Yeast transformation, yeast two-hybrid assay, and yeast complementation assay

The Matchmaker Gold Yeast Two‐Hybrid (Y2H) System (Clontech, Palo Alto, CA, USA) was used to verify proteins interaction. The bait (pGBKT7) and prey (pGADT7) plasmids were co‐transformed into yeast strain AH109 using the LiAc/SS carrier DNA/PEG method⁶⁷. Yeast transformants were screened on selective dropout (SD)/‐Trp‐Leu medium to select yeast cells containing the desired plasmids (pGADT7 and pGBKT7). Positive protein interactions were assessed on SD/‐Trp‐Leu‐His‐Ade medium supplemented with X‐α‐galactosidase (X‐α‐gal) after being incubated at 28°C for four days. For the yeast complementation assay, sensitivity of the different yeast strains to Congo red was determined as described previously²⁸. In brief, yeast cells were cultured in YPD or SD-Ura (for strains bearing YEp352 plasmid) medium at 24 °C overnight., The cells were collected by centrifugation at 5000 × g for 2 min, washed twice with ddH₂O, the cells were diluted to 3 × 10⁶ cells ml⁻¹ and then further five 1:5 serial dilutions. Cell suspension (five microliters) were spotted onto YPD medium and YPD supplemented with Congo red for 3 days at 30 °C.