CELLULAR PROTEINS INVOLVED IN TRANSPORT THROUGH

在植物中，细胞通过细胞间连接 连接，胞间连丝（PD）。 类似于细胞间 在动物的交流中，通过PD的运输起着至关重要的作用， 植物生理与发育 最近的证据表明，PD可以 主动运输大分子。 的分子机制 主动PD转运的发生是未知的。 一种方法来阐明这些 研究植物病原体如何通过宿主细胞传播的机制。 一般来说，入侵的微生物不会发明新的代谢产物， 途径，但适应现有的细胞过程，其生命周期。 的 病原体通过腹膜透析传播的最佳研究实例是细胞间传播 烟草花叶病毒（TMV）基因组RNA的转移， 一种病毒编码蛋白P30 在这里，P30将被用作研究的工具 参与PD转运的细胞蛋白。 拟议的研究已 四个具体目标。 (i)参与PD转运的P30细胞质受体的纯化。 与核输入类似，通过PD的主动转运可能需要 特异性细胞质受体。 为了验证这一假设， 将使用抗P30抗体鉴定和纯化P30细胞质受体。 抗独特型抗体以及与固定化P30的结合。 的基因 将通过免疫筛选cDNA克隆编码P30受体的基因 表达文库或在对纯化的表达文库进行微测序后， proteins. (ii)植物细胞壁相关蛋白激酶的纯化， 特异性磷酸化P30。 与许多其他交通工具类似 在这些过程中，磷酸化可能参与PD转运。 这里 细胞壁相关蛋白激酶，一种潜在的PD组分， 磷酸化P30，将被研究。 该蛋白质将通过以下方法纯化： 去除细胞壁基质并溶解，然后进行离子交换 和凝胶过滤色谱法。 纯化的蛋白激酶将 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。 的 将在体内研究纯化的受体的生物学作用 在显微注射植物细胞和P30受体相互作用后 将其特征在于在体外使用酵母遗传二杂交蛋白 检测系统、天然凝胶电泳和斑点印迹分析。 将确定所有纯化蛋白的亚细胞定位 使用电子显微镜。 潜在的开发监管 克隆的基因将通过北方分析、原位杂交 和使用转基因植物的遗传实验。 (iv)使用纯化的蛋白质开始表征细胞 大分子的PD转运途径。 使用蛋白质-蛋白质 在该建议中开发的相互作用测定中，纯化的蛋白质将 作为特异性探针，以识别 PD运输途径。 除了表征PD运输外， 拟议研究的结果可能具有一般生物学意义， 与大分子跨膜转运的相关性。

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.