MOLECULAR ANALYSIS OF FLAGELLAR DYNEIN FUNCTION

动力蛋白是一种基于微管的分子马达，涉及多种 细胞功能：如精子活力和细胞内小泡 运输。鞭毛衣藻中的动力蛋白尤其有用 作为一个模型系统，因为它们对生物化学和分子都是适用的 基因分析。动力蛋白外臂由3条重链组成(更大 大于或等于500 kDa)，2条69和78 kDa的中间链(IC69和 IC78)和10个轻链。重链包含了运动的活动 这个建筑群。这些IC彼此相关，并且与 胞质动力蛋白。以前的研究提出了一种可能性，即 这些IC元件参与调节ARM的活动和在 动力蛋白马达与适当货物的结合(在这种情况下 鞭毛动力蛋白的另一个微管)。建议的研究计划 有三个相关的具体目标。在第一阶段，IC78的区域 参与与微管的结合将由位点定向定义 诱变和体外结合试验。随后，职能作用 在外臂动力蛋白靶向的这些结构域中，将在 通过将改变的基因转化到衣藻属菌株而实现活体 此dynein组件的True Null。详细了解如何 实现微管结合还需要识别区域 牵涉到微管蛋白。第二个具体目标是确定IC78 微管蛋白结合部位的体外检测 体外合成的突变型微管蛋白或表达的微管蛋白片段 噬菌体展示技术。此分析最终将扩展为使用 在这里产生的试剂用于识别涉及到的微管蛋白区域 与包括马达在内的其他微管相关蛋白结合 动力蛋白和动蛋白的结构域。最后，还有分子遗传和 动力蛋白ICs可能参与调控的生化证据 外臂活性，并且一个IC可能包含一个核苷酸结合部位。 因此，这个项目的第三个具体目标是确定这个领域 用分子生物学和/或蛋白质化学在IC78中进行体外实验 接近了。那么，这个领域的功能意义将是 通过将改变的基因导入IC78-nullo在体内进行测定 背景资料。总而言之，这些研究将为我们提供对 动力蛋白ICs在动力蛋白复合体靶向其功能中的作用 货物，在调节马达活动方面也将提供洞察力 进入到微管蛋白的区域，这些微管蛋白参与调节结构和 机械力化学相互作用。

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.