Yeast Two-Hybrid (Y2H) Assays

The Y2H assays, using the Y2H Gold yeast strain (Clontech), were performed as described previously²⁶. Briefly, the donor vectors with cDNAs of bZIP60, HAC1 and XBP1 were recombined into pGBKT7-GW (bait) and pGADT7-GW (prey) vectors based on the LR reaction (Invitrogen, USA). The resulting constructs were transformed into the strain indicated above, and yeast cells were selected on a TRP- and LEU-deficient double dropout (DDO) media. Transformed colonies, confirmed by PCR and western blotting, were plated onto a HIS-, TRP-, LEU- and ADE-deficient quadruple dropout (QDO) media with the indicated concentrations of aureobasidin A (AbA) to test possible interactions.

The split-ubiquitin membrane Y2H assays were carried out on the NMY51 yeast strain as described previously⁶⁰. The encoding regions of IRE1 homologues, which were amplified from their donor vectors using the primers containing Sfi I site listed in Supplementary Table S1, were sub-cloned into the bait vector pBT3-SUC and the prey vector pPR3-SUC. The yeast cells co-transformed with the indicated bait-prey constructs were transformed into the yeast cells, and selected as described above. Baits expressing IRE1 orthologues against empty prey vector (pPR3-C) or the reciprocal combinations were also transformed into yeast cells test potential bait and prey auto-activation. The combination of pOST-NubI with each IRE1 bait vector was used as a positive reference. Interactions were determined by yeast growth on a synthetic triple dropout (TDO) media deficient in HIS, TRP and LEU supplemented with 1 mM, 5 mM, 10 mM and 25 mM 3-Amino-1,2,4-Triazole (3-AT) and the QDO media in the presence of 1 mM, 5 mM, and 10 mM 3-AT. The experiments were repeated at least three times.