Mechanism and Structure of the Cdc48 ATPase Complex

Abstract

The yeast ATPase Cdc48 and its mammalian homolog p97 are essential proteins that use the energy of ATP hydrolysis to perform work on polypeptide substrates. Cdc48 is a critical component of the ubiquitin-proteasome system and serves a general function as a “segregase,” separating polyubiquitinated substrates from membranes or macromolecular complexes prior to their degradation by the proteasome. Cdc48 is involved in diverse protein quality control pathways operating at the endoplasmic reticulum, mitochondrion, ribosome, and chromatin, and a wide variety of substrates have been shown to require Cdc48 for proper handling by the proteasome. Despite this broad set of cellular roles, Cdc48’s basic molecular mechanism has long been unclear. In this work, we use purified proteins to reconstitute Cdc48 substrate processing in vitro and interrogate its mechanism. We show that Cdc48, in cooperation with its essential cofactors Ufd1 and Npl4, recognizes and unfolds substrates that bear polyubiquitin chains of appropriate length and linkage. This unfolding reaction is mediated by substrate translocation through the central pore of the ATPase. Substrate release requires trimming of the polyubiquitin chain by a Cdc48-bound deubiquitinating enzyme, with concurrent passage of a portion of the chain through the central pore. Using cryo-electron microscopy and X-ray crystallography, we also provide the first high-resolution structures of the Cdc48/Ufd1/Npl4 complex. Unlike all previously characterized Cdc48 cofactors, Npl4 contains a zinc finger domain that anchors it to the top of Cdc48’s ATPase rings and positions the cofactor directly over the central pore in an arrangement that is likely to facilitate substrate insertion. Together, our results establish the essential structural and functional features of the Cdc48/Ufd1/Npl4 complex. These features are likely applicable to the handling of most or all of the polyubiquitinated substrates that depend on this ATPase in various cellular contexts.