The CENP-A recruiting activity resides within the N terminus of CAL1. (A) Diagram showing the three domains of CAL1 and respective known functions (see Results). (B) Representative chromosomes from cells expressing the N terminus (CAL1-N, aa 1–407) or the C terminus (CAL1-C, aa 700–979) of CAL1 fused to GFP-LacI (both in green). Although the N terminus (top row) recruited CENP-A and CENP-C (95%; n = 41; both shown in red), the C terminus did not recruit either protein (0%; n = 50). Green arrow, endogenous centromere; white arrow, ectopic lacO centromere. (C) Diagram summarizing the CENP-A recruitment efficiency of the various truncations tested. n = the number of GFP-lacO–positive cells counted in one experiment. Each truncation was assessed for CENP-A recruitment efficiency at least twice. (D) Metaphase chromosomes containing the indicated CAL1 truncations fused to GFP-LacI (green) targeted at the lacO array (arrow). CENP-A (red) was recruited weakly by residues 1–100, and more robustly by 1–192, but not by residues 1–50. DAPI is in blue. (E) A CAL1 mutant lacking aa 1–40 fused to GFP-LacI (green) failed to recruit CENP-A (red) at the lacO array (arrow). Bars, 1 µm.