Characterization of Ubiquitin Pathway in Higher Plants

9306759 Callis真核生物泛素途径涉及蛋白质泛素与细胞蛋白质的共价连接。 泛素化的蛋白质然后可以被靶向用于蛋白水解，或者泛素化可以导致稳定的翻译后修饰。 因此，泛素途径是真核生物中调节蛋白质水平及其活性的重要途径。 泛素修饰底物的分离和鉴定对于理解该途径的调节和特异性是必不可少的。 虽然基于免疫印迹研究，泛素化蛋白的数量似乎很大，但识别的体内底物的数量很少。 在高等植物中，只有一种内源性植物蛋白被鉴定为泛素途径的底物。 两种不同的方法对确定和分离高等植物在体内底物拟南芥作为模型系统进行了详细概述，并将进行。 鉴定出更多的体内底物将进一步加深我们对高等植物中ub途径作用的认识，并有助于理解泛素化的调控。 将克隆泛素途径的两个组分，这两个组分在高等植物中相对不具有特征，并且对于理解该途径的调节是重要的。 它们的分离也将有助于产生具有与体内发现的特异性相同的特异性的体外缀合系统的长期目标。 在高等植物中，泛素由一个多基因家族编码。 拟南芥中泛素基因家族的所有成员都已被分离。 多个泛素基因的作用将通过泛素基因表达的环境和发育变化的研究来解决。 此外，泛素基因在其他生态型中的表达将被确定，以协助鉴定功能重要的基因。 一 这两种方法的结合将增加我们对高等植物代谢、发育和生理中的泛素途径的理解，并确定其在介导植物对环境胁迫的响应中的作用。 ***

9306759 Callis The eukaryotic ubiquitin pathway involves the covalent attachment of the protein ubiquitin to cellular proteins. Ubiquitinated proteins can then be targeted for proteolysis, or ubiquitination can result in a stable post-translational modification. Therefore the ubiquitin pathway represents an important pathway regulating protein levels and their activities in eukaryotes. The isolation and identification of substrates for ubiquitin modification are essential for an understanding of the regulation and specificity of this pathway. While the number of ubiquitinated proteins appears to be large based on immunoblot studies, the number of identified in vivo substrates is small. In higher plants, only one endogenous plant protein has been identified as a substrate of the ubiquitin pathway. Two different approaches towards identifying and isolating higher plant in vivo substrates using Arabidopsis thaliana as the model system are outlined in detail and will be performed. Identification of additional in vivo substrates will further our knowledge of the roles of the ub pathway in higher plants and contribute toward an understanding of the regulation of ubiquitination. Two components of the ubiquitin pathway that are relatively uncharacterized in higher plants and are important for an understanding of the regulation of the pathway will be cloned. Their isoation will also contribute toward a long term goal of producing an in vitro conjugation system with specificity identical to that found in vivo. Ubiquitin is encoded by a multi-gene family in higher plants. All members of the ubiquitin gene family in Arabidopsis thaliana have been isolated. The roles of multiple ubiquitin genes will be addressed through studies of the environmental and developmental changes in ubiquitin gene expression. In addition, the expression of ubiquitin genes in other ecotypes will be determined to assist in the identification of functionally important genes. A combination of these two approaches will increase our understanding of the ubiquitin pathway in higher plant metabolism, development, and physiology, and define its role in mediating plant responses to environmental stresses. ***