A conserved SP site on the HJURP N-terminus regulates its interaction with CENP-C. (a) Schematic representation of HJURP truncations used to identify the CENP-C interacting region in b and c. Amino acids spanned by each truncation are shown to the left. (b) A fragment of HJURP spanning amino acids 1–400 binds to CENP-C. Interphase extract was supplemented with the FLAG-HJURP truncations specified at the top of the panel. Following FLAG-HJURP immunoprecipitation, co-precipitation of CENP-C was assayed by anti–CENP-C immunoblotting. For b, c, and e, mock precipitations using scrambled DNA translated in vitro (-IVT) or whole mouse IgG serve as negative controls. For b and c, the top panel shows 5% of the input material and the bottom panel shows the immunoprecipitates with the sizes of HJURP fragments indicated on the left. (c) HJURP amino acid residues 205–225 are required for its interaction with CENP-C. Interphase extract was supplemented with the specified FLAG-HJURP truncations. Coimmunoprecipitation of endogenous CENP-C was assayed by anti–CENP-C immunoblotting following FLAG-HJURP immunoprecipitation. (d) Vertebrate HJURP contains a conserved N-terminal S/P site. HJURP sequences are aligned using X. laevis HJURP^205-225 as a reference (blue row). (e) HJURP S220 (HJURP^S220) regulates association with CENP-C. Interphase and metaphase extracts were supplemented with the indicated FLAG-HJURP mutants. FLAG-HJURP was immunoprecipitated (IP) and coprecipitation of endogenous CENP-C was assayed by anti–CENP-C immunoblotting. The top panel shows 5% of the input material, the bottom panel shows the immunoprecipitates. Source data are available for this figure: SourceData F2.