Binding of the Fkh1 Forkhead Associated Domain to a Phosphopeptide within the Mph1 DNA Helicase Regulates Mating-Type Switching in Budding Yeast.
Binding of the Fkh1 Forkhead Associated Domain to a Phosphopeptide within the Mph1 DNA Helicase Regulates Mating-Type Switching in Budding Yeast.
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DOI：
10.1371/journal.pgen.1006094
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发表时间：
2016-06
期刊：
影响因子：
4.5
通讯作者：
Fox CA
中科院分区：
文献类型：
--
作者：
Dummer AM;Su Z;Cherney R;Choi K;Denu J;Zhao X;Fox CA
酿酒酵母 Fkh1 蛋白在细胞周期调节转录中发挥作用，并在交配类型转换期间的重组供体偏好中发挥非转录依赖性作用。 Fkh1 的保守 FHA 结构域通过将 III 号染色体上两个相距较远的区域并列来促进它们的重组来调节供体偏好。模型假设这种 Fkh1 介导的长程染色体并置需要 FHA 结构域和伴侣蛋白之间的相互作用，但迄今为止尚未描述相关的伴侣。在这项研究中，我们使用结构建模、2-杂交测定和突变分析来表明 Fkh1 的预测磷酸苏氨酸结合 FHA 结构域与多个伙伴蛋白相互作用。 Fkh1 FHA 结构域在细胞周期调节中发挥着重要作用，但没有单一的相互作用伙伴可以解释这一作用。相比之下，Fkh1 与 Mph1 DNA 修复解旋酶的相互作用调节交配类型转换期间的供体偏好。使用 2 杂交分析、免疫共沉淀和荧光各向异性，我们在这种相互作用所需的调节 Mph1 C 末端内绘制了一个离散肽，并鉴定了两个特别重要的苏氨酸。体外结合实验表明，这些苏氨酸中的至少一个必须被磷酸化才能有效地结合 Fkh1。用丙氨酸 (mph1-2TA) 取代这两个苏氨酸可特异性消除体内 Fkh1-Mph1 相互作用，并在交配类型转换过程中将供体偏好改变至与 mph1Δ 相同的程度。值得注意的是，mph1-2TA 等位基因在基因组稳定性方面保留了 Mph1 的其他功能。删除 YMR144W 编码的第二个 Fkh1 相互作用蛋白也会导致 Fkh1-FHA 依赖性供体偏好发生变化。我们将此基因命名为 FDO1，代表 Forkhead 一种参与供体偏好的相互作用蛋白。我们得出的结论是，Fkh1-FHA 和 Mph1 之间的磷酸苏氨酸介导的蛋白质-蛋白质界面有助于交配类型转换所需的特定长程染色体相互作用，但 Fkh1-FHA 还必须与其他几种蛋白质相互作用才能在此过程中实现全部功能。基因组远端区域之间的特定染色体相互作用允许正常细胞功能所需的DNA交换，但调节这种相互作用的蛋白质-蛋白质界面仍然很大程度上未知。芽殖酵母 Fkh1 蛋白利用其进化上保守的磷酸苏氨酸结合 FHA 结构域来调节称为交配型转换的长程 DNA 转换，使酵母细胞能够转换其性表型。在这项研究中，另一种保守的核蛋白 Mph1 DNA 修复解旋酶被证明可以直接与 Fkh1 的 FHA 结构域相互作用来调节交配类型转换。 Fkh1-Mph1 相互作用需要 Mph1 上的两个磷酸化苏氨酸，这对于许多其他 Mph1-蛋白质相互作用和其他 Mph1 染色体功能来说是可有可无的。因此，两个多功能染色体蛋白之间的离散蛋白质-蛋白质界面有助于定义对控制细胞行为很重要的长程染色体相互作用。
The Saccharomyces cerevisiae Fkh1 protein has roles in cell-cycle regulated transcription as well as a transcription-independent role in recombination donor preference during mating-type switching. The conserved FHA domain of Fkh1 regulates donor preference by juxtaposing two distant regions on chromosome III to promote their recombination. A model posits that this Fkh1-mediated long-range chromosomal juxtaposition requires an interaction between the FHA domain and a partner protein(s), but to date no relevant partner has been described. In this study, we used structural modeling, 2-hybrid assays, and mutational analyses to show that the predicted phosphothreonine-binding FHA domain of Fkh1 interacted with multiple partner proteins. The Fkh1 FHA domain was important for its role in cell-cycle regulation, but no single interaction partner could account for this role. In contrast, Fkh1’s interaction with the Mph1 DNA repair helicase regulated donor preference during mating-type switching. Using 2-hybrid assays, co-immunoprecipitation, and fluorescence anisotropy, we mapped a discrete peptide within the regulatory Mph1 C-terminus required for this interaction and identified two threonines that were particularly important. In vitro binding experiments indicated that at least one of these threonines had to be phosphorylated for efficient Fkh1 binding. Substitution of these two threonines with alanines (mph1-2TA) specifically abolished the Fkh1-Mph1 interaction in vivo and altered donor preference during mating-type switching to the same degree as mph1Δ. Notably, the mph1-2TA allele maintained other functions of Mph1 in genome stability. Deletion of a second Fkh1-interacting protein encoded by YMR144W also resulted in a change in Fkh1-FHA-dependent donor preference. We have named this gene FDO1 for Forkhead one interacting protein involved in donor preference. We conclude that a phosphothreonine-mediated protein-protein interface between Fkh1-FHA and Mph1 contributes to a specific long-range chromosomal interaction required for mating-type switching, but that Fkh1-FHA must also interact with several other proteins to achieve full functionality in this process. Specific chromosomal interactions between distal regions of the genome allow for DNA transactions necessary for normal cell function, but the protein-protein interfaces that regulate such interactions remain largely unknown. The budding yeast Fkh1 protein uses its evolutionarily conserved phosphothreonine-binding FHA domain to regulate a long-range DNA transaction called mating-type switching that allows yeast cells to switch their sexual phenotype. In this study, another conserved nuclear protein, the Mph1 DNA repair helicase, was shown to interact directly with the FHA domain of Fkh1 to regulate mating-type switching. The Fkh1-Mph1 interaction required two phosphorylated threonines on Mph1 that were dispensable for many other Mph1-protein interactions and other Mph1 chromosomal functions. Thus a discrete protein-protein interface between two multifunctional chromosomal proteins helps define a long-range chromosomal interaction important for controlling cell behavior.