Abstract
Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity.
Highlights
Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators
We systematically examined the interactions between protein-A-Renilla luciferase (PA-renilla luciferase (RL))-tagged fusion proteins and firefly luciferase (FL)-tagged fusion proteins in human embryonic kidney cells 293 (HEK293) cells
We examined whether ASPL-mediated p97 hexamer disassembly influences described functions of p97 such as its role in endoplasmic reticulum-associated protein degradation (ERAD)[39]
Summary
Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. The AAA þ ATPase p97, known as VCP (valosincontaining protein) in mammals or Cdc[48] in yeast, is a homohexameric ring-shaped molecular machine that together with partner proteins controls a plethora of essential cellular processes such as ubiquitin-dependent protein degradation[16], maintenance of quality control[17] or homotypic membrane fusion[18]. A large number of p97 interaction partners have been identified with proteomics methods and biochemical assays[25,26] This includes 13 ubiquitin regulatory X (UBX) domaincontaining proteins that commonly interact with the conserved N domain[27]. Information about the binding strengths of most known p97 interacting proteins is, not available
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
