Extended Data Fig. 8: The SIFI complex recognizes mitochondrial presequences.

a. Helical degrons in HRI and cDELE1 resemble mitochondrial presequences in amino acid composition (left panel) and structure (right panels). Presequences were aligned with COBALT (https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi). The structures of the HRI degron and the presequences of citrate synthase (CS) or COQ9 are AlphaFold2 models. The DELE1 helix is from its cryo-EM structure⁵⁶ and the ALDH2 presequence is its actual structure when bound to TOMM20⁵⁷. b. A prediction algorithm for mitochondrial presequences identifies the helical HRI and cDELE1 degrons. Internal MTS sequences were predicted using iMLP: iMTS-L predictor service (https://csb-imlp.bio.rptu.de/). A score above 0 is predictive of an internal MTS. Orange shaded boxes correspond to identified degrons in HRI and DELE1. c. The second HRI degron (helix 2) efficiently competes with mitochondrial presequences for access to the SIFI complex. A TAMRA-labeled COX8A presequence peptide (10 μM) was incubated with affinity-purified SIFI complex, E1, UBE2A and UBE2D3, and ubiquitin. As indicated, 100 μM of purified peptides encompassing the helices comprising degron 1 or degron 2 were added, and reaction products were analyzed after gel electrophoresis by fluorescence. Similar results in n = 2 independent experiments d. The SIFI complex, but not the quality control E3 ligase UBR5, ubiquitylates a presequence peptide. Ubiquitylation was analyzed as described above. Experiment performed once. e. The entire SIFI complex is required for presequence ubiquitylation. A TAMRA-labeled COX8A presequence peptide was incubated with affinity-purified SIFI complex purified from WT cells or cells carrying deletions of the endogenous KCMF1 binding-, calmodulin-, or UBR-domains in UBR4. E1, UBE2A and UBE2D3, and ubiquitin were added and reaction products were analyzed after gel electrophoresis by fluorescence. Similar results in n = 2 independent experiments. f. The SIFI complex ubiquitylates a TAMRA-labeled presequence peptide irrespectively of whether the E3 ligase had been purified from control cells or cells treated with arsenite (40 μM for 4 h). g. The SIFI complex modifies presequences with ubiquitin chains predominantly composed of K48-linkages. A TAMRA labeled COX8A presequence peptide was incubated with SIFI complex, E1, UBE2A and UBE2D3 and the indicated ubiquitin mutants (ubi-K0: all Lys residues mutated to Arg; ubi-K6only: all Lys residue except for K6 mutated to Arg), and reaction products were analyzed as above. Experiment performed once. h. The COX8A presequence is a SIFI-dependent degradation signal. The presequence was cloned as a fusion to GFP into the degradation reporter and assessed for its effects on protein stability by flow cytometry. As indicated, the proteasome inhibitor carfilzomib (CFZ) or the lysosome inhibitor bafilomycin A (BafA) were added. Note that only the cytoplasmic fraction of this fusion protein can be targeted via SIFI and the proteasome. Similar results in n = 2 independent experiments. i. A fusion between a COX8A presequence peptide carrying mutations in four Leu residues and GFP is not degraded through UBR4, the proteasome or the lysosome, as determined by flow cytometry. Similar results in n = 2 independent experiments. j. The mitochondrial import receptor TOMM20 competes with the SIFI complex for recognition of mitochondrial presequences. A TAMRA-labeled COX8A presequence was incubated with increasing concentrations of the cytoplasmic domain of TOMM20 or TOMM20^I74SV109S, which is incapable of binding presequences. The SIFI complex, E1, E2s, and ubiquitin were added, and ubiquitylation was monitored by gel electrophoresis and fluorescence imaging. Experiment performed once. k. Mutation of presequence residues required for TOMM20 binding also ablates ubiquitylation by the SIFI complex. Similar results in n = 2 independent experiments. l. Import inhibition leads to accumulation of mitochondrial precursor proteins that still contain their presequence. UBR4 deletion further increases precursor abundance, as seen by Western blotting after expressing of HA-tagged mitochondrial proteins in either WT or ΔUBR4 cells treated with mitochondrial import blocker oligomycin (1 μM, 16 h) and ISRIB. Similar results in n = 2 independent experiments. m. Inhibition of mitochondrial protein import upon depletion of TIMM16 stabilizes HRI. Similar results in n = 2 independent experiments. n. Inhibition of mitochondrial protein import upon depletion of TIMM16 stabilizes cDELE1, as determined by flow cytometry. Similar results in n = 2 independent experiments. o. Deletion of UBR4 partially stabilizes a presequence reporter if import was prevented by TIMM16 depletion, as seen by flow cytometry. Similar results in n = 2 independent experiments. p. Activation of ISR signaling in ΔUBR4 cells upon overexpression of the mitochondrial protein NIPSNAP1 is dependent on HRI and DELE1. HRI and DELE1 were depleted by specific siRNAs and ISR activation was monitored using the uORF-ATF4 reporter using flow cytometry. When indicated, NIPSNAP1 was overexpressed. Similar results in n = 2 independent experiments. For gel source data, see Supplementary Fig. 1.