The KEN box regulates Clb2 proteolysis in G1 and at the metaphase-to-anaphase transition

Clb2 mitotic cyclin inhibits cell cycle progression by preventing mitotic exit and DNA synthesis. To allow cell cycle progression, Clb2 proteolysis is triggered by Cdc20 during the metaphase-to-anaphase (M-A) transition and by Hct1 during mitotic exit and G1 [1-6]. A cis element called the destruction box is required for this proteolysis [7-11]. Recently, an additional cis element called the "KEN box" was also shown to be required for proteolysis of human CDC20 and Securin [3,12]. Using a novel color assay, we show that a Clb2 KEN box is required to target a fusion protein containing the first 124 amino acids of Clb2 for proteolysis. We further show that full-length Clb2 bearing mutations in the KEN box is degraded efficiently during the M-A transition, but poorly during G1. If the destruction box of Clb2 is mutated in combination with mutation of the KEN box, then this form of Clb2 is more stable than Clb2 bearing either mutation by itself during both M-A and G1. Our results show that the KEN box and the destruction box act together during both M-A and G1 to regulate Clb2 proteolysis.