Figure 3. Identification of flexible regions in IRE1^LD that are important for the regulation of IRE1 activity in cells.

(A) Left panel shows a bar diagram of the percentage of amide hydrogen exchange (%ex) of the indicated by IRE1^LD segments after 30 and 300 s incubation in D₂O. The amino acids (aa) covered by the peptic fragments are indicated on the left. Exchange was corrected for back exchange using a fully deuterated IRE1^LD preparation. Protein concentration was 5 µM. Shown are the data of three independent experiments (mean ± standard deviation). Right panel shows a cartoon of the IRE1^LD dimer (PDB: 2HZ6) with the left protomer coloured according to %ex at 30 s (areas with no sequence coverage are uncoloured). The location of the putative loop (residues 308–357) and the tail (residues 390–444) are schematically represented as dotted lines (see: Figure 3—source data 1) (B) Schematic description of a directed in vivo CRISPR-Cas9 mutagenesis strategy to probe regions of IRE1^LD for their relevance to regulating activity in CHO-K1 cells. Cas9 guides (red triangles) targeted sites across the Ern1 genomic locus encoding the protein’s region of interest. Transfection of individual or pairs of guides resulted in a collection of mutations (insertions and deletions, depicted as blue and red lines). Cell harbouring rare de-repressing mutations of IRE1 (blue) were selected by fluorescence-activated cell sorting (FACS) gated on XBP1s::Turquoise high and CHOP::GFP low signals. The resultant clones were isolated and genotyped. (C) Left panel is a histogram of XBP1s::Turquoise intensity of CHO-K1 dual UPR reporter cell populations transfected with guide-Cas9 encoding plasmids targeting a putative unstructured loop (aa 308–357) within IRE1^LD (identified in ‘A’). XBP1s::Turquoise bright cells within population 0 were collected by FACS (FACS1) yielding population 1, followed by a second round of enrichment for bright cells (FACS2 yielding population 2). Population 2 was treated with the IRE1 inhibitor 4µ8c to select against clones exhibiting IRE1-independent reporter activity. The final population was genotypically analysed (representative sequences are shown on the right). Frameshift mutations are coloured in blue and Cas9 cut sites are indicated below.

Figure 3—figure supplement 1. IRE1’s tail region is involved in maintaining the repressed state of IRE1 in vivo.

(A) Schematic representation of IRE1. The signal peptide (SP), luminal domain (LD), comprised of a structured core (CLD) and an unstructured tail, the transmembrane (TM) domain and the cytosolic effector domains are indicated with their corresponding amino acid (aa) resides below. (B) Representative sequences of clones that were selected as described in Figure 3C after transfection of cells with guide-Cas9 encoding plasmids targeting IRE1’s tail (residues 368–444). Frameshift mutations are coloured in blue and Cas9 cut sites are indicated by arrowheads below.