MOLECULAR ANALYSIS OF FLAGELLAR DYNEIN FUNCTION

动力蛋白是一种基于微管的分子马达，参与多种生物学过程。 细胞功能：例如精子运动和胞内囊泡 运输 来自衣原体鞭毛的动力蛋白特别有用 作为一个模型系统，因为它们可以在生物化学和分子生物学的 遗传分析 外动力蛋白臂由3条重链组成（更大）， 小于或等于500 kDa），2条69和78 kDa的中间链（IC 69和 IC 78）和10条轻链。 重链包含以下运动活性： 复杂的。 这些IC彼此相关，并且与 细胞质动力蛋白 先前的研究提出了一种可能性， 这些IC成分参与调节手臂活动， 动力蛋白马达与适当货物的结合（在这种情况下， 鞭毛动力蛋白是另一种微管）。 拟议的研究计划 有三个相关的具体目标。 首先，IC 78的区域， 参与结合微管将被定义为定点 诱变和体外结合测定。 其次，职能作用 这些结构域在外臂动力蛋白的靶向中的作用将在 通过将改变的基因转化到衣原体菌株中， 该动力蛋白成分为真空。 详细了解如何 实现微管结合还需要识别区域 tubulin相关。 第二个具体目标是确定IC 78 微管蛋白上的结合位点，使用体外测定来检查 在体外合成的突变微管蛋白或使用 噬菌体展示技术。 该分析最终将扩展为使用 这里产生的试剂用于鉴定参与微管蛋白区域， 与其他微管相关蛋白结合，包括马达 动力蛋白和驱动蛋白。 最后，还有分子遗传学， 生物化学证据表明，动力蛋白IC可能参与调节 外臂活性，并且一个IC可以含有核苷酸结合位点。 因此，本项目的第三个具体目标是确定这一领域 使用分子生物学和/或蛋白质化学在体外IC 78内 接近。 这个域的功能意义将是 通过将改变的基因转化到IC 78-nullo中在体内测定。 背景 总之，这些研究将提供深入了解 动力蛋白IC在动力蛋白复合物靶向其 货物，在运动活动的调节，也将提供洞察力 进入微管蛋白的区域，参与介导结构和 机械化学相互作用

Dyneins are microtuble-based molecular motors involved in a wide variety of cellular functions: e.g. sperm motility and intracellular vesicle transport. Dyneins from the Chlamydomonas flagellum are especially useful as a model system as they are amenable to both biochemical and molecular genetic analysis. The outer dynein arm consists of 3 heavy chains (greater than or equal to 500 kDa), 2 intermediate chains of 69 and 78 kDa (IC69 and IC78) and 10 light chains. The heavy chains contain the motor activity of the complex. The ICs are related to each other and to the ICs of cytoplasmic dynein. Previous studies have raised the possibility that these IC components are involved in the regulation of arm activity and in the binding of the dynein motor to the appropriate cargo (which in the case of a flagellar dynein is another microtubule). Thr proposed research plan has three related specific aims. In the first, the regions of IC78 that are involved in binding to microtubules will be defined by site-directed mutagenesis and in vitro binding assays. Subsequently, the functional role of these domains in the targeting of outer arm dynein will be assessed in vivo by transformation of altered genes into Chlamydomonas strains that are true nulls for this dynein component. Detailed understanding of how microtubule-binding is achieved also requires identification of the regions of tubulin involved. The second specific aim is to determine the IC78 binding sites on tubulin using in vitro assays to examine the binding of mutant tubulins synthesized in vitro or of tubulin segments expressed using phage display technology. This analysis ultimately will be extended using the reagents generated here to identify the tubulin regions involved in binding to other microtubule-associated proteins including the motor domains of dynein and kinesin. Finally, there is molecular genetic and biochemical evidence that dynein ICs may be involved in the regulation of outer arm activity and that one IC may contain a nucleotide binding site. Thus, the third specific aim of this project is to identify this domain within IC78 in vitro using molecular biological and/or protein chemistry approaches. The functional significance of this domain then will be determined in vivo by transforation of altered genes into the IC78-nullo background. In summary, these studies will provide insight into the functional roles of dynein ICs in targeting of the dynein complex to its cargo, in the regulation of motor activity and also will provide insight into the regions of tubulin involved in mediating both structural and mechanochemical interactions.