The cyclin-dependent kinase Cdc28p regulates distinct modes of Cdc6p proteolysis during the budding yeast cell cycle

Background: Cdc28p, the major cyclin-dependent kinase in budding yeast, prevents re-replication within each cell cycle by preventing the reassembly of Cdc6p-dependent pre-replicative complexes (pre-RGs) once origins have fired. Cdc6p is a rapidly degraded protein that must be synthesised in each cell cycle and is present only during the GI phase. Results: We found that, at different times in the cell cycle, there are distinct modes of Cdc6p proteolysis. Before Start, Gdc6p proteolysis did not require either the anaphase-promoting complex (APC/C) or the SCF complex, which mediate the major cell cycle regulated ubiquitination pathways, nor did it require Cdc28p activity or any of the potential Cdc28p phosphorylation sites in Cdc6p. In fact, the activation of B cyclin (Clb)-Gdc28p kinase inactivated this pathway of Cdc6p degradation later in the cell cycle. Activation of the G1 cyclins (Clns) caused Cdc6p degradation to become extremely rapid. This degradation required the SCFCDC4 and Cdc28p consensus sites in Cdc6p, but did not require Clb5 and Gibe. Later in the cell cycle, SCFCDC4-dependent Cdc6p proteolysis remained active but became less rapid. Conclusions: Levels of Gdc6p are regulated in several ways by the Gdc28p cyclin-dependent kinase. The Gin-dependent elimination of Gdc6p, which does not require the S-phase-promoting cyclins Clb5 and Clb6, suggests that the ability to assemble pre-RGs is lost before, not concomitant with, origin firing.