ROLE OF THE NUCLEAR ENVELOPE IN INTRACELLULAR PROTEIN SORTING

核运输通过调节细胞内的 转录因子、核激酶、类固醇的活性 激素受体和复制因子。核孔复合体 (NPC)介导mRNA和蛋白质跨核转运 信封.以前，我们描述了一个磷酸化的家族， 翻译后修饰出现的NPC糖蛋白 与核孔的组装和拆卸有关。我们 已经分子克隆并表征了人O-连接GlcNAc 负责糖基化核孔蛋白的转移酶。 使用小鼠和秀丽隐杆线虫的同源物， 我们正试图通过基因操作这种酶， 完整的有机体 使用毛地黄皂苷透化培养细胞，我们 已经研究了荧光缀合物的核和核仁转运 承载适当的定位信号。除了井 特征性的GTP依赖性核转运， 透化细胞，我们检测到一种核运输模式， 在细胞质钙浓度升高时不依赖GTP。 在这种条件下，核运输被钙调素阻断 抑制剂的 重组钙调素完全恢复ATP依赖性 在没有胞质溶胶的情况下的核运输。 钙调 小麦胚芽凝集素可抑制转运， 运输通过核孔进行。 我们建议释放 细胞激活后的细胞内钙储存抑制GTP- 依赖性核转运;然后升高的细胞质钙发挥作用 通过钙调素刺激新的GTP非依赖性模式， 运输 这提供了信号转导之间的直接联系 以及核运输的调节。

Nuclear transport serves a critical regulatory function by modulating the activities of transcription factors, nuclear kinases, steroid hormone receptors and replication factors. The nuclear pore complex (NPC) mediates the transport of mRNA and proteins across the nuclear envelope. Previously, we described a family of phosphorylated glycoproteins of the NPC whose posttranslational modifications appear to be associated with assembly and disassembly of nuclear pores. We have molecularly cloned and characterized the human O-linked GlcNAc transferase responsible for glycosylating nuclear pore proteins. Using both the mouse and Caenorhabditis elegans homologs of the enzyme we are attempting to genetically manipulate 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. In addition to the well characterized GTP-dependent nuclear transport observed in permeabilized cells, we detected a mode of nuclear transport that was GTP-independent at elevated cytoplasmic calcium concentrations. Nuclear transport under these conditions was blocked by calmodulin inhibitors. Recombinant calmodulin fully restored ATP-dependent nuclear transport in the absence of cytosol. Calmodulin-dependent transport was inhibited by wheat germ agglutinin suggesting that transport proceeded through nuclear pores. We propose that release of intracellular calcium stores upon cell activation inhibits GTP- dependent nuclear transport; the elevated cytosolic calcium then acts through calmodulin to stimulate the novel GTP-independent mode of transport. This provides a direct link between signal transduction and the modulation of nuclear transport.