Supplementary Information

Supplementary notes

Supplementary note 1

okp1-52 and ame1-2 kinetochores fail to achieve bipolarity but remain SAC competent

180 min after the release from G1 at 37oC the mutants had distributed their DNA but had segregated both CEN5 chromatids together to either mother or bud (Fig. S1A). Thus the mutants failed to achieve bipolar kinetochore attachment and displayed monopolar chromosome segregation. Despite this, okp1-52 and ame1-2 kinetochores maintained the capability to activate the SAC. When released from G1 at 37oC into nocodazole, both mutants arrested at G2/M due to a non-satisfied SAC. They maintained maximal Pds1 levels for several hours, indistinguishable to wild type cells (Fig. S1B), arrested with a 2N DNA content (whereas ∆mad2 cells re-replicate their DNA to 4N) (Fig. S1D) and localized Mad2 and Bub1 to the kinetochores (Fig. S1E). Thus, (at least some) okp1-52 and ame1-2 kinetochores are able to activate the SAC upon MT detachment. Consequently also the failure to establish bipolarity in the mutants might prolong SAC activation. Indeed, when released from G1 in the absence of nocodazole, both mutants maintained maximal Pds1 levels about 40min longer than wild type cells (Fig. S1C). Taken together, this data indicates that okp1-52 and ame1-2 cells fail to achieve bipolar kinetochore attachment and separate their spindle poles while SAC is partially active.

Supplementary note 2

ame1-2 cells don’t release Cdc14 from the nucleolus

When cells were released from a G1 arrest and Cdc14 localization was analyzed by time lapse microscopy (Fig. S2A-B; Video S1 and S2) wild type cells released and re-sequestered Cdc14 from/into the nucleolus in a time window of 90-150 min after the release. ame1-2 cells failed to do so, even when analyzed up to 240 min (not shown) after the G1 release. Thus ame1-2 and possibly okp1-52 cells separate spindle poles in the absence of Cdc14. Since Cdc14 is required for the formation of stable anaphase spindles, the lack of Cdc14 during spindle pole separation is likely to contribute to the okp1-52 and ame1-2 spindle defects.

Supplementary note 3

SAC inactivation or Cdc14 overexpression does not rescue the okp1-52 and ame1-2 spindle defect

We wondered whether lacking Cdc14 activity is the sole reason for the ame1-2 and okp1-52 spindle defect. If a partial active SAC prevents Cdc14 activation (see above) and Cdc14 is the sole cause for the defect, then the elimination of SAC signaling should rescue spindle formation in the mutants. Disrupting MAD2 in the mutants resulted in very sick cells unsuitable for analysis. We therefore depleted Mad2 during a G1 arrest. Efficient depletion was shown by the fact that the cells revealed no Mad2 by western analysis (not shown). Furthermore the Mad2 depleted cells re-replicated their DNA in the presence of nocodazole (FACS; data not shown) indicative of absent SAC function. When released from G1 the Mad2 depleted mutants still displayed severe spindle defects (Fig. S3A). Thus eliminating SAC did not rescue the okp1-52 and ame1-2 spindle defect.

A second way to rescue spindle defects that are due to a lack of Cdc14 is Cdc14 overexpression. This has been best demonstrated for cells carrying a TEV cleavage site in the cohesion Scc1 (strain Y1539: MET11-CDC20, SCC1TEV, GAL-TEV, GAL-CDC14; (Higuchi and Uhlmann 2005)). The expression of TEV protease in these cells initiates spindle pole separation. If Esp1 and Cdc14 are kept inactive by Cdc20 depletion during this pole separation, the cells show spindle defects. However, these defects can be rescued if Cdc14 is overexpressed in parallel to cohesion cleavage by TEV (Higuchi and Uhlmann 2005). When we integrated the ame1-2 mutation into Y1539 and repeated the above experiment the spindle defect was not rescued (Fig. S3B). Furthermore, we were also unable to rescue spindle formation by Cdc14 overexpression in okp1-52 cells (Fig. S3C). Taken together this data shows that the spindle defect in ame1-2 and okp1-52 persists even when Cdc14 activity is present during spindle pole separation. Thus there has to be an additional explanation besides the lack of Cdc14 for the okp1-52 and ame1-2 spindle defect.

Supplementary Methods

Yeast strains and growth conditions

Yeast strains and plasmids are described in Table SI. All strains were derived from YPH499 (Sikorski and Hieter 1989). Epitope tagging and promoter replacement of genes at endogenous loci as well as BUB1 disruption were performed using PCR-based methods (Janke et al. 2004). MAD2 disruption was performed using a disruption cassette from plasmid pJO608. Strains that exhibit CEN5-GFP were produced as described (Scharfenberger et al. 2003). For the construction of pGAL1-Ubiquitin-R-CSE4, pGAL1- Ubiquitin-R-STU1, pGAL1-Ubiquitin-R-MAD2 at the endogenous loci, a PCR-based method with plasmid pBL929 or pJO719 was performed. Construction of pGAL1-Ubiquitin-R-NDC80 was as described (Kemmler et al. 2009). For the construction of pGAL1-FLAG-STU1, pGAL1-FLAG-stu1(∆461-716) and pGAL1-FLAG-stu1(∆887-1513) strains, plasmid pJO1094, pJO1126 and pCF1148 were integrated at the LYS2 locus. The strain YCF1298 (stu1∆1175-1513) was produced by a PCR-based method using plasmid pCF1136 as a template. Temperature sensitive mutants were produced as described (Janke et al. 2002). Integrating plasmid pAW1035 at the AME1 locus of Y1539 produced strain YAW988.

Cultures were grown in 1% yeast extract (Y), 2% peptone (P) and dextrose (D) media or raffinose (Raf) and galactose (Gal) media when necessary. For cell synchronization in G1, exponentially growing ∆sst1 cells were incubated with alpha factor at 200ng/ml for 2-3h and released by washing and re-suspension in fresh medium. Nocodazole treatment was at 15µg/ml. Shut-down of pMET-CDC20 and subsequent induction of pGAL-CDC14 plus pGAL-TEV in strains Y1539 and YAW988 was as described (Higuchi and Uhlmann 2005). For shut-down of pGAL1-Ubiquitin-R-NDC80, pGAL1-Ubiquitin-R-MAD2, pGAL1-Ubiquitin-R-CSE4, pGAL1- Ubiquitin-R-STU1 and pGAL1-CDC20, cells were grown in YP-Raf (2%) medium containing 0.1%, 0.2%, 0.3%, 0,3% and 2% Gal respectively and shifted to YPD at 25ºC. To overexpress Cdc14 in strains YJO1177, YJO1178, YJO1234 and YJO1235, cells were released from a G1 arrest in YP-Raf (2%) and shifted to YP-Gal (2%) about 90 min after the release. Overexpression of Stu1 or mutated Stu1 was induced 1h before a G1 release by adding Gal (2%) to the YP-Raf (2%) medium.

Other methods

Quantification of Pds1 levels and FACS analysis has been described (Kemmler et al. 2009). ChIP was performed as described (Scharfenberger et al. 2003). To quantify the ChIP results for okp1-52 cells (Fig. S6A) the percentage of precipitated CEN3 relative to the input was determined by real-time PCR using the TagMan method (Heid et al. 1996) and normalized to the percentage of precipitated CEN3 in wilds type cells. Anti-Stu1 was produced against N- and C-terminal fragments, anti-Cse4 against an N-terminal fragment and all other antibodies against the full-length protein in rabbits.

Supplementary references

Heid, C.A., Stevens, J., Livak, K.J., and Williams, P.M. 1996. Real time quantitative PCR. Genome Res 6(10): 986-994.

Higuchi, T. and Uhlmann, F. 2005. Stabilization of microtubule dynamics at anaphase onset promotes chromosome segregation. Nature 433(7022): 171-176.

Janke, C., Magiera, M.M., Rathfelder, N., Taxis, C., Reber, S., Maekawa, H., Moreno-Borchart, A., Doenges, G., Schwob, E., Schiebel, E., and Knop, M. 2004. A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21(11): 947-962.

Janke, C., Ortiz, J., Tanaka, T.U., Lechner, J., and and Schiebel, E. 2002. Four new subunits of the Dam1-Duo1 complex reveal novel functions in sister kinetochore biorientation. Embo J 21: 181-193.

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(8): 1099-1110.

Scharfenberger, M., Ortiz, J., Grau, N., Janke, C., Schiebel, E., and Lechner, J. 2003. Nsl1p is essential for the establishment of bipolarity and the localization of the Dam-Duo complex. EMBO J 22(24): 6584-6597.

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(1): 19-27.

Supplementary Videos

Video S1: Cdc14-GFP release from nucleolus. Video of wild type cell shown in Fig. S2A. Speed is 600 times faster than the actual motion.

Video S2: Cdc14-GFP release from nucleolus. Video of ame1-2 cell shown in Fig. S2A. Speed is 600 times faster than the actual motion.

Video S3: Spindle formation (GFP-Tub1). Video of wild type cell shown in Fig. 1C. Speed is 120 times faster than the actual motion.

Video S4: Spindle formation (GFP-Tub1). Video of okp1-52 cell shown in Fig. 1C. Speed is 120 times faster than the actual motion.

Table SI. Yeast strains and plasmids
Name / Genotype / Figure
Yeast strains
YPH499 / MATa ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 (Sikorski and Hieter 1989)
YMS231 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801
YNG307 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52::GFP-TUB1::URA3 his3-∆200 lys2-801 / 1A,C,D, S5
YAW498 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1::ame1-2::LEU2 ura3-52::GFP-TUB1::URA3 his3-∆200 lys2-801 ame1::kanMX6 / 1A,C,D
YAW822 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52::GFP-TUB1::URA3 his3-∆200 lys2-801cdc15-1 / S5
YNG263 / MATa ∆sst1 ade2-101 ∆okp1::TRP1 leu2::okp1-52::LEU2 ura3::GFP-TUB1::URA3 his3 lys2-801ambre / 1A,C,D
YMS679 / MATa ∆sst1 ade2-101 leu2-∆1 ura3-52 his3-∆200 lys2-801ambre trp1::CFP-TUB1::TRP1
AME1-4GFP::kanMX6 / 1B
YMS680 / MATa ∆sst1 ade2-101 leu2-∆1 ura3-52 his3-∆200 lys2-801ambre trp1::CFP-TUB1::TRP1
OKP1-4GFP::kanMX6 / 1B
YMS552 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 801 ∆okp1::TRP1 leu2::okp1-52-LEU2 / S6A
YNG289 / MATa ∆sst1 ura3-52 his3-∆200 lys2-801 ∆okp1::TRP1 leu2::okp1-52- LEU2 ade2::TetR-GFP-ADE2 ura3:CEN5-tetO2x112::URA3 / S1A
YMS331 / MATa ∆sst1 trp1-∆63 leu2-∆1 his3-∆200 lys2-801ade2::tetR-GFP::ADE2 ura3::CEN5-tetO2x112::URA3 / S1A
YAW574 / MATa ∆sst1 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 ame1::kanMX6 ade2::tetR-GFP::ADE2 ura3::CEN5-tetO2x112::URA3 leu2::ame1-2::LEU2 / S1A
YMS299 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 PDS1-9MYC::klTRP1 / S1B,C
YJO371 / MATa ∆sst1 ade2-101 ura3-52 his3-∆200 lys2-801 ∆okp1::TRP1 leu2::okp1-52-LEU2 bar1::HIS3 PDS1::9Myc-kan / S1B,C
YAW523 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801∆ame1::KANMX6
leu2::LEU2-ame1-12 PDS1-9MYC::klTRP1 / S1B,C
YAW666 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 CDC14-GFP::HIS3MX6 / S2A,B
YAW667 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1::ame1-2::LEU2 ura3-52::GFP-TUB1::URA3 his3-∆200 lys2-801 ame1::kanMX6 CDC14-GFP::HIS3MX6 / S2A,B
YNG308 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 his3-∆200 lys2-801 ura3::GFP-TUB1::URA3 cdc20::KanMX6pGAL CDC20-3HA / 1E
YJO914 / MATa ∆sst1 ade2-101 his3-∆200 lys2-801 ∆okp1::TRP1 leu2::okp1-52::LEU2 ura3::GFP-TUB1::URA3 cdc20::HIS3 pGAL CDC20 / 1E
YJO1079 / MATa ∆sst1 ade2-101 lys2-801 ∆okp1::TRP1 leu2::okp1-52::LEU2 STU1-GFP::HIS3MX6 ura3::CFP-TUB1::URA3 mad2:KANMX6 pGAL-UbiR-MAD2 / S3A
YJO1080 / MATa ∆sst1 ade2-101 lys2-801 ∆ame1::kanMX6 leu2::LEU2-ame1-2 STU1-GFP::HIS3MX6 ura3::CFP-TUB1:URA3 mad2:KLTRP1 pGAL-UbiR-MAD2 / S3A
YAW988 / MATa scc1(TEV268)-3HA::LEU(1step) Gal-NLS-myc9-TEVprotease-NSL2::TRP1(x10) MET-HA3-Cdc20::TRP GAL-CDC14-pk::URA his3::TUB1p-yEGFP-TUB1::HIS3 ade2::ame1-2::ADE2 / S3B
Y1539 / MATa scc1(TEV268)-3HA::LEU(1step) Gal-NLS-myc9-TEVprotease-NSL2::TRP1(x10) MET-HA3-Cdc20::TRP GAL-CDC14-pk::URA his3::TUB1p-yEGFP-TUB1::HIS3 (Higuchi and Uhlmann 2005) / S3B
YJO1177 / MATa ∆sst1 trp1-∆63 leu2-∆1 lys2-801 ura3::CFP-TUB1::URA3 STU1-GFP::HIS3MX6 ade2::pGAL-CDC14-FLAG::ADE2 / S3C
YJO1178 / MATa ∆sst1 trp1-∆63 leu2-∆1 lys2-801 ura3::CFP-TUB1::URA3 STU1-GFP::HIS3MX6 ade2::pGAL-CDC14-FLAG::ADE2 ∆okp1::TRP1 leu2::okp1-52::LEU2 / S3C
YJO1091 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 ura3::CFP-TUB1::URA3 STU1-GFP::HIS3MX6 / 2A
YJO1117 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 STU1-GFP::HIS3MX6 AME1-GFP::kanMX6 SPC72-3mCherry::hph-NT1 / 2B, 3B, 7B
YJO1150 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 AME1-GFP::kanMX6
STU1-3mCherry::hphNT1 ura3::CFP-TUB1::URA3 / 2C, 3C, 4A
YJO1355 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 ∆okp1::TRP1 leu2::okp1-52::LEU2 ura3::GFP-TUB1::URA3 STU1-3mCherry::hphNT1 NDC10-eCFP::HIS3MX6 / 2D
YJO1238 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 ∆kip3::HIS3MX6 ura3::GFP-TUB1:URA3 ASE1-3mcherry:hphNT1 / 2E,F
YJO1192 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 ∆okp1::TRP1 leu2::okp1-52::LEU2 ura3::GFP-TUB1::URA3 ∆kip3::HIS3MX6 ASE1-3mCherry:hphNT1 / 2E,F
YJO1188 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 ura3::GFP-TUB1::URA3 ∆kip3::HIS3MX6 STU1-3mCherry::hphNT1 / 2E,F
YJO1182 / MATa ∆sst1 ade2-101 his3-∆200 lys2-801 ∆okp1::TRP1 leu2::okp1-52::LEU2 ura3::GFP-TUB1::URA3 ∆kip3::HIS3MX6 STU1-3mCherry:hphNT1 / 2E,F
YJO1235 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 ∆okp1::TRP1 leu2::okp1-52::LEU2 ura3::GFP-TUB1::URA3 ∆kip3::HIS3MX6 ASE1-3mCherry:hphNT1 ade2::pGAL-CDC14-FLAG::ADE2 / 2G,H
YJO1234 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 ∆okp1::TRP1 leu2::okp1-52::LEU2 ura3::GFP-TUB1::URA3 ∆kip3::HIS3MX6 STU1-3mCherry:hphNT1 ade2::pGAL-CDC14-FLAG::ADE2 / 2G,H
YJO1222 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 ura3::GFP-TUB1::URA3 ∆kip3::HIS3MX6 BIM1-3mCherry::hphNT1 / S4A,B
YJO1221 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 ∆okp1::TRP1 leu2::okp1-52::LEU2 ura3::GFP-TUB1::URA3 ∆kip3::HIS3MX6 BIM1-3mCherry::hphNT1 / S4A,B
YJO1220 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 ura3::GFP-TUB1::URA3 ∆kip3::HIS3MX6 SPC34-3mCherry::hphNT1 / S4A,B
YJO1219 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 ∆okp1::TRP1 leu2::okp1-52::LEU2 ura3::GFP-TUB1::URA3 ∆kip3::HIS3MX6 SPC34-3mCherry::hphNT1 / S4A,B
YJO1363 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 ura3::GFP-TUB1::URA3 ∆kip3::HIS3MX6 STU2-3mCherry::hphNT1 / S4A,B
YJO1414 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 ∆okp1::TRP1 leu2::okp1-52::LEU2 ura3::GFP-TUB1::URA3 ∆kip3::HIS3MX6 STU2-3mCherry::hphNT1 / S4A,B
YCF1354 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 ∆okp1::TRP1 leu2::okp1-52::LEU2 ura3::GFP-TUB1::URA3 STU1-3mCherry::hphNT1 cse4::KanMX6-pGAL-UbiR-CSE4 / 2I
YCF1330 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 ade2::tetR-GFP::ADE2 ura3::CEN5-tetO2x112:URA3 SPC72-3mCherry::hphNT1 / 3A
YCF1331 / MATa ∆sst1 ade2-101 trp1-∆63 ura3-52 his3-∆200 lys2-801leu2-∆1::LEU2 ade2::tetR-GFP:ADE2 ura3::CEN5-tetO2x112:URA3 SPC72-3mCherry::hphNT1 STU1-CFP::kanMX4 / 3D, 6
YJO1153 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 scc1-73 STU1-GFP::kanMX4 ura3::CFP-TUB1::URA3 / 3E
YJO1338 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801∆dad2::kan leu2::dad2-9::LEU2 SPC72-3mCherry::hphNT1 STU1-eCFP::klTRP1 / 3F
YJO1160 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 AME1-GFP::kanMX6 STU1-3mCherry::hphNT1 ura3::CFP-TUB1::URA3 cdc20::HIS3MX6-pGAL-CDC20 / 4B
YJO1090 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801∆okp1::TRP1 leu2::okp1-52::LEU2 STU1-GFP::HIS3MX6 cdc20::KanMX6pGAL1-3HA-CDC20 ura3::CFP-TUB1::URA3 / 4C
YMS696 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 STU1-GFP::HIS3MX6 / 5B
YJO1116 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 STU1-GFP::HIS3MX6 ura3::CFP-TUB1::URA3 cdc20::KanMX6-pGAL-CDC20 / 5C
YJO1334 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 ade2::tetR-GFP:ADE2 ura3::CEN5-tetO2x112:URA3 SPC72-3mCherry::hphNT1 stu1::TRP1kl-pGAL-UbiR-STU1-CFP::kanMX4 / 6
YJO1278 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 ∆okp1::TRP1 leu2::okp1-52::LEU2 NDC10-GFP::HIS3 STU1-3mcherry:hphNT1 SPC72-CFP::kan MX4 / S6B
YCF1360 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801
ade2::TETr-GFP::ADE2 ura3::1.4kb left of CENV::tetO2x112::URA3 ndc80::KANMX6-pGal-UbiR-NDC80-9MYC::TRP1 leu2::LEU2 STU1-3mCherry::hphNT1 lys2::CFP-TUB1::LYS2 / 7A
YCF1343 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 AME1-GFP::kanMX6
STU1-CFP::HIS3 SPC72-3mCherry::hph-NT1 ∆mad2::TRP1 / 7B
YCF1344 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 AME1-GFP::kanMX6
STU1-CFP::HIS3 SPC72-3mCherry::hph-NT1 ∆bub1::TRP1 / 7B
YCF1298 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 lys2::CFP-TUB1::LYS2
STU1::stu1∆1175-1513-NLS-GFP::klTRP1 BUB1-3mcherry::hphNT1 / 8A, B
YCF1359 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801
STU1-NLS-GFP::klTRP1 lys2::CFP-TUB1::LYS2 BUB1-3mcherry::hphNT1 / 8B
YCF1375 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-80
lys2::pGAL1-FLAG-STU1-CFP-TRP1::LYS2 SPC72-3mcherry::hphNT1 AME1-GFP::kanMX6 / 5A
YCF1361 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 ura3:TUBI-CFP:URA3
STU1-3mCherry::hphNT1 lys2:pGAL1-FLAG-stu1∆887-1513-NLS-GFP::LYS2 / 8C
YCF1342 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 STU1-GFP::HIS3MX6 lys2::pGAL-FLAG-stu1∆461-716-3mCherry-hphNT1::LYS2 SPC72-CFP::kanMX4 / 8C
YJO1212 / MATa ∆sst1 ade2-101 trp1-∆63 leu2-∆1 ura3-52 his3-∆200 lys2-801 trp1::TUB1-CFP::TRP1
OKP1-4GFP::kanMX6 lys2::pGAL-FLAG-stu1∆461-716-3mCherry-hphNT1-LYS2 / 8F
Plasmids
pAW945 / CFP-TUB1 in a URA3-based integration vector
pAW1035 / ame1-2(ORF)-ADE2(P-ORF-Term)- AME1 term seq. in pBSII-SK
pBL929 / kanMX6-pGAL1-Ubiquitin-R inpBS1539
pCF1136 / SV40-NLS in pYM29
pCF1148 / pGAL-FLAG-stu1∆(887-1513)-NLS-GFP in YDpK(LYS2) integration vector
pJO608 / mad2::TRP1 in pUC18
pJO719 / klTRP1-pGAL1-Ubiquitin-R in pBS1539
pJO1094 / pGAL-FLAG-STUI in a YDpK(LYS) integration vector
pJO1126 / pGAL-FLAG-∆stu1(461-716 Mt BD deleted ) in YpD-K(LYS2) integration vector