THE ROLE OF THE NUCLEAR ENVELOPE IN INTRACELLULAR PROTEIN SORTING

核膜在细胞内蛋白质分选中的作用

• 批准号: 3753959
• 负责人: J A HANOVER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3753959
• 关键词: biological signal transduction; calcium; cell cycle; chemical conjugate; fluorescent dye /probe; glycoproteins; glycosylation; guanosine triphosphate; heart cell; inositol phosphates; intracellular transport; ion transport; membrane channels; membrane potentials; membrane proteins; molecular cloning; nuclear membrane; phosphorylation; protein transport; recombinant proteins; tissue /cell culture; voltage /patch clamp

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.

控制基因表达的细胞外信号最终必须 传递到细胞核。 核运输在 调节转录因子的水平和活性，核 激酶、类固醇激素受体和复制因子。 核 孔复合物（NPC）促进mRNA和蛋白质的移动， 核膜。 之前，我们描述了一个家庭， 磷酸化的糖蛋白是NPC的组成部分。 主要 克隆了核孔蛋白p62，并以重组体形式表达 蛋白在大肠大肠杆菌和杆状病毒。 磷酸化位点， p62中的糖基化被定位到p62的中心富含Ser/Thr的区域。 分子。 有趣的是，磷酸化程度的变化 糖基化随细胞周期波动。 磷酸化 糖基化在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NPC的组装和分解相关。 利用毛地黄皂苷透化培养细胞，我们建立了研究方法， 核和核仁运输的荧光共轭物携带的 合适的定位信号。 这种缀合物集中在 细胞核和核仁，分别与细胞质， 分数和ATP。 核运输被强烈激活， Ca +2或GTP对耗尽的胞质溶胶的影响。 IP3和钙螯合剂抑制 运输; GTP-gamma-S也抑制核运输。 的 核转运对Ca +2和GTP的反应性表明， 转运可能与其他信号转导协调调节 细胞活化过程中的信号通路。 我们将这些研究 使用膜片钳电生理学检查调节的离子流通过 心肌细胞NE。 我们检测到大电导离子通道 NE处的活性类似于间隙连接通道的活性。 激活 这些通道需要胞质因子。 通道被堵住了 通过GTP-γ-S、麦胚凝集素和核孔特异性 抗体的 因此，大电导核离子通道活性是 可能是核孔所固有的。