Extended Data Fig. 7: UBE2A promotes ubiquitin chain elongation by SIFI.
From: Molecular basis of SIFI activity in the integrated stress response
a. KCMF1 does not determine linkage specificity of SIFI. SIFI was purified from WT or ΔKCMF1 cells and tested for its ability to catalyse ubiquitylation of a mitochondrial presequence. As indicated, either wt-ubiquitin or ubiquitin-K48R were used. Experiment performed once. b. KCMF1 can modify ubiquitin residues independently of a specific Lys residue. KCMF1 was affinity-purified from ΔUBR4 cells and incubated with E1, UBE2D3, and indicated ubiquitin variants. Ubiquitin polymers were detected by Western blotting. Similar results in n = 2 independent experiments. c. KCMF1 can introduce ubiquitin branches. KCMF1 was affinity-purified from ΔUBR4 cells and incubated with E1, UBE2D3, and indicated ubiquitin variants. Reaction products were visualized with either ubiquitin antibodies or with bispecific K11/K48-antibodies that preferentially detect K11/K48-branched ubiquitin molecules17. Experiment performed once. d. UBE2A and its close homologue UBE2B are required for HRI degradation. UBE2A and UBE2B were co-depleted from 293 T cells, and HRI stability was monitored using a HRI-GFP::mCherry reporter, as described before. Similar results in n = 2 independent experiments. e. UBE2A and UBE2B co-depletion results in increased activation of the integrated stress response, as determined by αATF4-Western blotting. Experiment performed once. f. UBE2A promotes ubiquitin chain elongation. SIFI was affinity-purified via UBR4FLAG and incubated with E1, ubiquitin and a mitochondrial presequence peptide (left) or HRI degron helix 2 peptide (right), resulting in short ubiquitin chains on the presequence substrate likely catalysed by an E2 that co-purified with SIFI. Addition of the E2 UBE2D3 results in an increase in short ubiquitin chains, indicative of a role of UBE2D3 in chain initiation. Addition of UBE2A leads to selective elongation of ubiquitin chains that had already been initiated. Similar results in n = 2 independent experiments. For gel source data, see Supplementary Fig. 1.
