Biochemical characterization and mutational analysis of the WDR62–katanin complex. (A) Schematic overview of the domain organization of katanin p60/p80 heterodimer and the deletion mutants and summary of their interactions with WDR62. WD40, WD-40 repeat domain; C, C-terminus; N, N-terminus; AAA, AAA-ATPase domain. (B) Streptavidin pull-down assays with extracts of HEK293T cells expressing Bio-GFP–tagged p60 or p80 subunit or the indicated fragments (bait) together with WDR62-GFP (prey), analyzed by Western blotting with GFP antibody. (C) Schematic overview of the domain organization of WDR62 and the deletion mutants and summary of their interactions with p80. CC, coiled-coil domain. (D) StrepTactin pull-down assays with extracts of HEK293T cells expressing Strep-p80 WD40 domain (WD; bait) together with GFP-tagged WDR62 or its indicated fragments (prey) analyzed by Western blotting with indicated antibodies. LZ, leucine zipper from GCN4, was used as a dimerization domain. (E) Alignment of katanin-binding region in WDR62 from five vertebrate species. The conserved arginine and tryptophan residues are indicated with asterisks. (F) Streptavidin pull-down assays with extracts of HEK293T cells expressing the Bio-GFP-p80 WD (bait) together with GFP-tagged WT WDR62 or its indicated mutants (prey) analyzed by Western blotting with GFP antibody. (G) Coomassie blue–stained gel of StrepTactin pull down using Strep-p80 WD or Strep-GFP purified from HEK293T cells and immobilized on StrepTactin beads as the bait and WT WDR62 M1 fragment or its W864A mutant purified from E. coli as the prey. (H and I) Immunofluorescence staining and quantification of p80 intensities at spindle poles in WDR62 knockout (KO) HeLa cells transiently transfected with GFP-tagged WT WDR62 or its indicated mutants or control GFP vector. The values were normalized to the intensity of control HeLa cells. For all conditions, n = 74 spindle poles. Scale bar, 2 µm. Data represent mean ± SD. ∗∗∗, P < 0.001; two-tailed t test.