Figure 2.

 Cells producing Far1-22p arrest in G₁ by inhibition of the G₁ kinase, Cdc28p–Clnp. (A,B) Cln2p tagged at its carboxyl terminus with three copies of the HA epitope (Cln2–HA) was immunoprecipitated from extracts prepared from cells (YMT263) expressing either Far1-22p or for control, an inactive mutant form of Far1p, Far1-22/Δ285–350, from the inducible GAL promoter. Cells were grown in media containing galactose (GAL, GAL promoter on) or glucose (GLU, GAL promoter off). Cln2–HAp-associated kinase activity was measured with histone H1 as a substrate and quantified (A). The kinase activity associated with Cln2–HAp from cells grown in glucose was normalized to 100%. Similar amounts of Cln2–HAp were immunoprecipitated in each assay as shown by immunoblotting (B). (C) Growth inhibition caused by production of Far1-22p was dependent on the ability of Far1p to bind to the Cdc28p–Clnp kinase. The following Far1p proteins were analyzed: wild-type Far1p, Far1-22p, Far1-Δ285–350, which is unable to bind to Cdc28p–Clnp (Peter et al. 1993), and Far1-22/Δ285–350. (D) Growth inhibition caused by expression of Far1-22p was rescued by co-overexpression of a stable G₁-cyclin, Cln3p (DAF1). DAF1-1 cells (IH2517), which express a stable form of the G₁ cyclin Cln3p or isogenic wild-type cells (IH2518), were transformed with plasmids expressing either wild-type Far1p, Far1-22p, or Far1-22/Δ285–350p from the inducible GAL promoter.