The Budding Yeast Point Centromere Associates with Two Cse4 Molecules during Mitosis

•Models of kinetochore function require accurate copy number of subunit proteins•Cse4 molecules per centromere determined with BiFC and stepwise GFP photobleaching•The budding yeast centromere binds two Cse4 molecules from S phase to anaphase•Cse4-binding capacity is fixed at point centromere, plastic at regional centromere

SummaryThe centromere is defined by the incorporation of the centromere-specific histone H3 variant centromere protein A (CENP-A). Like histone H3, CENP-A can form CENP-A-H4 heterotetramers in vitro [1]. However, the in vivo conformation of CENP-A chromatin has been proposed by different studies as hemisomes, canonical, or heterotypic nucleosomes [2, 3, 4, 5, 6, 7 and 8]. A clear understanding of the in vivo architecture of CENP-A chromatin is important, because it influences the molecular mechanisms of the assembly and maintenance of the centromere and its function in kinetochore nucleation. A key determinant of this architecture is the number of CENP-A molecules bound to the centromere. Accurate measurement of this number can limit possible centromere architectures. The genetically defined point centromere in the budding yeast Saccharomyces cerevisiae provides a unique opportunity to define this number accurately, as this 120-bp-long centromere can at the most form one nucleosome or hemisome. Using novel live-cell fluorescence microscopy assays, we demonstrate that the budding yeast centromere recruits two Cse4 (ScCENP-A) molecules. These molecules are deposited during S phase and they remain stably bound through late anaphase. Our studies suggest that the budding yeast centromere incorporates a Cse4-H4 tetramer.