Complex of Fas-associated Factor 1 (FAF1) with Valosin-containing Protein (VCP)-Npl4-Ufd1 and Polyubiquitinated Proteins Promotes Endoplasmic Reticulum-associated Degradation (ERAD)

Jae-Jin Lee,Joon Kyu Park,Jaeho Jeong,Hyesung Jeon,Jong-Bok Yoon,Eunice Eunkyeong Kim,Kong-Joo Lee

doi:10.1074/jbc.m112.417576

Abstract

Fas-associated factor 1 (FAF1) is a ubiquitin receptor containing multiple ubiquitin-related domains including ubiquitin-associated (UBA), ubiquitin-like (UBL) 1, UBL2, and ubiquitin regulatory X (UBX). We previously showed that N-terminal UBA domain recognizes Lys(48)-ubiquitin linkage to recruit polyubiquitinated proteins and that a C-terminal UBX domain interacts with valosin-containing protein (VCP). This study shows that FAF1 interacts only with VCP complexed with Npl4-Ufd1 heterodimer, a requirement for the recruitment of polyubiquitinated proteins to UBA domain. Intriguingly, VCP association to C-terminal UBX domain regulates ubiquitin binding to N-terminal UBA domain without direct interaction between UBA and UBX domains. These interactions are well characterized by structural and biochemical analysis. VCP-Npl4-Ufd1 complex is known as the machinery required for endoplasmic reticulum-associated degradation. We demonstrate here that FAF1 binds to VCP-Npl4-Ufd1 complex via UBX domain and polyubiquitinated proteins via UBA domain to promote endoplasmic reticulum-associated degradation.

Highlights

Fas-associated factor 1 (FAF1), which has multiple ubiquitin-like domains, interacts with various proteins (VCP, Hsp70, and polyubiquitinated proteins)
Binding of valosin-containing protein (VCP) to C-terminal ubiquitin regulatory X (UBX) Domain of FAF1 Is Necessary for the Recruitment of Polyubiquitinated Proteins to N-terminal UBA Domain—To investigate the effect of VCP binding on the biological function of FAF1, we designed a mutant of FAF1 defective in VCP binding
A previous structural study showed that UBX has a well conserved FPR motif in the S3/S4 loop that is a VCP binding motif [14, 24] and that FAF1 has an S3/S4 loop in UBX domain, 618TFPR621, that is a possible site for VCP binding of FAF1 UBX domain

Summary

Background

FAF1, which has multiple ubiquitin-like domains, interacts with various proteins (VCP, Hsp, and polyubiquitinated proteins). Results: Association of FAF1 UBX with VCP-Npl4-Ufd complex regulates ubiquitin binding to FAF1 UBA domain and promotes CD3␦ degradation in ERAD. We previously showed that N-terminal UBA domain recognizes Lys48-ubiquitin linkage to recruit polyubiquitinated proteins and that a C-terminal UBX domain interacts with valosin-containing protein (VCP). This study shows that FAF1 interacts only with VCP complexed with Npl4Ufd heterodimer, a requirement for the recruitment of polyubiquitinated proteins to UBA domain. C-terminal UBX domain of FAF1 binds to valosin-containing protein (VCP), a molecular chaperone in the ubiquitin-proteasome system. We previously demonstrated that UBX interacts with VCP in a stress-dependent manner and that FAF1 plays a key role as a ubiquitin receptor [1]. We found that recruitment of polyubiquitinated proteins to N-terminal UBA domain in FAF1 is regulated by the interaction of C-terminal UBX domain with VCP-Npl4-Ufd complex. We report here that FAF1 promotes the degradation of ERAD substrate CD3␦ in a VCP-Npl4-Ufd1-dependent manner

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION