Figure 6.

Cdc28p inhibits Fus2p nuclear export in G1/S through down-regulation of Fus3p. (A) Activation of Fus3p during G1/S allows Fus2p to exit before cell division. Fus2p-GFP localization was examined in STE5-8A as described in Fig. 1 B. Fus2p-GFP exited after cytokinesis in large-budded cells (top). Fus2p-GFP expressed from its own promoter was cytoplasmic and localized to the cortex in small-budded cells. Similar results were obtained when Fus2p-GFP was expressed from the GAL1 promoter. (B and C) Pheromone signaling in cells arrested at G2/M. cdc28-as1 (MY10451; B) or cdc28-as1 STE5-8A cells (MY10481; C) were arrested at G2/M by adding the Cdc28p-as1 inhibitor 1-NM-PP1 for 3 h. 85–92% of cells were budded under these conditions. After washing out inhibitor, the culture was split and treated with α factor alone (open symbols) or with Cdc28p-as1 inhibitor and α factor (closed symbols). Induction of pheromone-responsive genes was measured using fus1-LacZ expression (see Materials and methods). Activity is expressed relative to the 60-min time point of the arrested population. Error bars indicate SEM of three independent experiments. WT, wild type; β-Gal, β-galactosidase. Bar, 1 µm.