Figure 2. APC/C^Cdh1-dependent degradation of Fir1, Mps1, and Ybr138C in G1.

(A) Wild-type and cdc23-1 cells were arrested in G1 with alpha factor and transferred to 37°C to inactivate Cdc23-1, a core subunit of the APC/C. cdc28-13 cdh1Δ cells were arrested in G1 by incubation at the non-permissive temperature (37°C). The expression of FIR1, MPS1, and YBR138C was induced from GAL1p followed by addition of 2% dextrose and 500 µg/ml cycloheximide to terminate protein synthesis. Samples were withdrawn at the indicated times and processed for immunoblotting with PAP antibodies. (B) Cells carrying an empty vector (lanes 1–4) or GALLp-cdh1-m11 (lanes 5–8) were grown in the presence of raffinose and induced with 2% galactose for the indicated times. Cdh1-m11 lacks sites of inhibitory phosphorylation and is constitutively active. Samples were processed for immunoblotting to examine endogenous levels of Fir1, Mps1, and Ybr138C.