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An assay for de novo kinetochore assembly reveals a key role for the CENP-T pathway in budding yeast

  1. Jackie Lang
  2. Adrienne Barber
  3. Sue Biggins  Is a corresponding author
  1. Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, United States
  2. University of Washington, United States
Research Article
Cite this article as: eLife 2018;7:e37819 doi: 10.7554/eLife.37819
7 figures, 3 tables and 4 additional files

Figures

Figure 1 with 2 supplements
Assembly of kinetochores de novo on a centromere DNA template.

(A) A schematic of the budding yeast kinetochore. Inner kinetochore subcomplexes assemble onto centromeres, serving as the platform for outer kinetochore recruitment. The listed subcomplexes are ordered based on physical interactions, and the yeast proteins in each kinetochore subcomplex are shown on the right. (B) DNA templates for the assembly assay. The templates include 500 bp from the E. coli ampC gene that encodes for β-lactamase (green) as a negative control, the 117 bp chromosome III centromere (CEN3), or a mutant CEN3 (CEN3mut) containing three point mutations in the CBF3 binding site (red ‘X’). The three Centromere-Determining Elements (CDEs) are indicated and ~70 bp of flanking pericentromeric DNA on either side is shown (grey). The DNA templates also contain linker DNA (purple) before the biotinylation (red star) at the 3’ end of the centromere. (C) Kinetochores assembled in vitro are centromere-specific and span the entire kinetochore. The indicated DNA templates were incubated in WT whole cell extracts prepared from a CNN1-3V5 DSN1-3Flag DAM1-9myc strain (SBY17228) for the indicated time (min). DNA-bound proteins were analyzed by immunoblotting with the indicated antibodies. Extracts are shown in Figure 1—figure supplement 1. (D) Kinetochore assembly is inhibited in an ndc10-1 temperature sensitive mutant. Extracts from a DSN1-6His-3Flag strain (SBY8253) or a DSN1-6His-3Flag ndc10-1 strain (SBY8361) shifted to the non-permissive temperature were used for assembly assays. DNA-bound proteins were analyzed by immunoblotting with the indicated antibodies. Extracts in Figure 1—figure supplement 2.

https://doi.org/10.7554/eLife.37819.002
Figure 1—figure supplement 1
Whole cell extracts (left) and assembled kinetochores (right) from Figure 1C, immunoblotted with the indicated antibodies. * indicates a background band in the extract in all figures.
https://doi.org/10.7554/eLife.37819.003
Figure 1—figure supplement 2
Whole cell extracts (left) and assembled kinetochores (right) from Figure 1D, immunoblotted with the indicated antibodies.
https://doi.org/10.7554/eLife.37819.004
Figure 2 with 3 supplements
Assembled kinetochore particles contain a single, chaperone-dependent CENP-ACse4 nucleosome.

(A) Degradation of HJURPScm3 blocks assembly of the kinetochore beginning with CENP-ACse4. A DSN1-3Flag scm3-EGFP-AID strain (SBY16440) and a DSN1-3Flag scm3-EGFP-AID OsTIR1-myc strain (SBY16438) were treated with auxin and the extracts were used for assembly assays. DNA-bound proteins were analyzed by immunoblotting for the indicated proteins. Extracts in Figure 2—figure supplement 2. (B) Assembly on a centromeric DNA template of only 180 bp is similar to a 250 bp template. Extract from a DSN1-3Flag CNN1-3V5 DAM1-9myc (SBY17228) strain was used for assembly assays with the indicated DNA templates. DNA-bound proteins were analyzed by immunoblotting with the indicated antibodies. Extracts in Figure 2—figure supplement 3.

https://doi.org/10.7554/eLife.37819.006
Figure 2—figure supplement 1
Degradation of HJURPScm3 is lethal to cells.

The Scm3-AID protein is degraded in the presence of both auxin and OsTIR1, resulting in lethality. Saturated cultures were serial diluted and plated on the indicated media. The strains used are WT (SBY4), DSN1-3Flag (SBY14441), DSN1-3Flag scm3-EGFP-AID (SBY16440), and DSN1-3Flag scm3-EGFP-AID OsTIR1-myc (SBY16438).

https://doi.org/10.7554/eLife.37819.007
Figure 2—figure supplement 2
Whole cell extracts (left) and assembled kinetochores (right) from Figure 2A, immunoblotted with the indicated antibodies.
https://doi.org/10.7554/eLife.37819.008
Figure 2—figure supplement 3
Whole cell extracts (left) and assembled kinetochores (right) from Figure 2B, immunoblotted with the indicated antibodies.
https://doi.org/10.7554/eLife.37819.009
Figure 3 with 1 supplement
Kinetochore assembly in vitro is regulated by the cell cycle and phosphorylation.

(A) Assembly in vitro is most efficient in extracts made from mitotically arrested cells. Kinetochores were assembled using extract from WT cells (DSN1-3Flag CNN1-3V5 DAM1-9myc (SBY17227)) that were either asynchronously growing or arrested in G1 (with alpha factor), S phase (with hydroxyurea), or early mitosis (with benomyl). Diluted whole cell extracts (left) and DNA-bound proteins (right) were analyzed by immunoblotting with the indicated antibodies. (B) Outer kinetochore assembly is enhanced in dsn1-2D extracts. Assembly assays were performed using extracts prepared from benomyl-arrested DSN1-3Flag CNN1-3V5 DAM1-9myc (SBY17228) and dsn1-2D-3Flag CNN1-3V5 DAM1-9myc (SBY17234) strains on the indicated DNA templates. DNA-bound proteins were analyzed by immunoblotting with the indicated antibodies. Extracts in Figure 3—figure supplement 1. (C) dsn1-2D enhances the assembly of most outer kinetochore proteins by at least 5-fold. Assembly assays were performed using extracts from DSN1-3Flag (SBY14441) and dsn1-2D-3Flag (SBY14151) on CEN3 DNA. Assembled proteins were labeled with tandem mass tags and analyzed by quantitative mass spectrometry. For each protein, the relative abundance in dsn1-2D assembled kinetochores was divided by the relative abundance in WT to calculate the fold enrichment in the dsn1-2D assembled kinetochores.

https://doi.org/10.7554/eLife.37819.010
Figure 3—figure supplement 1
Whole cell extracts (left) and assembled kinetochores (right) from Figure 3B, immunoblotted with the indicated antibodies.
https://doi.org/10.7554/eLife.37819.011
Figure 4 with 1 supplement
Assembled kinetochore particles bind to microtubules.

(A) The Ndc80-3V5-AID and Sli15-3HA-AID proteins are degraded after one hour of auxin treatment as determined by immunoblotting of whole cell extracts. (B) Assembled kinetochores bind microtubules but not free tubulin. Assembly assays were performed using extracts from the following strains: dsn1-2D-3Flag DAM1-9myc OsTIR1 (SBY14343), dsn1-2D-3Flag DAM1-9myc OsTIR1 ndc80-3V5-AID (SBY14336), dsn1-2D-3Flag DAM1-9myc OsTIR1 sli15-3HA-AID (SBY14890), and dsn1-2D-3Flag DAM1-9myc OsTIR1 ndc80-3V5-AID sli15-3HA-AID (SBY17238). All strains were arrested in benomyl and treated with auxin before harvesting. The assembled kinetochores were then incubated with buffer, free tubulin, or taxol-stabilized microtubules. The free tubulin and the microtubules contained alexa-647-labeled tubulin. DNA-bound proteins were analyzed by immunoblotting with the indicated antibodies, and the tubulin and microtubules were analyzed by fluorescence imaging. The Ndc80-3V5-AID protein migrates slower than untagged Ndc80. Extracts and tubulin input in Figure 4—figure supplement 1.

https://doi.org/10.7554/eLife.37819.013
Figure 4—figure supplement 1
Whole cell extracts (left) and assembled kinetochores (right) from Figure 4B, immunoblotted with the indicated antibodies.

Free tubulin and microtubule inputs are loaded at 1:5 and 1:20 of the amount introduced to assembled kinetochores.

https://doi.org/10.7554/eLife.37819.014
CENP-TCnn1 localization to the kinetochore requires the CCAN.

(A–C) CENP-TCnn1 assembly occurs downstream of all other inner kinetochore components. Assembly assays were performed on the indicated DNA templates using extracts prepared from cells arrested in benomyl. The strains used in (A) were also shifted to the non-permissive temperature for three hours before harvesting: DSN1-3Flag CNN1-3V5 (SBY17230), DSN1-3Flag CNN1-3V5 cse4-323 (SBY17770), and DSN1-3Flag CNN1-3V5 mif2-3 (SBY17603). The strains used in (B) were treated with auxin for three hours before harvesting: DSN1-3Flag CNN1-3V5 (SBY17230), DSN1-3Flag CNN1-3V5 okp1-3V5-AID OsTIR1 (SBY17764), DSN1-3Flag CNN1-3V5 mcm22Δ (SBY17460), and DSN1-3Flag CNN1-3V5 chl4Δ (SBY17607). The strains used in (C) were benomyl treated only: DSN1-3Flag CNN1-3V5 (SBY17230) and DSN1-3Flag CNN1-3V5 mcm21Δ (SBY18304). (D) A schematic distinguishing the proteins involved in the CENP-TCnn1 and Mis12 recruitment pathways.

https://doi.org/10.7554/eLife.37819.015
Figure 6 with 1 supplement
Kinetochore assembly utilizes two pathways for Ndc80 recruitment.

Dsn1 and CENP-TCnn1 both contribute to Ndc80 recruitment. Kinetochores were assembled using extract from WT, dsn1-AID, cnn1Δ, or dsn1-AID cnn1Δ double mutant cells that were arrested in benomyl and treated with auxin: DSN1-3Flag Cnn1-3V5 OsTIR1 (SBY17548), DSN1-3Flag cnn1Δ OsTIR1 (SBY17546), dsn1-3HA-AID Cnn1-3V5 OsTIR1 (SBY17544), and dsn1-3HA-AID cnn1Δ OsTIR1 (SBY17380). Extracts in Figure 6—figure supplement 1.

https://doi.org/10.7554/eLife.37819.016
Figure 6—figure supplement 1
Whole cell extracts (left) and assembled kinetochores (right) from Figure 6, immunoblotted with the indicated antibodies.
https://doi.org/10.7554/eLife.37819.017
Figure 7 with 3 supplements
Cells require CENP-TCnn1 when the Mis12 pathway is impaired.

(A) The CENP-TCnn1 pathway is required for viability when the Mis12c assembly pathway is compromised. Dsn1-3A is synthetic lethal with cnn1Δ and deletions of other genes (MCM22 and CHL4) in the CENP-TCnn1 recruitment pathway. A dsn1-3A strain (SBY14170) was crossed to cnn1Δ (SBY13386), mcm22Δ (SBY6997), and chl4Δ (SBY8788). The meiotic products (tetrads) of the resulting diploids are oriented left to right, haploid spores were genotyped, and double mutants are indicated with circles. (B) A dsn1-3A mcm22-AID double mutant is lethal when treated with auxin. Serial dilutions of the following yeast strains were plated on the indicated media: WT (SBY3), dsn1-3A-3Flag (SBY14170), mcm22-3HA-AID OsTIR1 (SBY17982), and dsn1-3A-3Flag mcm22-3HA-AID OsTIR1 (SBY18171). (C) The CENP-TCnn1 pathway recruits Ndc80 when Mis12 complex assembly is compromised. Assembly was performed with extracts from HU-arrested strains that were treated with auxin: DSN1-3Flag OsTIR1 (SBY14131), DSN1-3Flag mcm22-3HA-AID OsTIR1 (SBY18044), dsn1-3A-3Flag OsTIR1 (SBY14169), and dsn1-3A-3Flag mcm22-3HA-AID OsTIR1 (SBY18034). Extracts in Figure 7—figure supplement 2. (D) WT (SBY18498) and dsn1-3A mcm22-3HA osTIR1 (SBY18324) cells containing MTW1-3GFP were released from G1 and kinetochores were analyzed by fluorescence microscopy during metaphase. The percentage of cells containing mono-lobed, bi-lobed or scattered kinetochores was quantified and a representative picture of the bi-lobed and scattered categories is shown above the graph. The p value for the difference between WT and the double mutant for bi-lobed kinetochores is 0.04 and for scattered kinetochores is 0.036. (E) The sequential order of kinetochore subcomplex recruitment to the DNA, as determined from our data and from (Pekgöz Altunkaya et al., 2016). Dotted lines indicate physical interactions.

https://doi.org/10.7554/eLife.37819.018
Figure 7—figure supplement 1
Mcm22-3HA-AID is efficiently degraded after 60 min of auxin treatment.

The strains are DSN1-3Flag CNN1-3V5 OsTIR1 (SBY18040), DSN1-3Flag CNN1-3V5 mcm22-3HA-AID OsTIR1 (SBY18042), and dsn1-3A-3Flag CNN1-3V5 OsTIR1 (SBY18028).

https://doi.org/10.7554/eLife.37819.019
Figure 7—figure supplement 2
Cnn1 recruitment is blocked in Mcm22-3HA-AID strains.

Whole cell extracts and assembly assays for strains DSN1-3Flag CNN1-3V5 OsTIR1 (SBY18040), DSN1-3Flag CNN1-3V5 mcm22-3HA-AID OsTIR1 (SBY18042), and dsn1-3A-3Flag CNN1-3V5 OsTIR1 (SBY18028). (C) Mcm22-3HA-AID degradation for the experiment in Figure 7C.

https://doi.org/10.7554/eLife.37819.020
Figure 7—figure supplement 3
Assembly assays were performed using the following strains that were arrested in hydroxyurea for two hours and then treated with IAA for one hour: WT (SBY14131), mcm22-3HA-AID OsTIR1 (SBY18453), dsn1-3A (SBY14169) and dsn1-3A mcm22-3HA-AID OsTIR1 (SBY18172).

The levels of Ndc10 and Ndc80 were quantified and the relative ratio was graphed. The standard deviation is indicated.

https://doi.org/10.7554/eLife.37819.021

Tables

Table 1
Components from each of the core subcomplexes are detected on assembled kinetochores.

Kinetochores were assembled on ampC, CEN3mut, or CEN3 DNA from an asynchronous WT DSN1-3Flag (SBY14441) extract and analyzed by LC/MS/MS mass spectrometry. The table indicates the human ortholog (if applicable) of each yeast protein, the percent coverage, and the number of unique and total peptides detected from each assembly. We included the only detected microtubule-associated protein.

https://doi.org/10.7554/eLife.37819.005
Table 1. WT assembled kinetochores
ampCampCampCCEN3mutCEN3mutCEN3mutCEN3CEN3CEN3
SubcomplexYeast ProteinHuman Protein% CoverageUnique PeptidesTotal Peptides% CoverageUnique PeptidesTotal Peptides% CoverageUnique PeptidesTotal Peptides
CPCIpl1Aurora BNot presentNot present23.7810
Sli15INCENP72214.86764.354113
Bir1Survivin15.1101122.6141759.270177
Nbl1BorealinNot presentNot present76.7611
CCANCbf1Cbf1Not present62.7307759.32960
Cbf3Ndc1011.47832.9232663.278194
Cep33.51215.38934.22576
Ctf132.71118.455462238
Skp128.43319.62241.81224
NucleosomeCse4CENP-A13.13324.56849.81031
Hta2H2A35.672035.651335.6619
Htb2H2B4581839.772939.7729
Hht1H35.1115.111Not present
Hhf1H445.671156.381946.6813
NucleosomePsh13.911Not presentNot present
AssociatedScm3HJURPNot present6.31128.3810
Mif2Mif2CENP-CNot present9.74458.72939
OAOkp1CENP-QNot present20.97942.62134
Ame1CENP-UNot present20.45661.42241
CMCtf19CENP-PNot present6.82244.72131
Mcm21CENP-ONot present10.33465.82639
Iml3Iml3CENP-LNot presentNot present60.81319
Chl4CENP-NNot present7.93337.11619
Nkp1Not present26.94657.61728
Nkp2Not present152455.6710
Ctf3Mcm16CENP-HNot present22.72348.6711
Ctf3CENP-INot present5.33323.71727
Mcm22CENP-KNot present25.53481.61827
Cnn1Cnn1CENP-TNot presentNot present45.71318
Wip1CENP-WNot presentNot present39.322
Mhf1CENP-S48.94448.9374025
Mhf2CENP-X43.84847.54628.834
Outer
Kt
Mtw1Mtw1Mis12Not presentNot present22.844
Nnf1PMF1Not presentNot present13.922
Nsl1Nsl1Not presentNot present24.133
Dsn1Dsn1Not presentNot present7.322
Ndc80Ndc80HEC1Not presentNot present28.41516
Nuf2NUF2Not presentNot present32.81213
Spc24SPC24Not presentNot present57.388
Spc25SPC25Not presentNot present27.155
Spc105Spc105KNL1Not presentNot present533
Kre28Zwint1Not presentNot presentNot present
Dam1Dam1Not presentNot present10.822
Dad126.61126.61226.611
Dad3Not presentNot presentNot present
Ask1Not presentNot present8.211
Duo1Not presentNot present7.311
Hsk315.91115.91115.911
Spc19Not presentNot present8.511
Spc34Not presentNot present4.712
Dad2Not presentNot presentNot present
Dad4Not presentNot presentNot present
MAPsStu2CHTOG3.522Not presentNot present
Table 2
Outer kinetochore assembly is enhanced by Dsn1 phosphorylation.

Kinetochores were assembled on the indicated DNA templates from an asynchronous dsn1-2D-3Flag (SBY14151) extract and analyzed by mass spectrometry as in Table 1. We included the detected microtubule-associated proteins.

https://doi.org/10.7554/eLife.37819.012
Table 2. dsn1-2D assembled kinetochores
ampCampCampCCEN3mutCEN3mutCEN3mutCEN3CEN3CEN3
SubcomplexYeast ProteinHuman Protein% CoverageUnique PeptidesTotal Peptides% CoverageUnique PeptidesTotal Peptides% CoverageUnique PeptidesTotal Peptides
CPCIpl1Aurora BNot presentNot present24.5915
Sli15INCENP114521.9101162.661221
Bir1Survivin20.2131525.5172064.371257
Nbl1Borealin23.31121.91161.6719
CCANCbf1Cbf1Not present65275359.33092
Cbf3Ndc1021.221424.5192258.968563
Cep320.68915.381034.521111
Ctf132.71112.35540.62171
Skp18.81222.23344.31132
NucleosomeCse4CENP-A20.555144649.31034
Hta2H2A35.651335.672035.6534
Htb2H2B39.771839.773131.3629
Hht1H3Not present5.111Not present
Hhf1H456.391156.381655.3822
Nucleosome
Associated
Psh17.422Not presentNot present
Scm3HJURPNot presentNot present30.5817
Mif2Mif2CENP-CNot present13.75555.72754
OAOkp1CENP-QNot present22.78943.62150
Ame1CENP-UNot present28.75554.91943
CMCtf19CENP-PNot present9.83342.31741
Mcm21CENP-ONot present25.87848.62342
Iml3Iml3CENP-LNot present15.53360.81328
Chl4CENP-NNot present7.233291221
Nkp1Not present26.546581835
Nkp2Not present152335.9514
Ctf3Mcm16CENP-H14.91119.92244.8612
Ctf3CENP-INot present42213.91219
Mcm22CENP-KNot present14.62274.91734
Cnn1Cnn1CENP-TNot presentNot present27.1811
Wip1CENP-WNot presentNot present21.122
Mhf1CENP-S48.93448.94921.122
Mhf2CENP-X62.57847.54428.823
Outer KTMtw1Mtw1Mis12Not present18.74448.11321
Nnf1PMF1Not present16.92230.31013
Nsl1Nsl1Not present15.322691521
Dsn1Dsn1Not present13.44439.22131
Ndc80Ndc80HEC1Not present18.58956.43763
Nuf2NUF2Not present15.766512742
Spc24SPC24Not present38.54563.41226
Spc25SPC25Not present8.11137.6810
Spc105Spc105KNL1Not present3.12245.94160
Kre28Zwint1Not present3.611722
Dam1Dam1Not presentNot present21.656
Dad126.61126.61137.224
Dad3Not presentNot present29.823
Ask1Not presentNot present21.234
Duo1Not presentNot present18.244
Hsk315.91115.91115.911
Spc19Not presentNot present30.346
Spc34Not presentNot present31.5611
Dad2Not presentNot presentNot present
Dad4Not presentNot presentNot present
MAPsStu2CHTOGNot presentNot present33.62331
Bim1Not presentNot present17.745
Slk19Not presentNot present1.611
Bik1Not presentNot present1034
Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Gene (S. cerevisiae)See supplementary file 1
Strain, strain
background
(Saccharomyces cerevisiae)
W303
Genetic reagent
(S. cerevisiae)
See supplementary file 1
Antibodyanti-Ndc10
(rabbit polyclonal)
Desai labOD1(1:5,000)
Antibodyanti-Cse4
(rabbit polyclonal)
Biggins lab9536(1:500)
Antibodyanti-Mif2
(rabbit polyclonal)
Desai labOD2(1:6,000)
Antibodyanti-Ctf19
(rabbit polyclonal)
Desai labOD10(1:15,000)
Antibodyanti-Ndc80
(rabbit polyclonal)
Desai labOD4(1:10,000)
Antibodyanti-Spc105
(rabbit polyclonal)
Biggins labPAC4065(1:10,000)
Antibodyanti-Ipl1
(rabbit polyclonal)
Desai labOD121(1:300)
Antibodyanti-HA
(mouse monoclonal)
Roche12AC5, Catalog #1-583-816(1:10,000)
Antibodyanti-V5
(mouse monoclonal)
InvitrogenCatalog #R960-25(1:5,000)
Antibodyanti-Flag
(mouse monoclonal)
Sigma-AldrichCatalog #F3165(1:3,000)
Antibodyanti-Myc
(mouse monoclonal)
Covance9E10, Catalog #MMS-150R(1:10,000)
Antibodyanti-mouse secondary
(goat monoclonal)
GE Healthcare BioSciencesNA931(1:10,000)
Antibodyanti-rabbit secondary
(goat monoclonal)
GE Healthcare BioSciencesNA934(1:10,000)
Recombinant
DNA reagent
See supplementary file 2
Sequence-based
reagent
See supplementary file 3
Chemical
compound, drug
α-factorUnited Biochemical Research Inc.10 mg/mL
Chemical
compound, drug
hydroxyureaSigmaH86270.2M
Chemical
compound, drug
benomylSigma381586–25G60 mg/mL
Chemical
compound, drug
indole-3-acetic acid (IAA)SigmaI3750-5G-A500 mM
OtherDynabeads M-280
Streptadivin
Invitrogen112-05D

Data availability

All data generated during this study are included in the manuscript and supplementary data.

Additional files

Supplementary file 1

Yeast strains used in this study.

Complete genotypes of the Saccharomyces cerevisiae strains used are listed along with the strain number to reference. Replicating plasmids are indicated in brackets. All strains are isogenic with W303.

https://doi.org/10.7554/eLife.37819.022
Supplementary file 2

Plasmids used in this study.

The relevant genes and markers on each plasmid used are listed.

https://doi.org/10.7554/eLife.37819.023
Supplementary file 3

DNA primers used in this study.

The sequence and purpose of each primer used is listed.

https://doi.org/10.7554/eLife.37819.024
Transparent reporting form
https://doi.org/10.7554/eLife.37819.025

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