Figure 5. Tuning degron strength in the Ame1 N-terminus suppresses a cdc4 mutant.

(A) The threonine phosphorylated peptide (VQPILTPPKL, cyan) establishes a strong network of conserved interactions involving its phosphorylated threonine and the conserved arginine residues of Cdc4 (yellow). This binding is further stabilized by several protein-peptide interactions (Figure 5—figure supplement 1). (B) Changing the phospho-degron motif 1 into a strong Cdc4-degron sequence (ILSSP to ILTPP) leads to a loss of detectable Ame1-CPD in the overexpression system. Also, note that Okp1 is not detectable anymore. The cdc4-1 mutant background stabilizes Ame1-CPD^ILTPP and Okp1. The cdc4-1 allele was used at the permissive temperature of 30°C. (C) Serial dilution assay of Ame1-WT or Ame1-CPD^ILTPP in a wild-type or cdc4-1 mutant strain background. Plates were photographed after 3 days of incubation at the indicated temperature. Note that Ame1-CPD partially suppresses the growth defect of cdc4-1 at 34°C or in the presence of benomyl (20 µg/ml) or hydroxyurea. (D) Serial dilution assays of Ame1-CPD^ILTPP combined with a ctf19 deletion at low temperature (20°C) or increasing benomyl concentrations. Plates were photographed after 2 days (30°C, benomyl) or 3 days (20°C) of incubation at the indicated temperature, respectively. Note the benomyl hypersensitivity of Ame1-CPD^ILTPP relative to the wild-type allele.

Figure 5—figure supplement 1. Analysis of an Ame1 peptide with increased degron strength.

(A) RMSD fluctuations displayed by Ame1-CPD^ILTPP complexed with Cdc4. (B) The representative frame from the dominating cluster of Ame1-CPD^ILTPP–Cdc4 complex simulations (peptide: VQPILTPPKL, shown in blue) is aligned with the crystal structure taken for the study (the peptide counterpart is shown in red ribbon). The population of the dominating cluster is also shown. (C) Interactions of phosphorylated threonine peptide with the Cdc4 protein.