(A) A Mot3p-reporter strain is Ura+ when Mot3p is inactive. A dan1::URA3/DAN1 mot3::KanMX4/MOT3 diploid was sporulated and tetrads dissected. Shown are five-fold serial dilutions of four spores from a tetratype tetrad, plated onto SD-CSM and SD-ura. Spore genotypes are as indicated.
(B) 5-fold serial dilutions of [mot3−] and [MOT3+] dan1::URA3 cells were spotted onto YPD and SD-ura.
(C) Lysates of diploid [mot3−] and [MOT3+] cells were investigated by SDD-AGE and Western blotting. Mot3p was detected via its naturally occurring 6xHis motif using an anti-His antibody.
(D) Wildtype HSP104 yeast cells and HSP104-deleted yeast cells, each carrying plasmids for galactose-inducible expression of either Mot3PrD-EYFP or control protein EYFP, were compared for [MOT3+] induction. Two transformants each were grown over night in galactose media, washed once in water, then plated at five fold serial dilutions to SD-CSM or SD-ura plates.
(E) A [MOT3+] isolate was passaged three times on YPD plates or YPD plates containing GdnHCl, and then grown over night in liquid YPD prior to spotting onto SD-ura plates.
(F) A [RNQ+] dan1::URA3 strain was converted to [rnq−] by four passages on GdnHCl-containing plates. The [RNQ+] and [rnq−] strains were transformed with a Mot3PrD-EYFP plasmid and assessed for [MOT3+] induction as in (D).