THE ROLE OF THE NUCLEAR ENVELOPE IN INTRACELLULAR PROTEIN SORTING

控制基因表达的细胞外信号最终必须是 与原子核进行通讯。核运输在以下方面发挥着关键作用 调节转录因子的水平和活性，核 激动酶、类固醇激素受体和复制因子。核电 孔道复合体(NPC)促进mRNA和蛋白质的跨膜转运 核包膜。在此之前，我们描述了一个家庭 作为鼻咽癌组成部分的磷酸化糖蛋白。少校 克隆并表达了核孔蛋白p62。 大肠杆菌和杆状病毒中的蛋白质。磷酸化和磷酸化部位 P62中的糖基化被定位于Ser/Thr富集区 分子。有趣的是，磷酸化程度的变化 糖基化随着细胞周期的变化而振荡。磷酸化 M期达到最大；S期达到高峰。 这种磷酸化和糖基化的相互调节可能是 与细胞周期中鼻咽癌的组装和拆解有关。 利用洋地黄素渗透培养的细胞，我们开发了研究手段 荧光偶联物的核和核仁运输 适当的定位信号。这类共轭化合物集中在 当供应胞浆时，核和核仁分别 分数和三磷酸腺苷。核运输被强烈地激活了 Ca+2或GTP转化为耗竭的胞浆。抑制IP3和钙络合剂 转运；GTP-伽马-S也抑制核转运。这个 核转运对Ca+2和GTP的反应性表明，核 运输可能与其他信号转导协调调节 细胞激活过程中的通路。我们延长了这些研究的时间 应用膜片钳电生理学检测调节离子流过 心肌细胞的去甲肾上腺素。我们探测到了大电导离子通道 NE的活动类似于缝隙连接通道的活动。激活 在这些通道中，需要胞质因子。通道被堵塞了 由GTP-γ-S、小麦胚芽凝集素和核孔专一性 抗体。因此，大电导核离子通道的活性是 很可能是核孔所固有的。

The extracellular signals controlling gene expression must ultimately be communicated to the nucleus. Nuclear transport plays a key role in regulating the levels and activities of transcription factors, nuclear kinases, steroid hormone receptors and replication factors. The nuclear pore complex (NPC) facilitates the movement of mRNA and proteins across the nuclear envelope. Previously, we described a family of phosphorylated glycoproteins which are components of the NPC. The major nuclear pore protein, p62, was cloned and expressed as a recombinant protein in E. coli and baculovirus. Sites of phosphorylation and glycosylation in p62 were mapped to the central Ser/Thr rich region of the molecule. Interestingly, changes in the degree of phosphorylation and glycosylation oscillated with the cell cycle. Phosphorylation reached a maximum during M phase; glycosylation peaked during S phase. This reciprocal regulation of phosphorylation and glycosylation may be associated with assembly and disassembly of NPC during the cell cycle. Using digitonin permeabilized cultured cells, we developed means of study nuclear and nucleolar transport of fluorescent conjugates bearing the appropriate localization signal. Such conjugates concentrated in the nucleus and nucleolus, respectively, when supplied with a cytosolic fraction and ATP. Nuclear transport was strongly activated by addition of Ca+2 or GTP to depleted cytosol. IP3 and calcium chelators inhibited transport; GTP-gamma-S also inhibited nuclear transport. The responsiveness of nuclear transport to Ca+2 and GTP suggests that nuclear transport may be coordinately regulated with other signal transduction pathways during cellular activation. We have extended these studies using patch-clamp electrophysiology to examine regulated ion flow through the NE of cardiac myocytes. We detected large conductance ion channel activity at the NE resembling that of gap junction channels. Activation of these channels required cytosolic factors. The channels were blocked by GTP-gamma-S, wheat germ agglutinin and nuclear pore-specific antibodies. Thus the large conductance nuclear ion channel activity is likely to be intrinsic to nuclear pores.