Figure 2.

Mitochondrial cyclophilin active-site mutant functions in yeast. (A) CPR3 complementation assay. cpr3 mutant strains (Eberhardt et al., 1992) were transformed with a centromeric plasmid expressing wild-type Cpr3 or the R73A or H144Q mutant proteins and grown on solid glucose or lactate medium at 30°C or 37°C for 96 h. (B) Expression and localization of Cpr3 wild-type and mutant proteins. Total cell extracts (T) and mitochondrial fractions (M) were prepared as described (Yaffe, 1991) from the cpr3::HIS3 strain JHY80–2B expressing the wild-type (lanes 1 and 2), R73A (lanes 3 and 4), or H144Q (lanes 5 and 6) Cpr3 protein tagged at the carboxyl terminus with the hemagglutinin (HA) epitope and analyzed by Western blot with anti-HA monoclonal antibodies to detect Cpr3p. Anti-COX2 antibodies (Molecular Probes, Eugene, OR) and FKBP12 antisera (Cardenas et al., 1995) served to detect mitochondrial and cytoplasmic marker proteins, respectively.