BIOCHEMICAL AND GENETIC ANALYSIS OF NUCLEAR TRANSPORT

许多核蛋白含有定位信号，并积累在 原子核以能量依赖的方式，然而，分子基础 这个过程是未知的。 我们建议进一步表征酵母 核被膜蛋白（p67），我们以前已经确定。P67 特异性识别核定位序列， 参与核运输起始的受体的候选者。 p67已被部分纯化，编码该蛋白的基因已 被分离出来并命名为NSB 1。 令人惊讶的是，NSB 1包含两个 RNA结合结构域以及酸性N-末端，其含有多个 丝氨酸簇和碱性C末端。 抗p67抗体染色， 核内的亚结构的外围，可能是核仁， 间接免疫荧光法。 NSB 1蛋白在 GAL 10启动子或在肽亲和柱上纯化，特别是 识别H2 B核定位序列。 基因正在被 被破坏以测试其本质。 NSB 1在核反应中的作用 将按照以下方式研究体内转运： 温度敏感性突变将被分离和表征， 核运输途径的缺陷。 的抑制剂 温度敏感表型将被分离，并有望成为新的 与NSB 1蛋白相互作用的核转运蛋白 将确定路径。 ATP在核反应堆中的作用 运输未知。 使用光亲和类似物，2-叠氮ATP，我们 已经在核膜上发现了特异性的ATP结合蛋白 在核运输过程中发挥作用。 抗体对抗 部分纯化的蛋白质被用来克隆这些基因， proteins.

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.