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Figure 4


Fig. 4. Cdc8 is acetylated in vivo. (A,B) cdc8-110 cells expressing additional integrant Tm genes under the control of the nmt41 promoter were grown at 36°C in EMM2 lacking thiamine. (A) The percentage of cells with two (black bars) or more than two (grey bars) nuclei were scored for each strain after 4 hours. (B) Growth curves generated for each strain over a 6 hour growth at 36°C. Only Cdc8 and smooth muscle Tm were capable of fully complementing the cdc8-110 mutation. Endogenous Cdc8 was purified from mid-log phase S. pombe cells and analysed alongside total protein extracts by silver staining of SDS-PAGE gels (C) and western blot analysis using Cdc8 antisera (D). A single doublet migrating at ~30kDa was revealed by silver staining (C), while Cdc8 antisera recognised identical doublets in each lane. (E) Mass spectroscopy analysis of the purified endogenous Cdc8 revealed ~20% of this protein had a mass of 18964.1 (predicted mass of Cdc8: 18,964.7 Da), while the remaining 80% had a mass pf 19005.5 Da, which corresponds to the predicted mass of acetylated Cdc8.





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