JCS03380 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

  Adobe PDF

• Supplemental Figure 1 -

  Fig. S1. Mutant and WT Clb2 localization to the spindle pole bodies (spb). (A) Comparison of the fluorescence intensity at the spb in the various mutant forms of Clb2. To quantify the fluorescence intensity at the spb, the mean value of a four-pixel square covering the spb was measured in anaphase cells using the ImageJ software. The background fluorescence, measured as the fluorescence level of a four-pixel square outside the cells was deducted from the spb values. All images were taken with the same optical settings and processed with identical parameters using Adobe PhotoShop. (B) Localization of the Clb2 nuclear spot relative to the spb in pre-anaphase cells co-expressing Spc29-CFP and Clb2 or Clb2^L303A-YFP. Quantification of the pattern of distribution is shown in the adjacent table. Cells were classified as having a YFP-associated Clb2 spot between the two spb, close to one of the spb or colocalizing with the CFP signal. Note that the fluorescent signals were lower with the YFP than with the GFP tag. Consequently, the single nuclear dot observed in clb2^ΔNLS-GFP cells before anaphase was not detected in this experiment. (C) WT and mutant Clb2 colocalize with a spb marker during anaphase. Cells co-expressing Spc29-CFP and Clb2, Clb2^L303A or Clb2^ΔNLS-YFP are shown. Bars, 2 μμ.

• Supplemental Figure 2 -

  Fig. S2. Forcing the cytoplasmic localization of Clb2^ΔNLS impairs anaphase initiation. (A) Addition of an ectopic NES signal prevents the nuclear localization of Clb2^ΔNLS. The NES-clb2^ΔNLS and mnes-clb2^ΔNLS constructs were subcloned pMJ200 (Bailly et al., 2003) to express GFP fusion proteins under the control of the GAL1 promoter. Cells grown in SC-raffinose were collected on nitrocellulose filters and induced for 1 hour on YEP-galactose plates before microscopic observation. Representative images of the localization of the NES-Clb2^ΔNLS and mnes-Clb2^ΔNLS-GFP cylins are shown at different stages of the cell cycle. The distributions of the WT, Clb2 and Clb2^ΔNLS proteins expressed under the same conditions are shown for comparison. Bars, 2 μμ. (B) Clb2^ΔNLS and derivatives were expressed at similar levels in metaphase-arrested cells. WT cells expressing the indicated alleles of CLB2 from pPC2 derived vectors were grown to mid log phase and blocked in metaphase by addition of 15 μg/ml nocodazole for 2 hours. Cells were processed for immunoblot with anti-HA antibodies. The loading control was a non specific protein recognized by the anti-HA antibodies. (C) W303 clb1Δ, clb3Δ, clb4Δ cells carrying a temperature-sensitive allele of CLB2 (Amon et al., 1993) were transformed with pPC2 vectors expressing the indicated alleles of CLB2. Cells were arrested in G1 with 5 μg/ml α-factor for 3 hours at 25°C, washed and synchronously released at 37°C. Aliquots were taken every 15 minutes and the percentage of binucleate cells determined after DAPI staining. More than 200 cells were counted for each time point.

• Supplemental Figure 3 -

  Fig. S3. Delayed phosphorylation of Net1 in clb2^ΔNLS-expressing cells. (A) Kinetics of Net1 phosphorylation in extracts from clb1Δ clb2^L303A and clb1Δ clb2^ΔNLS cells expressing a NET1-Myc allele. Total cellular proteins prepared at each time point after an α-factor released at 25°C were separated by a long migration on 8% polyacrylamide gels to detect Net1-Myc and by 10% SDS-PAGE for Clb2 and Cdc28. (B) Kinetics of Pds1 degradation. Complete time course of the experiment shown in Fig. 3C.

• Supplemental Figure 4 -

  Fig. S4. (A) Cdc28 binding and activation properties of Clb2^L303A. Cells expressing HA-tag fusions of Clb2 or Clb2^L303A from the chromosomal locus were arrested in metaphase by addition of 15 μg/ml nocodazole for 3 hours at 30°C. Cell lysates were processed for immunoprecipitation with anti-HA antibodies. The top panel shows an autoradiogram of the Clb2-associated histone H1 kinase activity present in the immunoprecipitates (Bailly et al., 2003). Clb2-HA (middle panel) and Cdc28 (bottom panel) in the input extracts were assayed by western blotting with anti-HA and anti-Cdc28 antibodies respectively. (B) WT, clb2^ΔNLS and clb2Δ cells expressing a CLN2-HA3 allele were released from an α-factor induced arrest at 30°C and analyzed at the indicated times for the amount of Cln2-HA, Clb2 and Cdc28 by western blot analysis.

• Supplemental Figure 5 -

  Fig. S5. Cell cycle progression in clb2^K459A,Y460C cells. (A) Conservation of amino acids at the C terminus of A type and B type cyclins. M.G is the sea urchin species Marthasterias glacialis. (B) Clb2^K459A,^Y460C-YFP colocalizes with Spc29-CFP during anaphase. (C) Clb2^K459A,^Y460C and the WT cyclin bind Cdc28 with similar affinity. Two mg total proteins from the WT strain and a strain where the clb2^K459A,Y460C allele was introduced at the CLB2 locus, were incubated with P13suc1 agarose beads for 2 hours as described by Lozano et al. (Lozano et al., 1998). The precipitates were analyzed for their Clb2 content and PSTAIRE epitope by western blotting. (D) WT and clb2^K459A,Y460C cells were arrested in G1 with 60 ng/ml of α-mating factor for 2 hours and released synchronously at 30°C in fresh medium. Aliquotes were taken every 15 minutes and analyzed microscopically for the percentage of budded cells and binucleate cells after DAPI staining.