Figure 1. S. pombe and S. cerevisiae Ire1 have functionally conserved stress sensing ER-lumenal domains and divergent cytosolic domains.

(A) Cartoon illustration of lumenal domain (LD), transmembrane/cytosolic linker domain (TMD + L) and kinase/RNase domain (KR) for S. pombe (Sp) (blue) and S. cerevisiae (Sc) Ire1 (orange). (B, C) Expression levels of S. cerevisiae Ire1 (128 kD), S. cerevisiae lumenal S. pombe cytosolic Ire1 (126 kD), S. pombe lumenal S. cerevisiae cytosolic Ire1 (125 kD) and S. pombe Ire1 (122 kD) in S. pombe (B) and S. cerevisiae cells (C). Extracts were immunoblotted for 3xFLAG-Ire1. Ponceau stain (B) or Pgk1 (C) was used as loading control. (D, E) Cell growth assay on tunicamycin (Tm) plates. Serial dilutions of S. pombe (D) or S. cerevisiae (E) cells, which expressed the indicated Ire1 constructs, were spotted onto plates containing 0.05 μg/ml (D) or 0.1 μg/ml (E) of Tm. Plates were photographed after incubation at 30°C for 4 days. (F, G) qPCR assay for S. pombe GAS2 (F) or BIP1 (G) mRNA fold change upon 1 μg/ml Tm treatment for 1 hr. Experiments were done in triplicates. In (G), uncleaved (dark grey) or total (light grey) BIP1 mRNA was detected using the corresponding PCR primers illustrated as arrows in the schematic insert. The red dashed line indicates the Ire1 cleavage position on BIP1 mRNA. (H) Detection of S. cerevisiae HAC1 mRNA splicing by RT-PCR across the splice junction. Cells were treated with or without 1 μg/ml of Tm for 1 hr.

Figure 1—figure supplement 1. Ire1 chimeras with S. pombe cytosolic domain cleave BIP1 and GAS2 mRNA in S. pombe.

Northern blots of S. pombe GAS2 (A) and BIP1 (B) mRNA. Cells were treated with 1 μg/ml of Tm for 1 hr.

Figure 1—figure supplement 2. Ire1 oligomeric state determines the HAC1 mRNA splicing dynamics in S. cerevisiae cells.

(A) Illustration of the HAC1 mRNA derived splicing reporter. The splicing reporter contains a part of the HAC1 mRNA 5’ exon replaced by the green fluorescent protein (GFP) coding sequence. In the unspliced reporter mRNA, translation is inhibited by a translation block formed by the intron and 5'UTR (indicated by the red arrow). Upon splicing, intron is removed and translation begins. (B) Measuring the HAC1 mRNA splicing dynamics in S. cerevisiae using automated flow cytometry. After 1.5 hr of incubation, either no Tm or 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml of Tm was added. Then, we monitored the splicing dynamics for 10 hr. The splicing dynamics under various conditions is plotted. Green lines represent the strains of interest, which expressed indicated Ire1 variants, and the black line represents WT control strain under the same condition. (C) Examining Ire1 foci formation in S. cerevisiae cells via fluorescence microscopy with or without 1 μg/ml Tm treatment for 20 min. (D) Growth assay on Tm plate for S. cerevisiae cells expressing Ire1 constructs with or without mCherry inserted into the cytosolic linker. The inserted mCherry does not affect Ire1's ability to alleviate ER stress.