Fig. 2. Loss of Orc2p function activates the budding-yeast metacaspase Yca1p. (A) Detection of signals by FACS in wild-type and GA1410 orc2-1 cells stained with propidium iodide (PI) and FITC-VAD-FMK, which stains cells containing activated caspases green. (B) The proportions of W303 wild-type and GA1410 orc2-1 cells with caspase activity before and after shifting to 35°C for 6 hours in cells from which the YCA1 metacaspase-encoding gene had (yca1) or had not been deleted. (C) Detection of caspase activity in wild-type and GA1410 orc2-1 cells with YCA1 deleted, and transformed with a plasmid expressing Yca1p or an empty vector control plasmid. The increase in caspase activation in controls for this experiment compared with the experiment presented in B is caused by culturing cells in defined medium required for plasmid maintenance instead of rich medium (data not shown). (D) Effect of yca1 on viability of GA1410 orc2-1 cells. Viability was measured as colony-forming units and was normalized to viability measured in cells maintained at 23°C compared with cells shifted to 35°C for indicated times.