Fission yeast condensin complex: essential roles of non-SMC subunits for condensation and Cdc2 phosphorylation of Cut3/SMC4
Fission yeast condensin complex: essential roles of non-SMC subunits for condensation and Cdc2 phosphorylation of Cut3/SMC4
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DOI：
10.1101/gad.13.17.2271
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发表时间：
1999-09-01
影响因子：
10.5
通讯作者：
Yanagida, M
中科院分区：
文献类型：
--
作者：
Sutani, T;Yuasa, T;Yanagida, M
青蛙提取物中的凝缩蛋白复合体含有两个 SMC（染色体结构维持）和三个非 SMC 亚基，可促进有丝分裂染色体凝缩，并且在有丝分裂过程中通过 Cdc2 磷酸化增强其超螺旋活性。在这里，我们报告裂殖酵母具有相同的五元缩合蛋白复合物，其中每一个对于有丝分裂缩合都是必需的。纯化凝缩蛋白复合物并通过微测序鉴定亚基。 Cnd1、Cnd2 和 Cnd3 这三个非 SMC 亚基与青蛙亚基表现出高度的序列保守性，对于生存能力至关重要，它们的基因破坏导致的表型与 cut3-477 和 cut14-208 中观察到的表型难以区分，而 cut3-477 和 cut14-208 是 SMC4 和 SMC2 样亚基中的已知突变。观察到用 GFP 标记的凝缩蛋白亚基在细胞周期中显着改变其定位，在有丝分裂期间富集在细胞核中，但在其他阶段富集在细胞质中。这种阶段特异性的定位改变需要 Cut3 中 T19 Cdc2 位点的有丝分裂特异性磷酸化。 T19 位点在体外被 Cdc2 激酶磷酸化，并在体内中期显示最大磷酸化。其丙氨酸取代突变体无法抑制 cut3-477 的温度敏感表型，并且显示出缩合缺陷，可能是因为 Cut3 T19A 仍然存在于细胞质中。因此，裂殖酵母凝缩蛋白的直接Cdc2磷酸化可能促进其在有丝分裂过程中的核积累。
The condensin complex in frog extracts, containing two SMC (structural maintenance of chromosomes) and three non-SMC subunits, promotes mitotic chromosome condensation, and its supercoiling activity increases during mitosis by Cdc2 phosphorylation. Here, we report that fission yeast has the same five-member condensin complex, each of which is essential for mitotic condensation. The condensin complex was purified and the subunits were identified by microsequencing. Cnd1, Cnd2, and Cnd3, three non-SMC subunits showing a high degree of sequence conservation to frog subunits, are essential for viability, and their gene disruption leads to a phenotype indistinguishable from that observed in cut3-477 and cut14-208, known mutations in SMC4 and SMC2-like subunits. Condensin subunits tagged with GFP were observed to alter dramatically their localization during the cell cycle, enriched in the nucleus during mitosis, but cytoplasmic during other stages. This stage-specific alteration in localization requires mitosis-specific phosphorylation of the T19 Cdc2 site in Cut3. The T19 site is phosphorylated in vitro by Cdc2 kinase and shows the maximal phosphorylation in metaphase in vivo. Its alanine substitution mutant fails to suppress the temperature-sensitive phenotype of cut3-477, and shows deficiency in condensation, probably because Cut3 T19A remains cytoplasmic. Therefore, direct Cdc2 phosphorylation of fission yeast condensin may facilitate its nuclear accumulation during mitosis.