br Acknowledgment br Introduction The yeast Cdc ATPase

Acknowledgment
Introduction The yeast Cdc48 ATPase and its metazoan ortholog p97 (or VCP) are critical components of many ubiquitin-dependent cellular pathways that require the segregation of individual proteins from binding partners or membranes (for review, see Buchberger, 2013, Meyer and Weihl, 2014, Xia et al., 2016). This ATPase is present in all eukaryotic GW3965 and is essential for their viability. The function of Cdc48/p97 is best understood in endoplasmic reticulum (ER)-associated protein degradation (ERAD), in which it extracts polyubiquitinated, misfolded proteins from the ER and transfers them to the proteasome for degradation (Christianson and Ye, 2014). Cdc48/p97 also functions in mitochondrion-associated protein degradation (Taylor and Rutter, 2011), ribosomal quality control (Verma et al., 2013), and the extraction of chromatin-bound proteins (Franz et al., 2016, Ramadan et al., 2007). Consistent with this central role in protein quality control, mutations in human p97 cause several neurodegenerative proteopathies (Chapman et al., 2011, Kimonis et al., 2008). Despite its biological significance, the mechanism of Cdc48/p97 action is poorly understood. Cdc48 belongs to the AAA+ family of ATPases (ATPases associated with various cellular activities), whose members use ATP hydrolysis to exert force on macromolecules (Erzberger and Berger, 2006, Sauer and Baker, 2011). Many of the AAA proteins form hexamers with either a single or double ring of ATPase domains (type I and II ATPases, respectively). Cdc48 is a type II ATPase consisting of an N-terminal (N) domain, two tandem AAA domains (D1 and D2) separated by a short linker, and a flexible C-terminal tail (DeLaBarre and Brunger, 2003). The AAA domains form two stacked rings surrounding a central pore. Both ATPase rings hydrolyze ATP (Chou et al., 2014, Ye et al., 2003), but their roles in substrate processing are unknown. ATP hydrolysis in the D1 ring seems to move the N domains from a so-called up-conformation in the ATP state to a position co-planar with the D1 ring in the ADP-bound state (down-conformation) (Banerjee et al., 2016). The function of this N domain movement is unclear. Cdc48 cooperates with a large number of protein cofactors that provide pathway selectivity and fine-tune substrate processing. One of the most important cofactors is the Ufd1/Npl4 heterodimer (UN), an essential complex that participates in many Cdc48-dependent processes, including ERAD (Ye et al., 2004). UN binds to the N domain of Cdc48 and recruits polyubiquitinated substrates to the ATPase (Ye et al., 2003). In a reconstituted in vitro system of partial ERAD reactions, a polyubiquitinated, misfolded protein could be extracted from the membrane in a UN- and Cdc48-dependent manner (Stein et al., 2014). However, most experiments with the Cdc48/p97 complex have been performed with intact cells, the complexity of which makes it impossible to obtain mechanistic insight. Dissection of Cdc48 function has also been hampered by a lack of suitable in vitro substrates. The only reported experiments employed non-ubiquitinated proteins (Baek et al., 2011, DeLaBarre et al., 2006), which are not the principal substrates in vivo. Cdc48’s physiological substrates are generally polypeptides modified with K48-linked polyubiquitin chains, which also serve as a major targeting signal for the proteasome (Pickart, 2000). In vitro experiments with the proteasome have employed polypeptides decorated with K63-linked polyubiquitin chains (Nathan et al., 2013), but these substrates are not appropriate for the more specific Cdc48 ATPase complex (Ye et al., 2003). One of the most important questions is how the Cdc48 ATPase can “pull” on a substrate, thereby releasing it from a protein complex or membrane. Some AAA ATPases, including the Clp ATPases in bacteria, the archaeal Cdc48 homologs, and the 19S proteasome, use a translocation mechanism (for review, see White and Lauring, 2007). In these cases, central loops with conserved aromatic residues are thought to contact a polypeptide chain and move in response to ATP hydrolysis, thereby dragging the substrate through the pore. Archaeal Cdc48 and some mammalian p97 mutants can also translocate polypeptides into associated 20S proteasomes under certain conditions (Barthelme and Sauer, 2012, Barthelme and Sauer, 2013), implying movement through the central pore. However, archaeal Cdc48 undergoes conformational changes that have never been observed with eukaryotic homologs (Huang et al., 2016), and wild-type yeast Cdc48 and mammalian p97 are thought not to use a translocation process (Banerjee et al., 2016, DeLaBarre and Brunger, 2005). A mechanism that does not involve translocation is also employed by Cdc48’s closest relative, the NEM-sensitive fusion protein (NSF) (Zhao et al., 2015). NSF is thought to bind the SNARE complex and the cofactor αSNAP with its N domains and to use ATP hydrolysis to unwind the supercoiled SNARE proteins without polypeptide passage through the central pore. A major argument against a translocation mechanism for Cdc48/p97 is that its central pore is very narrow or occluded in crystal or cryoelectron microscopy (cryo-EM) structures. These observations have led to the proposal of several alternative mechanisms. In one model, a substrate transiently enters the D2 ring or moves through the D2 ring, exiting between the D1 and D2 rings (DeLaBarre and Brunger, 2005). Other models postulate that the polypeptide chain inserts only shallowly into the D1 ring (for review, see Xia et al., 2016). Finally, it has been suggested that the large, nucleotide-dependent conformational changes of the N domains are sufficient to move a polypeptide chain (Schuller et al., 2016). In each of these cases, it is unclear how a continuous pulling force could be generated, although such a force would likely be required to extract a subunit from a tight multimeric complex or a multi-spanning protein from the ER membrane. Clearly, experiments are needed to test whether a translocation mechanism or any of the alternative models can explain the function of Cdc48.