BIOCHEMICAL AND GENETIC ANALYSIS OF NUCLEAR TRANSPORT

许多核蛋白含有定位信号，并在 原子核以能量依赖的方式，然而，分子基础 因为这个过程是未知的。我们建议进一步鉴定一种酵母菌。 核膜蛋白(P67)是我们以前发现的。P67页 专门识别核定位序列，是一种很好的 参与启动核运输的受体的候选者。 P67已经部分纯化，编码该蛋白的基因已经 已被分离，并命名为NSB1。令人惊讶的是，NSB1包含两个 RNA结合域以及含有数字的酸性N末端 丝氨酸簇和基本的C-末端。抗p67抗体染色 核内亚基结构的外围，可能是核仁， 通过间接免疫荧光法。NSB1蛋白在 GAL10启动子或在多肽亲和柱上纯化，具体 识别H2B核定位序列。这种基因正在被 被扰乱，以测试其本质。NSB1在核中的作用 体内转运将通过以下方式进行研究： 温度敏感型突变将被分离并鉴定为 核运输途径的缺陷。抑制者 温度敏感表型将被分离出来，有望成为新的表型 核转运中与NSB1蛋白相互作用的蛋白质 路径将被确定。在核过程中，ATP在哪里使用或如何使用 交通工具是未知的。使用光亲和类似物2-叠氮基ATP，我们 已经确定了核膜上特定的ATP结合蛋白 这可能会在核运输过程中发挥作用。抗人免疫球蛋白抗体 部分纯化的蛋白质正被用来克隆这些基因 蛋白质。

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.