CYTOPLASMIC DYNEIN LIGHT CHAIN STRUCTURE AND FUNCTION

细胞质动力蛋白是一种多亚基酶复合物，它将化学能转化为沿微管运动。它在内质网和高尔基复合体之间的运输以及膜细胞器的运输中起着广泛的作用。这种转运对于染色体的排列和分离、产生和维持细胞器的结构和位置以及促进细胞间物质的转移都是必不可少的。装配成功能马达的机制尚不清楚。此外，动力蛋白的任何亚基都没有高分辨率的结构。一个10 kDa的亚基，这里被称为DLC，在所有已知的动力蛋白中高度保守，在果蝇和人类之间具有94%的序列同源性，也是肌球蛋白v的一个组成部分。高度保守性表明该特定蛋白在各种生物体的运动复合物中具有共同功能。10 kDa亚基在几个细胞过程中被发现是必不可少的：果蝇的胚胎发育和卵发生，真菌的核迁移，血吸虫被盖的形成和维持，神经元一氧化氮合酶在轴突上的运输，以及病毒感染期间的转录调节。这些不同功能的机制尚不清楚。该项目的目的是确定DLC的结构特征如何有助于其多重调节作用。共价交联实验表明，这种多肽以二聚体结构存在于动力蛋白复合物内，并与另外两种动力蛋白成分结合。本文提出的实验将集中于利用核磁共振波谱技术来识别二聚体界面，二聚体的高分辨率结构和动力学，以及与蛋白质相互作用的潜在位点-在动力蛋白复合物内和与货物。未来的研究可能会集中在二聚化在体内复合物组装中的作用，动力蛋白其他亚基的表征，以及它们与DLC的相互作用。DLC的高分辨率结构不仅可以为其具体和各种功能提供线索，更重要的是可以为动力蛋白系统的结构表征提供途径。

Cytoplasmic dynein is a multisubunit enzyme complex that transforms chemical energy into motion along microtubules. It plays a wide range of roles in traffic between the endoplasmic reticulum and the Golgi complex and in the transport of membranous organelles. Such transport is essential for chromosome alignment and segregation, for generating and maintaining organelle structure and position, and for facilitating the transfer of material between compartments. The mechanism for assembly into a functional motor is not understood. Moreover there are no high resolution structures for any subunits of dynein. A 10 kDa subunit, here referred to as DLC, is highly conserved among all known dyneins, with 94% sequence identity between Drosophila and human, and is also a component of myosin V. The high conservation suggests a common function of this particular protein in motor complexes of various organisms. The 10 kDa subunit is found to be essential in several cellular processes: embryonic development and oogenesis in Drosophila, nuclear migration in fungus, formation and maintenance of the teguments in the blood fluke, transport of neuronal nitric oxide synthase across axons, and transcriptional regulation during viral infection. The mechanism for these various functions is not known. The purpose of this project is to determine how the structural characteristics of DLC contribute to its multiple regulatory roles. Covalent cross-linking experiments indicate that this polypeptide exists as a dimeric structure within the dynein complex and associates with two other dynein components. The experiments proposed here will focus on using nuclear magnetic resonance spectroscopy to identify the dimer interface, the high resolution structure and dynamics of the dimer, and potential sites of interactions with protein-within the dynein complex and with cargo. Future research is likely to focus on the role of dimerization on the assembly of the complex in vivo, characterization of other subunits of dynein, and their interactions with DLC. The high resolution structure of DLC will not only give clues to its specific and various functions, but more importantly may provide an avenue into structural characterization of the dynein system.