1. Cell Biology
  2. Chromosomes and Gene Expression

Cdc4 phospho-degrons allow differential regulation of Ame1CENP-U protein stability across the cell cycle

  1. Miriam Böhm
  2. Kerstin Killinger
  3. Alexander Dudziak
  4. Pradeep Pant
  5. Karolin Jänen
  6. Simone Hohoff
  7. Karl Mechtler
  8. Mihkel Örd
  9. Mart Loog
  10. Elsa Sanchez-Garcia
  11. Stefan Westermann  Is a corresponding author
  1. Department of Molecular Genetics I, Faculty of Biology, Center of Medical Biotechnology, University of Duisburg-Essen, Germany
  2. Department of Computational Biochemistry, Faculty of Biology, Center of Medical Biotechnology, University of Duisburg-Essen, Germany
  3. IMP - Research Institute of Molecular Pathology, Austria
  4. Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Austria
  5. Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna BioCenter (VBC), Austria
  6. Institute of Technology, University of Tartu, Estonia
https://doi.org/10.7554/eLife.67390
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Cite this article
  1. Miriam Böhm
  2. Kerstin Killinger
  3. Alexander Dudziak
  4. Pradeep Pant
  5. Karolin Jänen
  6. Simone Hohoff
  7. Karl Mechtler
  8. Mihkel Örd
  9. Mart Loog
  10. Elsa Sanchez-Garcia
  11. Stefan Westermann
(2021)
Cdc4 phospho-degrons allow differential regulation of Ame1CENP-U protein stability across the cell cycle
eLife 10:e67390.
https://doi.org/10.7554/eLife.67390
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12 figures, 4 videos, 4 tables and 1 additional file

Figures

Figure 1 with 1 supplement
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Phosphorylation analysis of the essential constitutive centromere-associated network (CCAN) subunit Ame1.

(A) Organization of the essential CCAN component Ame1CENP-U and localization of phosphorylation sites. Ame1 shows a Cdk1 phosphorylation cluster (T31, S41, S45, S52, S53, S59, S101) at the N-terminus. The first 15 amino acids are essential for Mtw1c binding, the coiled-coil region (aa 199–254) is required for heterodimerization with Okp1CENP-Q. Schematic overview on the right shows the four-protein complex COMA, consisting of Ame1CENP-U, Okp1CENP-Q, Ctf19CENP-P, and Mcm21CENP-O. The COMA complex binds to the outer kinetochore component Mtw1 complex and to the centromeric nucleosome. (B) In vitro kinase assay with recombinant Ame1-Okp1c with either Cdk1-Clb2 or Cdk1-Clb5. The migration pattern of Ame1 is shifted to a slowly migrating form when incubated with Cdk1-Clb2. Asterisk denotes a contaminating protein. (C) List of all mapped Ame1 phosphorylation sites either in vivo or in vitro. T31, S41, S45, S53, and S101 show the minimal motif for Cdk1 (S/TP). (D) Stably integrated Ame1 variants display distinct migration patterns in SDS-PAGE. Ame1-WT shows multiple slowly migrating forms that are eliminated in Ame1-7A and Ame1-7E. (E) Serial dilution assay of Ame1 variants using the FRB anchor-away system. Ame1-WT and both mutants can rescue the growth defect when endogenous Ame1 is anchored away from the nucleus. (F) Serial dilution assay of internal Ame1 truncation mutants in the anchor-away system.

Figure 1—source data 1

Mass spectrometry analysis of native constitutive centromere-associated network (CCAN) complexes.

https://cdn.elifesciences.org/articles/67390/elife-67390-fig1-data1-v2.xlsx
Figure 1—source data 2

Mass spectrometry analysis of in vitro phosphorylated COMA.

https://cdn.elifesciences.org/articles/67390/elife-67390-fig1-data2-v2.xlsx
Figure 1—figure supplement 1
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Quantitative phosphorylation analysis of recombinant Ame1-Okp1 by S-Cdk1 and M-Cdk1 complexes.

(A) In vitro phosphorylation of recombinant AO with Cdc28-Clb5 and Cdc28-Clb2 complexes or their hydrophobic patch mutants (hpm) that eliminate docking-dependent phosphorylation was analyzed. ΔN: N-terminal truncation mutant of Okp1. Ratio between phosphorylation of the different variants is given below. Quantification shows mean values of Ame1 or Okp1 phosphorylation (a.u.) and standard error of the mean is indicated, n = 3. (B) Multiple-sequence alignment of Ame1 N-terminus (aa 1–63) from different yeast species. Conserved residues are colored according to the ClustalW scheme. The identified phosphorylation sites are conserved in the most closely related Saccharomyces species.

Figure 2 with 2 supplements
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Identification of phospho-degron motifs in Ame1.

(A) Flag- and Myc-tagged versions of Ame1 and Okp1 were expressed from a two-micron plasmid under a bidirectional galactose-inducible promoter. Under normal growth conditions in YEP + dextrose or YEP + raffinose, no overexpression occurs, overexpression is only induced by adding galactose (GAL) to the medium. After 0, 2, and 5 hr in GAL, cell extracts were prepared and protein expression was followed using western blot analysis. (B) Western blot analysis of overexpressed Ame1-WT, -7A, and -7E variants in a wild-type strain background. (C) Quantification of protein levels of Ame1-WT and Ame1-7A after indicated times in galactose medium. Mean values and standard error of the mean are indicated, n = 7. (D) Overview of Ame1 phospho-mutants used for overexpression studies (E) or in vitro kinase assays (F). (E) Overexpression studies of individual Ame1 phospho-variants. Ame1 protein levels in this experiment are quantified below, the Ame1-WT level is set to 1. Okp1 levels are stable and used for normalization of Ame1 protein levels. (F) In vitro kinase assay of AO complexes using recombinant Cdk1-Clb2. Note reduced or lacking phosphorylation of Ame1-4A and -7A, respectively. Also Okp1 can be phosphorylated by Cdk1-Clb2. (G) Cdk1 target sites in Ame1 resemble two different types of phospho-degrons motifs that are recognized by the E3 ubiquitin ligase complex SCF-Cdc4.

Figure 2—figure supplement 1
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Additional analysis of Ame1 overexpression.

(A, B) Whole-cell extracts of overexpressed Ame1-WT and Ame1-7A in a wild-type background (A) or skp1-3 background (B) were treated with lambda phosphatase. The signal intensity of the collapsed signal was quantified yielding a ratio of Ame1-WT to Ame1-7A of 1:4 in a wild-type background. (C) Western blot analysis of overexpression of Ame1-WT and phosphorylation mutants from a galactose-inducible promoter in a mad1Δ background strain. Note that Ame1 levels remain low in this background. Ame1 phosphorylation mutants show the same accumulation effect as seen in wild-type background. (D) Same experiment as (C) in a checkpoint deficient psh1Δ strain background. (E) Overexpression of Ame1-WT and phosphorylation mutants in a deletion mutant of mub1, which is one subunit of the Ubr2/Mub1 ubiquitin ligase complex implicated in the regulation of Dsn1.

Figure 2—figure supplement 2
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Additional analysis of AO in vitro phosphorylation by Cdc28-Clb2.

Left panel: scheme of the used recombinant AO complexes. For the Okp1-1A mutant, the Cdk1 site Ser26 was mutated to alanine. Right panel: Coomassie-stained gel and corresponding autoradiography of in vitro kinase assays with the indicated AO variants. Note that Ame1-7A in combination with Okp1-1A completely eliminates phosphorylation.

Figure 3 with 1 supplement
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Gaussian-accelerated molecular dynamics simulations predict Ame1 peptide binding to Cdc4.

(A) Interactions between the conserved arginine residues of Cdc4 (yellow) and the phospho-serine residues of the doubly phosphorylated peptide (cyan). (B) The doubly phosphorylated Ser41/Ser45 peptide and Cdc4 establish an intense hydrogen bond network involving the phosphorylated residues of the peptide and the conserved arginine residues of Cdc4, as well as other residues.

Figure 3—figure supplement 1
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Additional analysis of peptide-Cdc4 interactions by Gaussian-accelerated molecular dynamics simulations.

(A) Interactions of the doubly phosphorylated Ame1 peptide Ser41/Ser45, as well as the monophosphorylated Ser41 and Ser45 peptides with the Cdc4 WD40 domain, are shown. (B) Root-mean-square deviation (RMSD) fluctuations for the doubly phosphorylated Ser41/Ser45 (P-Ser41/Ser45), monophosphorylated Ser41 (P-Ser41), and Ser45 (P-Ser45) complexed with Cdc4.

Figure 4
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SCF-Cdc4 regulates Ame1-Okp1 protein levels in vivo.

(A) Model of substrate binding to SCF complexes. SCF is composed of Skp1, Cdc53 (Cullin), Rbx1, an F-box protein (e.g., Cdc4 or Grr1), and here with the E2 enzyme Cdc34. Lower panel: overexpression of Ame1-WT leads to accumulation of the protein in a skp1-3 mutant strain over time as compared to a wild-type background. (B) Protein levels of Ame1-Okp1 in different SCF mutants after overexpression. Note that Ame1 levels remain low in the grr1Δ mutant (cytoplasmic F-box protein), and that Okp1 strongly accumulates in the cdc4-1 mutant. All alleles were used at the permissive temperature of 30°C. Quantification of Okp1 protein levels for this experiment is shown above, Okp1-WT signal was set to 1. (C) Serial dilution assay of overexpressed Ame1-Okp1 variants in wild-type or SCF mutant strain backgrounds (skp1-3 or cdc34-2). Plates were photographed after 2 days at the indicated temperature. (D) Serial dilution assay of overexpressed Ame1-Okp1 variants together with Ctf19-Mcm21 in a wild-type strain background. Plates were photographed after 2 days at the indicated temperature.

Figure 5 with 1 supplement
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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-CPDILTPP and Okp1. The cdc4-1 allele was used at the permissive temperature of 30°C. (C) Serial dilution assay of Ame1-WT or Ame1-CPDILTPP 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-CPDILTPP 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-CPDILTPP relative to the wild-type allele.

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

(A) RMSD fluctuations displayed by Ame1-CPDILTPP complexed with Cdc4. (B) The representative frame from the dominating cluster of Ame1-CPDILTPP–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.

Figure 6 with 1 supplement
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Analysis of endogenous Ame1 phospho-mutants over the cell cycle.

(A) Ame1-variants (Ame1-3A, CPD only, allowing phosphorylation at degron motifs 1 and 2 or eliminating it, Ame1-4A, CPD null) were expressed from the endogenous promoter as the sole copy, and phosphorylation was analyzed in different cell cycle arrests. Drugs used for the arrests: alpha-factor (1 mg/ml) for G1, hydroxyurea (0.2 M) for S-phase, and nocodazole (15 µg/ml) for M-phase. Ame1-3A shows one slowly migrating form in S-phase and two in M-phase, whereas Ame1-4A eliminates all slowly migrating forms. (B) Ame1-3A was released from an alpha-factor arrest, and phosphorylation was analyzed by western blotting. Right panel: DNA content analysis by FACS. Phosphorylation is maximal after 30 min when cells have completed S-phase (right: FACS analysis 15 + 30 min), and phosphorylated forms disappear when cells are in mitosis (45 + 60 min, dashed box). For phosphorylation pattern of Ame1-WT, see Figure 6—figure supplement 1A. (C, D) Cell cycle analysis of Ame1-3A (C) and Ame1-4A (D). (E) Analysis of Ame1-3A in the skp1-3 mutant at 34°C (semi-permissive). Note that phosphorylation at motif 1+ 2 persists in the mutant and cells remain in mitosis with 2C DNA content. (F) Analysis of Ame1-3A in the skp1-3 mutant at 37°C (restrictive). Note that under these conditions cells are delayed to complete replication and mainly a single phospho-form of Ame1 is found.

Figure 6—figure supplement 1
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Cell cycle analysis of Ame1-WT and phosphorylation mutants.

(A) Cells expressing Ame1-WT were released from alpha-factor arrest, and phosphorylation was followed by western blot analysis. Ame1 displays multiple slowly migrating forms that change over the cell cycle. (B) Cells expressing Ame1-4A were released from alpha-factor arrest, and phosphorylation was followed by western blot analysis. No slowly migrating forms are detectable, which can also be seen in Figure 6. (C) FACS analysis of Ame1-WT and Ame1-4A. Cells progress through the cell cycle similarly, both strains complete S-phase after 30 min. In Ame1-WT 1C cells reappear around min 60–75, whereas in Ame1-4A cells this is slightly delayed (75–90 min). (D) A Flag-tagged Ame1 variant in which only phosphorylation of motif 1 was permitted (Ame1-5A1) was followed over the cell cycle by western blotting after release from alpha-factor arrest. (E) A Flag-tagged Ame1 variant in which only phosphorylation of motif 2 was permitted (Ame1-5A2) was followed over the cell cycle by western blotting after release from alpha-factor arrest.

Figure 7 with 1 supplement
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Mtw1c binding shields the Ame1 phospho-degron from Cdk1 phosphorylation.

(A) Scheme of the kinase assay. Recombinant AO with Ame1-WT or Ame1-7A is used either alone or in combination with its binding partner Mtw1c. (B) In vitro kinase assay of Ame1-Okp1c alone or preincubated with Mtw1c and Cdk1-Clb2 shows decreased phosphorylation of Ame1-WT-Okp1c when bound to Mtw1c (lanes 3 + 4). Phosphorylation of Okp1 is overlapping with phosphorylation of Dsn1 (lanes 4 + 5 + 7). (C) Scheme of the FRB assay. An Mtw1-FRB strain was combined with Ame1-3A or Ame1-4A and Ame1 phosphorylation was analyzed after rapamycin addition. Rapamycin anchors Mtw1-FRB out of the nucleus to the ribosomal anchor RPL13-FKBP12. (D) Initial rapamycin assay to follow Ame1 phosphorylation over the time of 0, 90, and 180 min after rapamycin addition. Ame1-3A shows multiple slowly migrating forms that accumulate over time, whereas Ame1-4A eliminates all slowly migrating forms. See Figure 7—figure supplement 1A for a corresponding FACS analysis. (E) The rapamycin assay in combination with cycloheximide (CHX). Cultures were preincubated in YEPD for 2 hr and in YEPD + rapamycin for 180 min. New protein translation was inhibited by adding CHX (50 µg/ml) to the medium. Cell extracts were prepared after 0, 30, 60, 90, or 120 min after CHX addition, and Ame1-phosphorylation was analyzed by western blot analysis. Numbers below indicate signal intensities of the slowly migrating forms of Ame1 of the individual timepoints, normalized to timepoint 0.

Figure 7—figure supplement 1
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Additional analysis of Ame1 variants lacking binding to the Mtw1 complex.

(A) FACS analysis of cell cycle progression related to Figure 7D. The effect of rapamycin addition to Mtw1-FRB Ame1-3A-Flag strains was tested at timepoints 0, 90, and 180 min. Note that strains mostly display a 2C DNA content. The broad shoulder at t = 0 min for the +Rapamycin sample is likely an artifact because it is absent in the t = 90 min sample. (B) Western blot analysis of Ame1 variants lacking the Mtw1c binding motif (ΔN), schematically depicted on the left. Note that Ame1-3A variants display a slowly migrating form, which is eliminated in the 4A mutant.

Figure 8
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Model for SCF-mediated regulation of COMA assembly at the budding yeast kinetochore.

(A) Scheme illustrating stepwise phosphorylation of the degron motif on Ame1 by Cdk1 and protection from phosphorylation within the kinetochore. In addition to Cdk1, other kinases might be involved in addition, in particular in the phosphorylation of Okp1. (B) Cell cycle regulation of COMA complex stability; for details, see discussion. In S-phase, COMA is only partially phosphorylated, allowing assembly at the kinetochore. In M-phase, free COMA is fully phosphorylated and targeted for degradation, while kinetochore-bound COMA is protected.

Author response image 1
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Chromosome transmission fidelity experiment: Transmission of a chromosome fragment containing the SUP11 gene is assayed (Hieter et al.

, 1985). Loss of the fragment leads to red or red sectored colonies in an ade2-1 strain background. The ctf19 deletion mutant serves as a control.

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Western blot analysis of Ame1-3A (phosphorylation at S41/45 and S52/53 allowed) versus Ame1-5A1 (phosphorylation at S41 S45 prevented).

B. Quantification of Pds1 degradation in both samples. C. Corresponding FACS analysis, 0 min at bottom.

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Live cell microscopy of Ame1-WT and Ame1-7A tagged with GFP at metaphase and anaphase kinetochore clusters.

Scale bar: 5 mm. The intensity of 15-18 kinetochore clusters for each strain was quantified. P-values calculated from unpaired Student’s t-test.

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Multiple sequence alignment of Ame1 N-terminus from difference yeast species.

Conserved residues colored according to ClustalW scheme.

Videos

Video 1
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Gaussian-accelerated molecular dynamics (GaMD) simulation of the doubly phosphorylated (S41 and S45) Ame1 peptide (red) binding to WD40 domain of Cdc4 (blue).
Video 2
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Gaussian-accelerated molecular dynamics (GaMD) simulation of the monophosphorylated (S41) Ame1 peptide (red) binding to the WD40 domain of Cdc4 (blue).
Video 3
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Gaussian-accelerated molecular dynamics (GaMD) simulation of the monophosphorylated (S45) Ame1 peptide (red) binding to the WD40 domain of Cdc4 (blue).
Video 4
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Gaussian-accelerated molecular dynamics (GaMD) simulation of the Ame1-CPDILTPP peptide (red), phosphorylated at T52, binding to the WD40 domain of Cdc4 (blue).

Tables

Table 1
Analysis of Ctf19CCAN phosphorylation in yeast extracts.

Native constitutive centromere-associated network (CCAN) phosphorylation sites detected after purification of TAP-tagged kinetochore subunits from yeast extracts. For details, see Figure 1—source data 1.

S.c.CCAN subunitHuman homolog% sequence coverageTotal P-sites detectedMinimal Cdk1 sites detected (S/T)PFull Cdk1 sites detected (S/TP_K/R)
Ame1CENP-U7984 (T31, S41, S45, S53)-
Okp1CENP-Q856--
Mcm21CENP-O923-1 (S139)
Ctf19CENP-P801--
Nkp1-973-1 (S222)
Nkp2-89--
Chl4CENP-N9831 (S281)-
Iml3CENP-L95---
Ctf3CENP-I75---
Mcm22CENP-H97---
Mcm16CENP-K93---
Cnn1CENP-T90172 (T42, S192)2 (T21, S177)
Mhf1CENP-S9331 (T34)-
Mhf2CENP-X9511 (S60)-
Key resources table
Reagent type
(species) or resource		DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Saccharomyces cerevisiae)S288C
Recombinant DNA reagentSee Materials and methods, Table 2
Genetic reagent, gene (S. cerevisiae)See Materials and methods, Table 3
AntibodyAnti-Flag (monoclonal, peroxidase conjugated)Sigma AldrichA85921:10,000
AntibodyAnti-myc 9E10 (mouse monoclonal)BioLegendCatalog #6268011:1000
AntibodyAnti-mouse secondary (from sheep)Cytiva LifeSciencesCatalog #NA9311:10,000
AntibodyAnti-tubulin (monoclonal, peroxidase conjugated)Santa Cruz Biotechnologiessc-530301:1000
Chemical compound, drugα-factor1 mg/ml
Chemical compound, drugHydroxyureaUS Biological Life SciencesH91200.2 M
Chemical compound, drugNocodazoleSigma AldrichM1404-50MG15 µg/ml
Chemical compound, drugBenomylSigma Aldrich45339-250MG15–30 µg/ml
Chemical compound, drugRapamycinDiagonal370.940.0101 µg/ml
Chemical compound, drugCycloheximideSigma AldrichC7698-1G50 µg/ml
Table 2
Vectors for protein expression and yeast strain generation.
PlasmidDescriptionSource
Bacterial expression
pSW698pST39-Mtw1/Nsl1/Nnf1/6xHis-Dsn1Hornung et al., 2014
pSW900pST39-Okp1/Ame1-6xHisHornung et al., 2014
pMLU16pST39-Okp1/Ame1-7A-6xHis (T31A, S41A, S45A, S52A, S53A, S59A, S101A)This study
pMB91pST39-Okp1/Ame1-S52A+S53A-6xHisThis study
pMB92pST39-Okp1/Ame1-T31A-6xHisThis study
pMB93pST39-Okp1/Ame1-S41A+S45A-6xHisThis study
pMB94pST39-Okp1/Ame1-4A-6xHis (S41A, S45A, S52A, S53A)This study
pMB109pST39-Okp1-S26A/Ame1-6xHisThis study
pMB110pST39-Okp1-S26A/Ame1-4A-6xHis (S41A, S45A, S52A. S53A)This study
pMB111pST39-Okp1-S26A/Ame1-7A-6xHis (T31A, S41A, S45A, S52A, S53A, S59A, S101A)This study
pMB112pST39-Okp1-WT/Ame1-3A-6xHis (T31A, S59A, S101A)This study
Yeast expression
pESC-Clb2-TAPMorgan Lab
pESC-Clb5-TAPMorgan Lab
Yeast genetics
pMLU13Ame1-7A-6xFlag in pRS306 (T31A, S41A, S45A, S52A, S53A, S59A, S101A)This study
pMLU17Ame1-7E-6xFlag in pRS306 (T31E, S41E, S45E, S52E, S53E, S59E. S101E)This study
pMB54Ame1-WT-1xFlag + Okp1-WT-1xMyc in pESC-HISThis study
pMB55Ame1-7A-1xFlag + Okp1-WT-1xMyc in pESC-HISThis study
pMB56Ame1-7E-1xFlag + Okp1-WT-1xMyc in pESC-HISThis study
pMB64Ame1-TM3-6xFlag in pRS306 (Δ31–89)This study
pMB65Ame1-TM4-6xFlag in pRS306 (Δ31–116)This study
pMB66Ame1-TM5-6xFlag in pRS306 (Δ31–187)This study
pMB68Ame1-TM2-6xFlag in pRS306 (Δ31–75)This study
pMB72Ame1-S41A+S45A-1xFlag + Okp1-WT-1xMyc in pESC-HISThis study
pMB73Ame1-S52A+S53A-1xFlag + Okp1-WT-1xMyc in pESC-HISThis study
pMB84Ame1-ILTPP-1xFlag + Okp1-WT-1xMyc (optimal CPD, S52T + S53P) in pESC-HISThis study
pMB85Ame1-5A+ILTPP-1xFlag + Okp1-WT-1xMyc (optimal CPD, T31A, S41A, S45A, S52T, S53P, S59A, S101A)in pESC-HISThis study
pMB86Ame1-T31A-1xFlag + Okp1-WT-1xMyc in pESC-HISThis study
pMB87Ame1-S45A-1xFlag + Okp1-WT-1xMyc in pESC-HISThis study
pMB90Ame1-S41A-1xFlag + Okp1-WT-1xMyc in pESC-HISThis study
pMB98pRS306-Ame1-3A-6xFlag (T31A, S59A, S101A)This study
pMB99pRS306-Ame1-4A-6xFlag (S41A, S45A, S52A, S53A)This study
pMB104Ame1-7A-1xFlag + Okp1-S26A-1xMyc in pESC-HISThis study
pMB113pRS306-Ame1-5A1-6xFlag (T31A, S41A, S45A, S59A, S101A)This study
pMB114pRS306-Ame1-5A2-6xFlag (T31A, S52A, S53A, S59A, S101A)This study
pMB156pRS306-Ame1-3A-ΔN-6xFlag (Δ2–15, T31A, S59A, S101A)This study
pMB157pRS306-Ame1-4A-ΔN-6xFlag (Δ2–15, S41A, S45A, S52A. S53A)This study
pSW731pRS306-Ame1-WT-6xFlagHornung et al., 2014
pESC-HISpESC-HISAgilent Technologies
pESC-URApESC-URAAgilent Technologies
pDD526Pds1-13xMyc in pRS305Westermann lab
Table 3
Yeast strains.
Strain nameRelevant genotypeSource
SWY355Mat a; tor1-1, fpr1::loxP-LEU2-loxP, RPL13A-2xFKBP12::loxP-TRP1-loxPHaruki et al., 2008
SWY536Mat a; tor1-1 fpr1::loxP-LEU2-loxP RPL13A-2xFKBP12::loxP, Ame1-FRB::KanMXHornung et al., 2014
PSY1.1Mat a; tor1-1 fpr1::loxP-LEU2-loxP RPL13A-2xFKBP12::loxP, Mtw1-FRB::KanMXKillinger et al., 2020
MLY3Mat a; tor1-1 fpr1::loxP-LEU2-loxP RPL13A-2xFKBP12::loxP, Ame1-FRB::KanMX, Ame1-WT-6xFlag::URA3This study
MLY5Mat a; tor1-1 fpr1::loxP-LEU2-loxP RPL13A-2xFKBP12::loxP, Ame1-FRB::KanMX, Ame1-7A-6xFlag::URA3This study
MLY15Mat a, ade2-1, leu2-3,112, his3Δ200, ame1Δ::HIS3, ura3-52, Ame1-WT-6xFlag::URA3This study
MLY31Mat a; tor1-1 fpr1::loxP-LEU2-loxP RPL13A-2xFKBP12::loxP, Ame1-FRB::KanMX, Ame1-7E-6xFlag::URA3This study
MBY79Mat a, ade2-1, his3Δ200, ame1Δ::HIS3, leu2-3,112, ura3-52, Ame1-WT-6xFlag::URA3, Cse4-13xMyc::KanMXThis study
MBY81Mat a, ade2-1, his3Δ200, ame1Δ::HIS3, leu2-3,112, ura3-52, Ame1-7E-6xFlag::URA3, Cse4-13xMyc::KanMXThis study
MBY83Mat a, ade2-1, his3Δ200, ame1Δ::HIS3, leu2-3,112, ura3-52, Ame1-7A-6xFlag::URA3, Cse4-13xMyc::KanMXThis study
MBY153Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52 (two-micron-pESC-HIS) (pGAL-empty)This study
MBY155Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52 (two-micron pESC-HIS-Ame1-WT-1xFlag + Okp1-WT-1xMyc)This study
MBY156Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52 (two-micron pESC-HIS-Ame1-7A-1xFlag + Okp1-WT-1xMyc)This study
MBY157Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52 (two-micron pESC-HIS-Ame1-7E-1xFlag + Okp1-WT-1xMyc)This study
MBY158Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52, psh1Δ::NatNT2 (two-micron pESC-HIS)This study
MBY160Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52, psh1Δ::NatNT2 (two-micron pESC-HIS-Ame1-WT-1xFlag + Okp1-WT-1xMyc)This study
MBY161Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52, psh1Δ::NatNT2 (two-micron pESC-HIS-Ame1-7A-1xFlag + Okp1-WT-1xMyc)This study
MBY162Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52, psh1Δ::NatNT2 (two-micron pESC-HIS-Ame1-7E-1xFlag + Okp1-WT-1xMyc)This study
MBY163Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52, mad1Δ::KanMX (two-micron pESC-HIS)This study
MBY165Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52, mad1Δ::KanMX (two-micron pESC-HIS-Ame1-WT-1xFlag + Okp1-WT-1xMyc)This study
MBY166Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52, mad1Δ::KanMX (two-micron pESC-HIS-Ame1-7A-1xFlag + Okp1-WT-1xMyc)This study
MBY167Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52, mad1Δ::KanMX (two-micron pESC-HIS-Ame1-7E-1xFlag + Okp1-WT-1xMyc)This study
MBY225Mat α, his3Δ200, ura3-52, Ame1-TM2-6xFlag::URA3, lys2-801am, tor1-1 fpr1::loxP-LEU2-loxP RPL13A-2xFKBP12::loxP-TRP1-loxP, Ame1-FRB::KanMXThis study
MBY226Mat α, his3Δ200, ura3-52, Ame1-TM3-6xFlag::URA3, lys2-801am, tor1-1 fpr1::loxP-LEU2-loxP RPL13A-2xFKBP12::loxP-TRP1-loxP, Ame1-FRB::KanMXThis study
MBY227Mat α, his3Δ200, ura3-52, Ame1-TM4-6xFlag::URA3, lys2-801am, tor1-1 fpr1::loxP-LEU2-loxP RPL13A-2xFKBP12::loxP-TRP1-loxP, Ame1-FRB::KanMXThis study
MBY228Mat α, his3Δ200, ura3-52, Ame1-TM5-6xFlag::URA3, lys2-801am, tor1-1 fpr1::loxP-LEU2-loxP RPL13A-2xFKBP12::loxP-TRP1-loxP, Ame1-FRB::KanMXThis study
MBY241Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52 (two-micron pESC-HIS-Ame1-S52A+S53A-1xFlag + Okp1-WT-1xMyc)This study
MBY255Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52 (two-micron pESC-HIS-Ame1-ILTPP-1xFlag + Okp1-WT-1xMyc)This study
MBY256Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52 (two-micron pESC-HIS-Ame1-5A+ILTPP-1xFlag + Okp1-WT-1xMyc)This study
MBY273Mat a, ade2-1, leu2-3,112, his3Δ200, ame1Δ::HIS3, ura3-52, Ame1-WT-CPDILTPP-6xFlag::URA3This study
MBY275Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52 (two-micron pESC-HIS-Ame1-T31A-1xFlag + Okp1-WT-1xMyc)This study
MBY276Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52 (two-micron pESC-HIS-Ame1-S45A-1xFlag + Okp1-WT-1xMyc)This study
MBY278Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52 (two-micron pESC-HIS-Ame1-S41A+S45A-1xFlag + Okp1-WT-1xMyc)This study
MBY279Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52 (two-micron pESC-HIS-Ame1-S41A-1xFlag + Okp1-WT-1xMyc)This study
MBY292Mat a, ura3-52, lys2-801, ade2-101, his3Δ200, trp1Δ63, leu2Δ1, skp1-3::LEU2 (two-micron pESC-HIS-Ame1-WT-1xFlag-Okp1-WT-1xMyc)This study
MBY293Mat a, ura3-52, lys2-801, ade2-101, his3Δ200, trp1Δ63, leu2Δ1, skp1-3::LEU2 (two-micron pESC-HIS-Ame1-7A-1xFlag-Okp1-WT-1xMyc)This study
MBY310Mat a, cdc53-1 (ts), ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, psi+, ssd1-d2 (two-micron pESC-HIS-Ame1-WT-1xFlag-Okp1-WT-1xMyc)This study
MBY311Mat a, cdc53-1 (ts), ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, psi+, ssd1-d2 (two-micron pESC-HIS-Ame1-7A-1xFlag-Okp1-WT-1xMyc)This study
MBY312Mat a, cdc34-2 (ts), ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, psi+, ssd1-d2 (two-micron pESC-HIS-Ame1-WT-1xFlag-Okp1-WT-1xMyc)This study
MBY313Mat a, cdc34-2 (ts), ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, psi+, ssd1-d2 (two-micron pESC-HIS-Ame1-7A-1xFlag-Okp1-WT-1xMyc)This study
MBY314Mat α, cdc4-1 (ts), ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, psi+, ssd1-d2 (two-micron pESC-HIS-Ame1-WT-1xFlag-Okp1-WT-1xMyc)This study
MBY315Mat alpha, cdc4-1 (ts), ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, psi+, ssd1-d2 (two-micron pESC-HIS-Ame1-7A-1xFlag-Okp1-WT-1xMyc)This study
MBY316Mata, ade2-1, his3Δ200, trp1-1, ura3-52, grr1Δ::LEU2 (ts), lys2-801 (two-micron pESC-HIS-Ame1-WT-1xFlag-Okp1-WT-1xMyc)This study
MBY317Mata, ade2-1, his3Δ200, trp1-1, ura3-52, grr1Δ::LEU2 (ts), lys2-801 (two-micron pESC-HIS-Ame1-7A-1xFlag-Okp1-WT-1xMyc)This study
MBY322Mat a, ade2-1, leu2-3,112, his3Δ200, ura3-52, mub1Δ::natNT2 (two-micron pESC-HIS-Ame1-WT-1xFlag-Okp1-WT-1xMyc)This study
MBY323Mat a, ade2-1, leu2-3,112, his3Δ200, ura3-52, mub1Δ::natNT2 (two-micron pESC-HIS-Ame1-7A-1xFlag-Okp1-WT-1xMyc)This study
MBY324Mat a, his3Δ200, ame1Δ::HIS3, ura3-52, Ame1-3A-6xFlag::URA3, leu2-3-112This study
MBY327Mat a, his3Δ200, ame1Δ::HIS3, ura3-52, Ame1-4A-6xFlag::URA3, leu2-3-112This study
MBY331Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52 (two-micron pESC-HIS-Ame1-7A-1xFlag+Okp1-1A-1xMyc)This study
MBY333Mat a, ura3-52, lys2-801, ade2-101, his3Δ200, trp1Δ63, leu2Δ1, skp1-3::LEU2 (two-micron pESC-HIS-Ame1-7A-1xFlag+Okp1-1A-1xMyc)This study
MBY345Mat a, ame1Δ::HIS3, ura3-52, Ame1-5A1::URA3, ade2-3, leu2-3,112This study
MBY347Mat a, ame1Δ::HIS3, ura3-52, Ame1-5A2::URA3, ade2-3, leu2-3,112This study
MBY360Mat α, cdc4-1, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, psi+, ssd1-d2 (two-micron pESC-HIS-Ame1-CPDILTPP-1xFlag+Okp1-WT-1xMyc)This study
MBY361Mat α, cdc4-1, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, psi+, ssd1-d2 (two-micron pESC-HIS-Ame1-5A-CPDILTPP-1xFlag+Okp1-WT-1xMyc)This study
MBY371Mat a, his3Δ200, ame1Δ::HIS3, ura3-52, Ame1-3A-6xFlag::URA3, leu2-3,112, Pds1-13xMyc::LEU2This study
MBY372Mat α, his3Δ200, ame1Δ::HIS3, ura3-52, Ame1-4A-6xFlag::URA3, leu2-3,112, Pds1-13xMyc::LEU2This study
MBY373Mat a, his3Δ200, ame1Δ::HIS3, ura3-52, Ame1-5A1::URA3, ade2-3, leu2-3,112, Pds1-13xMyc::LEU2This study
MBY374Mat a, his3Δ200, ame1Δ::HIS3, ura3-52, Ame1-5A2::URA3, ade2-3, leu2-3,112, Pds1-13xMyc::LEU2This study
MBY380Mat a, his3Δ200, ame1Δ::HIS3, ura3-52, Ame1-3A-6xFlag::URA3, lys2-801, trp1-1, leu2-3,112, skp1-3::LEU2This study
MBY412Mat α, cdc34-2, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, psi+, ssd1-d2 (two-micron pESC-HIS-Ame1-7A-1xFlag+Okp1-1A-1xMyc)This study
MBY434Mat a, ade2-1, leu2-3,112, his3-11-15, ame1Δ::HIS3, ura3-52, Ame1-WT-CPDILTPP-6xFlag::URA3, psi1x, ssd1-d2, cdc4-1This study
MBY456Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52 (two-micron pESC-HIS, two-micron pESC-URA3)This study
MBY457Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52 (two-micron pESC-HIS, two-micron pGAL-URA-Mcm21-WT-3xHA+Ctf19-WT-1xMyc)This study
MBY462Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52 (two-micron pESC-HIS-Ame1-WT-1xFlag+Okp1-WT-1xMyc, two-micron pESC-URA)This study
MBY463Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52 (two-micron pESC-HIS-Ame1-WT-1xFlag+Okp1-WT-1xMyc, two-micron pESC-URA-Mcm21-WT-3xHA+Ctf19-WT-1xMyc)This study
MBY466Mat α, lys2-801am, leu2-3,112, his3Δ200, ura3-52 (two-micron pESC-HIS-Ame1-7A-1xFlag+Okp1-1A-1xMyc, two-micron pESC-URA)This study
MBY485Mat α, his3Δ200, ura3-52, Ame1-3A-6xFlag::URA3, lys2-801am, tor1-1 fpr1::loxP-LEU2-loxP RPL13A-2xFKBP12::loxP-TRP1-loxP, Mtw1-FRB::KanMXThis study
MBY486Mat α, his3Δ200, ura3-52, Ame1-4A-6xFlag::URA3, lys2-801am, tor1-1 fpr1::loxP-LEU2-loxP RPL13A-2xFKBP12::loxP-TRP1-loxP, Mtw1-FRB::KanMXThis study
MBY489Mat a, ade2-1, leu2-3,112, his3Δ200, ura3-52, Ame1-3A-ΔN-6xFlag::URA3This study
MBY490Mat a, ade2-1, leu2-3,112, his3Δ200, ura3-52, Ame1-4A-ΔN-6xFlag::URA3This study
MBY493Mat a, ade2-101, his3Δ200, trp1Δ63, lys2-801am, leu2Δ1, skp1-3::LEU2, ura3-52, Ame1-3A-ΔN-6xFlag::URA3This study
MBY494Mat a, ade2-101, his3Δ200, trp1Δ63, lys2-801am, leu2Δ1, skp1-3::LEU2, ura3-52, Ame1-4A-ΔN-6xFlag::URA3This study
MBY505.1Mat a, his3Δ200, ame1Δ::HIS3, ura3-52, Ame1-WT-6xFlag::URA3, ade2-1, lys2-801am, leu2-3,112, ctf19Δ::natNT2This study
MBY505.2Mat a, his3Δ200, ame1Δ::HIS3, ura3-52, Ame1-WT-6xFlag::URA3, ade2-1, leu2-3,112, ctf19Δ::natNT2This study
MBY509.2Mat a, his3Δ200, ame1Δ::HIS3, ura3-52, Ame1-CPDILTPP-6xFlag::URA3, leu2-3,112, ctf19Δ::natNT2This study
MBY510.1Mat α, his3Δ200, ame1Δ::HIS3, ura3-52, Ame1-CPDILTPP-6xFlag::URA3, ade2-1, lys2-801am, leu2-3,112, ctf19Δ::natNT2This study

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  1. Miriam Böhm
  2. Kerstin Killinger
  3. Alexander Dudziak
  4. Pradeep Pant
  5. Karolin Jänen
  6. Simone Hohoff
  7. Karl Mechtler
  8. Mihkel Örd
  9. Mart Loog
  10. Elsa Sanchez-Garcia
  11. Stefan Westermann
(2021)
Cdc4 phospho-degrons allow differential regulation of Ame1CENP-U protein stability across the cell cycle
eLife 10:e67390.
https://doi.org/10.7554/eLife.67390