Figure 5.

Active-site FKBP12 mutants are functional in vivo. Drug resistance. Strains JHY2–1c (fpr1; Heitman et al., 1991a,b) and CHY516 (vph6 fpr1) were transformed with a low-copy-number centromeric plasmid bearing the wild-type FPR1 gene (WT), the F43Y, D44V, or F106Y mutant genes, or no FPR1 gene (Δfpr1). Transformants were grown on synthetic medium lacking leucine without (− rapamycin) or with 0.1 μg/ml rapamycin (+ rapamycin), or with 1 μg/ml FK506. FK506 sensitivity was assayed in an vph6 fpr1 mutant strain in which calcineurin is essential for growth (Hemenway et al., 1995); drug-resistant growth and drug-sensitive growth are indicated by R and S, respectively. Growth rate. The doubling times (expressed in minutes) of the transformants were determined by monitoring the OD₆₀₀ of liquid cultures grown at 30°C. Protein expression. Protein extracts were analyzed by Western blot with an anti-FKBP12 polyclonal antisera (Cardenas et al., 1995).