FIGURE 2:
Overexpression of BRL1 mutants with defective AαH causes formation of petal-like structures at the NE. (A) Overexpression of brl1F391E and brl1L402E is toxic for cells. WT cells with the control plasmid PGal1 or PGal1-BRL1, PGal1-brl1F391E, PGal1-brl1L402E were spotted in 10-fold serial dilutions onto glucose (Glu) and galactose/raffinose (Gal/Raf) plates at 30°C. PGal1-APQ12 and PGAL1-apq12-ah were used as controls for overexpression toxicity and growth on galactose/raffinose (Gal/Raf) plates (Zhang et al., 2021). (B) BRL1 overexpression slightly suppresses PGal1-brl1F391E toxicity. WT yeast cells containing the indicated plasmids were grown on glucose or galactose/raffinose plates at 30°C. (C) Overexpression of brl1F391E and brl1L402E triggers the formation of petal-like structures along the NE. BRL1, brl1F391E, and brl1L402E were overexpressed under the galactose-inducible promoter PGal1 in cells carrying the dsRED-HDEL NE/ER marker for 0, 1, 2, 3, and 5 h. Cells were imaged by microscopy after galactose induction. Scale bar: 3 µm. (D) Graph showing the percentage of cells with NE petals upon BRL1, brl1F391E, brl1L402E overexpression over time. n = 3. Shown is the mean with SD. (E) brl1F391E localizes to sites of membrane overproliferation. Fluorescence image analysis of PGal1-yeGFP, PGal1-BRL1-yeGFP, and PGal1-brl1F391E-yeGFP cells carrying the dsRED-HDEL marker with (3 h) and without (0 h) promoter induction. Scale bar: 5 µm.