Figure 2. Identification and characterization CENP-N mutants defective in CENPA-nucleosome binding.

a) CENP-N mutants exhibit a range of affinities for CENP-A nucleosomes. CENP-N (wt) or the indicated CENP-N mutants were expressed and an equal concentration (~1nM) of each mutant was assayed for its ability to bind CENP-A nucleosomes (300nM). The control reaction (−) lacked CENP-A nucleosomes. b) Doseresponse experiments for each CENP-N mutant were performed as in (a) with increasing concentrations of CENP-A nucleosome added to each reaction (N=3, error bars represent SEM). c) Expression of CENP-N mutants in HEK293 cells. Nuclear extracts from stable HEK293 cell lines expressing GFP-CENP-N (wt) or the indicated GFP-CENP-N mutant were separated by SDS-PAGE and western blotted with the indicated antibodies. Two nonspecific bands (*) are present in the anti-CENP-N western blot. Upper and lower arrows indicate positions of GFP-CENP-N and endogenous CENP-N, respectively. Quantitation of each bands is presented in Figure S4a. d) Coimmunoprecipitation of CENP-H, K, and A from micrococcal-nuclease solubilized chromatin with CENP-N mutants. Anti-GFP immunoprecipitates from each cell line were probed with the indicated antibodies. 7X more nuclear extract from the CENP-NΔC cell line was required to achieve equal levels of CENP-N in the immunoprecipitations. Quantitation of each bands is presented in Figure S4b. e) CENP-N binds to CENP-L. 6xmyc-CENP-L, CENPL and CENP-N were expressed and labeled with ³⁵S-methionine and the indicated proteins were mixed at equal stoichiometry. 20% of each mixture was resolved as input (left panel). The remaining 80% was immunoprecipitated with anti-myc antibodies (right panel). Two background bands (*) are present in the input reactions. f) CENP-L binding requires the C-terminus of CENP-N. Immunoprecipitations are identical to those in (e), except that equal amounts of wildtype CENP-N (wt) or the indicated CENP-N mutant was used in each reaction.