Phosphorylation of MIND and relationship to the vertebrate Mis12 complex. (a) Phosphorylation of the MIND complex by the yeast Aurora B homologue, Ipl1, with and without the activating IN-box peptide. (b) ITC raw data and binding isotherms for Ser250 phosphorylated MIND and full-length NDC80 or Spc105^800–829. The red lines are normalized baselines for the experiment. (c) Comparison of the predicted structural features of five MIND orthologues. The bar length is proportional to the size of the protein. The purple boxes represent predicted coiled-coil segments (assessed using the COILS algorithm; Lupas et al., 1991), with the shading representing the confidence score. The positions of the proteolytic cleavage in the recombinant S. cerevisiae proteins are indicated with red arrowheads. (d) Limited proteolysis of the MIND complex using trypsin with the stable truncated products labeled. Molecular mass (MM) is indicated in kilodaltons. (e) Sequence alignment of five Mis12/Mtw1 orthologues. Both the budding and fission yeast proteins contain an extended C-terminal segment that is susceptible to proteolysis. The colors represent different classes of amino acid (turquoise, hydrophobic; purple, acidic; salmon, basic; orange, glycine; yellow, proline; and green, uncharged polar). (f) Model of the MIND complex showing predicted locations of the proteins based on the data presented in this work (left) and in vivo locations of the human Mis12 proteins derived from light microscopy (right).