VER2 and O-GlcNAc Signaling Figure 4. Detection of O-GlcNAc-modified proteins and their association with VER2 in vernalized and devernalized wheat plants. (A) SDS-PAGE results stained with coomassie blue. (B) Immunoblot analysis of O-GlcNAc-modified proteins in nonvernalized, vernalized and devernalized plants with O-GlcNAc site-specific antibody CTD110.6. Tubulin was immunoblotted as a loading control of total proteins with anti-tubulin polyclonal antibody. (C) Proteins from vernalized and devernalized wheat plants were immunoprecipitated with anti-VER2 antibody and detected with anti-VER2 and CTD110.6 antibodies, respectively. An O-GlcNAc modified protein with a molecular weight of about 35 kD was identified in VER2 immunoprecipitates from vernalized materials; no blotting signals for O-GlcNAcylated proteins were detected in devernalized materials. (D) VER2 was identified in O-GlcNAc immunoprecipitates from vernalized plants but not from devernalized plants. M, molecular weight markers; NV, nonvernalized; V, vernalized; DV, devernalized. IP, immunoprecipitate; WB, western blotting. doi:10.1371/journal.pone.0004854.g004 immunogold labeling was carried out using anti-GFP antibody and did not reveal a vacuolar distribution pattern of VER2-GFP as was reported for other lectins [37]. Gold particles located on the endoplasmic reticulum (ER) and Golgi apparatus and on the surface of uncoated vesicles (Figure 8A to D). This location pattern, combined with the ring punctate signals of VER2-GFP fluorescence (Figure 8E), suggests that VER2 targets to prevacuo- lar compartments (PVCs) reported in Arabidopsis [38,39]. Vacuolar sorting receptor (VSR) is a marker protein of PVC involved in vacuolar sorting [40]. Confocal immunofluorescence microscopy revealed that VER2-GFP colocalized in part with endogenous VSRAt immunolabeled with anti-VSR antibody in transgenic Arabidopsis expressing VER2-GFP (Figure 8F to H). Thus, punctate labeling of VER2-GFP indicated the ER, Golgi and PVCs, which are the components of the vacuolar trafficking pathway. The distribution of VER2 in the secretory pathway raises the possibility that VER2 acts as a sorting receptor of glycoproteins. The general domain organization of carbohydrate sorting receptors contains an exoplasmic domain with one or multiple carbohydrate recognition domains, a single transmembrane domain, and a cytoplasmic domain with sorting signals [41]. Nevertheless, unlike other known sorting receptors, such as ERGIC-53 and VSR, which have a typical transmembrane domain, VER2 did not display any of the main features of sorting receptors (http://www.cbs.dtu.dk/services/TMHMM/, http:// mobyle.pasteur.fr/cgi-bin/MobylePortal/portal.py?form = toppred, and http://www.cbs.dtu.dk/services/TargetP/). VER2 is therefore unlikely a sorting receptor of O-GlcNAcylated proteins in the secretory pathway. The effects of site-directed mutagenesis of phosphorylation sites on the compartmentalization of VER2 in Arabidopsis The amino acid sequence analysis of VER2 showed predicted serine and threonine phosphorylation sites and Yin-Yang sites, which have potential reciprocal dynamic phosphorylation and O- GlcNAc modification possibility (http://www.cbs.dtu.dk/services/ NetPhos/) (http://www.cbs.dtu.dk/services/YinOYang/). The serine at site 33 and threonine at site 209 of the amino acid sequence are predicted Yin-Yang sites. Phosphorylation on these two sites is important for protein functioning. We generated transgenic Arabidopsis overexpressing mutated VER2 of S33G and T209A to observe the compartmentalization of fluorescence of GFP. The location pattern of mutant VER2-S33G is different from that of VER2. VER2S33G was predominantly targeted to punctate structures, and very weak location signaling could be seen in nuclei and cytoplasm (Figure 9A). Transgenic Arabidopsis plants expressing VER2-T209A showed no obvious differences in GFP fluorescence distribution (Figure 9B). PLoS ONE | www.plosone.org 5 March 2009 | Volume 4 | Issue 3 | e4854