Figure 2.

Effect of temperature and time on yeast thermotolerance. (A) Yeast expressing the indicated Ydj1p variants were cultured to saturation in selective media, diluted into YPD, spotted using a multichannel pipettor onto YPD solid media, and plates incubated at indicated temperatures and times. A 1:20 dilution was the source for spots incubated at 25°, and a 1:2 dilution was used for spots incubated at other temperatures. (B) Yeast were cultured and processed as described for A, using the 40° condition with the following alterations: the 1:2 dilution was into YPD, incubation at 40° was for 72 hr followed by recovery at 25° for the indicated times. (C) Flow diagram of screen used to identify thermotolerant yeast expressing Ydj1p Cxxx variants in either the ydj1∆ or ydj1∆ ste14∆ background. (D) Examples of thermotolerance profiles observed and thermotolerance (T) scores assigned when expressed in ydj1∆ background; the controls and panel of mutants are replicated in Figure S2. Yeast thermotolerance assays were performed as described for Figure 1. (E) The thermotolerance profiles of all recovered mutants were scored relative to yeast expressing Ydj1p with a SASQ, CVIA, or CASQ motif, where the controls were assigned scores of 1, 3, and 5, respectively. The mutants were then binned according to their scores; see Materials and Methods for details on binning.