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: 5202061
• 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/5202061
• 关键词: adenosine triphosphate; agglutinins; atomic force microscopy; calcium; cell nucleus; cytoplasm; guanosine triphosphate; intracellular transport; membrane channels; myocardium; norepinephrine; nuclear membrane; protein transport; recombinant proteins; tissue /cell culture; transcription factor; voltage /patch clamp

Nuclear transport plays a key role in regulating the levels and activities of transcription factors, nuclear kinases, steroid hormone receptors and DNA replication factors. The nuclear pore complex (NPC) mediates the transport of mRNA and protein across the nuclear envelope. Previously, we described a family of phosphorylated glycoproteins which are components of the NPC. Interestingly, changes in the degree of phosphorylation and glycosylation occur during the cell cycle that may be associated with assembly and disassembly of nuclear pores. The major nuclear pore protein, p62 has been studied in greater detail. In addition, the enzyme which mediates O-linked GlcNAc addition to p62 has been purified and microsequenced. A homolog of the O-linked GlcNAC transferase has been identified in caenorhabditis elegans which may allow genetic manipulation of the enzyme in an intact organism. Using digitonin permeabilized cultured cells, we have studied 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 requires both Ca+2 and GTP suggesting 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. Using recombinant TATA-binding protein, c-Jun, NF-kappaB and SP1 it was possible to demonstrate transcription factor translocation through the nuclear pore channel by patch-clamp measurements and atomic force microscopy. These observations strong support the notion of a nuclear pore channel that can be measured using elecrophysiological means. Since these measurements can be made in real-time it should be possible to examine conformational alterations in the nuclear pore during transport.

核输运在调节核辐射水平和活动中起着关键作用 转录因子、核激酶、类固醇激素受体和 DNA复制因子核孔复合体（NPC）介导了 mRNA和蛋白质跨核膜的运输。此前我们 描述了一个磷酸化糖蛋白家族， NPC。有趣的是，磷酸化程度的变化， 糖基化发生在细胞周期中，可能与 核孔的组装和拆卸。主要核孔蛋白， p62已经被更详细地研究。此外， 介导的0-连接的GlcNAc添加到p62已经被纯化， 微测序O-连接的GlcNAC转移酶的同源物已经被发现。 在秀丽隐杆线虫中发现了这种基因， 在一个完整的有机体中的酶。 利用毛地黄皂苷透化培养的细胞，我们研究了细胞核和 核仁转运的荧光共轭物携带适当的 定位信号这种结合物集中在细胞核中， 核仁，分别与细胞质组分和ATP供应时。 核运输需要Ca +2和GTP，这表明核运输需要Ca +2和GTP。 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。我们扩展了这些研究， 膜片钳电生理学检查通过NE的调节离子流 心肌细胞。我们检测到大电导离子通道活性， NE类似于间隙连接通道的NE。激活这些 通道需要胞质因子。通道被GTP阻断了- γ-S、麦胚凝集素和核孔特异性抗体。因此，在本发明中， 大电导核离子通道活性可能是 是核孔的固有成分使用重组TATA结合蛋白，c-Jun， NF-κ B和SP1，可能证明转录因子 通过膜片钳测量通过核孔通道的移位 和原子力显微镜这些观察结果有力地支持了这样一种观点： 可以用电生理学方法测量的核孔通道 手段 由于这些测量可以实时进行， 可以检查核孔中的构象变化， 运输