Enzymatic activity and motility of recombinant Arabidopsis myosin XI, MYA1
Enzymatic activity and motility of recombinant Arabidopsis myosin XI, MYA1
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DOI：
10.1093/pcp/pcm054
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发表时间：
2007-06-01
影响因子：
4.9
通讯作者：
Yamamoto, Keiichi
中科院分区：
文献类型：
--
作者：
Hachikubo, You;Ito, Kohji;Yamamoto, Keiichi
我们表达了重组拟南芥肌球蛋白XI（MYA 1），其中MYA 1的马达结构域连接到由网骨藻α-辅肌动蛋白的三重螺旋重复序列组成的人工杠杆臂，以了解其马达活性和细胞内功能。在25 ℃时，重组MYA 1的肌动蛋白激活的Mg ~（2+）ATP酶活性的Vmax和K-actin分别为50.7 Pi头（-1）s（-1）和30.2 μ M。在体外运动实验中，重组MYA 1在25 ℃时能以1.8 μ m s（-1）的最大速度转位肌动蛋白丝。如果我们考虑杠杆臂长度的差异，则该值对应于天然MYA 1的3.2 μ m/s（-1）的运动性，并且该值非常接近拟南芥下胚轴表皮细胞中细胞质流动的速度。ADP对MYA 1运动的抑制程度与众所周知的进行性运动肌球蛋白V相似，表明MYA 1是一种进行性运动。ATP诱导的肌动蛋白-MYA 1-ADP复合物的解离速率（73.5 s（-1））和肌动蛋白激活的Mg ~（2+）ATP酶活性的Vmax值表明，MYA 1在其机械化学循环时间的70%左右处于肌动蛋白结合状态。肌动蛋白结合状态的高比率也是进行性运动的特征。我们的研究结果强烈表明，MYA 1是一个进行性电机和参与囊泡运输和/或细胞质流。
We expressed recombinant Arabidopsis myosin XI (MYA1), in which the motor domain of MYA1 was connected to an artificial lever arm composed of triple helical repeats of Dictyostelium alpha-actinin, in order to understand its motor activity and intracellular function. The V-max and K-actin of the actin-activated Mg2+ ATPase activity of the recombinant MYA1 were 50.7 Pi head(-1) s(-1) and 30.2 mu M, respectively, at 25 degrees C. The recombinant MYA1 could translocate actin filament at the maximum velocity of 1.8 mu m s(-1) at 25 degrees C in the in vitro motility assay. The value corresponded to a motility of 3.2 mu m s(-1) for native MYA1 if we consider the difference in the lever arm length, and this value was very close to the velocity of cytoplasmic streaming in Arabidopsis hypocotyl epidermal cells. The extent of inhibition by ADP of the motility of MYA1 was similar to that of the well-known processive motor, myosin V, suggesting that MYA1 is a processive motor. The dissociation rate of the actin-MYA1-ADP complex induced by ATP (73.5 s(-1)) and the V-max value of the actin-activated Mg2+ ATPase activity revealed that MYA1 stays in the actin-bound state for about 70% of its mechanochemical cycle time. This high ratio of actin-bound states is also a characteristic of processive motors. Our results strongly suggest that MYA1 is a processive motor and involved in vesicle transport and/or cytoplasmic streaming.