Figure 5.

Binding of CENP-T and the Mis12 complex to the Ndc80 complex is mutually exclusive. (A) L161R mutations in Spc25 disrupt the interaction with CENP-T. Phospho-mimetic chicken MBP-CENP-T^63–98 (T72D/S88D) and the mutant Spc24^125–195/Spc25^132–234 (L161R) complex were separated by gel filtration using a Superdex 75, analysed by SDS–PAGE, and stained with Coomassie. (B) I156R mutations in Spc25 disrupt the interaction with CENP-T. Phospho-mimetic MBP-CENP-T^63–98 (T72D/S88D) and the mutant Spc24^125–195/Spc25^132–234 (I156R) complex were analysed by gel filtration as in (A). (C) I149A L154A double mutants in human Spc25 disrupt binding to human CENP-T. Human phospho-mimetic CENP-T (MBP-hsCENP-T^76–106 T85D) and the wild-type Ndc80^Bonsai complex or the Ndc80^Bonsai I149A L154A mutant complex were mixed and analysed by gel filtration. Protein mixtures were incubated on ice for 30 min before conducting the chromatography using a Superose 6 column. Fractions were collected, analysed by SDS–PAGE, and stained with Coomassie. Elution profiles from the size-exclusion chromatography for the experiment are shown (top). Elution of proteins was monitored at A_{280 nm}. (D) The Ndc80^Bonsai complex or the Ndc80^Bonsai I149A L154A mutant complex and the human Mis12–KNL1^2106–2316 complex were mixed and analysed by gel filtration chromatography using a Superose 6 column as in (C). Elution profiles from the size-exclusion chromatography are shown (top). (E) CENP-T and the Mis12/KNL1^CT complex show mutually exclusive binding to the Ndc80^Bonsai complex. Human phospho-mimetic MBP-CENP-T^76–106(T85D), wild-type Ndc80^Bonsai complex, and the human Mis12-KNL1^2106–2316 complex were mixed in the indicated combinations and analysed by gel filtration. A large complex containing all components was not detected, although both CENP-T and KNL1/Mis12 bound individually to the Ndc80 complex based on altered migration.