Mechanistic insights into the UFM1 E3 ligase complex in ufmylation and ribosome-associated protein quality control

Abstract

Ubiquitin-fold modifier 1 (UFM1) is a ubiquitin-like protein covalently conjugated with intracellular proteins through ufmylation, similar to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control (RQC) at the ER (ER-RQC), and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Herein, we provide insights into the mechanism of the UFM1 E3 complex in not only ufmylation but also ER-RQC. The E3 complex consisting of UFL1 and UFBP1 interacted with UFC1, UFM1 E2, and subsequently CDK5RAP3, the last of which is an adaptor for ufmylating ribosomal subunit RPL26. When CDK5RAP3 was absent from the E3 complex, UFBP1 ufmylation occurred, a process thought to drive ER-phagy. Further, upon treatment with anisomycin, an inducer of disome formation, the UFM1 E3 complex associated with ufmylated RPL26 on the 60S ribosomal subunit through the UFM1-interacting region of UFBP1. Loss of E3 components or disruption of the interaction between UFBP1 and ufmylated RPL26 attenuated ER-RQC. These results clarify the molecular mechanism of the UFM1 system and provide new insights into the role of ufmylation.

Mono-UFMylation promotes misfolding-associated secretion of α-synuclein

“…3E). As reported previously, the C-terminal 40 amino acids harbor a UFL1 binding site (34), but its deletion did not completely abolish UFL1 binding, consistent with another study showing an interaction between UFBP1 and UFL1 independent of this sequence (21). Cellbased UFMylation assay showed that overexpression of UFBP1 or the UFBP1ΔC mutant did not reduce α-Syn UFMylation (fig.…”

The Epstein Barr virus deubiquitinase BPLF1 regulates stress-induced ribosome UFMylation and ER-phagy

“…Still, other interactions, either in the context of the trimolecular ligase complex, other ER membrane proteins, or the ER-associated ribosomes, are likely to contribute. It is noteworthy that while structural and functional data obtained in cells transfected with the interacting partners convincingly identify the ER-associated active ligase as a trimolecular complex of UFL1 with DDRGK1 and C53 30,31 , very little is known about the assembly of the endogenously expressed partners in cells. We found that catalytically active and inactive BPLF1 are coprecipitated by endogenously expressed UFL1, DDRKG1, and C53, but only DDRGK1 coprecipitated UFL1 and C53.…”

“…The UFMylation machinery is recruited to the ER by the adaptor protein DDRGK1 (also known as UFBP1) 27,29 . The interaction with DDRGK1 activates the E3 ligase 30,31 . In addition, the activity of the ligase is regulated by binding to CDK5RAP3 (also known as LZAP/C53, hereafter C53) 30,31 , which also serves as a speci c receptor for ribosome stress-induced ER-phagy 26 .…”

“…The interaction with DDRGK1 activates the E3 ligase 30,31 . In addition, the activity of the ligase is regulated by binding to CDK5RAP3 (also known as LZAP/C53, hereafter C53) 30,31 , which also serves as a speci c receptor for ribosome stress-induced ER-phagy 26 . The ER-associated ligase UFMylates the 60S subunit protein RPL26 24,25,27 on ribosomes that stall at the ER 12 , and several ER-localized proteins, including the oligosaccharyltransferase complex subunit RPN1 27 , and CYB5R3 that was recently proposed as a speci c regulator of ER-phagy 28 .…”

Structural study of UFL1‐UFC1 interaction uncovers the role of UFL1 N‐terminal helix in ufmylation

“…While UFC1 and UBA5 are freely present in the cytosol, UFL1 is associated with the ER membrane through its interaction with DDRGK1 (Liang et al , 2020). Additionally, recent findings indicate that LZAP, another partner of the UFL1‐DDRGK1 complex, also possesses a UFM1 binding site (Ishimura et al , 2023). This further assists in bringing UFC1 ~ UFM1 in proximity to UFL1, enhancing its ability to outcompete UBA5.…”