Determining the Function of O-GlcNAc Protein Modification in Signal Transduction

University Of Minnesota-Twin Cities, Minneapolis MN

Investigators

Abstract

Intellectual Merit: Post-translational modification of cytosolic and nuclear proteins with O-linked ß-D-Nacetylglucosamine(O-GlcNAc) affects signal transduction pathways in plants andmetazoans. In Arabidopsis thaliana, this modification is made by two O-linked N-acetylglucosaminetransferases (OGTs), SECRET AGENT (SEC) and SPINDLY (SPY). In arabidopsis, O-GlcNAc modification is involved in embryogenesis, meristem activity,circadian regulation and responses to gibberellin, cytokinin and light. While many plant proteins are likely O-GlcNAc modified, the identities of these proteins is largely unknown. A goal of a previous NSF award was to identify these O-GlcNAc modified proteins. This work has led to identification of proteins that are modified by E. coli expressed SEC and the development of methods that enrich O-GlcNAc modified peptides sufficiently for determining the peptide sequence and mapping the modification by mass spectrometry. The first specific aim of this award will employ these methods to determine what arabidopsis proteins are modified and map the modification(s). The second specific aim will employ molecular genetic approaches to determine the function of the modified proteins and the effect of O-GlcNAc modification on their activity. The nucleocytoplasmic ß-N-acetylglucosaminidase (OGA) that removes the modification has been identified in metazoans and deficiency in this enzyme has been shown to affect signaling pathways and to be associated with human diseases. While related OGAs are present in bacteria, in silico approaches do not identify a plant OGA suggesting that plants have an unrelated OGA. The final aim of this project will use biochemical and molecular biological approaches to identify the arabidopsis OGA and investigate its role in regulating plant processes. Broader Impacts: High school, undergraduate and graduate students have been trained under past awards and this training will be continued. The PI will participate in U of MN summer programs by providing laboratory research experiences for high school students, teachers and undergraduates. The PI will attempt to stimulate an interest in science in high school students by giving laboratory tours and presentations to students and teachers. The work of this award will contribute to scientific infrastructure by developing new methods for the identification of O-GlcNAc modified proteins and the mapping of the modification on these proteins.