Figure 3

Ama1p binding to the APC/C is not required for its degradation. A: ama1∆ strain (RSY562) harboring either Ama1p-T7 (pKC3036) or Ama1p^CB∆/IR∆-T7 (pKC3048) expression plasmids were induced to enter meiosis and timepoints taken as indicated. Immunoprecipitation and immunoblot analysis of protein extracts was conducted to detect Ama1p-T7 and Ama1p^CB∆/IR∆-T7. Immunoblot analysis of Tub1p was used as a loading control. B: Ama1p deleted for the CB and IR regions shows reduced binding to Cdc23p-9myc during meiosis. The Cdc27-9myc expressing strain (KCY328) harboring either the vector control, Ama1p-T7 or mutant versions of Ama1p as indicated were induced to enter meiosis and the cells harvested 12 h following transfer to SPM when both CDC27 and AMA1 are expressed. Immunoprecipitation and immunoblot analysis was conducted to detect the presence of both proteins. The top and middle panels control for protein expression (input). The bottom panel assays co-immunoprecipitation. C: The amino-terminal region (codons 1-200) of Ama1p is sufficient for APC/C association. The Cdc27-9myc expressing strain RSY1337 harboring either GST (lanes 3 and 4), GST-Ama1p^1-200 (lanes 1, 2 and 5) or GST-Ama1p^1-200CB∆ (lane 6) expression plasmids were grown in raffinose/galactose medium to induce the fusion genes. Immunoprecipitation and immunoblot analysis was conducted to detect the presence of both proteins. The top and middle panels control for protein expression (input). The bottom panel assays co-immunoprecipitation. [] represents the no antibody mock immunoprecipitation. The asterisk represents a background band. D: A wild-type strain (RSY750) harboring integrated AMA1-3HA and CDC20-18myc alleles were induced to enter meiosis and timepoints taken as indicated. Immunoblot analysis of immunoprecipitated protein extracts was conducted to detect Ama1p-3HA and Cdc20p-18myc. Immunoblot analysis of Tub1p was used as a loading control. In all experiments, the approximate times of meiosis I (MI) and meiosis II (MII) were determined by DAPI analysis.