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DOI: 10.1038/ncb2863

Gombos et al. Supplementary Figure 1

d

wt T146A

BF DAPI merge BF mergeDAPIclass1 class2 class3

5 10 15 20-0.0050.0000.0050.0100.0150.020

100 µM GTP standard73.3 µM γ-TuSC

V (ml)

A26

0

b

C13S T146A lethal lethal

lethal lethal

c

wtQ12

GC13

GC13

SD70

NE72

QS14

1AL22

4A

Q12G C

13G

D70N E72

Q

Q12G C

13G D

70N

Q12G C

13G S14

1AT14

6AN20

6A

S141A

T146A

C13S D

70N

T146A

L224A

Q12G C

13G T14

6A0

20

40

60

80

100no bud (G1)

small bud (G1/S)

large bud, 1 DAPI(metaphase)

large bud, 2 DAPI(ana-/telophase)

defect

Tub4GTP strain

% o

f cel

ls

a 10 20 30 40 50 60 1Z5V -PREIITLQLGQCGNQIGFEFWKQLCAEHGISPEAI------VTDRKD---VFFYQADDEHYIPRAVL Tub4 MGGEIITLQAGQCGNHVGKFLWSQLAKEHAIGTDGLSQLPDSSTERDDDTKPFFRENSRNKFTPRAIM ****** ***** * * ** ** * * * * ** *** 70 80 90 100 110 120 130 1Z5V LDLEPRVIHSILNSPYAKLYNPENIYLSG------NNWASGFSQGEKIHEDIFDIIDREADGSDSLEG Tub4 MDSEPSVIADVENT-FRGFFDPRNTWVASDGASAGNSWANGYDIGTRNQDDILNKIDKEIDSTDNFEG * ** ** * * * * ** * * ** ** * * * ** 140 150 160 170 180 190 200 1Z5V FVLCHSIAGGTGSGLGSYLLERLNDRYPKKLVQTYSVFPNQS-DVVVQPYNSLLTLKRLTQNADCLVV Tub4 FQLLHSVAGGTGSGLGSNLLEALCDRYPKKILTTYSVFPARSSEVVVQSYNTILALRRLIEDSDATVV * * ** ********** *** * ****** ****** * **** ** * * ** * ** 210 220 230 240 250 260 270 1Z5V LDNTALNRIATDRLHIQNPSFSQINQLVSTIMSASTTTLRYPGYMNNDLIGLIASLIPTPRLHFLMTG Tub4 FDNASLLNISGKVFRNPNIDLQHTNQLISTIISSVTNSIRFPSYMYSSMSSIYSTLIPSPELHFLSPS ** * * * *** *** * * * * ** *** * **** 280 290 300 310 320 330 340 1Z5V YTPLTTD---QSV--RKTTVLDVMRRLLQPKNVMVSTGT-NHCYIAILNIIQGEVDPTQVHKSLQRIR Tub4 FTPFTSDYIHDDIAHKGHSSYDVMLDLLDPSNSLVSTAMNNPTYFNVYNTIIGNVEPRQISRAMTKLQ ** * * *** ** * * *** * * * * * * * * 350 360 370 380 390 400 1Z5V ERKLANFIPWGPASIQVALSRKSPYLR-------VSGLMMANHTSISSLFERTCRQYDKLRKREAFLE Tub4 QR-I-KFPSWSSSAMHVNIGRRSPYLPLQPNENEVSGMMLSNMSTVVNVFENACNTFDKVFAKGAFLN * * * * * **** *** * * ** * ** *** 410 420 430 440 450 460 470 1Z5V QFRKEDMF---KDNFDEMDTSREIVQQLIDEYHAA---T----------------------------R Tub4 NYNVGDLFQSMQNVQDEFAESREVVQSLMEDYVAAEQDSYLDDVLVDDENMVGELEEDLDADGDHKLV * * ** *** ** * * **

Figure S1 GTP binding to yeast γ-tubulin Tub4. (a) Sequence alignment of Tub4 and human γ-tubulin from the 1Z5V crystal structure, which was used as a template for modelling. Conserved residues are marked with an asterisk. Residues contacting the bound GTPγS are highlighted in red. (b) γ-TuSC purified from insect cells contained 9.4% bound GTP as determined by RP-HPLC on a C18 column 1. Elution profiles of 20 µl of a 100 µM GTP

standard and of 73.3 µM γ-TuSC are shown. The protein concentration was determined using the calculated molar extinction coefficient. (c) Phenotypes of wild type TUB4 cells and nuclear migration defects of a representative ts tub4GTP strain (T146A). (d) Quantification of nuclear migration phenotypes of tub4GTP strains obtained from DAPI stained cultures grown overnight at 30°C.

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Gombos et al. Supplementary Figure 2

c

wt

0 100 200 3000

20406080

100

Time (min)

% o

f cel

ls

N228A

0 100 200 3000

20406080

100

Time (min)

% o

f cel

ls

N206A L224A

0 100 200 3000

20406080

100

Time (min)

% o

f cel

ls

C13S T146A

0 100 200 3000

20406080

100

Time (min)

% o

f cel

ls

null

0 100 200 3000

20406080

100

Time (min)

% o

f cel

ls

d

wt

N228A

C13S T14

6A

N206A

L224A null

0

20

40

60

80

100

bipolar spindle at bud neckcytoplasmic MT misguidancemislocalised spindlemonopolar/disassembled spindleother defect

tub4GTP strain

% o

f met

apha

se c

ells

e

f

1 1.8 3.0 2.9 2.0 2.0 1.4 63 47 19 54 2.4

YPH499

wt N228A

C13S T14

6A

N206A

L224

A

null

YPH499

wt N228A

C13S T14

6A

N206A

L224

A

null

Tub4

YPA+Glu(repression)

YPA+Raf/Gal(induction)

actin

normalized Tub4

g

YPA+Glu

wt

N228A

C13S T14

6A

N206A

L224A null

0

20

40

60

80

100

pGalS-tub4GTP strain

% o

f cel

ls

YPA+Raf/Gal

wt

N228A

C13S T14

6A

N206A

L224A null

0

20

40

60

80

100

no budsmall bud 1DAPIlarge bud 1 DAPIlarge bud 2 DAPImultiple bud/chain

pGalS-tub4GTP strain

% o

f cel

ls

a

inpu

tIP

no H

Awt T14

6AN20

6A

C13S D

70N

T146A

L224

A

Q12G C

13G T14

6A

Spc97-3HA

Tub4

Spc97-3HA

Tub4

wt

T146A

N206A

C13S D

70N

T146A

L224A

Q12G C

13G T14

6A0.0

0.5

1.0

1.5

Tub4GTP strain

Nor

mal

ized

Tub

4am

ount

b

anti-Spc97-3HA IP

brightfield GFP-Tub1Spc42-

mCherrymerge+DNA

wt

N22

8AC

13S

T146

AN

206A

L224

Anu

ll

100 kDa

55 kDa

55 kDa

100 kDa

55 kDa

55 kDa

Figure S2 tub4GTP levels and complex formation. (a) Spc97-3HA was immunoprecipitated (IP) by anti-HA antibodies from cell extracts of ts tub4GTP strains. Before immunoprecipitation, cells were synchronized with α-factor at 23°C and released at 37°C for 2 h. (b) Quantification of a. Error bars represent s.e.m. (normalized Tub4 values in Spc97-3HA Co-IPs of n = 3 independent experiments). See Supplementary Table S3 for raw data. Two-tailed, unpaired Student’s t-test was used to obtain P values. Tub4 in the γ-TuSC is not significantly different between tub4GTP and wild type cells (P ≥ 0.05). (c) TUB4-AID GFP-TUB1 SPC42-mCherry cells with pGalS (null), pGalS-TUB4 (wt) or pGalS-tub4GTP (N228A; C13S T146A; N206A L224A) were synchronised with α-factor, followed by both, galactose and IAA addition. Cells were released from the arrest in the presence of galactose and IAA and fixed at the time

points indicated. Cell cycle progression of DAPI stained cells was analysed. (phenotypes of n = 100 cells for each time point per experiment). (d) MT defects of TUB4-AID pGalS-tub4GTP cells. Cells were treated as in c and analysed 2 h after release, when entering metaphase. Bar, 5 µm. (e) Quantification of d (phenotypes of n = 100 cells per strain per experiment). (f) Overexpression of ectopic pGalS-tub4GTP in the presence of the wild type TUB4 allele in rich medium. Extracts of cells grown overnight at 30°C under repressive (glucose) or inducing (raffinose/galactose) conditions were analysed by immunostaining with the indicated antibodies. (g) Quantification of the nuclear migration phenotypes shows metaphase arrest of cells overexpressing ectopic pGalS-tub4GTP in the presence of the wild type TUB4 allele. Cells were grown as in f. DNA was stained with DAPI. (phenotypes of n = 100 cells per strain per experiment).

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Gombos et al. Supplementary Figure 3

a

b

Tub4

Tub4(+Spc42-yeGFP)+DNA

TUB

4-A

IDTU

B4-

AID

TUB

4Y

PH

499

YP

H50

1

c

d

Tub4Spc42-

mCherrymerge+DNA

wt

T146

AN

206A

Q12

G C

13S

T146

A

wt

0 50 100 150 200 250 300-0.4-0.20.00.20.40.60.81.0

whole cellFRAP ROI

Time (s)

Spc9

7-ye

GFP

fluor

esce

nce

at S

PB (a

.u.) C13S T146A

0 50 100 150 200 250 300-0.4-0.20.00.20.40.60.81.0

Time (s)

Spc9

7-ye

GFP

fluor

esce

nce

at S

PB (a

.u.)

e

-10 s 0 s 60 s 120 s 180 s 240 s 300 s-10 s 0 s 60 s 120 s 180 s 240 s 300 s

wt

T146A

N206A

Q12G C

13G T14

6A0

200

400

600

tub4GTP strain

Tub4

fluo

resc

ence

at S

PB (a

.u.)

Tub4-AID

Tub4-AID

TUB4

YPH499

YPH501

-200

0

200

400

600

800

1000

Strain

Tub4

fluo

resc

ence

at S

PB (a

.u.)

+IA

A

Figure S3 γ-TuSCGTP localizes to SPBs. (a) Calibration of anti-Tub4 immunostaining using a Δtub4 strain (TUB4-AID) in the presence of IAA), two haploid strains containing one ectopic copy of TUB4 (TUB4-AID/TUB4 in the presence of IAA) or the wild type TUB4 (YPH499) and a diploid strain carrying two copies of TUB4/TUB4 (YPH501). Cells were fixed 75 min after release from α-factor when entering metaphase. Bar, 2 µm. (b) Quantification of a shows linearity. Boxes represent upper and lower quartiles (25-75%) with a line at the median, whiskers extend from the minimum to the maximum. 5 independent experiments with each n = 50 cells per strain analyzed. (c) Tub4GTP (class 2: T146A; N206A, class 3: Q12G C13S T146A) localizes to SPBs by anti-Tub4 immunostaining. Conditional lethal tub4GTP cells were synchronized with

α-factor at 23°C and released at 37°C for 2 h. Bar, 2 µm. (d) Quantification of c. Boxes represent upper and lower quartiles (25-75%) with a line at the median, whiskers extend from the minimum to the maximum. 5 independent experiments with n = 50 cells analyzed per experiment. (e) FRAP experiments of Spc97-yeGFP in TUB4-AID cells with TUB4 and a representative tub4GTP mutant (C13S T146A) in the presence of IAA. Cells were released from α-factor arrest into nocodazole containing medium for 2 h. Normalized fluorescence intensity of Spc97-yeGFP at SPBs (top) and images of photobleached cells (bottom) are shown. ROI means region of interest – in this experiment the SPB. As a control the whole cell was bleached. None of the strains showed fluorescence recovery of SPB-associated Spc97-yeGFP. Bars, 5 µm.

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Gombos et al. Supplementary Figure 4

wt D70NT14

6AN20

6A

L224

AN22

8A

C13S D

70N

C13S T14

6A

D70N L2

24A

S141A

T146A

N206A

L224

A

Q12G C

13G T14

6A

kDa20015012010085

7060

50

40

Spc97/Spc98

Tub4

0.87:1

.0:1

0.94:1

.1:1

1.1:1.

4:1

0.76:1

.0:1

0.95:1

.1:1

0.71:0

.73:1

1.2:1.

2:1

0.97:1

.4:1

1.3:1.

3:1

0.99:1

.2:1

0.85:0

.76:1

1.0:1.

3:1Spc97:Spc98:Tub4

wt

C13

S T

146A

d

a

c

b

0.85 ml 1.45 ml 0.85 ml 1.45 ml

GS

T-S

pc11

01-22

0w

tT1

46A

N22

8AC

13S

T14

6AN

206A

L22

4A

Spc98Spc97

Tub4GST-Spc1101-220

Spc98Spc97

Tub4GST-Spc1101-220

Spc98Spc97

Tub4GST-Spc1101-220

Spc98Spc97

Tub4GST-Spc1101-220

Spc98Spc97

Tub4GST-Spc1101-220

GST-Spc1101-220

0.5 1.0 1.5 2.0 2.5-50

050

100150200

V (ml)

A 280

(mA

bs)

0.5 1.0 1.5 2.0 2.5-50

050

100150200

V (ml)

A 280

(mA

bs)

0.5 1.0 1.5 2.0 2.5-50

050

100150200

V (ml)

A 280

(mA

bs)

0.5 1.0 1.5 2.0 2.5-50

050

100150200

V (ml)A 2

80 (m

Abs

)

0.5 1.0 1.5 2.0 2.5-50

050

100150200

V (ml)

A 280

(mA

bs)

Tub4GTP complexGST-Spc1101-220

Tub4GTP complex +GST-Spc1101-220

GST-Spc1101-220/Tub4GTP complex GST-Spc1101-220+Tub4GTP complex

wt

T146

AN

228A

C13

ST1

46A

N20

6AL2

24A

100 kDa85 kDa

70 kDa60 kDa

50 kDa

100 kDa

85 kDa

70 kDa60 kDa

50 kDa

100 kDa85 kDa

70 kDa60 kDa

50 kDa

100 kDa85 kDa

70 kDa60 kDa

50 kDa

100 kDa85 kDa

70 kDa60 kDa

50 kDa

100 kDa85 kDa

70 kDa60 kDa

50 kDa

Figure S4 Analysis γ-TuSCGTP complexes purified from insect cells. (a) SDS-PAGE of purified γ-TuSC (wt) and γ-TuSCGTP. Tub4 gives two bands due to partial cleavage of Met1 as determined by mass spectrometry analysis. Bottom: band intensities of the Coomassie-stained gel were assessed by densitometry. (b) Size-exclusion chromatography of recombinant and purified GST-Spc1101-220 (top left), γ-TuSC (wt) and γ-TuSCGTP with (right) and without (left) the N-terminus of the receptor protein Spc110 (GST-Spc1101-220). γ-TuSC/γ-TuSCGTP and GST-Spc1101-220 were mixed

at a 1:2 molar ratio and analysed by gel filtration. SDS-PAGE of the peak fractions shows that γ-TuSC/γ-TuSCGTP co-elutes with GST-Spc1101-220. (c) Electron micrographs of negatively stained γ-TuSCGTP show typical Y-shaped complexes resembling wild type γ-TuSC 2. Bar, 10 nm. (d) Aligned and classified particles of negatively stained wt γ-TuSC and a representative TuSCGTP (C13S T146A). 1911 particles were analyzed for wild type and 1826 particles for the TuSCC13S T146A mutant and classified into 20 classes each. Bar, 10 nm.

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Gombos et al. Supplementary Figure 5

d

NE

nMTSPB

cMT

N CP

NE

nMT

SPBcMT

N

CP

NE

nMT

SPB

N

CP

NE

nMT

N

CP

wt

N20

6AT1

46A

Q12

G C

13G

T146

Ae

a b

c

wt

T146

AN

206A

Q12

G C

13G

T146

A

BFGFP-Tub1

+DNAwt

0 50 100 150 2000

20406080

100

Time (min)

% o

f cel

ls

T146A

0 50 100 150 2000

20406080

100

Time (min)

% o

f cel

ls

N206A

0 50 100 150 2000

20406080

100

Time (min)

% o

f cel

ls

Q12G C13G T146A

0 50 100 150 2000

20406080

100

Time (min)

% o

f cel

ls

YP

D+

15 µ

g/m

lbe

nom

yl in

DM

SO

YP

D +

DM

SO

14°C 23°C 30°C 33°C 35°C 37°C

wtT146AN206A

Q12G C13G T146AC13S D70N

S141A T146Awt

T146AN206A

Q12G C13S T146AC13S D70N

S141A T146A

f

Figure S5 MT organization defects of tub4GTP strains. (a) ts tub4GTP cells (class 2: T146A; N206A and class 3: Q12G C13G T146A) arrest in metaphase at the restrictive temperature. Cells were synchronized with α-factor at 23°C, released at 37°C and fixed at the time points indicated. Cell cycle progression of DAPI stained cells was analysed. (phenotypes of n = 100 cells per strain per experiment; 5 independent experiments). (b) MT defects of ts tub4GTP cells. Cells were treated as in a and analysed 2 h after release. Bar, 5 µm. (c) Quantification of b. (phenotype of n = 100 cells for each strain per experiment).

(d) Electron micrographs of thin sections stained with uranyl-acetate showing MT phenotypes of ts tub4GTP cells. Cells were treated as in a. Cartoons on the right shows the SPB and MTs of the micrograph on the left. CP, cytoplasm; N, nucleus; NE, nuclear envelope; cMT, cytoplasmic MT; nMT, nuclear MT. Bar, 200 nm. (e) Quantification of d. MT phenotypes of n = 9 cells for wt, 12 for T146A, 6 for N206A, and 11 for Q12G C13G T146A. (f) Benomyl rescue of ts tub4GTP cells. Cells were grown at 23°C and spotted onto benomyl containing plates or control plates and grown at the indicated temperatures.

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Gombos et al. Supplementary Figure 6

d

wt

T146

AN

228A

C13

ST1

46A

N20

6AL2

24A

ef

g

Tub4Spc42-

mCherrymerge+DNA

wt

T146

AN

206A

wt

T146A

N206A-200

0

200

400

600

800

1000

tub4GTP strain

Tub4

fluo

rsce

nce

at S

PB (a

.u.)

10 20 30 40 50 60 FtsZ LSP---------------------EDKELLEYLQQTKAKITVVGCGGAGNNTITRLKMEGIEGAKTV Tub1 MREVISINVGQAGCQIGNACWELYSLEHGIKPDGHLEDGLSKPKGGEEGFSTFFHETGYGKFVPRAI * * * * 70 80 90 100 110 120 130 FtsZ AINTDAQQLIRTKAD--------KKILIGKKLTRGLGAGGNPKIGEEAAKESAEEIKAAIQDSDMV- Tub1 YVDLEPNVIDEVRNGPYKDLFHPEQLISGKEDAANNYARGHYTVGREILGDVLDRIRKLADQCDGLQ ** * * * * * * 140 150 160 170 180 190 200 FtsZ --FITCGLGGGTGTGSAPVVAEISK----KIGALTVAVVTLPFVMEGKVRMKNAMEGLERLKQHTDT Tub1 GFLFTHSLGGGTGSGLGSLLLEELSAEYGKKSKLEFAVYPAPQVSTSVVEPYNTVLTTHTTLEHADC * ****** * * * * ** * * * * * * 210 220 230 240 250 260 FtsZ LVVIPNEKLFEIVPNMP--LKLAFKVADEVLINAVKGLVELITKDGLINVDFADVKA---------- Tub1 TFMVDNEAIYDMCKRNLDIPRPSFANLNNLIAQVVSSVTASLRFDGSLNVDLNEFQTNLVPYPRIHF ** * * ** *** 270 280 290 300 310 320 330 FtsZ -------------------------------------------------VMNNGGLAMIGIGESDSE Tub1 PLVSYSPVLSKSKAFHESNSVSEITNACFEPGNQMVKCDPRDGKYMATCLLYRGDVVTRDVQRAVEQ * 340 350 360 370 380 390 400 FtsZ KRAKEAVSMA------------LNSPLLDVDIDGATGALIHVMGPEDLTLEEAREVVATVSSRLDPN Tub1 VKNKKTVQLVDWCPTGFKIGICYEPPTATPNSQLATVDRAVCMLSNTTSIAEAWKRIDRKFDLMYAK * * * ** * ** 410 420 430 440 FtsZ ATII--WGATIDENLENTVRVLLVITGVQSRIEFTDTGLKRKKLE Tub1 RAFVHWYVGEGMEEGEFTEAREDLAALERDYIEVGADSYAEEEEF * * * **

GFP-Tub1Spc42-

mCherryBF

GFP-Tub1Spc42-

mCherry

GFP-Tub1Spc42-

mCherryBF BF

TUB

4-A

ID T

UB

4TU

B4-

AID

TUB

4-A

ID tu

b4N

228A

ND

C10

ND

C10

ND

C10

ND

C10

-AID

ND

C10

-AID

ND

C10

-AID

0 min 40 min 100 min

normal bipolar spindlecMT misguidancemislocalized spindlespindle with too few MTsserious spindle defect(broken/monopolar/no spindle)

0 20 40 60 80 1000

20406080

100

Time (min)%

cel

ls w

ithM

Ts a

t SPB

TUB4-AID TUB4TUB4-AID TUB4 NDC10-AID

0 20 40 60 80 1000

20406080

100

Time (min)

% c

ells

with

MTs

at S

PB

0 20 40 60 80 1000

20406080

100

Time (min)

% c

ells

with

MTs

at S

PBTUB4-A

ID TUB4

TUB4-AID

TUB4

NDC10-A

ID

020406080

100

tub4GTP strain

% m

itotic

cel

lsTUB4-A

ID

TUB4-AID

NDC10-A

ID0

20406080

100

tub4GTP strain%

mito

tic c

ells

TUB4-AID

tub4N

228A

TUB4-AID

tub4N

228A

NDC10-A

ID

0

20406080

100

% m

itotic

cel

ls

tub4GTP strain

TUB4-AIDTUB4-AID NDC10-AID

TUB4-AID tub4N228A

TUB4-AID tub4N228A NDC10-AID

c

bTub4

Spc42-yeGFP

merge+DNA

wt

N22

8AC

13S

T146

AN

206A

L224

Anu

ll

a

wt

N228A

C13S T14

6A

N206A

L224A null

-500

0

500

1000

1500

2000

tub4GTP strain

Tub4

fluo

resc

ence

at S

PB (a

.u.)

Figure S6 MT nucleation by tub4GTP. (a) Anti-Tub4 immunostaining of TUB4-AID cells (“null”) with TUB4 (wt) or tub4GTP at SPBs in the presence of IAA and nocodazole. Cells were treated as in Fig. 5a. Bar, 2 µm. (b) Quantification of a. Boxes represent upper and lower quartiles (25-75%) with a line at the median, whiskers extend from the minimum to the maximum. n = 50 cells analyzed per strain per experiment. 5 independent experiments were performed. Two-tailed, unpaired Student’s t-test was used to compare samples and to obtain P values. There are no significant differences between TUB4 and tub4GTP cells (P ≥ 0.05), while the null mutant is different from the wild type with P ≤ 0.001. (c) The kinetics of MT re-nucleation in tub4GTP cells is not influenced by functional kinetochores. TUB4-AID NDC10-AID cells with additional TUB4, tub4N228A or NDC10 (as indicated in the figure) were grown and treated with nocodazole as in Fig. 5c. The formation of MTs was followed over time. Middle: For each time point n = 100 cells were analyzed per strain per experiment for MTs at the SPB. The

right panel shows the quantification of cells for MT phenotypes (illustrated in right upper corner). n = 100 cells were analyzed for each time point and strain. (d) Electron micrographs of negatively stained rings of γ-TuSCGTP formed under low pH and low salt conditions. Bar, 25 nm. (e) Alignment of Tub1 and FtsZ by MODELLER. Note the long insertion in Tub1 between residues 261-319, which is not included in our model due to the lack of a homologous template. Conserved residues are marked with an asterisk. The two acidic residues that promote GTP hydrolysis are highlighted in red. (f) Anti-Tub4 immunostaining of ts tub4GTP cells at 37°C in the presence of nocodazole. Bar, 2 nm. (g) Quantification of f. Boxes represent upper and lower quartiles (25-75%) with a line at the median, whiskers extend from the minimum to the maximum. 5 independent experiments with n = 50 cells analyzed per experiment per strain. Two-tailed, unpaired Student’s t-test was used to obtain P values. There are no significant differences between TUB4 and tub4GTP cells (P ≥ 0.05).

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Gombos et al. Supplementary Figure 7

GDP exchangesagainst GTP

γ-tubulinbinds GTP

γ-TuSCassembly

GTP

GDP

γ-tubulin

α-tubulin

β-tubulin

Spc97

Spc98

Spc110 γ-tubulin:GTP binds tubulin with high affinity:

MT nucleation

assembly ofMT nucleation

platform

GTP independent γ-tubulin:GDP binds tubulin with low affinity

Figure S7 Model for the GTP/MT nucleation cycle of γ-tubulin. See Discussion for details.

© 2013 Macmillan Publishers Limited. All rights reserved.

S U P P L E M E N TA RY I N F O R M AT I O N

8 WWW.NATURE.COM/NATURECELLBIOLOGY

Gombos et al. Supplementary Figure 8

Fig. 2a

55 kDa

35 kDa

α-Tub4 55 kDa

35 kDa

α-Tub4

α-HAα-HA

250 kDa

130 kDa

100 kDa70 kDa

250 kDa

130 kDa

100 kDa70 kDa

input IP: α-HA

250 kDa130 kDa100 kDa

55 kDa

35 kDa

α-Tub4

250 kDa130 kDa

55 kDa

35 kDa

α-actin

130 kDa

100 kDa

55 kDa

35 kDa

α-Tub4

130 kDa

100 kDa

55 kDa

35 kDa

70 kDa

α-actin

α-HA

130 kDa100 kDa

35 kDa

55 kDa

130 kDa100 kDa

35 kDa

55 kDa

100 kDa

α-Tub4

α-Tub4

α-Tub4

α-HA130 kDa100 kDa

35 kDa

55 kDa

130 kDa100 kDa

35 kDa

55 kDa

YPAD YPAD + IAA

130 kDa100 kDa

55 kDa

35 kDa

130 kDa100 kDa

55 kDa

35 kDa

α-Spc98 α-Spc98

Fig. 2d

Supplementary Fig. S2a

Supplementary Fig. S2f

Figure S8 Full scans of blots. The background on the α-Spc98 blot from Fig. 2a is due to residual α-Tub4 staining after stripping.

© 2013 Macmillan Publishers Limited. All rights reserved.