Identification and analysis of BUB-1 mutations affecting MAD-1 kinetochore7 localization. (A) Identification of the C-terminal region of BUB-1 as the interaction domain for MAD-1 and results of a compensatory two-hybrid screen using MAD-1D423A. TPR, tetratricopeptide repeat. (B) Location of N781 and other activity-related residues in the BUB-1 kinase domain (Protein Data Bank accession no. 3E7E; Kang et al., 2008). Residue numbering: Ce N781 (Hs N879), Ce L777 (Hs L875), Ce K718 (Hs K821), Ce K847 (Hs K946), and Ce D814 (Hs D917). Panel on the right shows two-hybrid analysis of the MAD-1–BUB-1 interaction with N781K and L777K mutations. (C) Schematic of bub-1 transgene and analysis of embryo viability after endogenous BUB-1 depletion for the indicated variants. At least 7 worms and >445 total embryos were scored per condition. Error bars are the 95% confidence interval of the mean. Chr I, chromosome I. (D) Images and quantification of GFP::MAD-1 at unattached kinetochores for the indicated BUB-1 variants, normalized relative to the WT transgene control. n is number of embryos analyzed; error bars are the 95% confidence interval of the mean. Bar, 5 µm. (E) Kinase activity assay of WT and D814N mutant BUB-1 with GST-fused C. elegans histone H2a as substrate. The blot and gel image are representative of three experiments.
