Figure 3.

 Far1-22p has an increased half-life and is present throughout the cell cycle. (A) Cells (K699) that carry a plasmid coding for either wild-type Far1–GFPp or Far1-22p–GFP from the inducible GAL promoter were grown in raffinose and expression was induced by addition of galactose for 5 hr. Glucose was then added to shut off the GAL promoter, and samples were taken every 30 min as indicated and immunoblotted for the presence of Far1–GFP fusion protein (lanes 1–8). The specificity of the GFP antibodies was confirmed by including cells expressing an HA-tagged version of Far1p (lane 1). (B) Quantitation of the GAL shutoff experiments. The degradation of wild-type Far1p-GFP and Far1-22-GFP proteins was quantified by PhosphorImager and plotted against the time after repressing the GAL promoter. (C) Far1–22p, but not wild-type Far1p, is present throughout the cell cycle. EY957 cells producing wild-type Far1p–GFP (lanes 9–11) or Far1–22p-GFP (lanes 12–14) from the inducible GAL promoter were grown in medium containing raffinose (GAL promoter off), arrested in S phase with hydroxyurea (HU) or in mitosis with nocodazole (Noc), and then expression was induced by addition of galactose. Control cells were treated identically except that they were not exposed to the drugs (expo). After 5 hr, cells were analyzed for the presence of Far1p–GFP by immunoblotting with antibodies specific for GFP (top panel). Equal loading was confirmed by immunoblotting the samples with antibodies against actin (bottom panel).