Fig. 6. The interaction of UFBP1 with ufmylated RPL26 is indispensable for ER-RQC.
(A) UFBP1 KO cells were transfected with wild-type UFBP1 or UFM1 interaction–defective UFBP1. Twenty-four hours after transfection, the cells were treated with anisomycin (Ans, 0.1 μg/ml) for 30 min and then lysed. Ribosomes were separated by ultracentrifugation through sucrose density gradients. Protein samples prepared from gradient fractions were analyzed by immunoblot with indicated antibodies. Data shown are representative of three separate experiments. (B) UFBP1-KO cells expressing wild-type UFBP1, UFBP1UFIM mutant, or UFBP1K267R mutant were transfected with the HA-XBP1u-V5. Twenty-four hours after transfection, the cells were lysed. Top: The cell lysates were subjected to neutral PAGE followed by immunoblot with indicated antibodies. The free peptide and the peptidyl-tRNA (pep-tRNA) arrest products were detected by immunoblot with an anti–hemagglutinin (HA) antibody. Samples treated with RNase to digest the tRNA moiety of pep-tRNA are indicated by (+). Bottom: The relative levels of the HA-XBP1u arrest products with P values were determined by five independent experiments. (C) Schematic model of the enzymatic cascades during RPL26 ufmylation. Each protomer within the UBA5 homodimer binds UFM1 and UFC1 via UFIM and UBS, respectively, and catalyzes the formation of the UFM1-UFC1 thioester intermediate. UFL1-UFBP1 anchored to the ER recruits the UFM1-UFC1 intermediate via UFL1UBS binding to UFC1. Last, CDK5RAP3 is recruited to the E3 complex to complete the organization of the pentameric E3 complex on the ER. (D) Schematic model of substrate switching of the UFM1 E3 ligase. In the absence of CDK5RAP3, the UFM1-UFC1 intermediate can freely change its position and thereby access and ufmylate UFBP1 K267. Upon CDK5RAP3 binding, the position of the UFM1-UFC1 intermediate is locked away from UFBP1 K267, preventing its ufmylation and promoting RPL26 ufmylation instead. (E) Schematic model of ER-RQC through the UFM1 E3 ligase.