Fig 2: KCMF1 recruitment and function in SIFI.a. top: Schematic of domain arrangement in KCMF1. bottom: KCMF1 AlphaFold model suggests that the KCMF1 linker not observed in SIFI structures (orange) does not adopt a compact fold. b. The ZZ-domain of KCMF1, but not its linker, is required for substrate degradation. Stability of HRI or cleaved DELE1 was measured in ΔKCMF1 cells using flow cytometry. As indicated, WT-KCMF1, KCMF1ΔZZ, or KCMF1mutLink (containing 5 Q/E mutations in conserved residues), were expressed. Similar results in n = 2 independent experiments. c. The central Phe residue in the C-terminal KCMF1 helix is highly conserved. d. Deletion of the N-terminal ZZ domain or mutation of R20E, E38R, L61E, Y64E, R68E in the ZZ domain strongly impaired the interaction between KCMF1FLAG and endogenous UBR4, as shown by affinity-purification and Western blotting. The ZZ deletion also interfered with recognition of the N-degron substrate ABHD10, as described below. Similar results in n = 2 independent experiments. For gel source data, see Supplementary Fig. 1.

Fig 3: The DOC2 domain of UBR4 is important for SIFI function.a. Crosslinking of HRI~Ub or a presequence peptide to SIFI results in similar conformational rearrangements of the KCMF1 module composed of the N-terminal domains of KCMF1 and the DOC1 and UBR domains of UBR4, as determined by cryo-EM. fl: full-length; MTS: mitochondrial targeting sequence. All maps were Gaussian filtered with width sdev=2.0. b. SIFI binds endogenous mitochondrial proteins, when import has been disrupted by arsenite treatment. When SIFI was purified from cells expressing endogenous UBR4ΔDOC2, the binding of mitochondrial proteins was lost. Binding partners were determined after endogenous SIFI affinity-purification using mass spectrometry. Mitochondrial proteins, for which predicted presequence peptides could be identified, are shown in red. IP-MS samples were run as technical triplicates. c. The DOC2 domain of SIFI is not required for ABHD10 ubiquitylation. SIFI was purified from either WT or UBR4ΔDOC2 cells that also expressed ABHD10HA and incubated with E1, UBE2D3/UBE2A, and ubiquitin. Ubiquitylation of bound ABDH10 was detected by Western blotting. Experiment performed once. d. The DOC2 domain of SIFI is required for degradation of DELE1 or unimported mitochondrial proteins. The stability of DELE1 or the presequence of COX8A was determined in WT or UBR4ΔDOC2 cells by flow cytometry, as described before. Similar results in n = 2 independent experiments. e. Deletion of the DOC2 domain in UBR4 results in stronger activation of the integrated stress response. A reporter for integrated stress response activation based on uORFs of ATF4 was expressed in either WT, ΔUBR4, or UBR4ΔDOC2 cells, which were treated with sodium arsenite and analysed by flow cytometry. Similar results in n = 2 independent experiments. f. The DOC2 domain of UBR4 is essential for cell survival upon mitochondrial import stress. WT and UBR4ΔDOC2 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 the median of these experiments. For gel source data, see Supplementary Fig. 1.

Fig 4: The UBR boxes of UBR4 present substrates towards the interior of SIFI.a. The UBR box of UBR4 binds to DOC1 domain of UBR4 and the N-terminal domain of KCMF1, forming the KCMF1N138-DOC1-UBR subcomplex. X-ray structure of the UBR box of UBR1 (PDB 3NIH)35 reveals that its peptide binding groove is open. b. Modelling a N-degron peptide into the open substrate-binding groove of the UBR box of UBR4, based on the UBR1 X-ray structure (PDB 3NIH). c. Structure of SIFI showing the proximity of UBR box to other substrate-binding modules of SIFI, including the DOC2 domain of UBR4 and the ZZ domain of KCMF1.

Fig 5: The UBL domain of UBR4 is essential for ubiquitin chain elongation.a. Mass spectrometry of SIFIΔUBL indicates that the UBL domain is not required for structural integrity of SIFI. Endogenous SIFI was affinity-purified from WT, UBR4ΔUBL or UBR4M4444A/L4447E cells and bound proteins were detected by mass spectrometry. Technical replicates are shown. b. Western blotting of SIFIΔUBL shows that the UBL domain is not required for structural integrity of SIFI. Endogenous SIFI was affinity-purified from WT or UBR4ΔUBL 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 HRINT ~ 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 UBR4M4444A/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 UBR4ΔUBL 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.