Extended Data Fig. 5: 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 UBR4^ΔDOC2, 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 UBR4^ΔDOC2 cells that also expressed ABHD10^HA 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 UBR4^ΔDOC2 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 UBR4^ΔDOC2 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 UBR4^ΔDOC2 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.