FIGURE 9.
Activity of wild type IDE and ATP-binding site mutant IDEK898A,K899A,S901A in the yeast S. cerevisiae. A, immunoblot of yeast-expressed IDE and IDEK898A,K899A,S901A. Equivalent percentage amounts of total cellular extracts from yeast containing an empty expression vector or plasmids encoding IDE or the IDEK898A,K899A,S901A mutant were analyzed by SDS-PAGE and immunoblotting using an IDE-specific antibody. B, mating test for IDE activity. An excess of MATα yeasts were mixed with decreasing numbers of MATa cells deficient for Axl1p and Ste23p that were transformed with either an empty expression vector or a vector encoding IDE or IDEK898A,K899A,S901A as indicated. Mating, which leads to colony growth, is dependent on production of mature a-factor pheromone by plasmid-encoded IDE. C, spot halo assay for a-factor production. Concentrated samples of a-factor recovered from yeast bearing the indicated expression vector were spotted at the indicated dilutions on a lawn of RC757 MATα cells, which undergo growth arrest upon exposure to a-factor pheromone. The zones of growth arrest are the dark circular regions within the yeast lawn.