The p97-FAF1 Protein Complex Reveals a Common Mode of p97 Adaptor Binding

Caroline A Ewens,Silvia Panico,Patrik Kloppsteck,Ciaran Mckeown,Ima-Obong Ebong,Carol Robinson,Xiaodong Zhang,Paul S Freemont

doi:10.1074/jbc.m114.559591

Caroline A Ewens, Silvia Panico + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m114.559591

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Abstract

p97, also known as valosin-containing protein, is a versatile participant in the ubiquitin-proteasome system. p97 interacts with a large network of adaptor proteins to process ubiquitylated substrates in different cellular pathways, including endoplasmic reticulum-associated degradation and transcription factor activation. p97 and its adaptor Fas-associated factor-1 (FAF1) both have roles in the ubiquitin-proteasome system during NF-κB activation, although the mechanisms are unknown. FAF1 itself also has emerging roles in other cell-cycle pathways and displays altered expression levels in various cancer cell lines. We have performed a detailed study the p97-FAF1 interaction. We show that FAF1 binds p97 stably and in a stoichiometry of 3 to 6. Cryo-EM analysis of p97-FAF1 yielded a 17 Å reconstruction of the complex with FAF1 above the p97 ring. Characteristics of p97-FAF1 uncovered in this study reveal common features in the interactions of p97, providing mechanistic insight into how p97 mediates diverse functionalities.

Highlights

P97, known as valosin-containing protein, is a versatile participant in the ubiquitin-proteasome system. p97 interacts with a large network of adaptor proteins to process ubiquitylated substrates in different cellular pathways, including endoplasmic reticulum-associated degradation and transcription factor activation. p97 and its adaptor Fas-associated factor-1 (FAF1) both have roles in the ubiquitin-proteasome system during NF-␬B activation, the mechanisms are unknown
The active form of p97 is a homohexamer in which each monomer consists of three domains, two AAA domains, D1 and D2, arranged as two concentric rings, and an N-terminal domain that lies in the plane of the D1 ring in almost all crystal structures [2, 3]. p97 is involved in a large array of processes throughout the cell cycle [4], which include endoplasmic reticulum-associated degradation (ERAD) [5], autophagy [6], mito
Full-length FAF1 Binds p97 and Requires FAF1-ubiquitin-regulatory X (UBX) Domain— We first investigated the binding of recombinant full-length FAF1 to p97

Summary

Introduction

P97, known as valosin-containing protein, is a versatile participant in the ubiquitin-proteasome system. p97 interacts with a large network of adaptor proteins to process ubiquitylated substrates in different cellular pathways, including endoplasmic reticulum-associated degradation and transcription factor activation. p97 and its adaptor Fas-associated factor-1 (FAF1) both have roles in the ubiquitin-proteasome system during NF-␬B activation, the mechanisms are unknown. P97 interacts with a large network of adaptor proteins to process ubiquitylated substrates in different cellular pathways, including endoplasmic reticulum-associated degradation and transcription factor activation. P97 and its adaptor Fas-associated factor-1 (FAF1) both have roles in the ubiquitin-proteasome system during NF-␬B activation, the mechanisms are unknown. One well studied adaptor is Ufd1-Npl ( known as UN), which is required for p97 function in ERAD [1], but the largest group of p97 adaptor proteins are the ubiquitin-regulatory X (UBX) domain-containing proteins, which include p47 and Fas-associated factor 1 (FAF1) [11]. FAF1 [13] and p97 [17] have both been implicated in mediating I␬B␣ degradation, via unknown mechanisms, indicating the importance for the p97-FAF1 complex. Our data allow us to propose a common mode of interaction for p97 adaptors despite differences in domain architecture, suggesting a conserved mechanism of action for p97-adaptor complexes

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