Fig. 2.

The Mtw1 C terminus and a putative alpha-helix in Nsl1 are required for in vitro binding to the Ndc80 complex and are essential in vivo. (A) MIND complex protein sequences, with mutated amino acids shown in red. (B) Immunoprecipitation assays with FLAG–Ndc80 complex immobilized on anti-FLAG beads and the indicated MIND–His₆ complexes (WT, nsl1-4D, mtw1-220, and dsn1-2LD) added in solution. (Left) Coomassie-stained gel shows 7.5% input of protein complexes used in immunoprecipitation. (Right) Western blot of immunoprecipitation assay. Copurifying MIND complex was visualized by anti-His (staining Dsn1-His₆, green); Ndc80 complex was detected with anti-Ndc80 (red). (Below Right) Quantification of immunoprecipitation experiments. WT MIND binding was normalized to 100% for each experiment, and MIND mutant binding is shown as a percentage of WT binding (n = 2 and error bars denote SEM.) (C) Plasmid shuffle assay of cells spotted in 10-fold dilutions on synthetic complete (SC) media (Left) or SC media supplemented with 5-FOA (Right). (Top spots) nsl1Δ cells containing WT NSL1 on a URA3 plasmid and either WT or nsl1-4D on a LEU2 plasmid. WT NSL1 fully supports growth on 5-FOA media whereas nsl1-4D is lethal. (Bottom spots) mtw1Δ cells with WT MTW1 on a URA3 plasmid and either WT or mtw1-220 on a LEU2 plasmid. mtw1-220 cannot support growth on 5-FOA whereas MTW1 is viable.