model for protein extraction and segregation that is broadly applicable to the various Cdc48-dependent quality control systems.
    Results
    Discussion In the model, Cdc48 starts out with the D1 ATPases in the ADP-bound state (Figure 7, stage 1). The N domains are in the down-conformation, co-planar with the D1 ring. When the D1 domain binds ATP, the N domains move upward (stage 2). The UN complex can bind to either conformation, inhibiting the overall ATPase activity in the absence of substrate. Substrate is initially bound to the Cdc48 complex exclusively through an interaction of the attached K48-linked polyubiquitin chain with the UN cofactor (stage 3). Most of the UN cofactor in the cell is probably bound to Cdc48, which is present at high concentrations, so free UN would not compete for substrate. Substrate binding to the Cdc48 complex reduces ATPase activity in the D1 domain, biasing it toward its ATP-bound state with the N domain in its up-conformation. The UN cofactor and D1 ring ATPases might form a composite binding surface that can locally denature substrate without energy input, generating an unfolded polypeptide loop that can reach into the central pore. Substrate binding also stimulates ATP hydrolysis in the D2 domain. This activity allows pore loops in the D2 ring to move and drag the substrate polypeptide through the central pore (stage 4). The pulling force exerted by the D2 ATPases results in the unfolding of the substrate (stage 5). During translocation, most of the polyubiquitin moiety remains on the cis side, bound to the UN cofactor. However, a portion of the ubiquitin chain can enter the central pore along with the substrate (stage 5). The final step is substrate release. Once D1 has hydrolyzed ATP, the N domains convert back to the down-conformation, allowing access of the polyubiquitin chain to the deubiquitinating enzyme Otu1 (stage 6). When the ubiquitin chain has been shortened sufficiently, its affinity for the UN complex is reduced or lost, and the remaining ubiquitin moieties are unfolded and pulled through the central pore (stage 7). The ubiquitin probably refolds rapidly after translocation (Sivaraman et al., 2001), although this needs to be tested by future experimentation.