(A) Multiple sequence alignment of Cse4CENP-A proteins. Yeast protein sequences with the highest similarities to S. cerevisiae Cse4, three mammalian and the S. pombe homologous CENP-A protein sequences were included in the alignment. The amino acid (aa) patch, conserved in interrelated yeasts, is highlighted in pink (S. cerevisiae Cse4 aa 34–61). The RG motif in the mammalian sequences is indicated by arrowheads. Amino acid residues are colored and annotated according to the ClustalW color and annotation codes (S.: Schizosaccharomyces, C.: Candida, Z.: Zygosaccharomyces, L.: Lachancea). Residues that are identical among aligned protein sequences (*), conserved substitutions (:), and semiconserved substitutions (.) are indicated. (B) Scheme of the deletion mutants within the Cse4 N-terminus used in the SEC experiments in (C) and (D) and in the cell viability assays in (E). The conserved region (aa 34–61) is highlighted in pink. (C) SEC analysis of the indicated mixtures of recombinant Ame1/Okp1 (AO) and MTW1c and reconstituted H3-, Cse4-, Cse4Δ2–30- or Cse4Δ31–60-NCPs. Ame1/Okp1, MTW1c and the Cse4 proteins were mixed equimolar. Eluted proteins were visualized by SDS-PAGE and Coomassie staining. (D) SEC analysis of Ame1/Okp1 (AO) preincubated with Cse4Δ34–46- or Cse4Δ48–61-NCPs. Eluted complexes were analyzed by SDS-PAGE and Coomassie staining. (E) Left panel: Cell growth assay of Cse4 mutants in budding yeast using the anchor-away system. The Cse4 wild-type and indicated mutant proteins were ectopically expressed in a Cse4 anchor-away strain (Cse4-FRB) and cell growth was monitored by plating 1:10 serial dilutions on YPD medium at 30°C in the absence or presence of 1 µg/ml rapamycin. Right panel: Western blot analysis of the ectopically expressed Cse4 wild-type and mutant protein levels in the yeast strains shown on the left. Pgk1 levels are shown as loading control.