ROLE OF THE NUCLEAR ENVELOPE IN INTRACELLULAR PROTEIN SORTING

跨核膜的大分子运输起着中心作用。 在真核细胞生长发育的调控中发挥作用。这一机制 对核运输的调查正在进行中。最近，核定位 已经确定了选择性靶向蛋白质的序列 原子核。建立了一种体外结合和进口试验，该方法使用 分离的大鼠肝细胞核及其胶体标记的多肽结合物 GOLD或与(125)I具有与核结合的正确序列的结合物 其中Kd=10OnM。核膜是一种自组装结构； 将DNA添加到非洲爪哇卵的提取液中，形成核 与正常细胞相似。标记的共轭化合物进入这些 改造后的细胞核。该系统正在被用来分析需求 用于核运输。核孔复合体调节跨膜的运输 核膜。实验室已经证明，含有 胞质导向、O-连接的GlcNAc是细胞核的组成部分 毛孔复合体。检测这些糖蛋白在细胞核中的功能 运输，克隆了编码主要孔蛋白p62的CDNA。这个 编码p62的基因没有内含子，有两个潜在的转录。 启动站点。P62有两个结构域：一个肌球蛋白样结构域和一个胶原样结构域。通过 通过转染编码p62的表达载体，该蛋白已 在培养细胞中过表达。使用克隆的CDNA，它也被 证明了在体外，0-连接的GlcNAc加成可以在 兔网织红细胞裂解物。 目前正在努力鉴定p62的酵母同源物，以便酵母 遗传学可以用来确定p62的正常功能。这将是 结合涉及将重组大鼠p62添加到 非洲爪哇核重组试验。

Macromolecular transport across the nuclear membrane plays a central role in the regulation of eukaryotic cell growth and development. The mechanism of nuclear transport is under investigation. Recently, nuclear localization sequences have been identified which selectively target proteins to the nucleus. An in vitro binding and import assay has been developed using isolated rat liver nuclei and peptide conjugates labelled with colloidal gold or with (125)I. Conjugates having the proper sequence bind to nuclei with a Kd=lOOnM. The nuclear membrane is a self assembling structure; when DNA is added to extracts of eggs of Xenopus laevis, nuclei form which resemble those of normal cells. The labelled conjugates enter these reformed nuclei. This system is being exploited to analyze the requirements for nuclear transport. The nuclear pore complex mediates transport across the nuclear membrane. The laboratory has demonstrated that proteins bearing cytoplasmically oriented, O-linked GlcNAc are components of the nuclear pore complex. To examine the function of these glycoproteins in nuclear transport, the CDNA encoding the major pore protein p62 was cloned. The gene encoding p62 is devoid of introns and has two potential transcription start sites. p62 has two domains: one myosin-like and one collagen-like. By transfecting an expression construct encoding p62 this protein has been overexpressed in cultured cells. Using the cloned CDNA it has also been demonstrated that 0-linked GlcNAc addition can be catalyzed in vitro in a rabbit reticulocyte lysate. Efforts are underway to identify the yeast homologue of p62 so that yeast genetics can be used to determine the normal function of p62. This will be combined with studies involving the addition of recombinant rat p62 to the Xenopus nuclear reconstitution assay.