MECHANISM OF SEGREGATION OF MODIFIED TUBULIN IN VIVO

微管(MT)是一种细胞骨架元素，存在于 真核细胞，在那里它们在各种运动和 形态发生事件。所有MT均为微管蛋白聚合物 原构体，这是一种长寿的细胞蛋白质，其序列 在不同的生物体和细胞类型中是非常稳定的。 所提出的研究的长期目标是了解 微管蛋白或其相关蛋白的哪些生化变化 可以使MTS的功能发生变化。特别是， 转录后修饰似乎是好的。 可能会迅速和可逆地影响到 MT的组织或职能。微管蛋白受制于一对独特的 翻译后修饰，称为 酪氨酸化/去酪氨酸化，其中添加了酪氨酸残基 到阿尔法链的C-末端或从其移除。我们 先前发现MTS富含酪氨酸或 去酪氨酸化的微管蛋白组成细胞的互补亚群 MTS，并且这些微管蛋白物种通过循环分离 酪氨酸化/去酪氨酸化和微管蛋白的作用机制 聚合/解聚。我们还展示了 富含去酪氨酸微管蛋白的MTS表现不同寻常 属性和动力学。在这里，我们建议对此进行进一步的研究 不寻常的稳定的、退化的MTS种群，以及 确定这些MT的哪些属性是以下各项的函数 退化的原生生物的存在。此外，我们还将 对酪氨酸产生的酶(微管蛋白)进行详细研究 酪氨酸连接酶)和去酪氨酸(微管蛋白羧肽酶)α- 微管蛋白；纯化每个微管蛋白，分析其活性和在MT- 介导的细胞事件，推导出每个和 将其性质与其他相关酶的性质进行比较。 最后，对酶和不寻常的MTS进行了表征 他们创造的将使我们能够开发化学和反义RNA 每种酶的抑制剂，以确定其作用 MT功能的酪氨酸化/去酪氨酸化循环。我们的建议 关于这一独特的翻译后修改周期的工作将 使我们能够更好地了解MTS在 正常组织和病理性组织的分化和细胞分裂 设置。

Microtubules (MTs) are cytoskeletal elements found in all eukaryotic cells, where they function in a variety of motile and morphogenetic events. All MTs are polymers of tubulin protomers, which are long-lived cellular proteins whose sequence is remarkably constant among different organisms and cell types. The long-range goal of the research proposed is to understand what biochemical alterations in tubulin or its associated proteins can bring about changes in the function of MTs. In particular, post-transcriptional modifications would seem to be good candidates for alterations that could rapidly and reversibly affect MT organization or function. Tubulin is subject to a unique pair of post-translational modifications, called tyrosination/detyrosination, in which a tyrosine residue is added to or removed from the C-terminus of the alpha chain. We previously found that MTs enriched in tyrosinated or detyrosinated tubulin composed complementary subsets of cellular MTs, and that these tubulin species become segregated by a cyclic mechanism involving tyrosination/detyrosination and tubulin polymerization/depolymerization. We have also demonstrated that MTs enriched in detyrosinated tubulin exhibit unusual properties and dynamics. Here we propose further studies of this unusual population of stable, detyrosinated MTs, and a determination of which properties of these MTs are a function of the presence of detyrosinated protomers. In addition, we will perform detailed studies on the enzymes that tyrosinate (tubulin tyrosine ligase) and detyrosinate (tubulin carboxypeptidase) alpha- tubulin; purifying each, assaying its activity and levels during MT- mediated cellular events, deriving cDNA clones to each and comparing its properties to those of other related enzymes. Finally, characterization of the enzymes and the unusual MTs they create will allow us to develop chemical and anti-sense RNA inhibitors of each enzyme, in order to determine the role of the tyrosination/detyrosination cycle in MT function. Our proposed work on this unique cycle of post-translational modification will enable us to better understand the function of MTs during differentiation and cell division in both normal and pathological settings.