Chandra R, Salmon E D, Erickson H P, Lockhart A, Endow S A
Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710.
J Biol Chem. 1993 Apr 25;268(12):9005-13.
Nonclaret disjunctional (ncd) is a kinesin-related microtubule motor protein that is required for proper chromosome distribution in Drosophila. Despite its sequence similarity to kinesin heavy chain, ncd translocates with the opposite polarity as kinesin, toward microtubule minus ends. We have expressed different regions of the protein in bacteria and analyzed the proteins for function. Results indicate that ncd consists of three domains: a basic, proline-rich N-terminal "tail," a central alpha-helical coiled-coil stalk, and a C-terminal motor domain. The ncd N terminus proteins bundle microtubules in motility assays and show ATP-independent binding to microtubules in solution. Truncated proteins, lacking the tail but containing the predicted motor domain and differing lengths of the stalk, did not support microtubule gliding in in vitro assays but showed microtubule-stimulated MgATPase activity in solution. Addition of a nonspecific N terminus to two of the truncated proteins restored directional gliding and rotation of microtubules in motility assays, demonstrating that these properties map to the predicted mechanochemical domain of ncd. Physical properties of the C terminus proteins indicate that the stalk region is important for dimerization and that the ncd protein probably exists and functions as a dimer.
非红粒不分离（ncd）是一种与驱动蛋白相关的微管运动蛋白，在果蝇中，它是染色体正确分布所必需的。尽管其序列与驱动蛋白重链相似，但ncd与驱动蛋白的极性相反，向微管负端移动。我们已在细菌中表达了该蛋白的不同区域，并对这些蛋白的功能进行了分析。结果表明，ncd由三个结构域组成：一个碱性的、富含脯氨酸的N端“尾部”，一个中央α螺旋卷曲螺旋茎部，以及一个C端运动结构域。在运动分析中，ncd N端蛋白使微管成束，并在溶液中显示出与微管的ATP非依赖性结合。截短的蛋白，缺少尾部但含有预测的运动结构域和不同长度的茎部，在体外分析中不支持微管滑动，但在溶液中显示出微管刺激的MgATP酶活性。在其中两个截短蛋白上添加一个非特异性N端，在运动分析中恢复了微管的定向滑动和旋转，表明这些特性位于ncd预测的机械化学结构域。C端蛋白的物理特性表明，茎部区域对二聚化很重要，并且ncd蛋白可能以二聚体形式存在并发挥功能。
