Pro-UFM1 is cleaved by two specific proteases, UFSP1 and UFSP2, which converts it into the mature form by exposing the C terminus glycine residue essential for conjugation. The mature UFM1 is activated by the homodimer of UBA5, a UFM1-specific E1 enzyme, and transferred to UFC1, a UFM1-specific E2 enzyme in the UBA5-UFC1 complex. Subsequently, the UFM1-UFC1 thioester intermediate is transferred to a stable UFM1-specific E3 enzyme complex comprising UFL1 and UFBP1. UFBP1 contains an N-terminal transmembrane helix responsible for ER localization. In the absence of CDK5RAP3, the UFM1-UFC1 intermediate freely changes its position and thereby accesses and ufmylates UFBP1 and other ER-localizing proteins such as CYB5R3 for ER-phagy. Upon CDK5RAP3 binding, the position of the UFM1-UFC1 intermediate is locked, which promotes RPL26 UFMylation for ER-RQC. The UFMylated proteins are cleaved by UFSP2. UFSP2 forms a complex with UFBP1 and/or odorant response abnormal protein 4 (ODR4), a multi-pass membrane protein, to localize to the ER.
