PHOSPHORYLATION-DEPENDENT AND UBIQUITIN-DEPENDENT DEGRADATION OF THE CYCLIN-DEPENDENT KINASE INHIBITOR FAR1P IN BUDDING YEAST

Cyclin-dependent kinase inhibitors (CKIs) play key roles in controllin g the eukaryotic cell cycle by coordinating cell proliferation and dif ferentiation. Understanding the roles of CKIs requires knowledge of ho w they are regulated both through the cell cycle and in response to ex tracellular signals. Here we show that the yeast CKI, Far1p, is contro lled by ubiquitin-dependent proteolysis. Wild-type Far1p was stable on ly in the G(1) phase of the cell cycle. Biochemical and genetic eviden ce indicate that its degradation required the components of the G(1)-S ubiquitination system, Cdc34p, Cdc4p, Cdc53p, and Skp1p. We isolated a mutant form of Far1p (Far1p-22) that was able to induce cell cycle a rrest in the absence of alpha-factor. Cells that overexpress Far1-22p arrested in G(1) as large unbudded cells with low Cdc28p-Clnp kinase a ctivity. Wild-type Far1p, but not Far1-22p, was readily ubiquitinated in vitro in a CDC34- and CDC4-dependent manner. Far1-22p harbors a sin gle amino acid change, from serine to proline at residue 87, which alt ers phosphorylation by Cdc28p-Cln2p in vitro. Our results show that Fa r1p is regulated by ubiquitin-mediated proteolysis and suggest that ph osphorylation of Far1p by the Cdc28p-Clnp kinase is part of the recogn ition signal for ubiquitination.