Figure 4.

Identification of an essential Ndc80–Mtw1 interface. (A) Asci dissection of Δspc25/Spc25 Spc25^WT (left) and Δspc25/Spc25 Spc25^V159D (right). The viable Δspc25 Spc25^WT spores are highlighted with a light blue square. The Δspc25 Spc25^V159D spores, marked with a red rectangle, failed to be recovered, indicating that V159D is a lethal Spc25 mutation. (B) Elution profiles and Coomassie-stained gels of analytical size-exclusion chromatography performed with 4 μM Mtw1^{Dsn1(172–576)} complex (Mtw1C^{Dsn1(172–576)}), 4 μM wild-type Ndc80 complex (Ndc80C) and their combination (left); 4 μM Mtw1^{Dsn1(172–576)} complex, 4 μM Ndc80 complex including the Spc25^V159D mutant (Ndc80C^25V159D) and their combination (right). Complex formation is visualized by a shift to earlier elution volumes of both molecules. (C) Multiple-sequence alignment of a conserved C-terminal motif in Dsn1 proteins. Background colouring of residues is based on the Clustalx colouring scheme. Amino acids omitted before the stop are given in brackets. Amino acids mutated and tested in the plasmid shuffle assays are marked with an arrow. (D) Elution profiles and Coomassie-stained gels of analytical size-exclusion chromatography performed with 4 μM Mtw1C^{Dsn1(172–547)}, 4 μM wild-type Ndc80C and their combination. The lack of a shift to earlier elution volumes of both molecules indicates abolished complex formation. (E) Plasmid shuffle assays performed transforming Δdsn1 Dsn1:URA strain with Dsn1 wild type (WT), Dsn1^172–576 (172–576), Dsn1^1–547 (1–547) and the mutants Dsn1^{L562D, L563D} (L562D, L563D) and Dsn1^K564A (K564A). Single clones were spotted in two-fold dilutions on minimal medium lacking (left panel, SC) or containing (right panel) 5-FOA. Red rectangles highlight the lethality of mutants Dsn1^1–547 and Dsn1^{L562D, L563D}. (F) Plasmid shuffle assay executed as in (E) testing the functionality of a Dsn1 version where the last C-terminal 29 amino acids were substituted by the Cnn1 residues 60–84 (Dsn1 switch, in schematic representation underneath the panel). The red rectangle encloses the viable Dsn1 switch mutant.