NUCLEAR-CYTOPLASMIC TRANSPORT
核质运输
基本信息
- 批准号:2188744
- 负责人:
- 金额:$ 20.02万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1995
- 资助国家:美国
- 起止时间:1995-01-01 至 1998-12-31
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
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.
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
体外激酶底物,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
最近的发现强调了细胞生长的调节
核仁癌蛋白LYAR。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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U THOMAS MEIER其他文献
U THOMAS MEIER的其他文献
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{{ truncateString('U THOMAS MEIER', 18)}}的其他基金
Cellular impact of X-linked dyskeratosis congenita
X连锁先天性角化不良的细胞影响
- 批准号:
9861050 - 财政年份:2017
- 资助金额:
$ 20.02万 - 项目类别:
Cellular impact of X-linked dyskeratosis congenita
X连锁先天性角化不良的细胞影响
- 批准号:
9545059 - 财政年份:2017
- 资助金额:
$ 20.02万 - 项目类别:
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