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  • Our data indicate that the D domain only

    2024-05-15

    Our data indicate that the D1 domain only needs to hydrolyze ATP a few times, or perhaps even just once, during the processing of a given substrate molecule, whereas the D2 domain hydrolyzes ATP many times. Consistent with this model, only D2 contains the canonical aromatic pore loop residues implicated in substrate binding and translocation. However, the two ATPase rings likely communicate with each other. ATP hydrolysis in D1 not only controls the position of the N domains but also affects D2 activity, as substrate binding at the cis side stimulates the ATPase rate of D2. Inter-ring communication may also occur in the reverse direction, as our substrate release data indicate that the nucleotide state of D2 influences the activity of Otu1, which is bound to the N domain. It is also likely that the ATPase subunits influence one another within each ring when the subunits are in different nucleotide states. Because all members of the ring are forced to be in the same state in the presence of ADP or ATPγS or when Walker motifs are mutated, our model is likely a simplification; in the presence of ATP, only some of the six subunits of the D1 or D2 rings may undergo the described nucleotide binding and hydrolysis events in a synchronous manner. Several findings support the proposition that not only substrate but also some ubiquitin moieties are translocated by Cdc48 and released from the D2 side of the central pore. First, ubiquitin itself can serve as a translocation substrate, forming crosslinks to positions along the full length of the pore. Thus, at least one ubiquitin molecule completely traverses the double-ring ATPase. Second, a slowly hydrolyzing Cdc48 mutant retained oligoubiquitin chains, indicating that these are slowly translocated through the ATPase and released with delay. Third, fully released substrates are primarily oligoubiquitinated, rather than fully deubiquitinated. The last two points suggest that not only a single ubiquitin molecule but also a short ubiquitin chain can be translocated through the central pore. We attempted to confirm ubiquitin translocation by incubating Ub(n)-Eos with Cdc48-FtsH but were unable to reproducibly detect ATP-dependent aldose reductase inhibitors by mass spectrometry, perhaps because FtsH does not efficiently cleave ubiquitin. It should be noted that the proteasome can also translocate ubiquitin under a variety of circumstances (Shabek and Ciechanover, 2010). A translocation mechanism for Cdc48/p97 had previously been dismissed because the ATPase has a narrow central pore relative to other double-ring AAA ATPases of known structure, and its D1 pore is even occluded by a Zn2+ ion in a crystal structure (DeLaBarre and Brunger, 2005). However, our data now suggest that at least two polypeptide strands can be accommodated, as the ubiquitin attachment site on the substrate is likely moved into the pore. Our results thus imply that the pore diameter widens during translocation, although this has yet to be confirmed experimentally. A hairpin structure inside the central pore has been demonstrated for other AAA ATPases, and there is even evidence that three strands can be present (Burton et al., 2001). This mechanism differs from that of NSF, where the SNARE substrate is not translocated through the central pore, although the D1 ring has canonical pore residues (Zhao et al., 2015), and a single ATPase cycle disassembles the SNARE complex (Ryu et al., 2015). In our model, Cdc48 activity is coupled with substrate deubiquitination. Indeed, free Otu1 has much lower deubiquitination activity than Otu1 bound to the Cdc48 complex (Stein et al., 2014). This stimulation is in part due to the UN cofactor, which may present bound K48-linked ubiquitin molecules to Otu1 in an appropriate conformation. In addition, our data suggest that deubiquitination is delayed until the substrate is at least partially translocated. The slow deubiquitination and substrate release exhibited by the D1 Walker B mutant indicate that Otu1 may not have full access to the UN-associated ubiquitin chain while the D1 domains are in the ATP-bound state and the N domains in the up-conformation. Movement of the N domain to the down-conformation by ATP hydrolysis in D1 would relieve this inhibition and permit full deubiquitination. Efficient deubiquitination also requires ATP binding and hydrolysis in D2, ensuring that substrate translocation commences before Otu1 can act. Furthermore, the repression of D1 ATP hydrolysis by substrate gives the D2 ring a chance to translocate a substantial portion of the polypeptide before deubiquitination occurs. Taken together, these mechanisms allow substrate binding, translocation, and release to occur in a defined order. Our results show that Otu1 cooperates with Cdc48 and argue against an earlier model in which Otu1 antagonizes Cdc48 function by preventing substrate recognition (Rumpf and Jentsch, 2006).