FIGURE 2.

Ams2 is destroyed in G₁ in a KEN box-dependent manner in vivo. A and B, Ams2 stability in G₁ and G₂ phase was measured. Ams2 was expressed from the nmt1 (rep81) promoter in cdc10-129 (HYY96) or cdc25-22 (HYY8) cells that had been incubated at the restrictive temperature to arrest cells at G₁ or G₂, respectively. Then cycloheximide (a protein synthesis inhibitor) and thiamine (a repressor of the nmt1 promoter) were added at time 0. Samples were taken at the indicated times and analyzed by immunoblotting using indicated antibodies. As control, endogenous Cdc13 and Cdc2 were used. The graph represents relative levels of Ams2 and Ams2-dKm. C, cells expressing Myc-Ams2 (HYY908) and Myc-Ams2-dKm (HYY909) from the endogenous ams2⁺ promoter were grown in EMM2 (asynchronous, lane A), washed with EMM2-N and then cultured for 15 h at 25 °C. Upon addition of NH₄Cl₂ (nitrogen source), the cells were released from the G₁ block into the cell cycle. The samples were taken every 20 min and analyzed by immunoblotting.