The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3

Abstract

Protein modification by ubiquitin-like proteins (UBLs) amplifies limited genome information and regulates diverse cellular processes, including translation, autophagy and antiviral pathways. Ubiquitin-fold modifier 1 (UFM1) is a UBL covalently conjugated with intracellular proteins through ufmylation, a reaction analogous to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control at the ER and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Here we identify a UFM1 substrate, NADH-cytochrome b5 reductase 3 (CYB5R3), that localizes on the ER membrane. Ufmylation of CYB5R3 depends on the E3 components UFL1 and UFBP1 on the ER, and converts CYB5R3 into its inactive form. Ufmylated CYB5R3 is recognized by UFBP1 through the UFM1-interacting motif, which plays an important role in the further uyfmylation of CYB5R3. Ufmylated CYB5R3 is degraded in lysosomes, which depends on the autophagy-related protein Atg7- and the autophagy-adaptor protein CDK5RAP3. Mutations of CYB5R3 and genes involved in the UFM1 system cause hereditary developmental disorders, and ufmylation-defective Cyb5r3 knock-in mice exhibit microcephaly. Our results indicate that CYB5R3 ufmylation induces ER-phagy, which is indispensable for brain development.

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

“…1D). FLAG-tagged wild-type UFBP1 and UFBP1 UFL1 mutant were expressed in UFBP1 knockout (KO) HEK293T cells ( 27 ), and the cell lysates were immunoprecipitated with anti-FLAG antibody. The immunoprecipitant prepared from wild-type UFBP1-expressing cells contained endogenous UFL1, UFC1, and CDK5RAP3 (Fig.…”

“…UFBP1 possesses a signal peptide for the endoplasmic reticulum (ER) and a transmembrane helix localized on the ER ( 10, 26, 27 ), and UFL1 forms a stable complex with UFBP1 ( 13, 28 ), suggesting potential roles of the UFM1 system on the ER. Indeed, the UFM1 system has long been associated with ER stress and ER-associated degradation ( 7 ).…”

“…Indeed, the UFM1 system has long been associated with ER stress and ER-associated degradation ( 7 ). Further, accumulating evidence has recently indicated that ufmylation plays an important role in ribosome-associated protein quality control at the ER (ER-RQC) ( 29–31 ), as well as in selective autophagy of the ER (ER-phagy) ( 27, 32, 33 ). The former seems to be mediated by the ufmylation of ribosomal proteins such as RPL26 ( 29, 30 ), and the latter by the ufmylation of a UFM1 E3 component, UFBP1, and an ER-anchoring protein, CYB5R3 ( 10, 27, 34 ).…”

“…Further, accumulating evidence has recently indicated that ufmylation plays an important role in ribosome-associated protein quality control at the ER (ER-RQC) ( 29–31 ), as well as in selective autophagy of the ER (ER-phagy) ( 27, 32, 33 ). The former seems to be mediated by the ufmylation of ribosomal proteins such as RPL26 ( 29, 30 ), and the latter by the ufmylation of a UFM1 E3 component, UFBP1, and an ER-anchoring protein, CYB5R3 ( 10, 27, 34 ). However, aside from the X-ray crystal structures of UFM1 ( 35 ), UBA5 ( 36 ), UFC1 ( 37 ), and their complex ( 38, 39 ), the structure of the UFM1 E3 complex is unknown, and the UFM1 E3 components show no similarity to any previously identified E3 of ubiquitin or UBLs, making it difficult to infer their structure.…”

BioAFMviewer software for simulation atomic force microscopy of molecular structures and conformational dynamics

“…Applications of simulation AFM using the BioAFMviewer are rapidly increasing. The platform was used to construct atomic models of Annexin V protein lattices ( Marchesi et al, 2021 , Yamada et al, 2022 ) and to validate HS-AFM observations of aptamer-protein complexes ( Biyani et al, 2022 ), SARS-CoV-2 spike proteins ( Lim et al, 2021 ), actin-myosin complexes ( Moretto et al, 2022 , Matusovsky et al, 2022 ), and the NADH-cytochrome b5 reductase 3 enzyme ( Ishimura et al, 2022 ). In a recent application, fitting of available structures was applied to assign catalytic states to measured topographies of enzyme shapes, validating kinetic analysis of HS-AFM imaging ( Takeda et al, 2022 ).…”