Fig. 3: SIFI binds to substrates at the centre of its twisted-ring scaffold.
From: Molecular basis of SIFI activity in the integrated stress response
a, Overview of SIFI, highlighting a low-resolution density ascribed to ABHD10 dimers, with an ABHD10 AF2 model fitted in the map (Gaussian filter width 2.0, contour 0.052). b, AF2 model of ABHD10’s cleaved N terminus (cABHD10) fitting into the N-degron-binding pocket within the ZZ domain of KCMF1. c, ZZ-domain deletion or Asp29/Asp31 mutation in KCMF1 ablates recognition of ABHD10–HA, as seen by KCMF1–Flag immunoprecipitation from ΔUBR4 HEK293T cells. The experiment was performed once. d, Diagram of cross-linking MS analysis of the SIFI–HRI–Ub complex, highlighting cross-linked residues from UBR4, KCMF1 and HRI–Ub (left). Right, model of dimerized HRI–Ub placed at SIFI’s central cavity, with HRI located near to the DOC2 and KCMF1N138 domains, and fused ubiquitin near its cross-linked regions (red, salmon: cross-linked residues determined by MS). e, Conformational change of the KCMF1N138–DOC1–UBR subcomplex after SIFI binding to HRINT. f, SIFI was purified from WT or ΔDOC2 UBR4 cells and incubated with a fluorescently labelled MTS peptide, E1, E2 (UBE2D3 and UBE2A) and ubiquitin. Similar results were observed in n = 3 experiments. g, Deletion of the DOC2 domain in endogenous UBR4 stabilizes HRI to a similar extent to complete UBR4 inactivation. HRI stability was assessed by flow cytometry after HRI-GFP::mCherry expression. Similar results were observed in n = 3 experiments. h, DOC2 deletion leads to integrated stress response hyperactivation after mitochondrial stress induced by sodium arsenite (SA), as seen by induction of ATF4 using western blotting. Similar results were observed in n = 2 experiments. i, UBR4ΔDOC2 cells are hypersensitive to sodium-arsenite-induced stress, dependent on the integrated stress response. As indicated, the integrated stress response was inhibited using ISRIB. The fitness of GFP-labelled WT and mCherry-labelled UBR4ΔDOC2 cells was assessed through cell competition. n = 3 biologically independent replicates are presented together with the median of these experiments. Gel source data are provided in Supplementary Fig. 1.
