Department of Biology, University of North Carolina, Chapel Hill, NC 27599, USA.
J Cell Biol. 2011 Nov 14;195(4):573-82. doi: 10.1083/jcb.201106036.
Cse4 is the budding yeast homologue of CENP-A, a modified histone H3 that specifies the base of kinetochores in all eukaryotes. Budding yeast is unique in having only one kinetochore microtubule attachment site per centromere. The centromere is specified by CEN DNA, a sequence-specific binding complex (CBF3), and a Cse4-containing nucleosome. Here we compare the ratio of kinetochore proximal Cse4-GFP fluorescence at anaphase to several standards including purified EGFP molecules in vitro to generate a calibration curve for the copy number of GFP-fusion proteins. Our results yield a mean of ~5 Cse4s, ~3 inner kinetochore CBF3 complexes, and ~20 outer kinetochore Ndc80 complexes. Our calibrated measurements increase 2.5-3-fold protein copy numbers at eukaryotic kinetochores based on previous ratio measurements assuming two Cse4s per budding yeast kinetochore. All approximately five Cse4s may be associated with the CEN nucleosome, but we show that a mean of three Cse4s could be located within flanking nucleosomes at random sites that differ between chromosomes.
Cse4 是芽殖酵母同源物，是一种修饰组蛋白 H3，在所有真核生物中指定动粒的基础。芽殖酵母的独特之处在于每个着丝粒只有一个动粒微管附着位点。着丝粒由 CEN DNA、序列特异性结合复合物 (CBF3) 和含有 Cse4 的核小体指定。在这里，我们将后期动粒近端 Cse4-GFP 荧光与几个标准进行比较，包括体外纯化的 EGFP 分子，以生成 GFP 融合蛋白拷贝数的校准曲线。我们的结果得出的平均值约为 5 个 Cse4、3 个内动粒 CBF3 复合物和约 20 个外动粒 Ndc80 复合物。我们的校准测量结果表明，基于之前假设每个芽殖酵母动粒有两个 Cse4 的比例测量结果，真核动粒的蛋白拷贝数增加了 2.5-3 倍。所有大约 5 个 Cse4 可能与 CEN 核小体相关，但我们表明，3 个 Cse4 的平均值可以位于染色体之间不同的随机位置的侧翼核小体中。
