Journal Article

Substrate processing by the Cdc48 ATPase complex is initiated by ubiquitin unfolding

Wales, Thomas E., Rapoport, Tom A., Bodnar, Nicholas O., Engen, John R., Marto, Jarrod A., Ji, Zhejian, Twomey, Edward C., Ficarro, Scott B.

2019

Overview

Ubiquitin marks proteins for degradation by the proteasome. However, many substrates cannot be directly degraded because they are well folded or are located in cell membranes or in multimeric complexes. These proteins are first unfolded by the Cdc48 adenosine triphosphatase (ATPase), which forms a hexameric assembly that pulls polypeptides through its central pore. Twomey et al. determined structures of Cdc48 at an initiation stage of substrate processing. Surprisingly, a ubiquitin molecule in the substrate-linked polyubiquitin chain could be unfolded simply by binding to the Cdc48 complex. A segment of the unfolded ubiquitin inserts into the ATPase ring and initiates substrate unfolding. This explains why Cdc48 can deal with a broad range of substrates—even ones that are folded. Cooney et al. report the cryo–electron microscopy structure of Cdc48 in complex with an authentic substrate. In contrast to previously reported Cdc48 structures, an asymmetric spiraling assembly wraps around the extended substrate polypeptide. Thus, Cdc48 uses a hand-over-hand mechanism of translocation, which supports a common mechanism for protein substrate unfolding for AAA+ ATPases. Science , this issue p. eaax1033 , p. 502 The Cdc48 ATPase processes polyubiquitinated substrates by unfolding and translocating an attached ubiquitin molecule. The Cdc48 adenosine triphosphatase (ATPase) (p97 or valosin-containing protein in mammals) and its cofactor Ufd1/Npl4 extract polyubiquitinated proteins from membranes or macromolecular complexes for subsequent degradation by the proteasome. How Cdc48 processes its diverse and often well-folded substrates is unclear. Here, we report cryo–electron microscopy structures of the Cdc48 ATPase in complex with Ufd1/Npl4 and polyubiquitinated substrate. The structures show that the Cdc48 complex initiates substrate processing by unfolding a ubiquitin molecule. The unfolded ubiquitin molecule binds to Npl4 and projects its N-terminal segment through both hexameric ATPase rings. Pore loops of the second ring form a staircase that acts as a conveyer belt to move the polypeptide through the central pore. Inducing the unfolding of ubiquitin allows the Cdc48 ATPase complex to process a broad range of substrates.

• Publisher: American Association for the Advancement of Science,The American Association for the Advancement of Science
• Subject: Adenosine diphosphate / Adenosine triphosphatase / Adenosine triphosphate / Amino acids / Assembly / ATP / Beryllium / Beryllium fluorides / Binding sites / Biodegradation / Cell membranes / Chains / Crosslinking / Cryoelectron Microscopy / Degradation / Deuterium / Domains / Electron microscopy / Fluorides / Grooves / Hydrogen-deuterium exchange / Hydrolysis / Inserts / Macromolecules / Membranes / Microscopy / Molecular structure / Monomers / Multienzyme Complexes - chemistry / Mutation / Neurodegenerative diseases / Neurological diseases / Nucleocytoplasmic Transport Proteins - chemistry / Polypeptides / Polyubiquitin - chemistry / Proteasomes / Protein Domains / Protein folding / Protein Unfolding / Proteins / RESEARCH ARTICLE SUMMARY / Ring structures / Saccharomyces cerevisiae Proteins - chemistry / Saccharomyces cerevisiae Proteins - genetics / Substrate Specificity / Substrates / Translocation / Triphosphatase / Ubiquitin / Ubiquitination / Valosin Containing Protein - chemistry / Valosin Containing Protein - genetics / Valosin-containing protein / Vesicular Transport Proteins - chemistry / Yeast