Fig. 4: SIFI targets mitochondrial precursors.

a, Stability of HRI variants in WT and ΔUBR4 cells analysed by flow cytometry. Similar results in n ≥ 3 independent experiments. b, AlphaFold2 model of the amino-terminal HRI domain. c, Deletion of two helices protects HRI against UBR4-dependent degradation. Similar results in n = 3 independent experiments. d, Autoradiography image of HRI variants analysed for SIFI-dependent ubiquitylation. Similar results in n = 2 independent experiments. e, Fluorescence image of TAMRA-labelled HRI helix 2 peptides analysed for SIFI-dependent ubiquitylation. Similar results in n = 2 independent experiments. f, cDELE1 stability reporters were analysed in WT and ΔUBR4 cells by flow cytometry. ΔN, amino-terminal deletion (152-end); ΔOQC, deletion of putative orphan QC motif; ΔiMTS, deletion of the region overlapping with the presequence-like helix. Similar results in n = 2 independent experiments. g, Ubiquitylation of a TAMRA-labelled presequence (MTS) peptide by SIFI, E1, UBE2A and UBE2D3 was monitored by fluorescence imaging. Similar results in n = 3 independent experiments. h, Fluorescence image of a TAMRA labelled presequence ubiquitylated by SIFI purified from WT and ΔKCMF1 cells. Experiment was performed once. i, Fluorescence image of modification of a TAMRA-labelled presequence with ubiquitin mutants. Similar results in n = 2 independent experiments. j–l, Flow cytometry results for MTS. j, Depletion of HSPA9 destabilizes a presequence reporter that is partially dependent on SIFI. Similar results in n = 2 independent experiments. k, Depletion of both TIMM8A and TIMM8B (siTIMM8A/8B) destabilizes a presequence–GFP fusion in a SIFI-dependent manner. Similar results in n = 2 independent experiments. l, Cells were treated with oligomycin (1 μM, 16 h), and the stability of a presequence–GFP fusion was determined by flow cytometry. Similar results in n = 2 independent experiments. For gel source data, see Supplementary Fig. 1.