Recognition of the Centromeric Nucleosome By the Kinetochore in Budding Yeast

Abstract

Kinetochores mediate chromosome segregation during cell division. They assemble on centromeric nucleosomes and capture spindle microtubules. In budding yeast, a kinetochore links a single nucleosome, containing the histone variant Cse4 instead of H3, with a single microtubule. Conservation of most kinetochore components from yeast to metazoans suggests that the yeast kinetochore represents a module of the more complex metazoan arrangements, with multiple centromeric nucleosomes and multiple microtubules. I investigate here how the inner kinetochore attaches to the centromeric nucleosome. First, I describe an improved protocol for coexpression of soluble histone octamer that greatly facilitates sample preparation. My results establish bacterial coexpression as a convenient method to express large amounts of yeast centromeric histone octamer for structural studies. Next, I investigate the interaction of Mif2 with this Cse4-containing nucleosome using biochemistry and cryo-EM. I find that Mif2 binds the Cse4 nucleosome with a 2:1 stoichiometry just as human CENP-C binds the CENP-A nucleosome. Finally, I report an exploration of cryo-grid preparation and high-resolution structure of the yeast centromeric nucleosome determined by cryo-EM. Fewer base pairs are in tight association with the histone octamer than there are in canonical nucleosomes. Weak binding of the end sequences may contribute to specific recognition by other inner kinetochore components. The coexpression protocol for histone octamer, centromeric nucleosome structure and grid preparation strategies we describe will facilitate studies of many other aspects of kinetochore assembly and chromatin biochemistry.