Figure 1.
HJURP interaction with the CENP-C cupin domain is required for CENP-A assembly. (a) Schematic of CENP-C truncations and mutations. The CENP-C amino acid residue numbers for each truncation are listed to the left. The domains of CENP-C that interact with Mis12/CCAN, interact with CENP-A nucleosomes, and the cupin dimerization domain are highlighted in green, yellow, and red, respectively. The residue numbers of the boundaries of each domain are listed on top. Mutations that inhibit CENP-A nucleosome binding and dimerization are highlighted with red stars. (b) The C-terminus of CENP-C binds to HJURP. Interphase extract depleted of endogenous CENP-C was supplemented with Myc-CENP-C truncations. After immunoprecipitation (IP) of CENP-C truncations from interphase X. laevis egg extracts, coimmunoprecipitation of HJURP was assayed by anti-HJURP immunoblotting. The left panels show 5% of the input used in the immunoprecipitation, and the right panels show the CENP-C (top panels) and HJURP (bottom panels) in the immunoprecipitates. Each fragment or control (-IVT: scrambled DNA translated in vitro, IgG: nonspecific mouse IgG antibody) is listed above the panels, the amino acids encompassed by each fragment are shown to the left and the molecular weight to the right. (c) CENP-C cupin domain mutations affect HJURP interaction. Interphase extract depleted of endogenous CENP-C was supplemented with the specified FLAG-CENP-C mutants. Coimmunoprecipitation of endogenous HJURP with each FLAG-CENP-C protein was assayed by anti-HJURP immunoblotting (bottom panels). The levels of CENP-C in the extract and immunoprecipitation are shown in the top panels. Mock precipitations using scrambled DNA translated in vitro (-IVT) or whole mouse IgG are indicated. The left panels contain 5% of the input material and the right panels contain the immunoprecipitations. (d) CENP-C cupin domain mutations disrupt CENP-A assembly. Representative images of sperm nuclei incubated in CENP-C depleted interphase Xenopus egg extracts complemented with the CENP-C mutant indicated. Extracts were supplemented with RNA encoding V5-CENP-A and in vitro translated HJURP protein to assay CENP-A assembly. Immunolocalized protein is specified above. Scale bar, 5 µm. Insets are magnified 300%. (e) Quantification of V5-CENP-A assembly shown in d. Values are normalized to unsupplemented, mock-depleted extract. Plot shows mean V5-CENP-A signal on sperm chromatin ± SEM (n = 3; ***, P < 0.001; **, P < 0.01; Wilcox/Mann–Whitney test). Source data are available for this figure: SourceData F1.

HJURP interaction with the CENP-C cupin domain is required for CENP-A assembly. (a) Schematic of CENP-C truncations and mutations. The CENP-C amino acid residue numbers for each truncation are listed to the left. The domains of CENP-C that interact with Mis12/CCAN, interact with CENP-A nucleosomes, and the cupin dimerization domain are highlighted in green, yellow, and red, respectively. The residue numbers of the boundaries of each domain are listed on top. Mutations that inhibit CENP-A nucleosome binding and dimerization are highlighted with red stars. (b) The C-terminus of CENP-C binds to HJURP. Interphase extract depleted of endogenous CENP-C was supplemented with Myc-CENP-C truncations. After immunoprecipitation (IP) of CENP-C truncations from interphase X. laevis egg extracts, coimmunoprecipitation of HJURP was assayed by anti-HJURP immunoblotting. The left panels show 5% of the input used in the immunoprecipitation, and the right panels show the CENP-C (top panels) and HJURP (bottom panels) in the immunoprecipitates. Each fragment or control (-IVT: scrambled DNA translated in vitro, IgG: nonspecific mouse IgG antibody) is listed above the panels, the amino acids encompassed by each fragment are shown to the left and the molecular weight to the right. (c) CENP-C cupin domain mutations affect HJURP interaction. Interphase extract depleted of endogenous CENP-C was supplemented with the specified FLAG-CENP-C mutants. Coimmunoprecipitation of endogenous HJURP with each FLAG-CENP-C protein was assayed by anti-HJURP immunoblotting (bottom panels). The levels of CENP-C in the extract and immunoprecipitation are shown in the top panels. Mock precipitations using scrambled DNA translated in vitro (-IVT) or whole mouse IgG are indicated. The left panels contain 5% of the input material and the right panels contain the immunoprecipitations. (d) CENP-C cupin domain mutations disrupt CENP-A assembly. Representative images of sperm nuclei incubated in CENP-C depleted interphase Xenopus egg extracts complemented with the CENP-C mutant indicated. Extracts were supplemented with RNA encoding V5-CENP-A and in vitro translated HJURP protein to assay CENP-A assembly. Immunolocalized protein is specified above. Scale bar, 5 µm. Insets are magnified 300%. (e) Quantification of V5-CENP-A assembly shown in d. Values are normalized to unsupplemented, mock-depleted extract. Plot shows mean V5-CENP-A signal on sperm chromatin ± SEM (n = 3; ***, P < 0.001; **, P < 0.01; Wilcox/Mann–Whitney test). Source data are available for this figure: SourceData F1.

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