Condensin phosphorylated by the Aurora-B-like kinase Ark1 is continuously required until telophase in a mode distinct from Top2
Condensin phosphorylated by the Aurora-B-like kinase Ark1 is continuously required until telophase in a mode distinct from Top2
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DOI：
10.1242/jcs.078733
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发表时间：
2011-06-01
影响因子：
4
通讯作者：
Yanagida, Mitsuhiro
中科院分区：
文献类型：
--
作者：
Nakazawa, Norihiko;Mehrotra, Rajesh;Yanagida, Mitsuhiro
凝缩蛋白是一种保守的蛋白质复合物，在染色体凝缩和分离中发挥作用。之前尚未明确确定整个有丝分裂过程中是否需要凝缩蛋白。在这里，我们检查了粟酒裂殖酵母凝缩蛋白在有丝分裂过程中是否持续作用于染色体，并将其作用与 DNA 拓扑异构酶 II (Top2) 进行了比较。使用含有凝缩蛋白 SMC2 亚基 cut14 (cut14-208) 或 top2 的温度敏感等位基因的双突变体，以及冷敏感的 nda3-KM311 突变（在 β-微管蛋白中），进行了温度变化实验。这些实验允许凝缩蛋白或 Top2 在整个有丝分裂的各个阶段失活，甚至在后期后期也是如此。结果表明，有丝分裂染色体在整个有丝分裂过程中（甚至在末期）都需要凝缩蛋白和 Top2。然后我们证明凝缩蛋白（也称为 Barren）的 Cnd2 亚基是 Aurora-B 样激酶 Ark1 的靶亚基，并且 Ark1 介导的 Cnd2 磷酸化发生在整个有丝分裂过程中。通过质谱法测定 Cnd2 中的磷酸化位点，并构建了这些位点的丙氨酸和谷氨酸残基置换突变体构建体。 Cnd2 的丙氨酸取代突变体模仿未磷酸化的蛋白质，表现出广泛的有丝分裂缺陷，包括在末期，并且这些构建体的过度表达导致严重的显性失活效应。相比之下，模仿磷酸化蛋白的谷氨酸取代突变体减轻了 Ark1 抑制细胞中的分离缺陷。在末期，cut14-208突变体中的凝缩蛋白亚基聚集成块，其中含有未能分离的端粒DNA和蛋白质。因此，凝缩蛋白可能有助于在末期保持分离的染色体分开。
Condensin is a conserved protein complex that functions in chromosome condensation and segregation. It has not been previously unequivocally determined whether condensin is required throughout mitosis. Here, we examined whether Schizosaccharomyces pombe condensin continuously acts on chromosomes during mitosis and compared its role with that of DNA topoisomerase II (Top2). Using double mutants containing a temperature-sensitive allele of the condensin SMC2 subunit cut14 (cut14-208) or of top2, together with the cold-sensitive nda3-KM311 mutation (in beta-tubulin), temperature-shift experiments were performed. These experiments allowed inactivation of condensin or Top2 at various stages throughout mitosis, even after late anaphase. The results established that mitotic chromosomes require condensin and Top2 throughout mitosis, even in telophase. We then showed that the Cnd2 subunit of condensin (also known as Barren) is the target subunit of Aurora-B-like kinase Ark1 and that Ark1-mediated phosphorylation of Cnd2 occurred throughout mitosis. The phosphorylation sites in Cnd2 were determined by mass spectrometry, and alanine and glutamate residue replacement mutant constructs for these sites were constructed. Alanine substitution mutants of Cnd2, which mimic the unphosphorylated protein, exhibited broad mitotic defects, including at telophase, and overexpression of these constructs caused a severe dominant-negative effect. By contrast, glutamate substitution mutants, which mimic the phosphorylated protein, alleviated the segregation defect in Ark1-inhibited cells. In telophase, the condensin subunits in cut14-208 mutant accumulated in lumps that contained telomeric DNA and proteins that failed to segregate. Condensin might thus serve to keep the segregated chromosomes apart during telophase.