FIGURE 1:

Stress-inducible Btn2 and Cur1 interfere with prion inheritance. (A) A prion reporter based on the translation termination activity of the C-terminal domain of Sup35 was used to monitor [NRP1C+] in yeast (see the text for details). (B) [NRP1C+] yeast cells were incubated on YPD plates at 30°C or 37°C for 3 d. (C) Galactose-regulatable expression plasmids coding for BTN2 or CUR1 were introduced into [NRP1C+] yeast. The transformants were transferred onto galactose-containing plates and incubated for 3 d at 30°C. Subsequently, the strains were transferred onto fresh YPD plates to allow for colony color development. (D) Chromosomal BTN2 was tagged with GFP in the BY4741 strain background. CUR1 was modified with GFP in a strain carrying a temperature-sensitive mutation in the proteasome subunit CIM3. The strains were grown at 25°C, shifted to 39°C for 30 min (time points in red), and then again exposed to 25°C. Cell lysates were prepared at the indicated time points and analyzed by immunoblotting with an anti-GFP antibody. The extended presence of Cur1 is probably an artifact of the reduced proteasomal activity of the cim3-1 strain. (E) Cells were treated as in D except that Btn2-GFP and Cur1-GFP were expressed from a low-copy plasmid that carried a GPD promoter. (F) [NRP1C+] yeast carrying deletions of BTN2 and/or CUR1 were incubated at 37°C for 3 d.