Wigge P A, Kilmartin J V
Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom.
J Cell Biol. 2001 Jan 22;152(2):349-60. doi: 10.1083/jcb.152.2.349.
We have purified a complex from Saccharomyces cerevisiae containing the spindle components Ndc80p, Nuf2p, Spc25p, and Spc24p. Temperature-sensitive mutants in NDC80, SPC25, and SPC24 show defects in chromosome segregation. In spc24-1 cells, green fluorescence protein (GFP)-labeled centromeres fail to split during spindle elongation, and in addition some centromeres may detach from the spindle. Chromatin immunoprecipitation assays show an association of all four components of the complex with the yeast centromere. Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere. A human homologue of Nuf2p was identified in the expressed sequence tag database. Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells. Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.
我们从酿酒酵母中纯化出了一种包含纺锤体组件Ndc80p、Nuf2p、Spc25p和Spc24p的复合物。NDC80、SPC25和SPC24中的温度敏感突变体在染色体分离方面表现出缺陷。在spc24 - 1细胞中,绿色荧光蛋白(GFP)标记的着丝粒在纺锤体伸长过程中无法分开,此外一些着丝粒可能会从纺锤体上脱离。染色质免疫沉淀分析表明该复合物的所有四个组件都与酵母着丝粒相关。在粟酒裂殖酵母中发现了Ndc80p、Nuf2p和Spc24p的同源物,GFP标记显示它们位于着丝粒处。在表达序列标签数据库中鉴定出了Nuf2p的人类同源物。用抗人类Nuf2p和抗HEC(Ndc80p的人类同源物)进行免疫荧光染色表明,这两种蛋白质都存在于有丝分裂的HeLa细胞的着丝粒处。因此,Ndc80p复合物包含酵母和脊椎动物之间保守的着丝粒相关组件。