Ubiquitin and ATP-dependent unfoldase activity of P97/VCP·NPLOC4·UFD1L1 is enhanced by a mutation that causes multisystem proteinopathy
p97 is a segregase that plays a key role in numerous ubiquitin-dependent pathways, such as ER-associated degradation (ERAD). It has been hypothesized that p97 extracts proteins from membranes or macromolecular complexes to enable their proteasomal degradation; however, the complex nature of p97 substrates has made it difficult to directly observe the fundamental basis for this activity. To address this issue, we developed a soluble p97 substrate--Ub-GFP modified with K48-linked ubiquitin chains--for in vitro p97 activity assays. We demonstrate for the first time that wild type p97 can unfold proteins and that this activity is dependent on the p97 adaptor NPLOC4-UFD1L, ATP hydrolysis, and substrate ubiquitination, with branched chains providing maximal stimulation. Furthermore, we show that a p97 mutant that causes inclusion body myopathy, Pagets Disease of bone, and frontotemporal dementia (IBMPFD) in humans unfolds substrate faster, suggesting that excess activity may underlie pathogenesis. This work overcomes a significant barrier in the study of p97 and will allow the future dissection of p97 mechanism at a level of detail previously unattainable.