FIG. 2.
(A) The activities of wild-type Hap1 and various mutants at different concentrations of the heme analogue, deuteroporphyrin IX. Yeast cells bearing the UAS1/CYC1-lacZ reporter plasmid and the expression plasmid for wild-type Hap1 and mutants (Fig. 1) were grown to A0.5 and then induced with the indicated concentrations of dpIX for 7 h. The cells were subsequently collected, and β-galactosidase assays were performed. The data plotted here are averages of data from three experiments. The standard deviations are not shown, for clarity, but they were within 30%. (B) Western blot showing the protein levels of wild-type Hap1 and mutants in yeast cell extracts. Extracts were prepared from yeast cells expressing wild-type Hap1 (lane 7), H7-10A (lane 1), H7-1 (lane 2), H7-DH (lane 3), H7-NDH (lane 4), H7-N7 (lane 5), or H7-N (lane 6). Approximately 30 μg of total proteins from each extract was loaded onto 7.5% SDS-polyacrylamide gels, and the proteins were electrophoresed, transferred to PVDF membranes, and probed with an anti-Hap1 antibody (22). Hap1 proteins were visualized by using a chemiluminescence Western blotting kit (Boehringer Mannheim).