Poster: Protein modification

Abs # 807: SEC Exhibits O-GlcNAc Transferase Activity Towards Other Proteins

Presenter:	Scott, Cheryl L., scot0136@umn.edu
Authors	Scott, Cheryl L. (A)   Hartweck, Lynn M. (A)   Olszewski, Neil E. (A)
Affiliations:	(A): University Of Minnesota

The goal of our work is to understand the role of SPINDLY (SPY) And SECRET AGENT (SEC) in Arabidopsis. Genetic evidence has shown that SPY and SEC play a role in a number of plant responses. Both SPY and SEC are believed to be O-linked N-acetyl glucosamine (GlcNAc) transferases (OGTs). Loss of both SPY and SEC function causes embryonic lethality. Like their animal counterparts, SPY and SEC are nucleocytoplasmic glycosyltransferases that catalyze the addition of a single GlcNAc in an O-linkage to the hydroxyl groups of serine and threonine residues, and are capable of auto-glycosylation. Taken together, this indicates that SPY and SEC have overlapping functions and that OGT activity is essential in plants. We have developed an E. coli co-expression system that can be used to identify plant substrates of SEC. The proteins, GIGANTEA (GI), REPRESSOR OF ga-1-3 (RGA), and the coat protein (CP) of the Plum Pox Virus (PPV) were modified by SEC. GI has been shown to interact with SPY in both yeast two-hybrid and in vitro binding assays. RGA is a gene involved in the GA signal transduction pathway of Arabidopsis, and PPV is a Potyvirus. The future goals of this work are to biochemically characterize SEC and determine the function of GlcNAc modification of these proteins. Through fine scale deletion mapping, and mass spectroscopy the GlcNAc modification(s) will be mapped. The map positions will be confirmed by site directed mutagenesis. By studying the in planta functioning of proteins that cannot be modified, it may be possible to learn the function of GlcNAc modification. In addition, this work will provide well-described substrates that can be used to characterize the OGT activity of SEC.