JCS03342 Supplementary Material

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• Supplemental Table 1 -

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• Supplemental Table 2 -

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• Supplemental Figure 1 -

  Fig. S1. Spc110p phosphorylation levels in wild-type or cdc28-4 mutant cells. Extracts from the indicated strains were obtained from asynchronous cultures incubated at 25°C or following a shift to 37°C for the indicated times. Notice that, even at the permissive temperature, the hypomorphic cdc28-4 causes a mild decrease in phosphorylation relative to that in wild-type cells, however the additional effect of the temperature shift demonstrates that, in this mutant, Spc110p is still an efficient target for CDK at permissive temperature. As shown in Fig. 3C, however, deletion of CLB5 markedly reduced Spc110p phosphorylation in combination with cdc28-4, while only resulting in a modest reduction in an otherwise wild-type background. The effect of restoring Clb5p by overexpression is again more pronounced in the cdc28-4 clb5Δ background than in a single clb5Δ mutant. Based on this prominent genetic interaction, it follows that in vivo phosphorylation of Spc110p (partly due to CDK) behaves in a manner consistent with the premise that the cdc28-4 mutation sensitises cells to the loss of CLB5.

• Supplemental Figure 2 -

  Fig. S2. Analysis of cell cycle progression accompanying the time course in Fig. 3E. Samples from the following strains were used for the analysis of cell-cycle progression presented here in addition to the preparation of extracts for western blot analysis shown in Fig. 3E. Solid squares, wild-type SPC110; solid circles, spc110^36A; open triangles, spc110^91A; open diamonds, spc110^36A91A. Budding index was determined by microscopy analysis as described in Materials and methods. Analysis of DNA content by flow cytometry was carried out as previously described (Segal et al., 1998; Clarke et al., 2001).

• Supplemental Figure 3 -

  Fig. S3. Analysis of cell-cycle progression accompanying the time course in Fig. 4C. Samples from the following strains were used for the analysis of cell-cycle progression presented here in addition to the preparation of extracts for western blot analysis shown in Fig. 4C. Solid squares, spc110^mps1-; solid circles, spc110^mps1-,36A; open triangles, spc110^mps1-,91A; open diamonds, spc110^mps1-,36A91A. Other experimental details were as in Fig. S2.

• Supplemental Figure 4 -

  Fig. S4. Effect of S36A-S91A substitutions on Spc110^Δ13p phosphorylation in wild-type cells. Comparison of Spc110p phosphorylation levels in extracts from wild-type cells expressing the indicated forms of Spc110p (A) or Spc110^Δ13p (B) sampled from asynchronous (a) or nocodazole-arrested (Noc) cultures. Lane c, untagged strain control. In B, two different exposures of the same blot are shown. In the lighter exposure, a tight doublet and a fainter lower mobility band (the latter more obvious in the longer exposure) are apparent in extracts from asynchronous cells expressing Spc110^Δ13p. This pattern changes to a main fast migrating and a faint lower mobility band for Spc110^Δ13-36A91Ap. In both A and B, phosphorylation is increased by nocodazole for all forms of Spc110p shown. Judging by the magnitude of this effect, Mps1p-mediated phosphorylation may be less efficient for the Spc110^Δ13p construct that fails to localise to the SPB. Nevertheless, the dependency on the CDK sites shown here is also apparent in a cdc28-4 clb5Δ strain, demonstrating that these sites may be targeted for phosphorylation by alternative cyclins (the magnitude of this effect, may be in fact favoured again by the fact that Spc110^Δ13p is diffuse in the nucleus and accessible to CDKs that may not strongly localise to the SPB).

• Supplemental Figure 5 -

  Fig. S5. Clb5-GFP localisation in budded cells prior to spindle assembly. Representative image of cells expressing Clb5p-GFP (overlaid in green) and Spc29p-CFP (in red) following bud emergence. Arrows indicate Clb5-GFP labelling as lines extending from the unseparated SPBs, suggestive of a possible localisation to kinetochore microtubules. (a) DIC image; (b) overlay of fluorescence images; (c) Clb5-GFP; (d) Spc29-CFP. Experimental details were essentially as described for time-lapse analysis.