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. ***
