Fig 1.
Functional analysis of C-box peptides sufficient to inhibit APC/C activity.
A) A schematic diagram of the amino acid residues from the N-terminus of Cdc20 containing the region of the C-box, the KILR motif and the Mad2-binding motif [48]. The C-box and KILR motif regions, as well as the initial part of the Mad2-binding motif, interact with Cdc23 (APC8B in humans) based on structural analysis. The region in between the C-box and KILR-motif forms a loop that interacts with Apc1. The structure and potential binding site on the APC/C for the C-terminal portion of the Mad2-binding motif remains unknown (dashed line). The four C-box containing peptides AV16 (Cdc20 143–158), EV25 (Cdc20 134–158), C-box1 (130–167) or C-box2 (Cdc20 130–196) were designed to test their ability to inhibit APC/CCdc20 activity. B) Phosphor-images of APC/CCdc20 reaction time courses in the presence of buffer only or 220 μM of the C-box containing peptides AV16 (Cdc20 143–158), EV25 (Cdc20 134–158), C-box1 (130–167) or C-box2 (Cdc20 130–196). A significant level of APC/CCdc20 inhibition was observed only in the presence of the C-box2 peptide (n = 3). C) Phosphor-images of APC/CCdh1 reaction time courses in the presence of buffer or 220 μM C-box1 or C-box2 peptides. A significant level of APC/CCdh1 inhibition was observed in the presence of the C-box2 peptide (n = 3).
Fig 2.
Functional analysis of PQ65 peptides sufficient to inhibit APC/C activity.
A) A schematic diagram of the amino acid residues from the N-terminus of Cdc20 containing the region of the KILR motif and Mad2-binding motif. A 65 amino acid peptide PQ65 (Cdc20 169–232) was designed and synthesized around the most highly conserved regions within fungi. B) Phosphor-images of APC/CCdc20 reaction time courses in the presence of buffer only or 220 μM of peptides. A significant level of APC/CCdc20 inhibition was observed in the presence of the PQ65, PQ65-mut1 or PQ65-mut2 peptides (n = 3). Relative to the wild type PQ65 peptide, PQ65-mut1 peptide displayed a loss of inhibitory activity at the 30 and 60 minute time points (n = 3). C) Phosphor-image of a titration dilutions series using PQ65 where an IC50 of less than 50 μM was observed. D) Phosphor-images of APC/CCdh1 reaction time courses in the presence of buffer or 220 μM PQ65 peptide, where a significant level of APC/CCdh1 inhibition was observed (n = 3).
Fig 3.
Functional analysis of DQ36 peptides sufficient to inhibit APC/C activity.
A) A schematic diagram of the amino acid residues from the N-terminus of Cdc20 containing the region of the KILR motif and Mad2-binding motif. A 36 amino acid peptide DQ36 (Cdc20 197–232) was designed and synthesized around the most highly conserved regions within fungi. B) Phosphor-images of APC/CCdc20 reaction time courses in the presence of buffer only or 220 μM of peptides. C) Phosphor-images of APC/CCdh1 reaction time courses in the presence of buffer only or 220 μM of peptides. D) Phosphor-image of a titration dilutions series using DQ36 where an IC50 of about 150 μM was observed.
Fig 4.
Mad2 binding motif peptides bind to APC/C.
A) Wild type PQ65 and DQ36 pull-downs of APC/C at 20 μM peptide. Pre-coating the Avidin beads with biotin blocked the pull-down activity, but pre-coating APC/C with biotin had no effect. The addition of an excess of non-biotinylated DQ36 peptide was able to partially block the pull-down activity. B) Mutant DQ36-mut1 maintained the ability to pull-down APC/C, which was also partially blocked by an addition of excess DQ36 peptide. Mutant DQ36-mut2 was consistently observed to have partially lost APC/C binding activity relative to wild type DQ36 (n = 3).
Fig 5.
Functional analysis of PS46 and PL29 peptides sufficient to inhibit APC/C activity.
A) A schematic diagram of the amino acid residues from the N-terminus of Cdc20 containing the region of the KILR motif and Mad2-binding motif. A 46 amino acid peptide PS46 (Cdc20 167–213) containing the N-terminal region adjacent to the KILR-motif region was designed and tested, along with the 29 amino acid peptide PL29 (Cdc20 167–196) that only contained the N-terminal region in between the C-box and the KILR-motif. B) Phosphor-images of APC/CCdc20 reaction time courses in the presence of buffer only or 220 μM of peptides PQ65 and PS46. C) PS46 was consistently observed to have weaker APC/C binding activity relative to wild type DQ36 (n = 3). D) Phosphor-images of APC/CCdc20 reaction time courses in the presence of buffer only or 220 μM of peptides PS46 and PL29.
Fig 6.
Functional analysis of DK27, SQ19 and DS17 peptides for APC/C inhibition activity.
A) A schematic diagram of the amino acid residues from the N-terminus of Cdc20 containing the region of the KILR motif and Mad2-binding motif. A 27 amino acid peptide DK27 (Cdc20 197–223) containing KILR-motif region and only part of the C-terminal conserved region was designed and tested, along with the 19 amino acid peptide SQ19 (Cdc20 213–232) that only contained the C-terminal region, and the smaller DS17 (Cdc20 197–212) centered only on the KILR-motif and Mad2-binding motif. B) Phosphor-images of APC/CCdc20 reaction time courses in the presence of buffer only or 220 μM of peptides DK27, SQ19 and SQ19-mut1. C) DK27 and SQ19 peptides displayed the ability to bind APC/C similar to the control peptide DQ36, but SQ19-mut2 was consistently observed to have much weaker APC/C binding activity relative to wild type SQ19 (n = 3). D) Phosphor-images of APC/CCdc20 reaction time courses in the presence of buffer only or 220 μM of the peptide DS17 (Cdc20 197–212), where no inhibition was observed.
Fig 7.
Mad2-binding motif peptides induce sensitivity to the microtubule poison benomyl when over expressed in vivo, which requires the KILR-motif, and leads to a delay in Pds1 degradation upon recovery from benomyl.
A) Over expression of PQ65 or DQ36 in vivo leads to sensitivity to the microtubule poison benomyl. There was no significant difference observed in the absence of benomyl. B) The benomyl sensitivity phenotype was not observed when PQ65-mut1 or DQ36-mut1 peptides were over expressed where all 5 KILR-motif residues RILQY were changed to AAAAA. C) Over expression of DQ36 caused a delay in Pds1 degradation after cell cycle recovery from the microtubule poison benomyl as detected by Western blotting. Pds1 protein was detected using an anti-MYC antibody where the endogenous Pds1 had been epitope-tagged at the C-terminus with 13xMYC. An anti-actin antibody was used to probe for the levels of actin in each sample as a loading control.
Fig 8.
Summary of the functional and APC/C inhibitor analysis on the C-box and Mad2-binding motif regions of Cdc20.
A) The budding yeast KILR-motif is defined as RILQY. B) A summary of the APC/CCdc20 inhibition and APC/C binding data. The ability to inhibit the APC/C resides in both the N- and C-terminal regions of the PQ65 peptide, where both PL29 and SQ19 peptides contain an ability to inhibit APC/C even in the absence of the core KILR-motif region. In the DQ36-mut1 peptide, although the peptide does not fully inhibit the APC/C, the peptide retains the full ability to bind APC/C. In the DQ36-mut2 and SQ19-mut2 peptides there is a correlation between the loss of APC/C binding activity and a loss in the ability to inhibit APC/C.
Fig 9.
Identification of Tiny Yeast Comet 1 (TYC1).
A) BLAST search result between a segment of human p31comet and the predicted ORF YBR296C-A. The homologous region from p31comet was previously determined to mimic the “safety-belt” wrapping around the “pseudo-Cdc20” tail and contains β-sheet 7 and 8 from the structure. The structure of the predicted protein product from YBR296C-A is unknown. B) Detection of an endogenous mRNA product by RT-PCR. C) Detection of an endogenous epitope-tagged protein product produced from the native genomic promoter detected by Western blot. D) Detection of an over expressed epitope tagged protein from the genomic locus. E) A tyc1Δ strains are viable, where two independent clones are shown in a dilution series.
Fig 10.
Over expression of Tyc1 induced sensitivity to the microtubule poison benomyl.
A) Over expression of Tyc1 gave rise to a benomyl sensitive phenotype. B) Over expression of Tyc1 increased benomyl sensitivity in a mad2Δ background.
Fig 11.
A) Tyc1 proteins tested for their ability to inhibit APC/C activity. Tyc1-mut1 has 4 conserved amino acid residues changed to alanine. Tyc1-mut2 has 5 conserved residues changed to alanine. B) Phosphor-images show that 220 μM Tyc1 inhibits APC/CCdc20 activity. Tyc1-mut1 also fully inhibited APC/CCdc20. Tyc1-mut2 partially lost the ability to inhibit APC/CCdc20. C) 220 μM Tyc1 inhibits APC/CCdh1 activity. Tyc1-mut1 also fully inhibited APC/CCdh1. Tyc1-mut2 displayed a slight loss in the ability to inhibit APC/CCdh1. D) Phosphor-image of a titration dilutions series using Tyc1 where an IC50 of about 50 μM was observed.
Fig 12.
Tyc1 binds APC/C and blocks the ability of the Mad2 binding motif peptide DQ36 to bind to APC/C.
A) Biotinylated Tyc1 pull-downs of APC/C. The addition of an excess of non-biotinylated Tyc1 peptide was able to block the pull-down activity. B) Mutant Tyc1-mut1 and Tyc1-mut2 maintained the ability to pull-down APC/C (n = 3). C) An excess of Tyc1 blocked the ability of Mad2 binding motif peptide DQ36 from binding with the APC/C (n = 3). D) An excess of Tyc1 blocked the ability of Mad2 binding motif peptide DQ36-mut1 from binding with the APC/C (n = 3). E) An excess of Tyc1-mut1 retained the ability to block DQ36 peptide binding with APC/C. An excess of Tyc1-mut2 lost the ability to block DQ36 peptide binding with APC/C (n = 3). F) A summary of the activities of Tyc1 highlighting that the conserved TISLS residues are necessary for full APC/C inhibition and for blocking the binding between the Mad2 binding motif peptide DQ36 with the APC/C.
Fig 13.
The human peptide hp31 inhibits budding yeast APC/C activity and binds to yeast APC/C.
A) An alignment between budding yeast Tyc1 and the synthesized human hp31 peptide derived from human p31comet. B) Phosphor-images show that 220 μM human hp31 peptide inhibits yeast APC/CCdc20. C) Phosphor-images show that 220 μM human hp31 peptide inhibits yeast APC/CCdh1. D) The human-derived hp31 can pull-down budding yeast APC/C at 20 μM peptide (n = 3).
Fig 14.
Mad2-binding motif peptides and Tyc1 disrupt co-factor binding to APC/C and display an additive phenotype for microtubule poison sensitivity when co-ever expressed.
A) 35S-Methionine radio-labeled full-length Cdc20 or Cdh1 were made by IVT/T and pre-incubated with buffer only as a control, or Tyc1, DQ36 or PQ65 at a final concentration of 220 μM. Samples were mixed with enriched APC/C isolated on IgG beads and allowed to bind for 1 hour. Samples were washed and beads were boiled directly in protein sample buffer. The amount of APC/C-bound co-factor was measured by phosphor-imaging (n = 3). B) Co-over expression of PQ65 with Tyc1 in vivo leads to an increased sensitivity to the microtubule poison benomyl. In the presence of 12.5 μg/mL benomyl a significant decrease in the average colony number was observed (n = 5).