NUCLEAR-CYTOPLASMIC TRANSPORT

核仁和细胞质之间的大分子交换是一种 所有真核细胞的基本过程。我们之前确定了 Nopp140，一种穿梭的核定位信号结合蛋白 核仁和细胞质之间的高度定域化的轨迹。这个 这一巨大提议的长期目标是理解这种机制 以及对Nopp140介导的核仁-细胞质转运的调节。 具体：1.NAP57的特性和鉴定 额外的Nopp140相关蛋白。我们发现了一种Nopp140 定位于大鼠肝核抽提物中的相关蛋白NAP57 将其克隆到Nopp140样核内踪迹上，并克隆其cDNA。我们建议： (A)确定NAP57是否也穿梭于细胞核和细胞质之间。(B) 内源性和细菌表达的NAP57与NAP57相互作用研究 不是140。(C)通过以下方式确定额外的Nopp140相互作用蛋白 细胞组分的亲和层析 重组Nopp140和NAO57。2.Nopp140的结构和动力学 脚印。开发一种能够更容易地检测到 轨道，这将使我们能够同时操纵细胞 孵化条件，我们将通过激光扫描共聚焦进行可视化 光学切片的显微镜和三维重建： (A)免疫定位的Nopp140和NAP57，和(B)荧光标记的 微量注射或体外注射重组Nopp140和Nap57 核进口化验。3.Nopp140的磷酸化。我们之前 研究表明，Nopp140是一种高度磷酸化的蛋白质 细胞和核定位信号结合依赖于其 磷酸化状态。通过使用亚细胞分离和脉冲 Chase标签技术，它将决定是否以及如何 Nopp140的磷酸化状态因其亚细胞不同而不同 地点。因为我们已经证明了重组Nopp140可以作为 在体外，我们将利用它来鉴定和纯化 细胞Nopp140激酶(S)。4.Nopp140同源基因的鉴定 放在酵母里。初步数据显示，大鼠Nopp140的cDNA与 与特定的酵母mRNA杂交，多克隆抗体 抗哺乳动物蛋白与酵母核仁发生交叉反应 蛋白质。基于这些同源性，我们将克隆和测序 酵母Nopp140基因及其功能的进一步研究：(A) 研究基因破坏的影响。(B)分析其亚细胞 突变菌株的定位和磷酸化状态。(C)剥削 分子遗传学技术。最终，这些研究将有助于 对核糖体合成上调和核糖体表达的理解 导致核仁过度活动，这是主要特征 每一个癌细胞。事实上，核仁在生命周期中的重要性 最近发现的对细胞生长的调节已经被强调 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.