Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210, USA.
J Cell Biol. 2011 Nov 14;195(4):563-72. doi: 10.1083/jcb.201106078.
The stoichiometries of kinetochores and their constituent proteins in yeast and vertebrate cells were determined using the histone H3 variant CENP-A, known as Cse4 in budding yeast, as a counting standard. One Cse4-containing nucleosome exists in the centromere (CEN) of each chromosome, so it has been assumed that each anaphase CEN/kinetochore cluster contains 32 Cse4 molecules. We report that anaphase CEN clusters instead contained approximately fourfold more Cse4 in Saccharomyces cerevisiae and ~40-fold more CENP-A (Cnp1) in Schizosaccharomyces pombe than predicted. These results suggest that the number of CENP-A molecules exceeds the number of kinetochore-microtubule (MT) attachment sites on each chromosome and that CENP-A is not the sole determinant of kinetochore assembly sites in either yeast. In addition, we show that fission yeast has enough Dam1-DASH complex for ring formation around attached MTs. The results of this study suggest the need for significant revision of existing CEN/kinetochore architectural models.
使用组蛋白 H3 变体 CENP-A（在芽殖酵母中称为 Cse4）作为计数标准，确定了酵母和脊椎动物细胞中的动粒及其组成蛋白的化学计量。每个染色体的着丝粒（CEN）中都存在一个含有 Cse4 的核小体，因此人们一直认为每个后期 CEN/动粒簇包含 32 个 Cse4 分子。我们报告称，在酿酒酵母中，后期 CEN 簇实际上包含的 Cse4 分子数量比预期的多约四倍，在粗糙脉孢菌中则多约 40 倍。这些结果表明，CENP-A 分子的数量超过了每条染色体上动粒微管（MT）附着位点的数量，并且 CENP-A 不是酵母中动粒组装位点的唯一决定因素。此外，我们还表明，裂殖酵母具有足够的 Dam1-DASH 复合物用于围绕附着的 MT 形成环。本研究的结果表明，需要对现有的 CEN/动粒结构模型进行重大修订。
