Wilkerson C G, King S M, Koutoulis A, Pazour G J, Witman G B
Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
J Cell Biol. 1995 Apr;129(1):169-78. doi: 10.1083/jcb.129.1.169.
We have isolated and sequenced a full-length cDNA clone encoding the 78,000 Mr intermediate chain (IC78) of the Chlamydomonas outer arm dynein. This protein previously was shown to be located at the base of the solubilized dynein particle and to interact with alpha tubulin in situ, suggesting that it may be involved in binding the outer arm to the doublet microtubule. The sequence predicts a polypeptide of 683 amino acids having a mass of 76.5 kD. Sequence comparison indicates that IC78 is homologous to the 69,000 M(r) intermediate chain (IC69) of Chlamydomonas outer arm dynein and to the 74,000 M(r) intermediate chain (IC74) of cytoplasmic dynein. The similarity between the chains is greatest in their COOH-terminal halves; the NH(2)-terminal halves are highly divergent. The COOH-terminal half of IC78 contains six short imperfect repeats, termed WD repeats, that are thought to be involved in protein-protein interactions. Although not previously reported, these repeated elements also are present in IC69 and IC74. Using the IC78 cDNA as a probe, we screened a group of slow-swimming insertional mutants and identified one which has a large insertion in the IC78 gene and seven in which the IC78 gene is completely deleted. Electron microscopy of three of these IC78 mutants revealed that each is missing the outer arm, indicating that IC78 is essential for arm assembly or attachment to the outer doublet. Restriction fragment length polymorphism mapping places the IC78 gene on the left arm of chromosome XII/XIII, at or near the mutation oda9, which also causes loss of the outer arm. Mutants with defects in the IC78 gene do not complement the oda9 mutation in stable diploids, strongly suggesting that ODA9 is the structural gene for IC78.
我们已经分离并测序了一个全长cDNA克隆,它编码衣藻外臂动力蛋白的78,000 Mr中间链(IC78)。先前已表明该蛋白位于可溶的动力蛋白颗粒基部,并在原位与α微管蛋白相互作用,这表明它可能参与将外臂与双联微管结合。该序列预测了一个由683个氨基酸组成的多肽,质量为76.5 kD。序列比较表明,IC78与衣藻外臂动力蛋白的69,000 M(r)中间链(IC69)以及胞质动力蛋白的74,000 M(r)中间链(IC74)同源。这些链之间的相似性在其COOH末端的一半中最大;NH(2)末端的一半高度不同。IC78的COOH末端一半包含六个短的不完全重复序列,称为WD重复序列,被认为参与蛋白质-蛋白质相互作用。尽管以前没有报道过,但这些重复元件也存在于IC69和IC74中。使用IC78 cDNA作为探针,我们筛选了一组游动缓慢的插入突变体,并鉴定出一个在IC78基因中有大插入的突变体和七个IC78基因被完全删除的突变体。对其中三个IC78突变体的电子显微镜观察表明,每个突变体都缺少外臂,这表明IC78对于臂的组装或与外双联微管的附着至关重要。限制性片段长度多态性图谱将IC78基因定位在染色体XII/XIII的左臂上,位于oda9突变处或其附近,oda9突变也会导致外臂缺失。IC78基因有缺陷的突变体在稳定的二倍体中不能互补oda9突变,这强烈表明ODA9是IC78的结构基因。