Interaction of yeast kinetochore proteins with centromere-protein/transcription factor Cbf1
Interaction of yeast kinetochore proteins with centromere-protein/transcription factor Cbf1
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DOI：
10.1073/pnas.97.23.12583
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发表时间：
2000-11-07
影响因子：
11.1
通讯作者：
Diekmann, S
中科院分区：
文献类型：
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作者：
Hemmerich, P;Stoyan, T;Diekmann, S
酿酒酵母的着丝粒-动粒复合体是一种特殊的染色体亚结构，通过蛋白质-DNA和蛋白质-蛋白质相互作用的调节网络介导复制的染色体附着到有丝分裂纺锤体。我们已经使用体外试验来分析酵母着丝粒-动粒复合物组分之间的假定分子相互作用。谷胱甘肽S-转移酶下拉实验表明，在体外翻译的p110，p64，和p58的基本CBF3动粒蛋白复合物与Cbf1p，一个碱性区域螺旋-环-螺旋拉链蛋白（bHLHzip），特异性结合到着丝粒DNA上的CDEI区域的直接相互作用。此外，重组p64和p23各自刺激Cbf1p的体外DNA结合活性。p23的N-末端70个氨基酸足以介导这种作用。在着丝粒DNA存在的情况下，P64也能促进Cbf 1p的多聚化活性。这些结果显示了Cbf1p和CBF3亚基的直接物理相互作用，并提供证据表明CBF3组分可以促进Cbf1p与其在酵母动粒中的结合位点的结合。着丝粒结合蛋白与MET 16启动子结合的转录因子的功能比较揭示了着丝粒和MET 16启动子之间的强烈相似性。
The centromere-kinetochore complex of Saccharomyces cerevisiae is a specialized chromosomal substructure that mediates attachment of duplicated chromosomes to the mitotic spindle by a regulated network of protein-DNA and protein-protein interactions. We have used in vitro assays to analyze putative molecular interactions between components of the yeast centromere-kinetochore complex. Glutathione S-transferase pull-down experiments showed the direct interaction of in vitro translated p110, p64, and p58 of the essential CBF3 kinetochore protein complex with Cbf1p, a basic region helix-loop-helix zipper protein (bHLHzip) that specifically binds to the CDEI region on the centromere DNA. Furthermore, recombinant p64 and p23 each stimulated the in vitro DNA binding activity of Cbf1p. The N-terminal 70 amino acids of p23 were sufficient to mediate this effect. P64 could also promote the multimerization activity of Cbf1p in the presence of centromere DNA in vitro. These results show the direct physical interaction of Cbf1p and CBF3 subunits and provide evidence that CBF3 components can promote the binding of Cbf1p to its binding site in the yeast kinetochore. A functional comparison of the centromere binding proteins with transcription factors binding at MET16 promoters reveals the strong analogy between centromeres and the MET16 promoter.