Figure 8.

S. cerevisiae Spc105, a component of the conserved Ndc80 kinetochore complex, rescues mal3 mutants. (A) Spc105 copurifies with the Ndc80 complex. Protein A-tagged Spc24 was purified from 4000 OD of cells by affinity chromatography with IgG-agarose, and the complete preparation was subjected to SDS-PAGE. Copurifying proteins ⁵⁷⁸were identified from Coomassie-stained bands by peptide mass finger printing (MALDI-TOF). Bands that are not labeled represent contaminants. (B) TBZ hypersensitivity of the S. pombe mal3-1 and Δmal3 strains and the S. cerevisiae Δbim1 strain is partially rescued by overexpression of SPC105. Panels show serial dilution patch tests (10⁴ to 10¹ cells) of mal3-1, Δ mal3, and Δ bim1 transformants grown under selective conditions in the absence (–TBZ) or presence (+TBZ) of TBZ. Plates were incubated for 5 d at 24°C (mal3 strains) or 3 d at 30°C (Δbim1 strain). Vector control indicates plasmid without insert. (C) Serial dilution patch tests of Δbim1, tetO-CYC1/SPC105 single and double mutant strains on YPD with doxycyline with or without 50 μg/ml TBZ. Strains were grown at 30°C for 48 h.