CELLULAR PROTEINS INVOLVED IN TRANSPORT THROUGH

参与转运的细胞蛋白

• 批准号: 2187902
• 负责人: VITALY H CITOVSKY
• 金额: $ 19.68万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 1996-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2187902
• 关键词: active transport; cell wall; electron microscopy; gel filtration chromatography; genetically modified plants; immunocytochemistry; in situ hybridization; intercellular connection; ion exchange chromatography; laboratory rabbit; macromolecule; membrane transport proteins; microinjections; molecular cloning; nucleic acid metabolism; phosphorylation; plant physiology; protein kinase; protein sequence; receptor binding; tobacco mosaic virus; virus RNA; virus protein

In a plant, cells are linked with each other by intercellular connections, the plasmodesmata (PD). Similar to intercellular communication in animals, transport through PD plays a vital role in plant physiology and development. Recent evidence suggests that PD can actively transport macromolecules. The molecular mechanisms by which active PD transport occurs are unknown. One approach to elucidate these mechanisms is to study how plant pathogens spread through host cells. Generally, the invading microorganism does not invent novel metabolic pathways but adapts existing cellular processes for its life cycle. The best studied example of pathogen spread through PD is cell-to-cell movement to tobacco mosaic virus (TMV) genomic RNA which is mediated by a virus-encoded protein, P30. Here, P30 will be used as a tool to study cellular proteins involved in PD transport. The proposed research has four specific aims. (i) Purification of P30 cytoplasmic receptors involved in PD transport. By analog to nuclear import, active transport through PD may require specific cytoplasmic receptors. To test this hypothesis, the potential P30 cytoplasmic receptors will be identified and purified using anti-P30 antiidiotype antibodies as well as binding to immobilized P30. The genes coding for P30 receptors will be cloned by immunoscreening of cDNA expression libraries or following microsequencing of the purified proteins. (ii) Purification of a plant cell wall-associated protein kinase that specifically phosphorylates P30. Similar to many other transport processes, phosphorylation may be involved in PD transport. Here, the cell wall-associated protein kinase, a potential PD component capable of phosphorylating P30, will be studied. This protein will be purified by removal of cell wall matrix and solubilization followed by ion exchange and gel filtration chromatography. The purified protein kinase will be used to clone its encoding gene. (iii) Study of biochemistry and cell biology of the purified P30 cytoplasmic receptors and cell wall-associated protein kinase. The biological role of the purified receptors will be studied in vivo following their microinjection plant cells, and P30-receptor interaction will be characterized in vitro using yeast genetic two hybrid-protein detection system, native gel electrophoresis and dot blot assay. Subcellular localization of all purified proteins will be determined using electron microscopy. Potential development regulation of the cloned genes will be examined by northern analysis, in situ hybridization and genetic experiments using transgenic plants. (iv) Use of the purified proteins to begin characterization of cellular pathways for PD transport of macromolecules. Using protein-protein interaction assays developed in this proposal, the purified proteins will serve as specific probes to identify additional cellular components of the PD transport pathway. In addition to characterizing PD transport, the results of the proposed research may have a general biological relevance for transmembrane transport of macromolecules.

在植物中，细胞通过细胞间相互联系 连接，plasmodesmata（PD）。 类似于细胞间 动物的交流，通过PD运输在 植物生理和发育。 最近的证据表明PD可以 积极运输大分子。 分子机制 主动PD传输发生尚不清楚。 一种阐明这些的方法 机制是研究植物病原体如何通过宿主细胞扩散。 通常，入侵的微生物不会发明新型代谢 途径但可以使现有的蜂窝过程适应其生命周期。 这 最好研究的病原体通过PD传播的例子是细胞到细胞 运动转向烟草病毒（TMV）基因组RNA，该基因组RNA介导 病毒编码的蛋白质，P30。 在这里，P30将用作研究的工具 涉及PD转运的细胞蛋白。 拟议的研究已经 四个具体目标。 （i）与PD转运有关的P30细胞质受体的纯化。 通过类似于核进口，通过PD的主动运输可能需要 特异性细胞质受体。 为了检验这一假设，潜力 将使用抗P30鉴定并纯化P30细胞质受体 抗体型抗体以及与固定的P30结合。 基因 P30受体编码将通过cDNA的免疫统计来克隆 表达式文库或遵循纯化的微链测序 蛋白质。 （ii）纯化植物细胞壁相关的蛋白激酶 特异性磷酸化P30。 类似于许多其他运输 过程，PD转运可能涉及磷酸化。 在这里， 细胞壁相关蛋白激酶，一种能够的潜在PD成分 将研究磷酸化P30。 该蛋白将被纯化 去除细胞壁基质和溶解，然后进行离子交换 和凝胶过滤色谱法。 纯化的蛋白激酶将是 用于克隆其编码基因。 （iii）纯化P30的生物化学和细胞生物学研究 细胞质受体和细胞壁相关蛋白激酶。 这 将研究纯化受体的生物学作用 跟随其微注射植物细胞和P30受体相互作用 将使用酵母遗传两种杂交蛋白在体外表征 检测系统，天然凝胶电泳和DOT印迹测定。 将确定所有纯化蛋白的亚细胞定位 使用电子显微镜。 潜在的发展调节 克隆的基因将通过北方分析进行检查，原位杂交 和使用转基因植物进行遗传实验。 （iv）使用纯化的蛋白质开始表征细胞 大分子PD转运的途径。 使用蛋白质蛋白质 在本提案中开发的相互作用分析，纯化的蛋白质将 作为识别其他蜂窝成分的特定探针 PD传输途径。 除了表征PD传输外， 拟议研究的结果可能具有一般的生物学 与大分子的跨膜转运相关。