Bub1, Sgo1 and Mps1 mediate a distinct pathway for chromosome bi-orientation in budding yeast

Supplementary figure legend Supplementary Fig. 1

The localization of Sgo1-GFP to pre-anaphase spindles is abolished in cells lacking the kinase domain of Bub1. An Spc29-RFP fusion construct was used to visualize the spindle poles. The series of images in the lower panel were captured by live-cell imaging and show the spindle recruitment and subsequent removal of Sgo1-GFP over the course of a single mitosis.

Supplementary Fig. 2

A.IPL1 over-expression cannot rescue the growth defect of sgo1 or bub1 tetraploids. B. Alleles of BIR1 that code for a mutant protein unable to interact with Sli15 (BIR1⁹⁰¹) or unable to interact with both Sli15 and Ipl1 (BIR1^931,935) cannot suppress the growth defect of tetraploids lacking either BUB1 or SGO1.

Supplementary Fig. 3

A. Tetraploid cells lacking SGO1 do not exhibit defects in sister-chromatid cohesion during mitosis, in agreement with previous results in haploid cells. We used tetraploid cells expressing TetR-GFP and marked a single centromere by introducing a TetO array at chromosome IV. Cells were arrested in G1 and in metaphase (M) and scored for the number of distinct GFP foci. B. A mutant Sgo1 protein containing isoleucine instead of arginine in position 51 no longer interacts with the phosphatase PP2A, but it can still partially restore the viability of the sgo1 tetraploids.

Supplementary Fig. 4

A. The benomyl-sensitivity of cells lacking Cdc55 (a regulatory subunit of the phosphatase PP2A) cannot be suppressed by the over-expression of BIR1 or SLI15. B.Cdc55 is not required for the ability of over- expressed SGO1 to suppress the benomyl sensitivity of haploid bub1 cells.

Supplementary Fig. 5

A . Over-expression of MPS1 on a 2-micron plasmid partially rescues the growth defect of sgo1 tetraploids. B. Over-expression of MPS1 on a 2-micron plasmid partially rescues the sensitivity of sgo1 haploid to benomyl. C. Over-expression of MPS1 rescues the benomyl sensitivity of the checkpoint- competent haploid strain bub1K (lacking the kinase domain) but not the checkpoint-deficient strain bub1.

Sgo1-GFP SPC29-RFP

BUB1 Empty vector

BIR1 BIR1

BIR1 901

931,935

bub1 4N

Empty vector

BIR1 BIR1

BIR1 901

931,935 SGO1

sgo1 4N

A

Supplementary Figure 2, Storchova, Becker et al.

bub1 4N

BUB1 Empty vector (aurora B)IPL1

SGO1

sgo1 4N

vector (aurora B)IPL1 Empty

Δ

37 C ^o

37 C ^o

B

DIC CENIV SPB Merged

5 10 15 20 25

G1 M

% c ells with 2 dots

M

B

wt sgo1

wt sgo1

sgo1 4N

sgo1 4N

vector SGO1

sgo1N51I

benomyl 9

benomyl 15

CDC55

BIR1 (su rvivin)

SLI15 (INCENP)

cdc55 Δ ^1N

vector

bub1

bub1

SGO1

bub1

Kcdc55

bub1

cdc55

bub1 cdc55

SGO1

SGO1 bub1

Kcdc55

bub1

BUB1

benomyl 5

benomyl 10

control

A

B

Supplementary Figure 4, Storchova, Becker et al.

24 C

24 C

SGO1

BIR1 SLI15 Empty vector

MPS1

sgo1 4NΔ

37 C

BUB1

bub1ΔK

MPS1

MPS1 SGO1 Empty vector

Empty vector SGO1 bub1Δ

bub1 1N

24 C

C

sgo1 1NΔ

10 g/ml benomyl

control

MPS1

Empty vector SGO1

24 C