BIOCHEMICAL AND GENETIC ANALYSIS OF NUCLEAR TRANSPORT

核输运的生物化学和遗传学分析

• 批准号: 3304226
• 负责人: TERI MELESE
• 金额: $ 16.26万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3304226
• 关键词: adenylate kinase; cell free system; immunofluorescence technique; intracellular transport; molecular cloning; nuclear membrane; nucleic acid sequence; nucleoproteins; pore forming protein; protein sequence; receptor binding; temperature sensitive mutant; tissue /cell culture; transport proteins; yeasts

Many nuclear proteins contain localization signals and accumulate in the nucleus in an energy dependent fashion, however, the molecular basis for this process is unknown. We propose to further characterize a yeast nuclear envelope protein (p67) we have previously identified. p67 specifically recognizes nuclear localization sequences and is a good candidate for a receptor involved in the initiation of nuclear transport. p67 has been partially purified, and the gene encoding this protein has been isolated, and designated NSB1. Surprisingly, NSB1 contains two RNA-binding domains as well as an acidic N-terminus containing a number of serine clusters, and basic C-terminus. Anti-p67 antibodies stain the periphery of a substructure within the nucleus, possibly the nucleolus, by indirect immunofluorescence. The NSB1 protein either expressed under the GAL10 promoter or purified on a peptide affinity column, specifically recognizes the H2B nuclear localization sequence. The gene is being disrupted to test its essential nature. The role of NSB1 in nuclear transport in vivo will be studied in the following manner: Temperature-sensitive mutations will be isolated and characterized for defects in the nuclear transport pathway. Suppressors of the temperature-sensitive phenotype will be isolated and hopefully new proteins interacting with the NSB1 protein in the nuclear transport pathway will be identified. Where or how ATP is used during nuclear transport is unknown. Using the photoaffinity analog, 2-azido ATP, we have identified specific ATP-binding proteins at the nuclear envelope that may function during nuclear transport. Antibodies against the partially purified proteins are being used to clone the genes for these proteins.

许多核蛋白包含定位信号并积聚 然而，以能量依赖性的核是分子基础 因为这个过程是未知的。 我们建议进一步描述酵母 我们先前已经鉴定出的核包膜蛋白（p67）。 P67 专门识别核定位序列，是一个好的 参与核运输的受体的候选者。 p67已部分纯化，编码该蛋白的基因已有 被隔离并指定为NSB1。 令人惊讶的是，NSB1包含两个 RNA结合域以及含有数量的酸性N端 丝氨酸簇和基本C末端。 抗P67抗体染色 核内的一个子结构的周围，可能是核仁， 通过间接免疫荧光。 NSB1蛋白在 GAL10启动子或在肽亲和柱上纯化，特别是 识别H2B核定位序列。 基因正在 被破坏以测试其本质。 NSB1在核中的作用 体内运输将以以下方式进行研究： 温度敏感的突变将被隔离并表征 核运输途径的缺陷。 抑制器 温度敏感的表型将是孤立的，希望是新的 与NSB1蛋白相互作用的蛋白质中的蛋白质 将确定途径。 核过程中的ATP在哪里或如何使用 运输是未知的。 使用光亲和力类似物，2-齐多ATP，我们 已经确定了核包膜处的特定ATP结合蛋白 这可能在核运输过程中起作用。 反对抗体 部分纯化的蛋白用于克隆这些基因 蛋白质。