A Cdc28 mutant uncouples G(1) cyclin phosphorylation and ubiquitination from G(1) cyclin proteolysis

Proteolysis of the yeast G, cyclins is triggered by their Cdc28-dependent p hosphorylation. Phosphorylated Cln1 and Cln2 are ubiquitinated by the SCF-G rr1 complex and then degraded by the 26 S proteasome. In this study, we ide ntified a cak1 allele in a genetic screen for mutants that stabilize the ye ast G(1) cyclins. Further characterization showed that Cln2HA was hypophosp horylated, unable to bind Cdc28, and stabilized in cak1 mutants at the rest rictive temperature. Hypophosphorylation of Cln2HA could thus explain its s tabilization. To test this possibility, we expressed a Cak1-independent mut ant of Cdc28 (Cdc28-43244) in cak1 mutants and found that Cln2HA phosphoryl ation was restored, but surprisingly, the phospho-Cln2HA was stabilized. Wh en bound to Cdc28-43244, Cln2HA was recognized and polyubiquitinated by SCF -Grr1. The Cdc28-43244 mutant thus reveals an unexpected complexity in the degradation of polyubiquitinated Cln2HA by the proteasome.