Protein Modification and Turnover
Hartweck, Lynn M , Oldenhof, Harriette , Olszewski, Neil E .
Analysis of O-GlcNAc modification GIGANTEA.
Protein function can be greatly affected by cytoplasmic/nuclear posttranslation modification with O-linked N-acetylglucosamine (O-GlcNAc) to serine or threonine amino acids. Mutant studies with the two arabidopsis O-GlcNAc transferases, SPY and SEC also indicate that O-GlcNAc modification is involved in responses to the hormones gibberellin and cytokinin, responses to light, circadian regulation, meristem, gamete and embryo development and viral pathogenesis. To determine how modification of individual proteins affects these processes, we developed an E. coli-based co-expression system to identify SEC substrates and map the modification sites. Plum pox virus (PPV) capsid protein, a protein shown previously to be modified, was modified by SEC in E. coli with the same specificity as in plants. O-GlcNAc modification of PPV appears to affect viral pathogenesis. We also identified GIGANTEA (GI) as a SEC substrate and mapped the O-GlcNAc modification to a single site, threonine 829. GI is a protein that is central to the circadian clock and most GI mutations result in a late flowering phenotype. To examine the function of O-GlcNAc modification of GI, we created transgenic plants that would express wildtype or non-modifiable mutant versions of GI under the control of the 35S promoter. Although we were able to obtain over 25 independent transformants that were had normal flowering times with the wildtype GI sequence, very few plants were obtained that expressed RNA for the non-modifiable mutant protein. To continue testing O-GlcNAc modification of GI, we have taken three strategies. In case overexpression caused secondary effects, new constructs using the GI promoter are under construction. Since GI and SPY interact physically and genetically, GI might also be modified by SPY. To test whether modification by SPY is also affecting GI, the new constructs will be tested in plants doubly mutant for gi and spy. Using the E. coli-based co-expression system we will examine GI is modified at other sites by hybrid SEC/SPY proteins.
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1 - University of Minnesota, Plant Biology, 250 Biological Sciences Center, 1445 Gortner Ave, St. Paul, MN, 55108, USA
2 - University of Minnesota, Plant Biology
Post Translational Modification
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM