FIG. 7.

Heterologous IRP1 activities under various iron growth conditions in wild type and dre2-137 mutant strains. The CDV38a wild type (strain 84-1) and dre2-137 mutant (strain 97-55) were transformed with a plasmid (YCplac22-GPDprom-IRP1, plasmid 18-3) for expression of human cytosolic aconitase IRP1. Cells were grown to stationary phase in defined selective glucose medium to maintain the IRP1 plasmid and diluted to an OD₆₀₀ of 0.1 in growth medium of the same composition supplemented with various amounts of iron added as ferrous ammonium sulfate. After four doublings, cells were harvested and digested with zymolyase, and 100 μg of cell lysate was separated by native gel electrophoresis. Aconitase activities on lysates separated by native gel electrophoresis were revealed by an in-gel assay as described previously (2, 43). Positions of the endogenous mitochondrial aconitase activity (Aco1) and plasmid-expressed cytoplasmic aconitase activity (IRP1) are indicated. Lanes 1, 2, 3, 4, and 5 are wild-type lysates grown in 0, 100 nM, 1 μM, 10 μM, and 20 μM added iron, respectively. Lanes 6, 7, 8, 9, and 10 are dre2-137 strain lysates grown in 0, 100 nM, 1 μM, 10 μM, and 20 μM added iron, respectively. Lane 11 contains 100 μg of purified mitochondria.