Importance of the monomer-dimer-tetramer interconversion of the clock protein KaiB in the generation of circadian oscillations in cyanobacteria
Importance of the monomer-dimer-tetramer interconversion of the clock protein KaiB in the generation of circadian oscillations in cyanobacteria
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DOI：
10.1111/gtc.12211
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发表时间：
2015-03-01
期刊：
影响因子：
2.1
通讯作者：
Ishiura, Masahiro
中科院分区：
文献类型：
--
作者：
Iida, Takahiro;Mutoh, Risa;Ishiura, Masahiro
蓝藻生物钟振荡器的分子机制由三种蛋白质KaiA、KaiB和KaiC组成，它们在ATP的存在下相互作用以产生生物钟振荡（体外KaiABC时钟振荡器）。KaiB包含四个亚基，其组织为二聚体的二聚体。我们先前的研究表明，在与KaiC的相互作用中，四聚体KaiB分子解离成两个二聚体KaiB分子。KaiB是否也作为单体存在以及KaiB单体是否可以驱动正常的昼夜节律振荡尚不确定。为了解决这些问题，我们构建了具有N-末端缺失的新KaiB寡聚体突变体KaiB（10-108）。KaiB（10-108）在4 ℃时为单体，但在35 ℃时为二聚体。KaiB（10-108）能够在25 ℃下在体外重建的KaiABC时钟振荡器中驱动正常的时钟振荡，但它不能在41 ℃下驱动蓝藻细胞中的正常昼夜基因表达节律。野生型KaiB存在于二聚体和四聚体之间的平衡，在较低的KaiB浓度或在1 m NaCl的存在下。我们的研究结果表明，KaiB是在蓝藻细胞中的单体，二聚体和四聚体之间的平衡。
The molecular machinery of the cyanobacterial circadian clock oscillator consists of three proteins, KaiA, KaiB and KaiC, which interact with each other to generate circadian oscillations in the presence of ATP (the in vitro KaiABC clock oscillator). KaiB comprises four subunits organized as a dimer of dimers. Our previous study suggested that, on interaction with KaiC, the tetrameric KaiB molecule dissociates into two molecules of dimeric KaiB. It is uncertain whether KaiB also exists as a monomer and whether the KaiB monomer can drive normal circadian oscillation. To address these questions, we constructed a new KaiB oligomer mutant with an N-terminal deletion, KaiB(10-108). KaiB(10-108) was a monomer at 4 degrees C but a dimer at 35 degrees C. KaiB(10-108) was able to drive normal clock oscillation in an in vitro reconstituted KaiABC clock oscillator at 25 degrees C, but it was not able to drive normal circadian gene expression rhythms in cyanobacterial cells at 41 degrees C. Wild-type KaiB existed in equilibrium between a dimer and tetramer at lower KaiB concentrations or in the presence of 1m NaCl. Our findings suggest that KaiB is in equilibrium between a monomer, dimer and tetramer in cyanobacterial cells.