NUCLEAR-CYTOPLASMIC TRANSPORT

核仁和细胞质之间的大分子交换是一个重要的生物学过程。 所有真核细胞的基本过程。 我们之前发现 Nopp 140是一种核定位信号结合蛋白， 在核仁和细胞质之间的高度局部化的轨道上。 的 这个巨大的提案的长期目标是了解这个机制 和Nopp 140介导的核质转运的调节。 具体而言：1. NAP 57的表征和NAP 57的鉴定 其他Nopp 140相关蛋白。 我们发现了一个诺普140 相关蛋白NAP 57在大鼠肝细胞核提取物中的定位， 克隆了Nopp 140的cDNA。 我们建议： (a)确定NAP 57是否也在细胞核和细胞质之间穿梭。 （B） 研究内源性和细菌表达的NAP 57与 没有140。 (c)通过以下方法鉴定其他Nopp 140相互作用蛋白： 细胞级分的亲和层析， 重组Nopp 140和NAO 57。 2. Nopp 140的结构和动力学 跟踪. 为了开发一种系统，可以更容易地检测 这将使我们能够同时操纵细胞 在孵育条件下，我们将通过激光扫描共聚焦 光学切片的显微镜和三维重建： (a)免疫定位的Nopp 140和NAP 57，和（B）荧光标记的 重组Nopp 140和Nap 57，显微注射后或体外 核进口分析。 3. Nopp 140的磷酸化。 我们之前 Nopp 140是人类中磷酸化程度最高的蛋白质之一。 细胞与核定位信号结合依赖于其 磷酸化状态 通过采用亚细胞分离和脉冲 追逐标签技术，它将被确定，如果和如何 Nopp 140的磷酸化状态根据其亚细胞 位置. 由于我们已经证明重组Nopp 140可以作为 激酶底物，我们将利用它来鉴定和纯化 细胞Nopp 140激酶。 4. Nopp 140同源物的鉴定 在酵母中。 初步数据显示，大鼠Nopp 140 cDNA跨- 与特异性酵母mRNA杂交，多克隆抗体 提出了针对哺乳动物的蛋白质交叉反应与酵母核仁 proteins. 基于这些同源性，我们将克隆和测序 酵母Nopp 140基因，并通过以下方法进一步了解其功能：（a） 研究基因破坏的影响。 (b)分析其亚细胞 突变株中的定位和磷酸化状态。 (c)利用 分子遗传学技术 最终，这些研究将有助于 对核糖体合成上调的理解， 导致核仁过度活跃，这是 每个癌细胞 事实上，核仁在细胞中的重要性 细胞生长的调节由于最近发现的 核仁癌蛋白LYAR。

Exchange of macromolecules between the nucleolus and the cytoplasm is an essential process of all eukaryotic cells. We previously identified Nopp140, a nuclear localization signal binding protein that shuttles between the nucleolus and the cytoplasm on highly localized tracks. The long-term objective of this giant proposal is to understand the mechanism and regulation of Nopp140 mediated nucleolar-cytoplasmic transport. Specifically: 1. Characterization of NAP57 and identification of additional Nopp140 associated proteins. We have identified a Nopp140 associated protein, NAP57, in rat liver nuclear extracts that localized to Nopp140 like intranuclear tracks, and cloned its cDNA. We propose to: (a) Determine if NAP57 also shuttles between nucleus and cytoplasm. (b) Study the interaction of endogenous and bacterially expressed NAP57 with Nopp140. (c) Identify additional Nopp140 interacting proteins by affinity chromatography of cellular fractions using columns of recombinant Nopp140 and NAO57. 2. Structure and dynamics of Nopp140 tracks. To develop a system that allows an easier detection of the tracks and that will enable us to simultaneously manipulate cellular incubation conditions, we will visualize them by laser scanning confocal microscopy and three dimensional reconstruction of optical sections of: (a) immunolocalized Nopp140 and NAP57, and (b) fluorescently labeled recombinant Nopp140 and Nap57, after microinjection or in in vitro nuclear import assays. 3. Phosphorylation of Nopp140. We previously showed that Nopp140 is one of the most highly phosphorylated proteins in the cell and that nuclear localization signal binding is dependent on its phosphorylation state. By employing subcellular fractionation and pulse chase labeling techniques, it will be determined if and how the phosphorylation state of Nopp140 varies according to its subcellular location. Since we have shown that recombinant Nopp140 can serve as kinase substrate in vitro, we will exploit it to identify and purify the cellular Nopp140 kinase(s). 4. Identification of the Nopp140 homolog in yeast. Preliminary data show that the rat Nopp140 cDNA cross- hybridizes with a specific yeast mRNA, and that polyclonal antibodies raised against the mammalian protein crossreact with yeast nucleolar proteins. Based on these homologies, we will clone and sequence the yeast Nopp140 gene and obtain further insight into its function by: (a) Studying the effects of gene disruption. (b) Analyzing its subcellular location and phosphorylation state in mutant strains. (c) Exploiting molecular genetic techniques. Ultimately, these studies will contribute to the understanding of the upregulation of ribosome synthesis and the resulting nucleolar hyperactivity which are major characteristics of every cancer cell. In fact, the importance of the nucleolus in the regulation of cell growth has been emphasized by the recent discovery of the nucleolar oncoprotein LYAR.