Quantitative Proteomic Analysis of Purified Yeast...

S1

SUPPLEMENTAL DATA

Quantitative Proteomic Analysis of Purified Yeast Kinetochores

Identifies a PP1 Regulatory Subunit

Bungo Akiyoshi, Christian R. Nelson, Jeffrey A. Ranish and Sue Biggins

Supplemental Experimental Procedures

Supplemental Figures and Legends

Figure S1. Kinetochore proteins associated with minichromosomes

Figure S2. Fin1 localizes at metaphase and anaphase kinetochores

Figure S3. The 36 kDa Fin1-binding protein is Bmh2

Figure S4. Deletion of FIN1 mildly suppresses the temperature sensitivity of ipl1-321

Figure S5. Fin1-5A cells move the entire nucleus into the bud

Figure S6. Fin1-5A cells do not separate sister chromatids during nocodazole arrest

Figure S7. Fin1-5A expression from the endogenous FIN1 promoter does not cause

obvious growth defects

Figure S8. Expression of Fin1-WT redistributes Glc7 onto spindles during anaphase

Figure S9. A positive feedback model for the Fin1/PP1 phosphatase complex

Figure S10. Ipl1 phosphorylates Fin1 in vitro

Supplemental Tables

Table S1. Proteins identified in the WT centromere minichromosome sample

S2

Table S2. Proteins identified in the mutant centromere minichromosome sample

Table S3. Kinetochore proteins identified in the WT centromere minichromosome

sample

Table S4. Identified phosphorylation sites in kinetochore proteins

Table S5. Proteins identified in the SILAC quantitative MS experiment

Table S6. Proteins identified in the Fin1-FLAG immunoprecipitation sample

Table S7. Yeast strains used in this study

Table S8. Plasmids used in this study

S3

Supplemental Experimental Procedures

Yeast Strain Construction

The ndc10-1 (Goh and Kilmartin 1993), glc7-12 (MacKelvie et al. 1995), and ipl-

321 (Biggins et al. 1999) alleles were crossed to make strains for this study. Strains

containing PDS1-18Myc::LEU2, GFP-TUB1::LEU2, pGAL-∆176-CLB2::LYS2 , pCMV-

LacI-3FLAG::URA3, and FIN1-3GFP::HIS3 were made by integrating plasmid pSB205

digested with HindIII at the PDS1 locus, pSB340 digested with AgeI at the LEU2 locus,

pSB102 digested with BspEI at the LYS2 locus, pSB737 digested with StuI at the URA3

locus, and pSB1169 digested with EcoRI at the FIN1 locus, respectively. Deletions, as

well as the 3FLAG, 13Myc and 3HA epitope tags, were made using a PCR-based

integration system and confirmed by PCR (Longtine et al. 1998; Gelbart et al. 2001).

Specific primer sequences are available upon request.

Strains containing minichromosomes were made as follows to reduce the size of

the minichromosome. The bacterial sequences in pSB963 (WT CEN3) and pSB972

(mutant CEN3) were removed by digesting the plasmids with EcoRI. The resulting

fragment was ligated and transformed into yeast. Correct transformation of the

minichromosome was confirmed by PCR.

Plasmid Construction

To generate the minichromosome (pSB963), a 278 bp fragment containing CEN3

was PCR amplified using primers SB744 and SB745 that contained engineered NgoMIV

restriction sites and cloned into pGEM-T (Promega) to generate pSB751. The CEN3

fragment was then introduced into a unique NgoMIV site of the TRP1-ARS1-8lacO

S4

plasmid (TALO8, gift from T. Tsukiyama) to create pSB963. The minichromosome with

the mutant CEN3 (pSB972) was made by mutating the conserved CCG site in the CDEIII

domain of pSB963 to AGC by Quickchange site-directed mutagenesis (Stratagene). The

FIN1-3GFP plasmid (pSB1169) was made by PCR amplification of the carboxy-terminal

573 bp of FIN1 using primers SB1815 and SB1816 that have ClaI and BamHI restriction

sites engineered, respectively. The resulting PCR product was digested with ClaI-BamHI

and ligated into the same sites of pSB623 (kind gift of D. Pellman). pSB1194 (pGALS-

FIN1-WT, URA3), pSB1195 (pGALS-fin1-5A, URA3) and pSB1215 (pGALS-fin1-5Aglc7-,

URA3) were made by introducing a stop codon just prior to the GFP ORF of pSB1185,

pSB1186 and pSB1212, respectively, using site-directed mutagenesis with primers

SB1891 and SB1892.

Fin1glc7- mutants were made by site directed mutagenesis of the five putative PP1

binding sites in Fin1 as follows: site 1, L121 and F123 (using primers SB1927 and

SB1928), site 2, F142 (primers SB1929 and SB1930), site 3, F182 (primers SB1907 and

SB1908), site 4, F236 (primers SB1909 and SB1910) and site 5, V240 and F242 (primers

SB1893 and SB1894) were all changed to alanine to make pSB1237 (pGALS-fin1glc7--

GFP, URA3) and pSB1212 (pGALS-fin1-5Aglc7--GFP, URA3) from pSB1185 and

pSB1186, respectively.

Fin1-GFP plasmids containing the endogenous FIN1 promoter were constructed

in multiple steps as follows. First, a LEU2-marked pGALS-FIN1-WT-GFP plasmid was

constructed by PCR amplification of the pGALS-FIN1-WT-GFP fragment from pSB1185

with primers SB1976 and SB1977 that have NotI and HindIII sites engineered,

respectively. Second, the fragments were ligated into the same sites of pRS415 (Sikorski

S5

and Hieter 1989) to make pSB1227. Third, the FIN1 promoter region was amplified by

PCR with primers SB1992 and SB1993 that have SacII and SpeI restriction sites

engineered, respectively. Fourth, the resulting PCR product was cloned into pGEM-T

(Promega) to make pSB1250 which was subsequently digested with SacII and SpeI and

the fragment was used to replace the GALS promoter of pSB1227 to generate pSB1252

(pFIN1-FIN1-WT-GFP, LEU2). pFIN1-fin1glc7--GFP (pSB1267) was made similarly by

first constructing a LEU2 plasmid (pSB1261) containing pGALS-fin1glc7--GFP from

pSB1237 (URA3) and then replacing the GALS promoter with the endogenous promoter

to create pSB1267 (pFIN1-fin1glc7--GFP, LEU2). The pFIN1-fin1-5A-GFP plasmid

(pSB1359) was made similarly except that the FIN1 promoter was PCR amplified from

pSB1252 with primers SB2051 and SB1993 that have NotI and SpeI sites engineered,

respectively, and cloned into pSB1228 (pGALS-fin1-5A-GFP, LEU2) that was

constructed by PCR amplification of the gene from pSB1186 and cloned into pRS415.

The pFIN1-fin1-5Aglc7--GFP (pSB1361) was made similarly to pSB1359 by first making

pSB1212 (pGALS-fin1-5Aglc7--GFP, URA3) from pSB1186 (see above), and then the

promoter in pSB1260 (pGALS-fin1-5Aglc7--GFP, LEU2) was replaced to generate

pSB1359. In addition, there was an extra mutation in pSB1359 and pSB1361 (Woodbury

and Morgan 2007) that created translation problems (data not shown) that we restored to

WT by site-directed mutagenesis.

Large-scale minichromosome preparation for MS analysis

To prepare large-scale mitotic cultures containing minichromosomes, synthetic

media without tryptophan containing 120 µg/ml benomyl was prepared by adding

S6

benomyl (30 mg/ml stock solution dissolved in DMSO) to boiling media and cooled to

room temperature. Equivalent amounts of benomyl media were added to growing cell

cultures (at OD600 ~0.7) so that the final benomyl concentration was 60 µg/ml. Cells

were grown for 3 additional hours and harvested when greater than 90 % of cells were

large-budded.

Buffer H/0.15 (25 mM HEPES pH 8.0, 2 mM MgCl2, 0.1mM EDTA pH 8.0, 0.5

mM EGTA pH 8.0, 0.1 % NP-40, 150 mM KCl, and 15 % Glycerol) supplemented with

protease inhibitors (10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml chymostatin, and

0.2 mM PMSF) and phosphatase inhibitors (1 mM sodium pyrophosphate, 2 mM Na-

beta-glycerophosphate, 0.1 mM NA3VO4, 5 mM NaF, 100 nM microcystin-LR) was used

in all purification steps unless stated otherwise. Typically, 10 liters of mitotic culture

were harvested by centrifugation and subsequent steps were performed at 4 ˚C. Cells

were washed once with dH2O supplemented with 0.2 mM PMSF, and once with BH/0.15.

Cell pellets (~18 g) were frozen in liquid nitrogen and stored at -80 ˚C. To efficiently

prepare yeast extracts from large cell pellets, we mechanically broke cells in dry ice with

a blender for 25 min. BH/0.15 (50 ml) was then added to the cell powder and the sample

sat for 25 min to allow lysis. The lysed cells were centrifuged at 24,000 rpm for 90 min

at 4 ˚C in a SW41 rotor (Beckman). Soluble cell extract was collected through a syringe.

Total protein concentration of the extract was determined by BCA protein assay (Pierce).

Typically, 40 ml of 12–16 mg/ml extract was obtained. Anti-FLAG immunoprecipitation

was performed using 300 µg of FLAG M2 antibodies (Sigma-Aldrich) that had been pre-

conjugated to 600 µl slurry of Protein G magnetic beads (Dynal) with DMP according to

manufacturer’s instructions. The use of magnetic beads significantly reduced non-

S7

specific binding relative to agarose beads ((Alber et al. 2007) and our unpublished data).

Beads were incubated with extract for three hours with constant rotation, followed by six

washes with BH/0.15 that included 2 mM DTT. Beads were further washed three times

with pre-elution buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 1 mM EGTA).

Associated proteins were eluted from the beads by gentle agitation of beads in 110 µl of

elution buffer (50 mM Tris-HCl pH 8.3, 1 mM EDTA, 0.1 % SDS) for 30 min at room

temperature. 10 µl of the sample was used to verify the immunoprecipitation and

determine the amount of protein by comparison to BSA standards. The rest of the sample

(100 µl) was frozen in liquid nitrogen and stored at -80 ˚C.

Preparation of peptides for LC-MS/MS

The sample was adjusted to 0.2 % SDS and incubated at 100 ˚C for 5 min.

Proteins (approximately 20 µg from 10 L culture) were reduced with 5 mM DTT at 37 ˚C

for 30 min, diluted with 100 µl of 50 mM Tris-HCl pH 8.3 and 1 mM EDTA, and then

alkylated with 10 mM iodoacetamide at 37 ˚C for 30 min. The reaction was quenched by

adding 10 mM DTT at 37 ˚C for 30 min, and 200 µl of 20 mM Tris-HCl pH 8.3 was

added to reduce the SDS concentration to 0.05 %. Proteins were digested overnight at 37

˚C with 0.2 µg of endo-proteinase Lys-C (Roche) and 1 µg of trypsin (Promega). We

diluted the sample with 600 µl of Buffer A (0.5 % acetic acid and 2 % acetonitrile) and

adjusted the pH to 2.7 with 10 % trifluoroacetic acid. Peptides were manually loaded

onto an SCX column (1 mm I.D. X 2 cm Upchurch packed with Partisphere SCX resin,

Whatman) equilibrated with Buffer A at a flow rate of 75 µl/min, and peptides were

fractionated by eluting with Buffer A containing increasing concentrations of ammonium

S8

acetate (e.g. 10, 20, 27, 35, 50, and 120 mM to prepare six fractions). In addition to

reducing sample complexity, SCX also removed undesirable chemicals such as SDS.

Fractionated peptides were desalted over a Vydac C18 column (Nest group), dried in

vacuo and resuspended in Buffer A' (0.1 % formic acid, 2 % acetonitrile) for LC-MS/MS

analysis.

LC-MS/MS analysis, database search and data analysis

Peptides were separated by online reversed-phase microcapillary liquid

chromatography (LC) and analyzed by electrospray tandem mass spectrometry using an

LCQ-DecaXP ion-trap mass spectrometer (ThermoScientific) for qualitative experiments

as described (Kim et al. 2007), and an LTQ-Orbitrap (ThermoScientific) for quantitative

MS experiments. The precolumn was packed with 5 mm, 200 Å Magic C18AQ resin

(Michrom Bio-Resources), and the peptides were separated on a microcapillary column

packed with 5 mm, 100 Å Magic C18AQ using a linear gradient from 98 % solvent A

(0.1 % formic acid) and 2 % solvent B (99.9 % acetonitrile, 0.1 % formic acid) to 30 %

solvent B over 60 min at a flow rate of ~0.2 µl/min. Using data dependent settings, each

MS1 scan (from m/z 300 to 1,800) acquired in the Orbitrap with a resolution of 60,000 at

m/z 400 was followed by collision induced dissociation, acquired in the LTQ of the three

most abundant precursor ions with a normalized collision energy of 35 %. Ions selected

for MS2 were dynamically excluded for 60 s. Ions with singly charged as well as

unassigned charge states were also rejected.

Peptides were identified by searching MS/MS spectra against a yeast protein

database (Saccharomyces Genome Database; http://www.yeastgenome.org/) with

SEQUEST with carbamidomethyl cystein (57.0215 Da) as fixed modification. One

S9

missed cleavage was allowed. Monoisotopic parent masses were used for high accuracy

spectra derived from Orbitrap. Oxidized-methionine (15.9949 Da), phosphorylation

(79.9663 Da), [13C6 15N2] lysine (8.0142 Da), and [13C6 15N4] arginine (10.0083 Da) were

searched as variable modifications as appropriate. The relative abundance ratio of each

peptide and protein was calculated using XPRESS (Han et al. 2001). Similar results were

obtained using ASAPRatio (Li et al. 2003) (data not shown). Data analysis was

performed by using a suite of software tools including INTERACT (Han et al. 2001),

PeptideProphet (Keller et al. 2002), and ProteinProphet (Nesvizhskii et al. 2003) as

described (Kim et al. 2007). The data sets were filtered by using ProteinProphet

probability of 0.95 so that the predicted error rate was less than 0.005. All of the MS data

is available upon request.

S10

SUPPLEMENTAL FIGURES AND LEGENDS

Figure S1. Kinetochore proteins associated with minichromosomes.

WT and mutant CEN3 minichromosomes were purified as in Fig. 1C. Immunoblots were

performed using anti-Ctf19, anti-Mif2, and anti-Ndc10 antibodies.

S11

Figure S2. Fin1 localizes to metaphase and anaphase kinetochores.

Centromeric minichromosomes were purified from cells containing Fin1-Myc, Ndc80-

Myc, and galactose inducible non-degradable clb2 (SBY6464) that had been treated with

either nocodazole (metaphase arrest) or galactose (late anaphase arrest) for 2.5 hours.

Samples were analyzed by immunoblots using anti-Myc and anti-Cse4 (loading control)

antibodies.

S12

Figure S3. The 36 kDa Fin1-binding protein is Bmh2.

The Fin1-FLAG purification was repeated in cells containing Bmh2 fused to 13 copies of

the Myc epitope-tag (30 kDa) (SBY6368). The 36 kDa band shifted to 66 kDa,

confirming that it is Bmh2.

S13

Figure S4. Deletion of FIN1 mildly suppresses the temperature sensitivity of ipl1-321.

Serial dilutions (5-fold) of WT (SBY3), fin1∆ (SBY6875), ipl1-321 (SBY5429), fin1∆

ipl1-321 (SBY7515) were plated at 23 ˚C (permissive temperature) and 33 ˚C (semi-

permissive temperature for ipl1-321).

S14

Figure S5. Fin1-5A cells move the entire nucleus into the bud.

Cells in Figure 5A were fixed 160 min after release and stained with DAPI. Examples of

proper segregation (left), and mis-segregation to the daughter cell (middle) or mother cell

(right) are shown. The scale bar represents 5 µm.

S15

Figure S6. Fin1-5A cells do not separate sister chromatids during a nocodazole arrest.

Cells in Figure 5D were fixed 180 min after release into nocodazole and the separation of

chromosome IV was quantified. The scale bar represents 5 µm.

S16

Figure S7. Fin1-5A expression from the endogenous FIN1 promoter does not cause

obvious growth defects.

A. Serial dilutions (5-fold) of cells expressing FIN1-WT-GFP (SBY7275), fin1-5A-GFP

(SBY7544), or fin1-5Aglc7--GFP (SBY7545) from the endogenous promoter were plated

at 23 ˚C. SBY7546 contains a control vector. B. Lysates were prepared and the

expression of Fin1 proteins was examined by immunoblots using anti-GFP and anti-

Tubulin (loading control) antibodies.

S17

Figure S8. Expression of Fin1-WT redistributes Glc7 onto spindles during anaphase.

Cells containing Glc7-GFP and either empty vector (SBY6800) or pGAL-FIN1-WT

(SBY6483) were released from G1 into galactose and fixed 140 min after release. 74%

of Fin1-WT cells exhibited strong Glc7 signal on anaphase spindles.

S18

Figure S9. A positive feedback model for the Fin1/PP1 phosphatase complex.

(A) Aberrant attachments are destabilized by Ipl1/Aurora B-mediated kinetochore

phosphorylation. Ipl1 may also phosphorylate Fin1 to reduce its affinity for kinetochores

S19

and keep the counteracting phosphatase activity low. During this time, the bulk of Fin1

is sequestered by 14-3-3 proteins to avoid PP1 misregulation. (B) Once kinetochores are

fully phosphorylated and become unattached, Aurora B activity may be reduced by

substrate inhibition (Rosasco-Nitcher et al. 2008). At this time, Fin1/PP1 may start to

dephosphorylate kinetochores as well as Fin1 (C), inducing a positive feedback loop that

increases Fin1/PP1 at kinetochores to stabilize attachments (D). As kinetochores

biorient, Aurora B becomes spatially separated from the key targets at kinetochores (Liu

et al. 2009), allowing the Fin1/PP1 phosphatase complex to maintain the

dephosphorylated state of the kinetochore and silence the spindle checkpoint (E).

S20

Figure S10. Ipl1 phosphorylates Fin1 in vitro.

Fin1-3GFP was purified (SBY6209) with anti-GFP antibodies in the absence of

phosphatase inhibitors and an Ipl1 in vitro kinase assay was performed using recombinant

GST-Ipl1 and GST-Sli15. An untagged strain (SBY3) was used as a control.

S21

Table S1. Proteins identified in the WT centromere minichromosome sample Name Function Coverage

(%) Unique peptides

Total peptides

Protein probability

LacI LacI 64.4 63 331 1 NET1 50.5 74 204 1 HTB2 H2B 50.4 16 97 1 ASG1 28.8 25 81 1 RVB1 51.2 26 77 1 RVB2 43.9 28 75 1 MOT1 16.1 29 73 1 SSB1 41.3 25 72 1 NDC80 Kinetochore 28.4 26 65 1 RPA190 15.6 27 65 1 VPS1 31 26 57 1 RPA135 15.4 23 56 1 TAF5 33.8 23 56 1 CST6 42.6 23 54 1 DSN1 Kinetochore 35.9 22 51 1 INO80 13.6 18 48 1 REB1 23.2 18 45 1 CDC14 23.8 16 44 1 MPS1 Spindle checkpoint,

Kinetochore 28.8 18 42 1

SPC24 Kinetochore 57.3 14 42 1 SSA2 37.6 17 42 1 RSC8 28.7 19 41 1 SPC105 Kinetochore 19.6 13 40 1 RSC58 37.5 17 39 1 HHF1, HHF2

H4 36.9 14 38 1

SPC25 Kinetochore 41.2 13 38 1 TAF12 34 18 37 1 SWI5 31.6 18 36 1 PMA1 18.4 15 35 1 DUO1 Kinetochore 25.5 12 34 1 ISW1 15.1 15 34 1 MSN4 30.5 17 33 1 NUF2 Kinetochore 30.2 13 33 1 SKO1 28.4 12 32 1 STH1 13 14 32 1 NSL1 Kinetochore 38.9 10 31 1 RSC2 22.5 12 31 1 RSC6 30 10 30 1 RPC40 41.2 13 28 1 SUM1 22.4 13 28 1 ABF1 26.7 12 27 1

S22

ACT1 33.6 13 27 1 HTA1, HTA2

H2A 32.6 6 27 1

RSC4 12.3 11 26 1 CSE4 Kinetochore 26.6 7 25 1 DIG1 28.8 10 25 1 RSC3 16.8 12 25 1 RTG3 28.2 13 25 1 TEF2 28.2 15 25 1 IES1 16.3 9 24 1 SPC34 Kinetochore 47.1 11 24 1 HHT1, HHT2

H3 21.3 7 23 1

NNF1 Kinetochore 50.7 10 23 1 RNQ1 22.7 8 23 1 ARP8 14.3 9 22 1 RPS14A 49.6 7 22 1 SPT15 20.8 6 22 1 IES2 36.2 9 21 1 MTW1 Kinetochore 24.2 9 21 1 NPL6 19.5 7 21 1 GAT1 18.6 10 20 1 HMO1 26.8 8 20 1 MIF2 Kinetochore 19.5 11 20 1 CRZ1 14.9 6 19 1 RTT102 52.9 8 19 1 ABF2 27.3 8 18 1 NDC10 Kinetochore 15.2 11 18 1 FIN1 Fin1 30.2 8 18 1 HAP1 8.4 9 18 1 NHP10 39.9 7 18 1 RSC30 13.7 7 18 1 SGF73 21.2 10 18 1 SIR2 18.9 9 18 1 SPC19 Kinetochore 25.5 7 18 1 SPT20 16.6 8 18 1 WAR1 14.4 10 18 1 ACE2 12.1 7 17 1 ARP5 15.5 10 17 1 DAM1 Kinetochore 23.3 6 17 1 MBP1 7.9 6 17 1 HAA1 12 8 16 1 NKP1 Kinetochore 28.2 7 16 1 RPA49 10.4 5 16 1 RPC19 47.9 6 16 1 GIS2 40.5 8 15 1

S23

PAB1 20.1 8 15 1 RPA43 18.4 7 15 1 TFC3 12.8 9 15 1 TRA1 3.2 9 15 1 ARP9 16.5 6 14 1 BAS1 11 7 14 1 RIM1 28.1 3 14 1 IES3 25.6 7 13 1 RPA14 24.8 7 13 1 RPL20A 37.4 6 13 1 TAF14 13.5 2 13 1 ASK1 Kinetochore 24 5 12 1 DID2 26 5 12 1 IES5 44 5 12 1 RPA12 45.6 4 12 1 RPL36B 46 7 12 1 RSC9 8.8 4 12 1 SKN7 14 5 12 1 SNF7 26.7 5 12 1 YAP1 14.9 7 12 1 MSN2 17.5 6 11 1 RPS3 17.1 3 11 1 TAF10 20.4 4 11 1 YDR532C Kinetochore 14.3 5 11 1 YTA7 7.1 4 11 1 CHD1 5 5 10 1 SFH1 9.9 4 10 1 SPT23 11.2 7 10 1 STE12 14 7 10 1 TAF6 16.7 6 10 1 ARP4 11.7 4 9 1 HSK3 Kinetochore 47.8 4 9 1 PDC2 9.9 4 9 1 RPC10/RPB12

51.4 4 9 1

RPL19B 29.1 4 9 1 RSC1 5.8 3 9 1 SPT7 6.5 7 9 1 SPT8 14.1 5 9 1 SRP1 5.9 3 9 1 STU2 MT binding,

Kinetochore 12.3 7 9 1

TDH3 19.3 4 9 1 TOP1 5.1 4 9 1 YRF1-1 7.9 4 9 1 ADR1 4.8 4 8 1

S24

DED1 17.5 5 8 1 FKH2 15.4 7 8 1 HSF1 9 5 8 1 MET4 8.5 5 8 1 NOP1 13.1 4 8 1 OKP1 Kinetochore 13.8 5 8 1 RAP1 11 6 8 1 RPL8A 11.3 5 8 1 RPS26A 26.9 4 8 1 RPS6B 18.2 3 8 1 TAF1 7.1 6 8 1 UME6 10.3 5 8 1 YLR278C 8.7 5 8 1 YMR111C 14.9 4 8 1 ARP7 11.5 3 7 1 ENO2 9.8 4 7 1 GAL11 4.3 4 7 1 GAL4 4.9 3 7 1 INO4 29.1 4 7 1 ITC1 4 5 7 1 KAR2 11.1 5 7 1 LDB7 33.9 4 7 1 LRS4 Kinetochore 13.8 3 7 1 MCD1 10.4 3 7 1 MCM21 Kinetochore 8.7 3 7 1 MSN1 17.3 3 7 1 NGG1 8.3 5 7 1 PDR1 8.1 5 7 1 RET1 3.7 3 7 1 RPL4A 25.4 6 7 1 SSA1 35.4 4 7 1 BUR6 14.8 2 6 1 CDC19 10.4 4 6 1 CEP3 Kinetochore 6.1 3 6 1 CNN1 Kinetochore 12.2 3 6 1 DAD1 Kinetochore 33 3 6 1 FET4 7.1 2 6 1 GCN5 12.8 3 6 1 IES6 26.5 4 6 1 IML3 Kinetochore 10.6 2 6 1 INO2 9.5 2 6 1 ISW2 2.1 2 6 0.9962 RPL25 24.6 2 6 1 RPL33B 24.3 3 6 1 RPP0 14.1 4 6 1 RPS13 35.1 3 6 1

S25

SGF11 41.4 3 6 1 SMC2 4.3 3 6 1 SMC3 4.6 5 6 1 AZF1 5.9 4 5 1 BIR1 Chromosome

passenger, Kinetochore

7.5 5 5 1

CBF1 11.4 3 5 1 FKH1 6.8 3 5 1 GSP1 9.6 3 5 1 HTZ1 H2AZ 17.2 2 5 1 IOC3 5.3 3 5 1 MGM101 13.4 3 5 1 NHP6A 21.5 2 5 0.9995 PGD1 5.8 3 5 1 RGR1 2.9 3 5 1 ROS17B 20.6 2 5 1 RPA34 24 3 5 1 RPL12B 26.1 4 5 1 RPL17B 13.6 3 5 1 RPL18B 14.5 2 5 1 RPL23A 5.8 2 5 1 RPL31B 31.9 3 5 1 RPS15 16.9 3 5 1 RTG1 23.7 3 5 1 SPT3 13.4 2 5 1 SRM1 12 4 5 1 SSL2 3.7 2 5 1 STP3 9.6 3 5 1 TAF7 4.2 2 5 1 TFC7 8.7 3 5 1 YEL007W 10.5 3 5 1 AME1 Kinetochore 11.7 2 4 1 BOP3 10.4 2 4 1 CAC2 10.7 3 4 1 CHL4 Kinetochore 3.7 2 4 1 CTF19 Kinetochore 3.3 1 4 0.9858 IOC2 4.7 2 4 1 MED2 8.6 4 4 1 MGA2 4 3 4 1 MOT3 2 2 4 1 MVP1 3.7 1 4 0.9811 NHP6B 13.1 1 4 0.9881 NKP2 Kinetochore 7.8 1 4 0.9881 ORC1 3.7 3 4 1 PDC1 8.5 3 4 1

S26

RPB8 15.1 2 4 1 RPI1 12.8 3 4 1 RPL26B 18.9 4 4 1 RPL3 5.4 2 4 1 RPL6A 30.1 3 4 1 RPO31 2.5 3 4 1 RPS7B 7.9 1 4 0.9881 SEF1 4.3 3 4 1 SIS1 8.8 3 4 1 SNF12 2.8 1 4 0.9881 SRL2 6.6 2 4 1 SRP54 3.3 1 4 0.9881 SSB2 41.3 3 4 1 STB3 5.3 2 4 1 TFB1 4.2 2 4 0.9996 TFC6 7.4 2 4 1 ADA2 6.9 2 3 1 BMH1 11.6 2 3 0.9996 BMH2 11.4 2 3 0.9996 BRF1 7.4 2 3 1 CIN5 8.1 2 3 1 DAD3 Kinetochore 35.1 3 3 1 DAL82 6.3 1 3 0.9881 DID4 3.9 1 3 0.9869 EGD2 21.3 2 3 0.9997 FOB1 6.4 2 3 1 GCR1 5.5 2 3 1 MCM22 Kinetochore 15.9 3 3 1 PHO4 13.1 2 3 0.9998 POB3 5.4 2 3 1 RCS1 4.1 2 3 1 RPB2 1.1 1 3 0.9651 RPL15A 14.7 3 3 1 RPL21A 13.1 2 3 1 RPL30 34.3 2 3 0.9998 RPL5 8.4 3 3 1 RPL7A 12.7 2 3 0.9994 RPO26 12.3 2 3 1 RPS24A 17.8 2 3 1 RPS8B 21 3 3 1 RPS9B 16.9 2 3 1 SFI1 0.7 2 3 0.9987 SIN3 3.3 2 3 1 SIN4 4.5 2 3 1 SSN2 2 2 3 1 SWI1 1.2 1 3 0.9869

S27

SWI6 2.2 1 3 0.9881 TBF1 5 2 3 1 TFA2 8.2 2 3 1 VPS72 5 3 3 1 WWM1 25.6 2 3 1 YAP6 6.5 2 3 0.9992 YDL156W 5.2 2 3 0.9999 YIL148W 21 1 3 0.9881 BUB1 Spindle checkpoint,

Kinetochore 2.5 2 2 0.9975

CCT8 1.6 1 2 0.9823 CTF13 Kinetochore 4.2 2 2 0.9989 CTF3 Kinetochore 1.9 1 2 0.9881 DBF4 3.1 1 2 0.9881 DPB4 6.6 1 2 0.9881 EAF3 3.2 2 2 0.9991 ECM22 2.6 1 2 0.9777 EMG1 6.3 1 2 0.9881 IES4 10.3 1 2 0.9881 IRR1 2.1 1 2 0.9811 MCM1 3.8 1 2 0.9881 MED1 2.1 1 2 0.9881 MED6 16.6 2 2 1 MED8 9 1 2 0.9881 MET31 5.1 1 2 0.9869 NOP58 3.3 1 2 0.9881 NPL3 3.1 1 2 0.9881 RCO1 1.8 1 2 0.9754 RDS2 3.1 1 2 0.9881 RFA1 2.4 1 2 0.9881 RFA3 14.8 1 2 0.9881 RFC1 3.3 2 2 1 RLM1 3.1 1 2 0.9923 ROX3 14.5 1 2 0.9881 RPB5 7 1 2 0.9881 RPC82 2 2 2 0.9998 RPL11B 8 1 2 0.9881 RPL16A 7.5 1 2 0.9881 RPL16B 13.6 1 2 0.9948 RPL9A 9.9 1 2 0.9881 RPP1B 15.1 1 2 0.9881 RPP2A 11.3 1 2 0.9858 RPS1A 3.1 1 2 0.9518 SCD6 3.7 1 2 0.9869 SKP1 Kinetochore 7.2 1 2 0.9881 SMC1 2.2 1 2 0.9881

S28

SMC4 1.8 1 2 0.9881 SNF5 1.4 1 2 0.9881 SNU13 7.9 1 2 0.9858 SPT5 2.9 1 2 0.9881 SRB7 7.9 1 2 0.9846 SSC1 5.7 2 2 1 STB5 3.4 2 2 1 STP4 7.8 2 2 0.9999 SWI3 1.6 1 2 0.9869 TAF11 3.2 1 2 0.9869 TAF8 5.5 2 2 0.9999 TFB3 4.4 1 2 0.9846 TOP2 1.3 2 2 0.9984 UBP8 2.5 1 2 0.9881 UGA3 2.7 1 2 0.9811 VHR1 5.8 1 2 0.9881 VID21 1.3 1 2 0.9881 YMR075C-A

13.2 1 2 0.9858

YOR338W 9.4 2 2 0.9563

S29

Table S2. Proteins identified in the mutant centromere minichromosome sample Name Function Coverage

(%) Unique peptides

Total peptides

Protein probability

LacI LacI 66.8 70 511 1 HTB2 H2B 79.4 23 324 1 YTA7 39.8 80 266 1 ISW1 41 68 230 1 HHF1 H4 64.1 34 202 1 RSC8 61.4 49 168 1 STH1 31.1 58 161 1 RVB2 61.6 43 152 1 RSC2 34.9 38 123 1 RSC58 62 36 114 1 RVB1 50.5 28 109 1 IOC2 37.9 38 107 1 RSC9 34.4 28 102 1 ASG1 39 34 101 1 ABF1 43.1 34 101 1 ORC1 30.6 35 99 1 CBF1 49 28 96 1 NPL6 35.4 25 94 1 SPT16 31.1 32 91 1 ABF2 43.2 19 82 1 HHT1 H3 33.8 12 79 1 SSB2 42.3 28 79 1 HTA2 H2A 43.9 17 77 1 SMC6 28.5 28 73 1 RSC4 27.7 20 71 1 CHD1 18.7 31 70 1 SMC5 21 24 68 1 TEF2 39.5 26 66 1 RSC6 37.3 20 61 1 GAL4 21.2 23 61 1 ORC2 18.9 20 57 1 RTT102 63.1 14 57 1 HHO1 H1 31 13 57 1 RSC3 28 23 55 1 ARP9 38.1 21 55 1 SRM1 25.7 20 54 1 VPS72 30.4 18 53 1 ARP7 24.7 18 53 1 SIR3 21.6 20 52 1 SWR1 17.1 19 48 1 SSA2 36.6 14 45 1 BDF1 27.7 18 45 1 MOT1 9.9 16 44 1

S30

ORC4 36.9 18 44 1 ACT1 43.2 16 39 1 GSP1 32.4 12 39 1 YLR247C 11.1 16 38 1 REB1 17.9 14 36 1 VPS1 19.6 13 36 1 GAL80 29.2 12 35 1 HTZ1 H2AZ 29.9 12 35 1 MCM3 25.2 14 34 1 MCM6 12.4 9 34 1 RSC30 17.4 14 34 1 TAF12 26.5 12 33 1 NET1 18.5 19 33 1 TAF5 18 12 32 1 POB3 27.5 15 31 1 AHC1 29.7 11 30 1 SWC3 32.6 18 29 1 ORC3 17.4 10 28 1 ARP4 21.5 11 27 1 ORC6 24.1 10 26 1 ASF2 18.9 9 25 1 RPS14A 47.4 6 24 1 TDH3 26.5 9 24 1 SUM1 20.4 11 23 1 IOC4 15.2 11 23 1 YDR210C-D 16.2 13 22 1 INO80 10.5 13 22 1 CDC48 16.3 11 21 1 MMS21 37.8 11 21 1 IOC3 12.6 8 21 1 SFH1 28.6 10 21 1 CDC46 20.9 12 20 1 GIS2 47.7 9 20 1 ORC5 22.1 10 19 1 TOP1 13.4 10 19 1 CDC47 16.3 10 18 1 DED1 13.2 5 18 1 CDC54 13.6 12 18 1 RIM1 34.8 5 17 1 RSC1 12.2 9 17 1 MVP1 6.1 2 17 1 LDB7 43.3 7 16 1 NOP1 25.7 8 16 1 HMO1 22 8 16 1 SPT15 17.9 6 16 1 SWC4 15.5 9 16 1

S31

SPO14 1.6 3 16 0.9996 RPO31 10.4 10 16 1 RET1 7 7 16 1 SWC5 11.2 4 15 1 PAB1 17.5 8 15 1 PMA1 12 9 15 1 SFI1 0.7 2 15 0.9997 NSE4 20.1 7 14 1 MOT3 18.8 10 14 1 SRP1 14.6 7 14 1 RPS18A 36.3 5 13 1 VPS71 31.8 7 13 1 HOS3 13.8 6 13 1 IES1 12.6 6 12 1 NSR1 26.1 9 12 1 CST6 24.4 9 12 1 SWC7 28 4 12 1 ASE1 1.8 2 12 0.9907 ISW2 7.4 7 12 1 HTL1 21.8 3 11 1 RPS6B 19.9 4 10 1 RNQ1 19.5 4 10 1 MSN4 5.7 3 10 1 SPT5 8.2 6 10 1 RPA190 5.2 7 10 1 RPC40 23 5 10 1 NSE3 14.2 4 9 1 TAF6 11.8 5 9 1 RPL40A 44.6 4 9 1 RPL26A 37.8 5 9 1 RPC19 19 4 9 1 SSA1 36.8 4 8 1 MCM2 10.9 6 8 1 RPO21 4.2 5 8 1 INO2 19.4 5 8 1 NGG1 9.8 4 8 1 KAR2 12.5 4 8 1 NSE1 7.7 2 8 1 YLR278C 5.6 5 8 1 INO4 20.5 2 8 1 NHP6A 22.6 3 8 1 SCD6 13.5 3 8 1 RPC82 7.5 4 8 1 AKL1 8.8 6 7 1 ADR1 4.6 4 7 1 ADA2 6.5 2 7 1

S32

GAT1 9.6 3 7 1 TAF1 6.4 5 7 1 RPC10/RPB12

41.4 3 7 1

SWI6 3.4 2 7 1 TAF4 18.8 5 7 1 RPL20A 25.9 4 7 1 TOP2 3.5 3 7 1 YAF9 27.4 5 7 1 RPB2 2.6 3 7 1 TAF14 19.3 3 7 1 CDC19 2.8 1 6 0.9762 SGF29 12.4 2 6 1 DPB4 19.4 2 6 1 RPL12B 9.1 2 6 1 CDC14 10 5 6 1 SKN7 3.4 2 6 1 SNF7 10.4 2 6 1 SIK1 9.7 4 6 1 MET4 6.8 3 6 1 RPS11B 25.6 4 5 1 RPB5 7.4 2 5 0.9996 NHP10 23.2 3 5 1 SSB1 42.3 2 5 1 TAF10 28.2 3 5 1 RPB7 14.6 2 5 1 RPS17B 33.8 3 5 1 RPL8A 17.2 3 5 1 IES3 21.2 4 5 1 TAF9 21.7 3 5 1 FET4 3.3 1 5 0.9816 RPL25 18.3 3 5 1 RFC1 6.9 3 5 1 RPB8 15.1 2 5 1 RPL33A 20.6 2 5 1 RPA135 4.3 4 5 1 NHP6B 13.1 1 4 0.9816 SIR4 2.7 3 4 1 SNU13 7.9 1 4 0.9816 RPL30 28.6 2 4 0.9992 RPL7A 12.7 3 4 1 ITC1 1.8 2 4 0.9994 RPL11B 14.4 1 4 0.9816 GCN5 2.7 1 4 0.9816 SSC1 5.8 3 4 1 RPC37 10.3 2 4 1

S33

BAS1 2.5 2 4 0.9996 ARP6 6.6 2 4 1 TAF11 9.8 3 4 1 ARP5 6.1 3 4 1 RPS3 21.7 3 4 1 YNL284C-B 7.7 2 4 0.9729 AZF1 3.5 3 4 1 ARP8 3.2 4 4 1 SNF2 1.5 1 4 0.9884 EAF3 9.7 3 4 0.9999 TFC3 2.2 2 3 0.9999 RPL21A 5.6 2 3 1 SIR2 5.3 2 3 1 YDL156W 4.4 2 3 1 SWI5 2.4 1 3 0.9816 UME6 4.3 2 3 0.9991 GCD11 5.7 2 3 1 SMC1 3.8 2 3 1 IMD2 7.8 2 3 0.9958 MCM10 3.7 2 3 0.9798 IML2 4.8 3 3 0.985 RPL17B 20.1 2 3 1 RFC2 9.9 3 3 1 RPL43B 27.2 2 3 0.9945 RPL15A 7.8 2 3 0.9976 PDC1 4.8 3 3 1 SPT8 3.8 2 3 1 MSN2 11.8 3 3 1 RCO1 4.8 2 3 1 RRP5 1.7 3 3 0.9796 VID27 7 3 3 1 RPC34 10.1 3 3 1 SPN1 3.7 1 3 0.978 IMD1 14.1 2 2 0.9616 RTG3 8.2 2 2 1 SPT7 2.6 2 2 0.9746 VMA2 3.7 1 2 0.978 MBP1 3.1 1 2 0.9917 RPS29B 17.9 1 2 0.978 RPS13 6.6 1 2 0.9673 PDC2 2.9 2 2 1 YRA1 8.8 2 2 0.9998 NCB2 7.5 1 2 0.9638 YEL007W 7.1 1 2 0.9884 RAD23 12.8 2 2 1 WWM1 13.3 1 2 0.9816

S34

EPL1 4.8 2 2 1 PHO4 13.1 2 2 0.9999 STE12 4.1 2 2 0.9999 TRA1 0.7 2 2 0.999 ENO2 6.9 2 2 1 RPB3 9.7 2 2 0.9794 SSL2 3.4 2 2 0.9991 GLG2 2.4 1 2 0.9816 RPA12 25.6 2 2 1 MGM101 7.1 1 2 0.978 DEF1 4.5 2 2 0.9993 MIF2 Kinetochore 3.1 2 2 0.9944 ACE2 3.9 2 2 1 TAF8 3.1 1 2 0.9816 RPL16B 12.6 2 2 0.9997 RPL18B 7 1 2 0.9638 GAL11 1.2 1 2 0.9816 IES4 20.7 2 2 0.9975 RIS1 1.7 2 2 0.9643 NOP58 2.5 1 2 0.9816 YPR022C 2.3 2 2 0.997 SRP54 3.3 1 2 0.9744 RPO26 12.3 1 2 0.9762

S35

Table S3. Kinetochore Proteins Identified by MS in the WT CEN Minichromosome Sample

Number of peptides Complex Protein Mammalian homologue

Coverage (%) Unique Total

Cse4 CENP-A 26.6 7 25 Mif2 CENP-C 19.5 11 20 CBF3 Ndc10 15.2 11 18 Skp1 7.2 1 2 Cep3 6.1 3 6 Ctf13 4.2 2 2 COMA Okp1 13.8 5 8 Ame1 11.7 2 4 Mcm21 CENP-O 8.7 3 7 Ctf19 3.3 1 4 NDC80 (N) Spc24 Spc24 57.3 14 42 Spc25 Spc25 41.2 13 38 Nuf2 Nuf2 30.2 13 33 Ndc80 Hec1 28.4 26 65 MIS12 (M) Nnf1 Nnf1 50.7 10 23 Nsl1 Nsl1 38.9 10 31 Dsn1 Dsn1 35.9 22 51 Mtw1 Mis12 24.2 9 21 SPC105 (K) Spc105 KNL1/Blinkin 19.6 13 40 Ydr532c 14.3 5 11 DAM1 Hsk3 47.8 4 9 Spc34 47.1 11 24 Dad3 35.1 3 3 Dad1 33 3 6 Duo1 25.5 12 34 Spc19 25.5 7 18 Ask1 24 5 12 Dam1 23.3 6 17 Dad2 - - N.D. Dad4 - - N.D. Others Iml3 10.6 2 6 Chl4 CENP-N 3.7 2 4 Ctf3 CENP-I 1.9 1 2 Mcm22 15.9 3 3 Cnn1 12.2 3 6 Nkp1 28.2 7 16 Nkp2 7.8 1 4 Mcm16 - - N.D. MS statistics Number of peptides

Number of proteins Unique Total

WT centromere 329 1924 4560 Mutant centromere 249 2256 6715

S36

Table S4. Identified Phosphorylation Sites in Kinetochore Proteins

Protein Phospho site Phosphopeptide sequence Reference if previously identified

Ame1 S59 LANpSFEFPITTNNVNAQDR (Albuquerque et al. 2008)

Cnn1 T21 AAGNNENTEVSEIRpTPFRER Novel Dsn1 T380 or T386 ILDNTENYDDTELR Novel Ndc80 S242 or T248 SLINQNTQEITILSQPLK (Kemmler et al. 2009) Spc105 S109 TSTNpSPTKISSQEEPLVTSTQIDDARTEEK Novel Spc105 T172, T167 or

S168 ASQHDPTSMEMTEVFPR Novel

Spc105 T356 KLDTVSDYAASVTpTPVKEAK (Li et al. 2007; Albuquerque et al. 2008; Holt et al. 2009)

Spc105 S380 MpSPITFSDVDNK (Li et al. 2007; Albuquerque et al. 2008; Holt et al. 2009)

Spc19 S116 NISMDDDDALNpSPDMGQEYEGR (Cheeseman et al. 2002; Albuquerque et al. 2008; Holt et al. 2009)

Spc24 T130 DSEIISpTPNGSKIK (Albuquerque et al. 2008)

S37

Table S5. Proteins identified in the SILAC quantitative MS experiment Name Function Coverage

(%) Unique peptides

Total peptides

Ratio (H/L)

Standard deviation

Protein probability

BUB1 Spindle checkpoint, Kinetochore

24.4 21 35 59.35 46.50 1

BUB3 Spindle checkpoint, Kinetochore

42.2 14 29 31.19 14.98 1

MCM3 Replication 20.6 14 27 26.25 17.00 1 CHL4 Kinetochore 18.3 8 16 20.75 10.98 1 MCM22 Kinetochore 44.4 10 13 20.00 11.83 1 MCM2 Replication 18.8 14 21 18.87 11.31 1 CDC46 Replication 14.3 10 17 17.48 5.48 1 CNN1 Kinetochore 17.5 7 8 15.73 7.31 1 MCM6 Replication 14.5 12 16 14.92 5.12 1 YDR532c Kinetochore 40.3 19 56 14.23 6.46 1 CTF3 Kinetochore 12.6 7 12 14.08 1.29 1 FIN1 Microtubule binding 26.8 9 15 13.73 1.98 1 MCM16 Kinetochore 13.3 3 6 12.65 1.62 1 DAD3 Kinetochore 59.6 6 10 12.63 2.02 1 GAL4 Transcription 5.3 3 3 12.56 2.56 1 ASK1 Kinetochore 33.2 8 18 12.44 2.67 1 LEU1 Amino acid

biosynthesis 23 14 20 11.73 6.54 1

AME1 Kinetochore 46.9 24 50 11.02 3.85 1 NSL1 Kinetochore 67.6 25 71 10.88 3.37 1 MTW1 Kinetochore 45.7 24 96 10.46 2.83 1 DSN1 Kinetochore 34.4 31 117 10.42 1.98 1 IML3 Kinetochore 41.2 9 26 10.33 1.69 1 SPC25 Kinetochore 33.5 16 48 10.29 0.92 1 DAD1 Kinetochore 37.2 6 11 10.17 0.42 1 SPC105 Kinetochore 48.1 58 156 9.88 2.40 1 NUF2 Kinetochore 51 47 143 9.79 1.77 1 CDC54 Replication 21 15 19 9.67 2.60 1 HSK3 Kinetochore 33.3 4 8 9.65 0.21 1 SPC24 Kinetochore 59.2 20 61 9.48 1.90 1 NNF1 Kinetochore 57.2 23 68 9.37 2.38 1 OKP1 Kinetochore 30.5 23 54 9.31 3.94 1 NDC80 Kinetochore 57 69 235 9.29 1.71 1 SPC34 Kinetochore 49.2 22 51 9.25 1.04 1 SPC19 Kinetochore 50.9 14 33 9.19 1.23 1 CDC47 Replication 16.8 13 16 9.13 2.58 1 MCM21 Kinetochore 21.7 9 18 8.92 1.19 1 NKP1 Kinetochore 46.2 14 25 8.87 1.23 1 CTF19 Kinetochore 26.8 12 24 7.92 1.77 1 STU2 Microtubule binding 30.6 27 50 7.50 2.69 1 DAD2 Kinetochore 6.8 2 6 6.85 0.52 0.9998

S38

DUO1 Kinetochore 18.6 6 8 6.73 0.31 1 CSE4 Kinetochore 21 8 10 4.77 0.63 1 NKP2 Kinetochore 15 4 9 4.54 2.69 1 GCN4 Transcription 33.8 9 12 4.46 0.13 1 SUI2 15.1 4 4 3.38 0.37 1 CTF13 Kinetochore 12.1 8 12 3.25 0.25 1 CBF1 40.5 22 65 3.17 0.17 1 SKP1 Kinetochore 7.2 2 4 3.15 0.04 1 NDC10 Kinetochore 26.3 33 64 3.04 0.62 1 CEP3 Kinetochore 17.9 16 28 2.77 0.83 1 ACS2 5.7 5 14 2.65 0.12 1 EDC3 7.8 3 3 2.56 1.27 1 GCD11 12.3 6 7 2.48 0.38 1 YEF3 4.4 5 6 2.46 0.25 1 BMH1 31.5 16 31 2.35 0.12 1 RAD7 12.4 4 4 2.19 0.13 1 EGD2 21.3 4 6 2.17 0.08 1 SHM2 10.9 6 6 2.15 0.17 1 GDH1 17.4 5 5 2.15 0.10 1 LYS21 22.5 15 18 2.13 0.08 1 SNU13 19.8 6 25 2.13 0.23 1 SRC1 2.8 3 3 2.10 0.19 1 POB3 19.4 14 19 2.08 0.13 1 THS1 7.6 8 9 2.06 0.08 1 PDS5 3.5 5 6 2.06 0.12 1 SPT16 19.5 23 35 2.02 0.17 1 ABF1 45.4 51 130 2.02 0.10 1 NOP15 10 3 3 1.98 0.02 1 NHP2 16 3 3 1.98 0.02 1 PRP43 11.7 10 11 1.98 0.08 1 PIP2 6.3 7 10 1.98 0.23 1 ARO8 5.4 3 4 1.96 0.15 1 RAD23 10.6 4 4 1.94 0.17 1 SRP54 5.2 5 6 1.90 0.10 1 ORC1 Replication 17.6 18 20 1.87 0.67 1 RPS14A 41.6 17 60 1.85 0.06 1 PAB1 30.3 23 43 1.77 0.10 1 HIR1 5.2 3 3 1.77 0.31 1 TAF2 10.6 21 31 1.77 0.10 1 TAF11 16.5 11 17 1.77 0.10 1 TAF8 9.8 5 6 1.77 0.12 1 RPS9B 36.9 11 25 1.77 0.75 1 TAF3 25.2 10 15 1.75 0.15 1 RPL23A 43.1 12 32 1.75 0.10 1 TAF4 37.1 27 49 1.73 0.15 1 OAF1 7.9 8 8 1.73 0.10 1

S39

CAC2 25.6 16 29 1.71 0.12 1 PHO2 7.9 4 4 1.71 0.27 1 TEA1 24.6 16 22 1.71 0.12 1 CCT8 4.8 3 3 1.69 0.15 1 HRR25 5.7 3 3 1.67 0.06 1 FOL2 42.4 17 37 1.60 0.10 1 RPS27B 46.3 9 27 1.60 0.33 1 RPS10B 14.3 3 6 1.60 0.02 1 ORC6 Replication 12.2 5 7 1.58 0.10 1 ORC5 Replication 16.9 8 8 1.58 0.10 1 ORC4 Replication 31.9 20 29 1.56 0.12 1 YMR233W 11.1 5 7 1.56 0.06 1 HAS1 6.1 3 3 1.56 0.13 1 REB1 45.4 71 235 1.54 0.13 1 ORC3 Replication 7.3 5 6 1.52 0.04 1 RPS3 46.2 20 65 1.52 0.17 1 RPL38 19.2 4 10 1.50 0.04 1 RPS7B 31.6 11 32 1.50 0.15 1 HTZ1 H2AZ 22.4 5 32 1.48 0.10 1 UTP22 0.7 2 4 1.48 0.08 1 UGA3 2.7 2 4 1.46 0.02 1 RPS1B 42.4 20 45 1.46 0.06 1 TAF5 44.7 54 161 1.44 0.42 1 DCP2 6.8 4 4 1.44 0.17 1 ACA1 10.6 6 9 1.44 0.31 1 MRPL38 32.6 6 7 1.44 0.13 1 MCD1 13.6 7 11 1.42 0.08 1 RPL31A 27.4 5 8 1.42 0.40 1 RPS24A 36.3 10 29 1.42 0.08 1 BAS1 30.1 30 66 1.42 0.25 1 RPS15 14.1 5 15 1.42 0.08 1 YMR144W 23.7 6 9 1.40 0.10 1 NHP6B 21.2 2 10 1.40 0.06 1 RXT2 9.1 3 5 1.40 0.87 1 SWR1 12.4 16 32 1.40 0.71 1 SWI6 22 29 54 1.40 0.17 1 SMC1 19.1 33 41 1.38 0.06 1 MSI1 6.6 3 4 1.38 0.19 1 RPS1A 39.2 3 4 1.38 0.12 1 YDJ1 17.1 9 12 1.38 0.08 1 SSB2 58.6 72 403 1.38 0.02 1 RPL33B 38.3 2 3 1.38 0.04 1 TAF9 44.6 14 39 1.37 0.06 1 SWC5 17.2 4 7 1.37 0.50 1 RPL9A 24.6 9 27 1.37 0.10 1 TAF6 32.9 36 90 1.35 0.06 1

S40

SMC3 22.4 34 49 1.35 0.21 1 SEF1 9.6 14 16 1.35 0.10 1 RPS13 43.7 10 37 1.35 0.31 1 IMD3 27.2 4 6 1.35 0.04 1 RAP1 37.8 43 96 1.35 0.13 1 HMS1 6.2 4 4 1.35 0.04 1 BRN1 23.1 24 36 1.33 0.15 1 SSB1 60.7 76 443 1.33 0.04 1 RPS22A 46.9 13 26 1.33 0.06 1 TCB3 3.9 3 4 1.33 0.13 1 MOT3 8.2 9 24 1.33 0.31 1 RRP5 8.6 14 17 1.33 0.60 1 EFT2 8.6 7 10 1.31 0.58 1 RFC3 16.5 4 4 1.31 0.44 1 TBF1 27.8 25 72 1.31 0.17 1 TAF12 44.3 45 135 1.29 0.06 1 YCS4 12.6 17 22 1.29 0.15 1 RPL32 23.8 5 9 1.29 0.60 1 RPC37 22 5 6 1.29 0.21 1 SUS1 63.5 12 22 1.29 0.21 1 TFC4 25.9 39 73 1.27 0.21 1 RPS4B 34.5 20 36 1.27 0.10 1 RDS2 17 9 16 1.27 0.06 1 TEF2 25.5 27 209 1.25 0.17 1 RPL27B 27.2 9 23 1.25 0.10 1 MGM101 8.9 3 4 1.25 0.27 1 FBA1 23.4 8 11 1.25 0.04 1 GPM1 36.4 13 18 1.25 0.13 1 RPS29A 26.8 3 5 1.25 0.02 1 RPL20A 33.9 10 38 1.23 0.08 1 TFC7 36.6 18 31 1.23 0.06 1 RPL33A 38.3 7 19 1.23 0.04 1 SAR1 24.7 5 7 1.21 0.06 1 SPT7 26.7 44 74 1.21 0.46 1 GCR1 32.6 29 50 1.21 0.17 1 MCM1 3.8 2 12 1.19 0.04 1 HHF1 H4 52.4 27 405 1.19 0.13 1 TFC1 18.8 15 30 1.19 0.06 1 SIS1 21.9 9 9 1.19 0.10 1 TFC8 15.3 10 12 1.19 0.08 1 HTB2 H2B 51.1 15 125 1.17 0.08 1 HTA2 H2A 23.5 4 23 1.17 0.02 1 UTP8 4.9 4 4 1.17 0.04 1 VHR1 20.5 12 26 1.17 0.08 1 RPS5 29.8 13 38 1.17 0.13 1 VID22 23.9 31 78 1.17 0.44 1

S41

SMC2 18.5 25 34 1.15 0.12 1 RPC11 30.9 4 6 1.15 0.12 1 RPS18A 50.7 19 95 1.15 0.15 1 RSC1 27 35 82 1.15 0.29 1 TDH1 30.1 3 5 1.15 0.44 1 SRP1 21.4 18 58 1.15 0.08 1 YRR1 2.5 3 3 1.15 0.06 1 RPC82 22.2 15 21 1.15 0.27 1 SMC4 16.4 29 35 1.13 0.12 1 RPL14B 21.7 7 16 1.13 0.04 1 RPL21A 35 8 28 1.13 0.17 1 RPS29B 28.6 5 11 1.13 0.06 1 UTP9 7.5 5 5 1.13 0.06 1 SKN7 23.6 27 66 1.13 0.04 1 UTP10 3.4 5 5 1.13 0.13 1 RPL43B 41.3 8 26 1.13 0.04 1 YKR064W 10 10 17 1.13 0.06 1 NHP6A 29 10 27 1.13 0.08 1 RPC17 25.5 3 6 1.12 0.06 1 HSF1 29.7 28 49 1.12 0.08 1 RPS2 16.5 8 18 1.12 0.04 1 ITC1 33.5 65 125 1.12 0.17 1 FKH1 18.4 11 21 1.12 0.10 1 VPS71 17.1 4 6 1.12 0.06 1 ISW2 27.3 52 111 1.12 0.21 1 VMA2 45.3 34 57 1.10 0.15 1 RSC6 43.7 37 132 1.10 0.12 1 SPT3 30.3 15 35 1.10 0.04 1 RPL30 45.7 6 10 1.10 0.06 1 RPS20 38.8 10 23 1.10 0.12 1 SRL2 23.2 11 25 1.10 0.04 1 FKH2 20.1 22 39 1.10 0.08 1 RPS7A 22.6 3 6 1.10 0.00 1 RIM1 51.1 11 29 1.08 0.17 1 TFC6 12.4 8 18 1.08 0.04 1 RPS17B 44.1 10 27 1.08 0.06 1 BRF1 29 21 33 1.08 0.17 1 RSC4 34.2 42 124 1.08 0.10 1 RSC58 55 51 159 1.08 0.06 1 SFH1 43.7 36 79 1.08 0.06 1 ADH1 36.8 19 47 1.08 0.12 1 TFB2 24.4 13 21 1.08 0.19 1 NAN1 3.6 3 3 1.08 0.08 1 HFI1 18.4 14 28 1.08 0.04 1 TAF14 36.9 20 64 1.06 0.10 1 HHT1 H3 10.3 4 128 1.06 0.08 1

S42

SGF29 23.2 10 16 1.06 0.23 1 DPB4 59.2 15 25 1.06 0.10 1 ADA2 27.2 13 28 1.06 0.04 1 RSC8 51.7 70 289 1.06 0.10 1 NPL6 54.7 46 183 1.06 0.06 1 IES4 50.9 14 23 1.06 0.17 1 RET1 16.8 21 32 1.06 0.21 1 SGF11 48.5 7 14 1.06 0.02 1 TFB4 18.6 7 9 1.06 0.19 1 TSA1 24 4 5 1.04 0.25 1 RPC31 23.5 3 3 1.04 0.10 1 HTL1 53.8 8 16 1.04 0.06 1 NGG1 34.2 31 56 1.04 0.08 1 PDR1 20.4 30 51 1.04 0.12 1 YGR071C 23.8 21 31 1.04 0.06 1 GCN5 33.3 19 48 1.04 0.08 1 RSC9 36.8 46 114 1.04 0.06 1 RPL36A 33 6 13 1.04 0.06 1 RPS19B 39.6 11 25 1.04 0.06 1 RFM1 20.3 10 16 1.04 0.06 1 YCG1 19.4 22 33 1.02 0.29 1 LDB7 21.7 3 7 1.02 0.08 1 NHP10 51.2 22 65 1.02 0.06 1 SPT8 20.8 20 45 1.02 0.06 1 RSC2 33.7 48 121 1.02 0.06 1 IMD4 20.4 11 20 1.02 0.06 1 UBP8 20.4 13 16 1.02 0.04 1 DAL82 12.5 4 4 1.02 0.02 1 TAF10 31.6 11 26 1.00 0.38 1 CDC19 39.8 24 50 1.00 0.21 1 PDC2 28.4 44 117 1.00 0.04 1 SUM1 43.2 62 149 1.00 0.10 1 RPC10/RPB12

51.4 6 18 1.00 0.04 1

HIT1 23.2 4 4 1.00 0.13 1 SSA2 63.4 50 169 1.00 0.15 1 RPP0 36.2 14 27 1.00 0.19 1 RPL16B 23.2 6 10 1.00 0.04 1 RPC34 15.5 6 11 1.00 0.10 1 RPL8B 21.5 9 22 0.98 0.04 1 RPL15A 27.5 5 7 0.98 0.19 1 SSA1 52.6 43 143 0.98 0.06 1 RSC3 35.7 60 180 0.98 0.10 1 YEL007W 13.2 6 7 0.98 0.13 1 IES1 36.6 51 133 0.98 0.08 1 TRA1 21.7 128 266 0.98 0.06 1

S43

SSC1 27.8 21 28 0.98 0.12 1 RPC25 9.9 3 3 0.98 0.02 1 IES3 30 15 49 0.98 0.04 1 SWC7 54.5 8 11 0.98 0.12 1 ABF2 48.1 40 439 0.98 0.06 1 FPR1 28.9 4 7 0.98 0.04 1 RFA1 7.9 6 8 0.96 0.42 1 SSL2 16.5 17 28 0.96 0.08 1 YLR247C 15.3 31 42 0.96 0.08 1 MOT1 36.2 136 432 0.96 0.21 1 ARP7 39 34 88 0.96 0.06 1 IES5 69.6 15 47 0.94 0.04 1 RPL7A 23.8 9 19 0.94 0.12 1 RPL26B 27.1 9 24 0.94 0.02 1 RTT102 58 18 60 0.94 0.06 1 RSC30 23.7 32 64 0.94 0.06 1 BDF1 7.1 4 4 0.92 0.04 1 GCR2 21.3 13 30 0.92 0.10 1 RPD3 34.2 16 22 0.92 0.08 1 RPO31 21.7 35 52 0.92 0.08 1 ARP8 36.9 59 158 0.92 0.06 1 RPL35B 19.2 5 10 0.90 0.06 1 TFB5 12.5 2 3 0.90 0.02 1 TUB2 6.6 3 4 0.90 0.02 1 RPL11B 12.1 3 9 0.90 0.12 1 ASG1 41.8 58 153 0.90 0.06 1 SSL1 20.2 11 20 0.90 0.06 1 ARP9 40.5 39 132 0.90 0.21 1 BOP3 9.6 3 4 0.90 0.06 1 NOG2 15.6 10 14 0.90 0.04 1 HST1 14.3 7 10 0.90 0.27 1 CHA1 21.9 13 17 0.88 0.08 1 RTG3 29.8 22 59 0.88 0.06 1 RPL13A 24.6 7 15 0.88 0.23 1 RPS16B 35.7 6 9 0.88 0.04 1 RPS21A 52.9 7 19 0.88 0.12 1 RPL25 24.6 7 18 0.88 0.04 1 CIN5 14.9 5 9 0.88 0.27 1 RPL4A 21.3 10 17 0.87 0.04 1 RPS6B 30.9 13 52 0.87 0.10 1 RPL1B 12 4 5 0.87 0.15 1 HAL9 7.8 6 8 0.87 0.08 1 PGK1 19.7 8 13 0.85 0.12 1 ISW1 40.3 99 329 0.85 0.08 1 PFK1 4.7 4 5 0.85 0.04 1 ARP4 29.9 29 90 0.85 0.06 1

S44

PDC1 22.4 14 23 0.85 0.02 1 RPL22A 41.3 6 8 0.85 0.12 1 IOC2 15.9 19 51 0.85 0.04 1 YLR278C 26 40 78 0.85 0.25 1 YMR111C 30.1 13 20 0.85 0.12 1 RTG1 32.2 8 12 0.85 0.04 1 FAS2 3.4 3 3 0.83 0.37 1 SNF5 12.4 7 7 0.83 0.21 1 MBP1 28.5 26 40 0.83 0.17 1 RPL12B 44.2 9 26 0.83 0.04 1 IES6 31.3 10 22 0.83 0.13 1 ARP6 8 4 4 0.83 0.10 1 ARP5 31 51 118 0.83 0.19 1 RPC19 45.8 10 22 0.83 0.06 1 RPL18B 22.6 7 18 0.83 0.04 1 SIN3 11.3 17 23 0.83 0.08 1 INO4 51.7 11 22 0.83 0.04 1 RPL5 27.9 8 18 0.83 0.12 1 UME1 14.8 7 12 0.83 0.06 1 RFC5 17.5 6 8 0.81 0.04 1 TFB1 13.1 10 12 0.81 0.13 1 RAD3 8.4 7 10 0.81 0.08 1 SWC4 12.6 7 11 0.81 0.04 1 STH1 36.7 88 263 0.81 0.08 1 DID4 22.8 4 6 0.81 0.50 1 YAF9 9.3 2 5 0.81 0.02 0.9933 SKO1 40.5 29 61 0.81 0.29 1 ATP2 7 5 5 0.79 0.15 1 CDC48 6.9 5 7 0.79 0.12 1 INO2 12.5 6 19 0.79 0.02 1 KAR2 28 19 35 0.79 0.06 1 SIK1 32.1 26 49 0.79 0.38 1 RPS28B 52.2 7 15 0.79 0.02 1 MET4 16.4 10 25 0.79 0.06 1 MET31 37.3 7 12 0.79 0.12 1 STB3 5.1 2 3 0.77 0.02 0.9998 RPL28 10.1 2 5 0.77 0.00 1 RPL17A 23.4 8 17 0.77 0.12 1 VPS66 17.7 6 8 0.77 0.04 1 GAT1 2.4 2 3 0.75 0.04 1 VID21 16.2 14 18 0.75 0.06 1 RPP2B 33.6 5 16 0.75 0.02 1 RFC2 12.7 6 9 0.75 0.58 1 HAP1 19.9 32 53 0.75 0.04 1 RPS12 22.4 6 12 0.75 0.02 1 RPA135 30.8 41 73 0.75 0.08 1

S45

RPC40 20 10 35 0.75 0.06 1 FHL1 8.8 7 9 0.73 0.08 1 RPP1B 15.1 2 8 0.73 0.06 1 SWI4 10.8 12 16 0.73 0.08 1 TDH3 44.9 27 56 0.73 0.00 1 TUB1 13.9 6 9 0.73 0.08 1 VID27 24.7 17 26 0.73 0.12 1 RPP2A 34.9 5 10 0.73 0.02 1 RPA43 30.7 13 29 0.73 0.04 1 RPS0B 17.5 5 8 0.71 0.02 1 FKS1 5.1 8 10 0.71 0.12 1 HOS3 6 3 3 0.71 0.02 1 ARO1 14.7 22 34 0.71 0.08 1 ASC1 24.1 10 18 0.71 0.06 1 RPL3 14.5 7 11 0.71 0.04 1 NOP58 9.4 5 5 0.71 0.02 1 RPA190 33.7 75 155 0.71 0.08 1 BIR1 7.4 8 11 0.69 0.13 1 RPA14 24.1 4 5 0.69 0.02 1 SCP160 6.7 6 7 0.69 0.10 1 RPB5 28.8 8 21 0.69 0.04 1 SWI3 17.8 15 28 0.69 0.27 1 TDH2 44.6 26 55 0.69 0.02 1 WAR1 19.3 26 51 0.69 0.06 1 KRE33 4.2 4 5 0.69 0.48 1 RPA49 29.6 24 50 0.69 0.10 1 RFC4 14.2 4 5 0.69 0.06 1 BFR1 16.4 7 11 0.69 0.02 1 RPL6A 9.7 3 6 0.67 0.02 0.9978 SWI1 8.4 9 11 0.67 0.12 1 FOB1 9.2 4 7 0.67 0.08 1 YRF1-3 26.3 7 12 0.67 0.08 1 RPA12 12 2 4 0.67 0.02 1 MSN1 25.4 8 18 0.67 0.10 1 RPB8 25.3 6 11 0.67 0.06 1 RNQ1 12.1 14 43 0.65 0.08 1 TFB3 27.7 7 8 0.65 0.06 1 SPT15 42.9 18 99 0.65 0.04 1 ENO2 27.7 19 40 0.65 0.02 1 RPO26 12.3 2 6 0.65 0.04 1 CDC10 12.4 3 3 0.63 0.10 1 RPS23A 18.6 4 4 0.63 0.00 1 RFA3 41.8 5 6 0.63 0.19 1 TFA1 18.5 5 5 0.63 0.12 1 KAP95 5.5 3 4 0.63 0.10 1 CDC28 8.7 3 5 0.62 0.04 1

S46

SSN3 8.1 3 3 0.62 0.31 1 YAP6 16.4 5 5 0.62 0.06 1 RPL34A 12.4 2 6 0.62 0.04 1 TFA2 15.9 5 6 0.62 0.19 1 RPL10 26.2 10 13 0.62 0.04 1 RPL2A 22.8 9 24 0.60 0.04 1 ENO1 25.9 2 2 0.60 0.00 1 RPB2 15.5 17 31 0.60 0.13 1 RPS8A 25 6 12 0.58 0.04 1 RPS11B 6.4 2 6 0.58 0.02 0.9999 CUP9 22.2 6 8 0.58 0.06 1 RPL19B 23.3 6 13 0.56 0.02 1 MED6 14.6 6 10 0.56 0.27 1 FAS1 1.3 2 3 0.56 0.08 1 SNF12 10.2 4 5 0.56 0.04 1 YDR458C 8 4 5 0.54 0.06 1 SAH1 10.7 5 8 0.54 0.48 1 SNF6 7.2 2 3 0.54 0.02 1 TFC3 31.6 42 75 0.54 0.08 1 SWP82 12 5 8 0.52 0.02 1 STE23 15.9 14 22 0.52 0.06 1 EAF3 24.9 9 14 0.52 0.15 1 SPT6 2.4 3 3 0.50 0.04 1 SIR3 12.1 8 10 0.50 0.12 1 YAP1 50 30 51 0.50 0.12 1 LRS4 34 14 23 0.48 0.08 1 LacI LacI 53.9 58 314 0.48 0.06 1 UBP15 2.4 3 3 0.48 0.17 1 MED7 15.3 4 6 0.48 0.08 1 UTP5 4.4 2 2 0.48 0.31 1 YER064C 15.4 6 8 0.48 0.19 1 SRB4 17.9 9 11 0.46 0.10 1 YLR108C 5.6 2 4 0.46 0.12 1 GSP1 27.4 7 26 0.46 0.06 1 GAL11 13.9 15 23 0.44 0.06 1 IRR1 4.3 4 4 0.44 0.04 1 SRB6 22.3 3 3 0.44 0.06 1 NCB2 28.8 11 34 0.44 0.06 1 ACT1 32.5 25 100 0.44 0.33 1 MLC1 18.1 3 4 0.44 0.00 1 YFR038W 6.9 5 6 0.42 0.17 1 CAD1 18.6 7 12 0.42 0.19 1 PGD1 12.3 5 7 0.42 0.04 1 YHR020W 7 4 4 0.42 0.27 1 SRB2 54.3 10 19 0.42 0.10 1 RVB2 59.4 76 250 0.42 0.06 1

S47

YDL156W 15.7 7 10 0.40 0.02 1 MED4 18.7 5 9 0.40 0.08 1 MED2 16.5 5 6 0.40 0.02 1 BUR6 26.1 8 27 0.40 0.06 1 SRM1 13.3 8 12 0.40 0.12 1 VPS24 24.1 3 4 0.40 0.02 1 RSF1 18.4 8 16 0.40 0.08 1 GIS2 44.4 23 157 0.40 0.04 1 CSM1 52.6 9 18 0.38 0.21 1 ROX3 27.3 3 3 0.38 0.04 1 FET4 21.7 13 32 0.38 0.06 1 SRB7 20 2 3 0.37 0.00 1 CSE2 19.5 3 5 0.37 0.08 1 RVB1 63.1 56 238 0.37 0.08 1 SRB5 21.2 10 14 0.37 0.06 1 RPL40A 56.4 6 24 0.37 0.23 1 SPT20 13.9 6 10 0.35 0.08 1 RPI1 11.3 9 21 0.35 0.17 1 DID2 47.1 11 23 0.35 0.04 1 SNF7 22.5 5 10 0.35 0.02 1 RGR1 20.1 23 30 0.35 0.04 1 SIN4 22.1 20 38 0.35 0.02 1 TOP1 24.2 30 81 0.35 0.06 1 MED1 13.6 8 12 0.35 0.08 1 SAM1 9.2 3 5 0.33 0.08 1 AZF1 20.2 16 28 0.33 0.17 1 MED11 28.2 3 4 0.33 0.00 1 YBL100W 2.9 4 6 0.31 0.08 1 SIR2 23.8 16 42 0.31 0.06 1 WWM1 26.5 3 5 0.29 0.02 1 NUT1 18.9 18 26 0.29 0.13 1 STB5 8.2 6 8 0.29 0.04 1 FAA1 5.7 5 5 0.29 0.35 1 HMO1 33.3 19 99 0.27 0.08 1 RCS1 5.5 4 6 0.27 0.02 1 TOA2 60.7 5 7 0.25 0.02 1 MGA2 12.1 8 10 0.23 0.17 1 INO1 6.9 3 3 0.23 0.08 1 RRN9 21.6 6 8 0.23 0.08 1 RFC1 4.2 3 3 0.23 0.04 1 TAF1 12.9 11 15 0.23 0.12 1 VPS1 48.3 70 305 0.23 0.06 1 TOA1 13.6 4 5 0.23 0.08 1 DED1 17.7 7 10 0.21 0.15 1 INO80 27.7 42 114 0.21 0.06 1 TOP2 9.7 13 18 0.19 0.12 1

S48

RRN10 17.9 2 5 0.17 0.02 1 RPO21 16.7 24 38 0.17 0.12 1 CDC14 23.8 20 93 0.17 0.06 1 CST6 29.3 14 25 0.17 0.06 1 CHD1 16 21 30 0.15 0.08 1 IOC3 22 17 34 0.15 0.06 1 PMA1 30.3 47 175 0.15 0.08 1 ZRT1 7.4 3 8 0.15 0.02 1 NET1 13.3 14 26 0.13 0.12 1 TOF2 14.3 8 11 0.12 0.04 1 LEU3 4.2 3 3 0.12 0.00 1 SUA7 13.9 3 4 0.12 0.02 1 SSN2 3.9 5 8 0.10 0.08 1 ACE2 9 6 10 0.10 0.08 1 RCO1 7.6 2 2 0.10 0.02 1 TYE7 15.5 3 7 0.08 0.02 1 SRB8 4.2 4 6 0.04 0.04 1 STE12 5.1 3 3 0.04 0.04 1 IES2 21.2 8 16 0.04 0.02 1

S49

Table S6. Proteins identified in the Fin1-FLAG immunoprecipitation sample Name Function Coverage

(%) Unique peptides

Total peptides

Protein probability

FIN1 Fin1 45.4 19 79 1 BMH1 14-3-3 53.9 25 73 1 BMH2 14-3-3 49.8 5 38 1 SSB1 22.2 10 18 1 TEF2 11.6 7 16 1 AKL1 16.9 9 15 1 RPS14A 35 6 15 1 YIL002W-A 39.1 3 12 1 NDC80 Kinetochore 8.5 4 11 1 RPL20A 28.7 6 10 1 NSR1 18.1 6 9 1 DEF1 9.1 3 8 1 SSA2 16.4 5 8 1 RPL23A 11.7 3 8 1 RPL6B 18.2 3 8 1 TIF3 10.3 4 7 1 RPS3 12.5 2 7 1 RPL25 18.3 3 7 1 RPL4A 11.6 2 7 0.9999 DSN1 Kinetochore 5.7 2 6 1 RPA135 4.3 4 6 1 RPL11B 20.1 4 6 1 YDR210C-D 4.1 4 6 0.9938 CDC19 5.4 2 5 1 RPS11B 13.5 3 5 1 RPL21A 23.8 3 5 1 RPL16B 12.1 2 5 1 PAB1 8.5 3 4 1 SPT5 0.9 2 4 1 PHO84 2.7 1 4 1 MLF3 8.2 2 4 1 DED1 2.6 1 4 1 NEW1 3.4 2 4 1 RPL19B 18.5 3 4 1 RPS6B 11.9 2 4 1 RPS9B 12.8 2 4 1 RPS26B 20.2 2 4 1 RPS20 21.5 3 4 1 YBL054W 5.9 2 4 1 TIF4631 2.4 3 4 1 RPS13 7.3 1 4 0.9996 NUF2 Kinetochore 7.1 1 4 0.9992 ASK1 Kinetochore 6.5 1 3 1

S50

GCD11 2.7 1 3 1 TDH3 9.6 2 3 1 STM1 4.8 2 3 1 RPP0 7.1 2 3 1 SUB1 9.9 2 3 1 RPL26B 12.6 2 3 1 RPL17B 6.5 2 3 1 NOG2 5.1 2 3 1 NOP1 6.4 2 3 0.9977 RPL15A 11.3 2 3 0.9931 RPL36A 9 1 3 0.9852 SPC19 Kinetochore 10.3 2 2 1 MCM21 Kinetochore 4.1 1 2 1 SSB2 20.1 1 2 1 PRT1 1.7 1 2 1 RPL12B 11.5 1 2 1 RPS7B 7.9 1 2 1 YBL104C 3.3 2 2 1 BEM2 2.3 2 2 1 YHR020W 2.8 1 2 1 CBK1 3 1 2 1 REX4 4.5 1 2 1 RPL1B 6.5 2 2 0.9999 PMA1 1.7 2 2 0.9999 RPL13A 5 1 2 0.9996 RPL8A 4.3 1 2 0.9996 FET4 1.8 1 2 0.9992 RPS15 7.7 1 2 0.9992 GCD10 4.2 1 2 0.9992 RPL35B 10.8 1 2 0.9984 RPS17B 11.8 1 2 0.998 DAM1 Kinetochore 2.9 1 2 0.9976 RPL7A 4.1 2 2 0.9975 LSB3 1.8 1 2 0.9956 RPS4B 4.2 1 2 0.9925 RPL10 5.9 1 2 0.991 YDR365W-B,YER138C

2.7 1 2 0.9852

SBP1 2.7 1 2 0.9802 RPS16B 6.3 1 2 0.9741 RPL31A 14.2 1 2 0.9711 SPC105 Kinetochore 2.4 1 2 0.9703 RPS24A 6.7 1 2 0.9692

S51

Table S7. Yeast strains used in this study. All strains are isogenic with the W303

background. Plasmids are indicated in brackets.

Strain Genotype

SBY3 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 LYS2 can1-100 bar1Δ

SBY625 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 LYS2 bar1Δ

GLC7-3HA::HIS3

SBY5218 MATa ura3-1::pCMV-LacI-3FLAG::URA3 leu2,3-112 his3-11 trp1-1 ade2-

1 can1-100 LYS2 BAR1 NDC80-13myc::KAN [WT CEN3 Minichromosome,

TRP1]


1 can1-100 LYS2 BAR1 NDC80-13myc::KAN [mutant CEN3

Minichromosome, TRP1]

SBY5429 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 LYS2 can1-100 ipl1-321

SBY5962 MATα ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 bar1Δ LYS2

FIN1-3FLAG::KAN


1 can1-100 LYS2 bar1Δ NDC80-13myc::KAN FIN1-13myc::HIS3 [WT

CEN3 Minichromosome, TRP1]


1 can1-100 LYS2 bar1Δ NDC80-13myc::KAN FIN1-13myc::HIS3 [mutant



1 can1-100 LYS2 NDC80-13myc::KAN [CEN, TRP1 (pRS314)]


1 CAN1 lys2Δ arg4Δ::HIS3 bar1Δ NDC80-13myc::KAN [WT CEN3



1 CAN1 lys2Δ arg4Δ::HIS3 bar1Δ NDC80-13myc::KAN [mutant CEN3



S52

FIN1-3GFP::HIS3

SBY6368 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 lys2Δ bar1Δ

GLC7-3HA::HIS3 BMH2-13myc::TRP1 FIN1-3FLAG::KAN


GLC7-3HA::HIS3 BMH2-13myc::TRP1


BMH2-13myc::TRP1 FIN1-3FLAG::KAN

SBY6452 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 lys2Δ::pGAL-

Δ176-clb2::LYS2 bar1Δ GLC7-3HA::HIS3 BMH2-13myc::TRP1 FIN1-

3FLAG::KAN

SBY6458 MATa ura3-1 leu2,3-112 his3-11::pCUP1-GFP12-lacI12:HIS3 trp1-

1:256lacO::TRP1 ade2-1 lys2Δ can1-100 bar1Δ PDS1-myc18:LEU2

[pGALS-FIN1-WT, CEN, URA3 (pSB1194)]



[pGALS-fin1-5A, CEN, URA3 (pSB1195)]


1 NDC80-13myc::KAN FIN1-13myc::HIS3 lys2Δ::pGAL-Δ176-clb2::LYS2

[WT CEN3 Minichromosome, TRP1]


GLC7-3GFP::HIS3 [pGALS-FIN1-WT, CEN, URA3 (pSB1194)]


GLC7-3GFP::HIS3 [pGALS-fin1-5A, CEN, URA3 (pSB1195)]


GLC7-3HA::HIS3 BMH2-13myc::TRP1 FIN1-3FLAG::KAN [pGALS-

FIN1-WT-GFP, CEN, URA3 (pSB1185)]


GLC7-3HA::HIS3 BMH2-13myc::TRP1 FIN1-3FLAG::KAN [pGALS-fin1-

5A-GFP, CEN, URA3 (pSB1186)]


S53


SPC42-CFP::KAN [pGALS-FIN1-WT, CEN, URA3 (pSB1194)]



SPC42-CFP::KAN [pGALS-fin1-5A, CEN, URA3 (pSB1195)]


1 can1-100 LYS2 NDC80-13myc::KAN FIN1-13myc::HIS3 ndc10-1 [WT



1 can1-100 LYS2 NDC80-13myc::KAN FIN1-13myc::HIS3 NDC10 [WT



GLC7-3HA::HIS3 BMH2-13myc::TRP1 FIN1-3FLAG::KAN [pGALS-fin1-

5Aglc7--GFP, CEN, URA3 (pSB1212)]


GLC7-3GFP::HIS3 [pGALS-fin1-5Aglc7-, CEN, URA3 (pSB1215)]



SPC42-CFP::KAN [pGALS-fin1-5Aglc7-, CEN, URA3 (pSB1215)]


GLC7-3GFP::HIS3 [pGAL, 2 micron, URA3 (pSB819)]

SBY6809 MATa ura3-1 leu2,3-112::GFP-TUB1::LEU2 his3-11 trp1-1 ade2-1 LYS2

can1-100 bar1Δ [pGALS-fin1-5A, CEN, URA3 (pSB1195)]


PDS1-myc18:LEU2 mad2ΔKAN [pGALS-fin1-5Aglc7-, CEN, URA3

(pSB1215)]

SBY6825 MATa ura3-1 leu2,3-112::GFP-TUB1::LEU2 his3-11 trp1-1 ade2-1 LYS2

can1-100 bar1Δ [pGALS-FIN1-WT, CEN, URA3 (pSB1194)]



S54

[pGALS-fin1-5Aglc7-, CEN, URA3 (pSB1215)]


1:256lacO::TRP1 ade2-1 lys2Δ can1-100 bar1Δ PDS1-myc18:LEU2 ipl1-

321 [pGALS-fin1-5Aglc7-, CEN, URA3 (pSB1215)]


fin1Δ::HIS3


[pGALS-FIN1-WT-GFP, CEN, URA3 (pSB1185)]


[pGALS-fin1-5A-GFP, CEN, URA3 (pSB1186)]


[pGALS-fin1-5Aglc7--GFP, CEN, URA3 (pSB1212)]


[pFIN1-Fin1-WT-GFP, CEN, LEU2 (pSB1252)]




[CEN, LEU2 (pRS415)]


bar1Δ fin1::HIS3


[pFIN1-fin1-5A-GFP, CEN, LEU2 (pSB1359)]


[pFIN1-fin1-5Aglc7--GFP, CEN, LEU2 (pSB1361)]






[CEN, LEU2 (pRS415)]

S55


1 can1-100 lys2Δ bar1Δ fin1Δ::HIS3 [WT CEN3 Minichromosome, TRP1]








SBY7609 MATα ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 can1-100 lys2Δ bar1Δ

GLC7-3HA::HIS3 BMH2-13myc::TRP1 fin1Δ::HIS3 [pFIN1-Fin1-WT-

GFP, CEN, LEU2 (pSB1252)]


GLC7-3HA::HIS3 BMH2-13myc::TRP1 fin1Δ::HIS3 [pFIN1-fin1-5A-GFP,

CEN, LEU2 (pSB1359)]


GLC7-3HA::HIS3 BMH2-13myc::TRP1 fin1Δ::HIS3 [pFIN1-fin1-5Aglc7--

GFP, CEN, LEU2 (pSB1361)]


GLC7-3HA::HIS3 BMH2-13myc::TRP1 fin1Δ::HIS3 [pFIN1-fin1glc7--GFP,

CEN, LEU2 (pSB1267)]



[pFIN1-fin1glc7--GFP, CEN, LEU2 (pSB1267)]

SBY7841 MATa ura3-1 leu2,3-112 his3-11 trp1-1::glc7-12::TRP1 ade2-1 can1-100

lys2Δ bar1Δ BMH2-13myc::TRP1 FIN1-3FLAG::KAN glc7::LEU2

SBY7895 MATa ura3-1 leu2,3-112 his3-11 trp1-1 ade2-1 lys2Δ can1-100 bar1Δ

GLC7-3HA::HIS3 fin1Δ::HIS3 NDC80-13myc::KAN


GLC7-3HA::HIS3 FIN1 NDC80-13myc::KAN

S56


GLC7-3HA::HIS3 fin1Δ::HIS3 DSN1-3FLAG::KAN


GLC7-3HA::HIS3 FIN1 DSN1-3FLAG::KAN


FIN1-13myc::HIS3 DSN1-3HA::HIS3

S57

Table S8. Plasmids used in this study.

Plasmid Description

pRS314 CEN, TRP1

pRS415 CEN, LEU2

pSB102 pGAL-∆176-CLB2, LYS2 (integrating)

pSB340 GFP-TUB1, LEU2 (integrating)

pSB623 3GFP, HIS3 (gift from David Pellman, PB1585)

pSB737 pCMV-LacI-3FLAG, URA3 (integrating) (gift from Toshio Tsukiyama)

pSB751 CEN3

pSB819 pGAL, 2 micron, URA3

pSB963 WT CEN3, 8LacO, TRP1

pSB972 Mutant CEN3, 8 LacO, TRP1

pSB1169 FIN1-3GFP, HIS3 (integrating)

pSB1185 pGALS-FIN1-WT-GFP, CEN, URA3 (gift from David Morgan, pDM300)

pSB1186 pGALS-fin1-5A-GFP, CEN, URA3 (gift from David Morgan, pDM301)

pSB1194 pGALS-FIN1-WT, CEN, URA3

pSB1195 pGALS-fin1-5A, CEN, URA3

pSB1212 pGALS-fin1-5Aglc7--GFP, CEN, URA3

pSB1215 pGALS-fin1-5Aglc7-, CEN, URA3

pSB1227 pGALS-FIN1-WT-GFP, CEN, LEU2

pSB1228 pGALS-FIN1-5A-GFP, CEN, LEU2

pSB1237 pGALS-fin1glc7--GFP, CEN, URA3

pSB1250 pFIN1

pSB1252 pFIN1-Fin1-WT-GFP, CEN, LEU2

pSB1260 pGALS-fin1-5Aglc7--GFP, CEN, LEU2

pSB1261 pGALS-fin1glc7--GFP, CEN, LEU2

pSB1267 pFIN1-fin1glc7--GFP, CEN, LEU2

pSB1359 pFIN1-fin1-5A-GFP, CEN, LEU2

pSB1361 pFIN1-fin1-5Aglc7--GFP, CEN, LEU2

S58

Supplemental References

Alber, F., Dokudovskaya S., Veenhoff L.M., Zhang W., Kipper J., Devos D., Suprapto

A., Karni-Schmidt O., Williams R., Chait B.T. et al. 2007. Determining the

architectures of macromolecular assemblies. Nature 450: 683-94.

Albuquerque, C.P., Smolka M.B., Payne S.H., Bafna V., Eng J., and Zhou H. 2008. A

multidimensional chromatography technology for in-depth phosphoproteome

analysis. Mol Cell Proteomics 7: 1389-96.

Biggins, S., Severin F.F., Bhalla N., Sassoon I., Hyman A.A., and Murray A.W. 1999.

The conserved protein kinase Ipl1 regulates microtubule binding to kinetochores

in budding yeast. Genes Dev 13: 532-44.

Cheeseman, I.M., Anderson S., Jwa M., Green E.M., Kang J., Yates J.R., Chan C.S.,

Drubin D.G., and Barnes G. 2002. Phospho-Regulation of Kinetochore-

Microtubule Attachments by the Aurora Kinase Ipl1p. Cell 111: 163-172.

Gelbart, M.E., Rechsteiner T., Richmond T.J., and Tsukiyama T. 2001. Interactions of

Isw2 chromatin remodeling complex with nucleosomal arrays: analyses using

recombinant yeast histones and immobilized templates. Mol Cell Biol 21: 2098-

106.

Goh, P.Y. and Kilmartin J.V. 1993. NDC10: a gene involved in chromosome segregation

in Saccharomyces cerevisiae. J. Cell Biol. 121: 503-512.

Han, D.K., Eng J., Zhou H., and Aebersold R. 2001. Quantitative profiling of

differentiation-induced microsomal proteins using isotope-coded affinity tags and

mass spectrometry. Nat Biotechnol 19: 946-51.

S59

Holt, L.J., Tuch B.B., Villen J., Johnson A.D., Gygi S.P., and Morgan D.O. 2009. Global

analysis of Cdk1 substrate phosphorylation sites provides insights into evolution.

Science 325: 1682-6.

Keller, A., Nesvizhskii A.I., Kolker E., and Aebersold R. 2002. Empirical statistical

model to estimate the accuracy of peptide identifications made by MS/MS and

database search. Anal Chem 74: 5383-92.

Kemmler, S., Stach M., Knapp M., Ortiz J., Pfannstiel J., Ruppert T., and Lechner J.

2009. Mimicking Ndc80 phosphorylation triggers spindle assembly checkpoint

signalling. EMBO J 28: 1099-110.

Kim, B., Nesvizhskii A.I., Rani P.G., Hahn S., Aebersold R., and Ranish J.A. 2007. The

transcription elongation factor TFIIS is a component of RNA polymerase II

preinitiation complexes. Proc Natl Acad Sci U S A 104: 16068-73.

Li, X., Gerber S.A., Rudner A.D., Beausoleil S.A., Haas W., Villen J., Elias J.E., and

Gygi S.P. 2007. Large-scale phosphorylation analysis of alpha-factor-arrested

Saccharomyces cerevisiae. J Proteome Res 6: 1190-7.

Li, X.J., Zhang H., Ranish J.A., and Aebersold R. 2003. Automated statistical analysis of

protein abundance ratios from data generated by stable-isotope dilution and

tandem mass spectrometry. Anal Chem 75: 6648-57.

Liu, D., Vader G., Vromans M.J., Lampson M.A., and Lens S.M. 2009. Sensing

chromosome bi-orientation by spatial separation of aurora B kinase from

kinetochore substrates. Science 323: 1350-3.

Longtine, M.S., McKenzie A., 3rd, Demarini D.J., Shah N.G., Wach A., Brachat A.,

Philippsen P., and Pringle J.R. 1998. Additional modules for versatile and

S60

economical PCR-based gene deletion and modification in Saccharomyces

cerevisiae. Yeast 14: 953-61.

MacKelvie, S.H., Andrews P.D., and Stark M.J. 1995. The Saccharomyces cerevisiae

gene SDS22 encodes a potential regulator of the mitotic function of yeast type 1

protein phosphatase. Mol Cell Biol 15: 3777-85.

Nesvizhskii, A.I., Keller A., Kolker E., and Aebersold R. 2003. A statistical model for

identifying proteins by tandem mass spectrometry. Anal Chem 75: 4646-58.

Rosasco-Nitcher, S.E., Lan W., Khorasanizadeh S., and Stukenberg P.T. 2008.

Centromeric Aurora-B activation requires TD-60, microtubules, and substrate

priming phosphorylation. Science 319: 469-72.

Sikorski, R.S. and Hieter P. 1989. A system of shuttle vectors and yeast host strains

designed for efficient manipulation of DNA in Saccharomyces cerevisiae.

Genetics 122: 19-27.

Woodbury, E.L. and Morgan D.O. 2007. Cdk and APC activities limit the spindle-

stabilizing function of Fin1 to anaphase. Nat Cell Biol 9: 106-12.

Quantitative Proteomic Analysis of Purified Yeast...

Documents

Transcript of Quantitative Proteomic Analysis of Purified Yeast...