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.