Figure 3.

Isoprenylation and cleavage properties of Ydj1p Cxxx mutants identified through thermotolerance screening. (A and B) Yeast expressing the indicated Ydj1p mutants were evaluated by a gel-shift assay as described in Figure 1C. The sequences evaluated were either randomly identified from (A) the CASQ-like and CASQ-like (weak) groups or (B) reflect the combined set of sequences presenting with CVIA-like and CVIA-like (weak) thermotolerance phenotypes. Reference controls included on the blots are: farnesylated and uncleaved Ydj1p (CASQ); farnesylated, cleaved, and carboxymethylated Ydj1p (CVIA); unmodified Ydj1p (SASQ). Unmodified Ydj1p (open triangle) migrates at a larger apparent kDa than prenylated Ydj1p (closed triangle). The values in B reflect the thermotolerance scores observed for the corresponding mutant. (C) Yeast expressing the indicated a-factor Cxxx variants in a mfa1∆ mfa2∆ background were assessed for mating competence using a serial dilution mating assay (panel) and quantitative mating assay (values). Values represent mating efficiency relative to wild-type CVIA, which was set to 100%; values were determined using four or more replicates from two or more individual experiments. The panel is representative of one of the replicates.