Fig. 5. Ace2p localization and regulation. (A) The ace2-GFP (KGY4385) strain was grown at 25°C and confocal images of live cells were obtained. (B) The ace2-GFP sid4-GFP (KGY1311) strain was fixed in ethanol and the spindle pole body marker Sid4p was used to determine cell-cycle stage. 100 cells at each cell-cycle stage were examined to determine the percentage of cells expressing Ace2p-GFP. Representative images of different stages are shown. (C) Ace2p-HA₃ was immunoprecipitated from native protein lysates prepared from wild-type (KGY246; lane 1), ace2-HA₃ (KGY1123; lane 2), ace2-HA₃ fkh2 (KGY5388; lane 3) and ace2-HA₃ mbx1 (KGY5394; lane 4) strains with the 12CA5 antibody. Immunoprecipitates were resolved as in Fig. 4B as well as an aliquot of protein lysate set aside prior to immunoprecipitation. Immunoblotting was performed using antibodies against HA (12CA5; upper panel) or against Cdc2p (PSTAIRE; lower panel) to demonstrate that immunopreciptation was performed on equal amounts of protein within the lysates. (D) ace2-gfp fkh2 (KGY5361) (left panel) or ace2-gfp mbx1 (KGY5393) (right panel) cells were grown in at 32°C. Confocal images of live cells were obtained. Bars, 5 µm.