Fig. 7: The hydrophobic crest is essential for NC-interface integrity in yeast septins.

A Addition of the α0-helix to the Cdc10 subunit of the truncated tetramer employed for crystallization is sufficient to restore an octamer. Shown are analytical size exclusion runs with normalized peak intensity. B Mutations of the conserved hydrophobic crest positions in Cdc10 interrupt NC-interface integrity leading to a tetrameric elution profile (indicated by the red dashed line). Mutation at the non-conserved L26 retains an octameric elution profile (indicated by the blue dashed line). The reference chromatograms for tetramer and octamer (dashed lines) are shown in Supplementary Fig. 6. Shown are analytical size exclusion runs with normalized peak intensity. C Evaluation of hydrophobic crest mutants in Cdc10 in the context of the living yeast cell. Cdc10 mutants are expressed from a centromeric plasmid in a CDC10 knock out strain after kick-out of a rescue plasmid on FOA medium. F15A, I18A and Δα0 mutants are lethal. V13A, I22A and F31A show a slight growth defect and L26A and L27A are indistinguishable from wildtype. ΔΔG values (kcal/mol) predicted by FoldX are provided. The higher the value, the higher the disruptive potential of the introduced mutation. D Introduction of the I18A mutation into a disulfide mutant in Cdc10 prevents successful crosslinking under oxidizing conditions whereas L26A and L27A do not interfere with crosslinking. Shown is a Western blot detecting the S-tag fused to Cdc10 in an octameric septin rod preparation. Labeling as in Fig. 4.