Extended Data Fig. 9: The UBL domain of UBR4 is essential for ubiquitin chain elongation.

a. Mass spectrometry of SIFI^ΔUBL indicates that the UBL domain is not required for structural integrity of SIFI. Endogenous SIFI was affinity-purified from WT, UBR4^ΔUBL or UBR4^{M4444A/L4447E} cells and bound proteins were detected by mass spectrometry. Technical replicates are shown. b. Western blotting of SIFI^ΔUBL shows that the UBL domain is not required for structural integrity of SIFI. Endogenous SIFI was affinity-purified from WT or UBR4^ΔUBL cells and bound proteins were detected by Western blotting. Experiment performed once and validated by mass spectrometry. c. Mutation of the UBL domain in UBR4 prevents ubiquitin chain elongation, as seen with a TAMRA-labelled presequence peptide as substrate. Similar results in n = 2 independent experiments. d. Mutation or deletion of the UBL domain in UBR4 both prevent ubiquitin chain elongation by SIFI, as seen with ³⁵S-labelled HRI^NT ~ SUMO as a substrate. e. Deletion of the UBL prevents ubiquitin chain elongation, even if initiation was performed independently of SIFI by a 2 h preincubation with E1/E2. Similar results in n = 2 independent experiments. f. The UBL domain of UBR4 is required for degradation of DELE1 or unimported mitochondrial proteins. Protein stability was determined by flow cytometry, as described before. Similar results in n = 2 independent experiments. g. Mutation of the UBL domain stabilizes cDELE1 and unimported mitochondrial proteins, as seen by flow cytometry with two independent clones expressing endogenous UBR4^{M4444A/L4447E}. Similar results in n = 2 independent experiments. h. Mutation of the UBL domain in UBR4 results in increased stress signalling, as seen by an increase in ATF4 levels after import was compromised with arsenite. Experiment validated with two independent clones. i. Deletion of the UBL domain in UBR4 increases stress response activation upon sodium arsenite treatment, as detected by uORF-ATF4 reporters in flow cytometry as described above. Similar results in n = 2 independent experiments. j. Mutation of the UBL domain in UBR4 increases stress response activation, as measured by flow cytometry. Experiment validated with two independent clones. k. Mutation of the UBL domain in UBR4 compromises cell survival upon arsenite-induced import stress, as seen in a competition experiment with WT cells. n = 3 independent replicates shown with median of these experiments. l. The UBL domain of UBR4 is essential for cell survival upon mitochondrial import stress. WT and UBR4^ΔUBL cells were labelled with GFP and mCherry, respectively, mixed, and depleted of either TIMM8A or TIMM8A/HRI, as indicated. After 12 d of co-culture, the ratio between cell types was determined by flow cytometry. n = 3 independent replicates shown with median of these experiments.