Supplementary table 1

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1 Supplementary table 1 S. pombe strain list Fig. 1A JX38 h + ade6-m216 nda3-km311 PX476 PW775 PX545 PX546 h- ade6-m216 sgo2::ura4 + nda3-km311 h 9 mad2::ura4 + nda3-km311 h + ade6-m21 nda3-km311 rad21 + -GFP<<kan r h + ade6-m21 sgo2::ura4 + nda3-km311 rad21 + -GFP<<kan r 1 PZ631 h + leu1 cut9-665 cen2-gfp<<kan r <<ura4 + PT582 h + leu1 sgo2::kan r cut9-665 cen2-gfp<<kan r <<ura4 + PT583 h + leu1 swi6::kan r cut9-665 cen2-gfp<<kan r <<ura4 + Fig. 2A PX543 h 9 leu1 nda3-km311 cen2-gfp<<kan r <<ura4 + PX544 h 9 leu1 sgo2::kan r nda3-km311 cen2-gfp<<kan r <<ura4 + 2 PW373 h 9 ade6 leu1 psc3-1t<<kan r cut2 + -GFP<<kan r sad1 + -GFP<<kan r PW376 PW378 h 9 ade6 leu1 mad2::ura4 + psc3-1t<<kan r cut2 + -GFP<<kan r sad1 + -GFP<<kan r h 9 ade6 leu1 sgo2::ura4 + psc3-1t<<kan r cut2 + -GFP<<kan r sad1 + -GFP<<kan r 2C,D PT151 h- leu1 mad2 + -mcherry<<kan r cut2 + -GFP<<kan r sad1 + -GFP<<kan r PT194 PT196 h 9 leu1 psc3-1t<<kan r mad2 + -mcherry<<kan r cut2 + -GFP<<kan r sad1 + -GFP<<kan r h 9 leu1 sgo2::ura4 + psc3-1t<<kan r mad2 + -mcherry<<kan r cut2 + -GFP<<kan r sad1 + -GFP<<kan r Fig. 3A PT585 h- ade6 leu1 sgo2 + -mcherry<<kan r ark1 + -GFP<<kan r prep81(cfp-atb2 + ) 3 PW776 h 9 ade6-m216 leu1 ark1 + -GFP<<kan r prep81(cfp-atb2 + ) PX481 h- ade6-m216 leu1 sgo2::ura4 + ark1 + -GFP<<kan r prep81(cfp-atb2 + ) 3C PX827 h- ade6-m216 nda3-km311 ark1 + -GFP<<kan r PW1 JX37 h- ade6-m216 nda3-km311 sgo2::ura4 + ark1 + -GFP<<kan r h- ade6-m216 nda3-km311 3D PT139 h- leu1 sgo2 + -mcherry-kan r cut2 + -GFP<<kan r sad1 + -GFP<<kan r PT19 h- leu1 ark1-t7<<kan r sgo2 + -mcherry<<kan r cut2 + -GFP<<kan r sad1 + -GFP<<kan r 3E PX365 h 9 ade6-m216 leu1 sgo2 + -GFP<<kan r prep81(cfp-atb2 + ) PW723 h 9 leu1 bir1-t1<< kan r sgo2 + -GFP<<kan r prep81(cfp-atb2 + ) PX419 PW883 JX37 h + nda3-km311 sgo2 + -GFP<<kan r h- nda3-km311 bir1-t1<< kan r sgo2 + -GFP<<kan r h- ade6-m216 nda3-km311 1

2 Fig 4A,,D,E JY45 h 9 ade6-m216 leu1 PY615 h 9 ade6-m21 leu1 ura4-d18 swi6::ura4 + PZ865 h 9 ade6-m21 leu1 ura4-d18 sgo2::ura4 + PW54 PZ917 PV35 PV269 h 9 ade6-m216 leu1 bir1-t1<< kan r h 9 ade6-m21 leu1 sgo2::ura4 + swi6::kan r h 9 ade6-m216 leu1 ark1-t8-gfp<< kan r h- leu1 pic1-t269<< hyg r PW17 h- ade6 leu1 ark1-t8-gfp<< kan r swi6::ura4 + PV269 h- bir1-t1<< kan r swi6::ura4 + PR62 h- leu1 pic1-t269<< hyg r swi6::ura4 + PT63 PT64 PR65 h 9 ade6-m21 leu1 sgo2::ura4 + swi6::kan r lys1:: P adh81-bir1 + -CFP-2CD<<hyg r h 9 ade6-m21 leu1 sgo2::ura4 + swi6::kan r lys1:: P nmt1<<hyg r h 9 ade6-m21 leu1 sgo2::ura4 + swi6::kan r lys1:: P adh81-bir1 + -3pk<<hyg r 4C PT621 h- cnp3 + -tdtomato<<kan r lys1::p adh81-bir1 + -CFP-2CD<<hyg r PT613 h- sgo2::ura4 + cnp3 + -tdtomato<<kan r lys1::p adh81-bir1 + -CFP-2CD<<hyg r PR84 h 9 leu1 cnp3 + -tdtomato<<kan r lys1::p adh81-bir1 + -CFP-2CD<<hyg r prep81(gfp-atb2 + ) PR85 h 9 leu1 sgo2::ura4 + cnp3 + -tdtomato<<kan r lys1::p adh81-bir1 + -CFP-2CD<<hyg r prep81(gfp-atb2 + ) Fig. 5A PX993 h 9 leu1 cut2 + -GFP<<kan r sad1 + -GFP<<kan r PW37 PW359 PW375 h 9 leu1 rec12-15::leu2 + cut2 + -GFP<<kan r sad1 + -GFP<<kan r h 9 leu1 rec12-15::leu2 + mad2::ura4 + cut2 + -GFP<<kan r sad1 + -GFP<<kan r h 9 leu1 rec12-15::leu2 + sgo2::kan r cut2 + -GFP<<kan r sad1 + -GFP<<kan r 5 PT416 h 9 ade6 leu1 cen2-gfp<<kan r <<ura4 + PT625 PT626 PR66 h 9 ade6-m21 leu1 ura4-d18 sgo2::ura4 + lys1::p adh41-bir1 + -3pk<<hyg r cen2-gfp<<kan r <<ura4 h 9 ade6-m21 leu1 ura4-d18 sgo2::ura4 + lys1:: P nmt1<<hyg r cen2-gfp<<kan r <<ura4 h 9 leu1 bir1-t1<< kan r cen2-gfp<<kan r <<ura4 5C PV488 h 9 leu1 P rad21-slp1 + <<kan r P rad21-cut23 + <<kan r ark1 + -GFP<<kan r prep81(cfp-atb2 + ) PV486 h 9 leu1 sgo1::ura4 + P rad21-slp1 + <<kan r P rad21-cut23 + <<kan r ark1 + -GFP<<kan r prep81(cfp-atb2 + ) PV489 h 9 leu1 sgo2::ura4 + P rad21-slp1 + <<kan r P rad21-cut23 + <<kan r ark1 + -GFP<<kan r prep81(cfp-atb2 + ) 5D PV362 h 9 ade6-m216 leu1 P rad21-slp1 + <<kan r P rad21-cut23 + <<kan r par1 + -GFP<<hyg r prep81(cfp-atb2 + ) PV363 h 9 ade6 leu1 sgo1::ura4 + P rad21-slp1 + <<kan r P rad21-cut23 + <<kan r par1 + -GFP<<hyg r prep81(cfp-atb2 + ) 2

3 PV474 h 9 ade6-m216 leu1 sgo2::nat r P rad21-slp1 + <<kan r P rad21-cut23 + <<kan r par1 + -GFP<<hyg r prep81(cfp-atb2 + ) Fig. 6A PT18 h- ade6 nda3-km311 sgo myc<<kan r ark1 + -GFP<<kan r lys1::p adh81-pic1 + -3HA<<hyg r 6D PT19 h 9 ade6-m216 leu1 sgo2 + -GFP<<kan r prep41 PT2 PT3 h 9 ade6-m216 leu1 sgo2 + -GFP<<kan r prep41(bir1-δc) h 9 ade6-m216 leu1 sgo myc<<kan r prep41(bir1- C) 6E PT1 h 9 ade6-m216 leu1 prep41 PT11 PT16 PT17 h 9 ade6-m216 leu1 prep41(bir1-δc) h 9 ade6-m21 leu1 ura4-d18 sgo2::ura4 + prep41 h 9 ade6-m21 leu1 ura4-d18 sgo2::ura4 + prep41(bir1-δc) Sup. Fig. 1 PX746 h- leu1 ade6-m216 plo1 + -GFP<<kan r SH211 h + leu1 ade6-m216 nda3-km311 plo1 + -GFP<<kan r SH51 h 9 leu1 ade6 sgo2::kan r plo1 + -GFP<<kan r SH515 SH516 h + leu1 ade6-m216 nda3-km311 mad2::ura4 + plo1 + -GFP<<kan r h + leu1 ade6-m21 nda3-km311 sgo2::ura4 + plo1 + -GFP<<kan r SH521 h- plo1 + -GFP<<kan r bir1-t1<< kan r SH526 h + leu1 nda3-km311 bir1-t1<< kan r plo1 + -GFP<<kan r Sup. Fig. 2A PX543 h 9 leu1 nda3-km311 cen2-gfp<<kan r <<ura4 + PX544 h 9 leu1 sgo2::kan r nda3-km311 cen2-gfp<<kan r <<ura4 + PT68 h- ade6-m216 leu1 bir1-t1<< kan r nda3-km311 cen2-gfp<<kan r <<ura4 + 2 PW373 h 9 ade6 leu1 psc3-1t<<kan r cut2 + -GFP<<kan r sad1 + -GFP<<kan r PW378 PW423 PV391 h 9 ade6 leu1 sgo2::ura4 + psc3-1t<<kan r cut2 + -GFP<<kan r sad1 + -GFP<<kan r r h 9 ade6 leu1 ark1-t7-gfp<<kan r psc3-1t<<kan r cut2 + -GFP<<kan r sad1 + -GFP<<kan r h 9 leu1 bir1-t1<< kan r psc3-1t<<kan r cut2 + -GFP<<kan r sad1 + -GFP<<kan r Sup. Fig. 3A PT614 h 9 leu1 sgo2 + -mcherry<<kan r taz1 + -GFP<<kan r prep81(cfp-atb2 + ) 3 PT611 h 9 leu1 sgo2 + -GFP<<kan r cnp3 + -tdtomato<<kan r prep81(cfp-atb2 + ) Sup. Fig. 4A PT139 h- leu1 sgo2 + -mcherry<<kan r cut2 + -GFP<<kan r sad1 + -GFP<<kan r PR73 h- leu1 ark1-t8-gfp<< kan r sgo2 + -mcherry<<kan r cut2 + -GFP<<kan r sad1 + -GFP<<kan r 4 PX365 h 9 ade6-m216 leu1 sgo2 + -GFP<<kan r prep81(cfp-atb2 + ) PR67 h 9 leu1 bir1(cut17)-275 sgo2 + -GFP<<kan r prep81(cfp-atb2 + ) 4C PT139 h- leu1 sgo2 + -mcherry<<kan r cut2 + -GFP<<kan r sad1 + -GFP<<kan r PT624 h- leu1 pic1-t269<< hyg r sgo2 + -mcherry<<kan r cut2 + -GFP<<kan r sad1 + -GFP<<kan r 3

4 4D PW776 h 9 ade6-m216 leu1 ark1 + -GFP<<kan r prep81(cfp-atb2 + ) PT3 h 9 leu1 bir1-t1<< kan r ark1 + -GFP<<kan r prep81(cfp-atb2 + ) Sup. Fig. 5A JY45 h 9 ade6-m216 leu1 PZ865 h 9 ade6-m21 leu1 ura4-d18 sgo2::ura4 + PY532 h - ade6-m21 psc3-1t<<kan r PW359 h 9 ade6-m21 leu1 psc3-1t<<kan r sgo2::ura4 + 5 PV388 h 9 ade6-m216 leu1 ark1 + -GFP<<kan r prep41(cfp-atb2 + ) PW9 h - ade6-m216 leu1 swi6:: ura4 + ark1 + -GFP<<kan r prep41(cfp-atb2 + ) Sup. Fig. 6A JY45 h 9 ade6-m216 leu1 PT63 PT64 PR65 PR82 h 9 ade6-m21 leu1 sgo2::ura4 + swi6::kan r lys1:: P adh81-bir1 + -CFP-2CD<<hyg r h 9 ade6-m21 leu1 sgo2::ura4 + swi6::kan r lys1:: P nmt1<<hyg r h 9 ade6-m21 leu1 sgo2::ura4 + swi6::kan r lys1:: P adh81-bir1 + -3pk<<hyg r h 9 ade6-m21 leu1 sgo2::ura4 + swi6::kan r lys1:: P adh81-cfp-2cd <<hyg r 6 PT571 h 9 ade6-m216 leu1 lys1:: P adh81-bir1 + -3pk<<hyg r PR59 PT12 PT572 PR6 PT123 h 9 ade6-m216 leu1 lys1:: P adh81-bir1 + -CFP-2CD<<hyg r h 9 ade6-m216 leu1 lys1:: P nmt1<<hyg r h 9 ade6-m21 leu1 ura4-d18 sgo2::ura4 + lys1:: P adh81-bir1 + -CFP-2CD<<hyg h 9 ade6-m21 leu1 ura4-d18 sgo2::ura4 + lys1:: P adh81-bir1 + -3pk<<hyg r h 9 ade6-m21 leu1 ura4-d18 sgo2::ura4 + lys1:: P nmt1<<hyg r Sup. Fig. 7A S11 h 9 leu1 ade6 (ura4-d18) sgo2::kan r ura4 + <<GFP-bir1 + S13 h 9 leu1 (ade6-m216) ura4-d18 ura4 + <<GFP-bir1 + 7 PV482 h 9 leu1 P rad21-slp1 + <<kan r P rad21-cut23 + <<kan r sgo1 + -FLAG-GFP prep81(cfp-atb2 + ) PV483 h 9 leu1 P rad21-slp1 + <<kan r P rad21-cut23 + <<kan r P rad21- bir1-t1<<hyg r <<kan r sgo1 + -FLAG-GFP prep81(cfp-atb2 + ) PV484 h 9 leu1 P rad21-slp1 + <<kan r P rad21-cut23 + <<kan r sgo2 + - GFP<<kan r prep81(cfp-atb2 + ) PV485 h 9 leu1 P rad21-slp1 + <<kan r P rad21-cut23 + <<kan r P rad21- bir1-t1<<hyg r <<kan r sgo2 + - GFP<<kan r prep81(cfp-atb2 + ) Sup. Fig. 1 JY45 h 9 ade6-m216 leu1 PZ865 h 9 ade6-m21 leu1 ura4-d18 sgo2::ura4 + PV323 PV324 h 9 ade6 leu1 ura4-d18 sgo2(53sa) h 9 ade6 leu1 ura4-d18 sgo2(53sa 542TA) 4

5 7 hr release from HU DIC Plo1-GFP Plo1-GFP positive cells (%) time after release from HU [h] nda3 nda3 nda3 mad2δ Plo1-GFP positive cells (%) time after release from HU [h] bir1-t1 nda3 bir1-t1 Supplementary Figure 1 Sgo2 is not required for the mitotic checkpoint sensing the absence of attachment. The indicated strains were arrested at G1/S phase by HU at the permissive temperature for nda3- KM311, and released from the arrest to the restrictive temperature (18 C). Samples were collected up to 9 hr after release and fixed with paraformaldehyde. The number of cells exhibiting Plo1-GFP signal was determined as an indicator of mitosis (n > 2 per time point). Example pictures are shown at the top. Note the presence of two plo1-gfp dots, indicating separated SPs, in nda3 + cells. In contrast, nda3-km311 cells show only one dot, since microtubules do not form and the SPs fail to separate.

6 (%) cen2-gfp non-disjunction + bir1-t1 cen2-gfp DNA DNA bir1-t1 nda3-km311 (%) Prometaphase cells (SP duplicated, securin positive) psc3-1t 1 psc3-1t ark1-t7 5 psc3-1t bir1-t1 psc3-1t (min) HU release Supplementary Figure 2 S.pombe Aurora passenger complex is required for preventing cosegregation and activating the spindle checkpoint. (A) Quantification of cen2-gfp non-disjunction in sgo2 + nda3-km311, nda3-km311 and bir1-t1 nda3-km311 cells after block and release to 28 C (n>2). Examples of bir1-t1 cells are shown (arrowhead indicates non-disjunction of cen2-gfp). () The indicated cells were arrested at G1/S phase by adding HU at 25 C and released to 36 C. Prometaphase cells (SP duplicated and securin/cut2-gfp positive) were counted at each time point for each strain (n>15). Note that like cells both ark1-t7 and birt1-t1 cells impair the activation of the spindle checkpoint sensing the loss of tension.

7 Inter. Meta. Inter. Meta. Sgo2-mCh Taz1-GFP Cnp3-tdT Taz1-GFP Cnp3-tdT Sgo2-mCh Sgo2-mCh CFP-Tubulin CFP-Tubulin Supplementary Figure 3 Localization of Sgo2 changes between interphase and mitosis. Relative position of Sgo2-mCherry and telomeres (Taz1-GFP) (A) or and centromeres (Cnp3-tdTomato) () were examined at interphase and metaphase. Microtubules were visualized by expressing CFP-atb2 +. Note that in interphase Sgo2 always co-localizes with one to three of the Taz1 dots but not all of them, whereas metaphase Sgo2 does not co-localize with Taz1 at all.

8 Sgo2-mCh DNA Sad1-GFP Cut2-GFP Sgo2-mCh (AU) 12 8 Sgo2-mCh fluorescence CFP-Tubulin DNA (AU) 12 8 fluorescence ark1-t8 36 C 4 ark1-t8 bir C 4 bir1-275 C Sgo2-mCh DNA Sad1-GFP Cut2-GFP Sgo2-mCh D Ark1-GFP CFP-Tubulin DNA Ark1-GFP pic1-t C bir1-t1 metaphase pic1-t C bir1-t1 (AU) Sgo2-mCh fluorescence pic1-t269 (AU) C 36 C 8 4 pic1-t269 (AU) anaphase Ark1-GFP fluorescence metaphase (AU) 12 anaphase 8 4 bir1-t1 bir1-t1 Supplementary Figure 4 Localization of Ark1 or Sgo2 in passenger complex mutants. (A) Sgo2-mCherry was detected at metaphase in wild-type and ark1-t8 cells after shift to 36 C for 3 hr. Spindles or SPs were visualized by expressing Cut2-GFP and Sad1-GFP. DNA was stained by DAPI. Signal intensity of Sgo2-mCherry was measured. Error bars represent s.e.m. (n>14). () was detected at metaphase in wild-type and bir1-275 cells expressing CFP-atb2 + and cultured at 25 C. Signal intensity of was measured. Error bars represent s.e.m. (n=1). (C) Wild-type and pic1- T269 cells were arrested by HU for 3 hr at 25 C and 1 hr at 36 C, then released at 36 C for 1hr and detected for Sgo2-mCherry, Cut2-GFP (spindle and nuclear signals), and Sad1-GFP (SP signals). DNA was stained by DAPI. Signal intensity of Sgo2-mCherry was measured. Error bars represent s.e.m. (n>4, 25 C; n=3, 36 C). (D) Centromeric localization of Ark1-GFP is reduced in bir1-t1 cells cultured at 25 C. Spindles were visualized by expressing CFP-atb2 + and DNA was stained by Hoechst Signal intensity of Ark1-GFP was measured. Error bars represent s.e.m. (n=15, metaphase; n=4, anaphase).

9 ir1 DNA Tubulin psc3-1t psc3-1t Signal intensity of centromeric ir1 (AU) * * ir1 4 2 C swi6δ swi6δ Ark1-GFP CFP-Tubulin DNA Ark1-GFP Fluorescence Ark1-GFP (AU) (Metaphase) n= 32, 26, 28, 1 swi6δ psc3-1t psc3-1t Supplementary Figure 5 ir1 localization is more extensively impaired in the psc3-1t double mutant than in the single mutant. (A) ir1 was monitored at prometaphase in wild-type,, psc3-1t and psc3-1t cells. Cells were arrested by HU at 25 C for 3 hr and shifted to 36 C for an additional 1 hr, then released (HU washed out) to 36 C for 6 min ( and ) or 9 min (psc3-1t and psc3-1t), fixed with and stained for tubulin and ir1. Representative images from the indicated strains with short spindles (prometaphase or metaphase) are shown. The strongest ir1 signals along the metaphase spindle (centromeric signals) are marked by an arrowhead. Note that displaced ir1 in the mutant cells is dispersed into nucleolus. () Immunofluorescence of centromeric ir1 signals was quantified in promertaphase cells. For this, the strength of centromeric ir1 signals was subtracted by the average signal strength of the whole nucleus (including nucleolus). Error bars represent s.e.m. All difference among these mutants are statistically significant (asterisk, p<.5) except between and psc3-1t (p=.1). The partial reduction of ir1 in psc3-1t cells may explain why the spindle checkpoint was not fully activated in all cells (~4% of psc3-1t cells delayed the onset of anaphase, Figure 2D). Nevertheless, these results argue that Sgo2 dependent localization of the passenger complex plays a crucial role in activating the spindle checkpoint at least in these 4% cells. (C) Ark1-GFP was detected at metaphase and anaphase in wild-type and swi6δ cells. Spindles were visualized by expressing CFP-atb2 + and DNA was stained by Hoechst Signal intensity of Ark1-GFP at metaphase was measured. Error bars represent s.e.m. (n>18).

10 - bir1 + -CD swi6δ bir1 + CD bir1 + -CD bir1 + + bir1 + -CD bir1 + + TZ 12.5µg/ml Supplementary Figure 6 Suppression of by ir1-cd expression. (A) The indicated strains were streaked onto a YEA plate and incubated at 3 C. Note that the expression of ir1-cd, but neither ir1 nor CD alone, suppressed the groh defect of swi6δ. () Serial dilutions of the indicated strains were spotted onto a YEA plate or a YEA plate containing 12.5µg ml -1 TZ and incubated at 3 C. Note that the TZ-sensitivity of cells was partly suppressed by ir1-cd expression as well as by the ir1 expression. However, since ir1-cd causes some deleterious effects on the TZ-sensitivity even of wild type cells, the suppression effect of ir1-cd in cells could be underestimated relative to ir1 in this assay. One reason for this could be that the constitutive, rather than prometaphase-specific, centromeric localization of ir1 may reduce functionality of ir1-cd.

11 Meta-I Ana-I Meta-II Ana-II GFP-ir1 GFP-ir1 max. intensity (AU) Meta I Ana I Meta II Ana II Fluorescence (AU) Sgo1-GFP CFP-tubulin Intensity of Sgo1-GFP Prad21 -bir1-t Intensity of Sgo1 -GFP Fluorescence (AU) CFP-tubulin Prad21 -bir1-t1 Sgo2 -GFP Supplementary Figure 7 Analyses in meiosis. (A) GFP-ir1 intensity in meiosis was measured in a sgo2 + and a strain. To assess specifically the intensity on centromeres, an area was placed over the middle of the spindle (not including the SPs), and the maximum intensity in this area was recorded. For anaphase cells, the area was placed on the strongest visible signal in the middle of the spindle. Each dot in the graph represents the measurement from one spindle and the bars indicate the average maximum intensity for each strain and meiotic stage. Exemplary pictures are shown on top. () To stringently examine the requirement of ir1 for Sgo1 or Sgo2 localization, we repressed bir1-t1 expression (which is transcriptionally enhanced in meiosis I) by replacing the promoter with the mitosis-specific promoter of rad21+ (Kitajima 24). Indeed meiotic defects were enhanced in the resultant Prad21-bir1-T1 cells compared to bir1-t1 cells (data not shown). Cells of wild-type and Prad21-bir1-T1 were arrested at metaphase I by repressing APC activation (slp1 + and cut23 + expression) and observed microscopically. The intensity of Ark1- GFP at metaphase I was measured. Error bars represent s.e.m. (n>3). Representative images of each strain are shown at the bottom.

12 GST-ir1 fragments 5% input His-Sgo2-N His-Sgo2-C ir IR IR NLS Supplementary Figure 8 Fine mapping of the binding region of ir1 to Sgo2. The indicated GST-ir1 fragments, His-Sgo2-N (a.a.1 to 333) and His-Sgo2-C (a.a.334 to 647) recombinant proteins were expressed and purified from E. coli. His-Sgo2-N and His-Sgo2-C were incubated with each GST protein bound to glutathione sepharose beads. ead-bound fractions were separated by SDS-PAGE and analyzed by Western blotting using antibodies against His. Schematic drawing of the fragments of ir1 used in this study is shown. lue bars, but not grey bars, indicate an ability to bind Sgo2. Light blue regions mark probable binding sites on ir1. The N-terminal region of Sgo2 interacts with ir1.

13 hsgo1 Aurora ACA DNA hsgo1 Aurora ACA control Survivin RNAi hsgo1 RNAi Aurora centromeric localization Ratio control Survivin RNAi hsgo1 Survivin Aurora ACA DNA Survivin Aurora ACA control Survivin RNAi Aurora RNAi hsgo1 RNAi Survivin centromeric localization 5 Ratio control Survivin Aurora RNAi hsgo1 Supplementary Figure 9 hsgo1 is not required for localization of the passenger complex. (A) HeLa cells were mock transfected or transfected with sirnas against Survivin or hsgo1, and stained with anti-hsgo1, anti-aurora and ACA. DNA was counterstained with Hoechst The scale bar represents 1µm. () HeLa cells after RNAi against the indicated proteins were stained with anti-survivin (green), anti-aurora (red) and ACA (blue). DNA was counterstained with Hoechst The scale bar represents 1µm. (A, ) Quantification of centromeric signals of Aurora and Survivin are shown on the right. For each cell, the fluorescent intensities of Aurora or Survivin at 1 centromeres were related to the intensities of the corresponding ACA signals. The means and standard deviations of 1 mitotic cells are shown.

14 N C CN CC Sgo2 coiled-coil basic His-Sgo2 GST-Sgo2 N C CN CC His-Sgo2 GST-Sgo2 N C CN CC C + GST-Sgo2- CC 52 SA 53 SA TA TA SA SA GST -Ark1 D sgo2(53sa) + TZ 1 µg/ml sgo2(53sa 542TA) Supplementary Figure 1 Phosphorylation of Sgo2 by Ark1 at residues Ser53 and Thr542 is dispensable for the function of Sgo2. (A) Schematic drawing of the Sgo2 fragments used in the in vitro phosphorylation assay. () The indicated His/GST-Sgo2 fragments, GST-Ark1 and GST-Pic1-C (a.a.51 to 118) proteins were expressed and purified from E. coli. GST/His-Sgo2 fragments were incubated with GST-Ark1 and GST- Pic1-C (this peptide potentiates Ark1 activity in vitro) in the presence of [γ- 32 P] ATP for 3 min at 3 C. Incorporation of the radioactive phosphate group was visualized by autoradiography (right) and protein loading analyzed by Coomassie blue (left). His-Sgo2-C and GST-Sgo2-CC were efficiently phosphorylated by Ark1 in vitro. (C) The indicated mutants of GST-Sgo2-CC were similarly incubated and incorporation of the radioactive phosphate group was visualized by autoradiography (bottom). Protein loading was analyzed by Coomassie blue (top). Phosphorylation of GST-Sgo2-CC by Ark1 was largely reduced by the 53SA mutation, in which Serine 53 was replaced by Alanine, and almost completely abolished by further mutation of 542TA. (D) Chromosomal sgo2 + was replaced by the mutant alleles sgo2(53sa) or sgo2(53sa 542TA). Serial dilutions of the indicated strains were spotted onto a YEA plate or a YEA plate containing 1µg ml -1 TZ and incubated at 3 C. Neither point mutotion caused any defects in the spindle checkpoint sensing loss of tension (data not shown) or resistance to TZ.

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