Genetically enconded peptides as tools for studying the post-translational modification in Arabidopsis O-glycoproteins

ESTEVEZ J. M. & C. SOMERVILLE.

Copenhagen

Congreso; Plant Cell wall Meeting; 2007

Cell Wall Meeting

One of the many challenges in understanding the synthesis and function of Hydroxiproline-Rich Glycoproteins (HRGPs) like Arabinogalactan associated Proteins (AGPs) and extensins is that they are typically very highly glycosylated. The high degree of glycosylation combined with the large number of species makes it difficult to purify a specific HRGP for structural analyses. For this reason we are using genetically encode peptides (GEP) composed of repetitive proline units containing AGP- and extensin-motifs to study the prolyl hydroxylation and subsequent O-glycosylation of the hydroxyproline residues. GEP were expressed in planta (Arabidopsis) and the amount of hydroxylation and glycosylation of the various constructs were analyzed on pure isolated GEP. In addition, the accumulation of the glicoproteins exhibited a high degree of cell-type specificity within various tissues due to post-transcriptional effects. The observations reveal a high level of complexity in the synthesis, secretion, and turnover of these HRGPs. The expression of GEP was also visualized in live cells by confocal microscopy following labelling with the small FlAsH- or ReAsH-EDT2 reagents together with Fluorescent proteins (GFP, YFP, and mCherry). Peptides with a repetitive (Ser-Pro) units linked to a signal sequence and a tetra-cysteine tag (TC) were expressed transiently in tobacco and transiently and stably in Arabidopsis under control of a strong constitutive promoter. The results obtained indicated that GEP tag with FlAsH-TC and ReAsH-TC were correctly targeted to the ER and Golgi bodies but also secreted to the plasmamembrane. FlAsH-TC showed better signal noise ratio compared with the ReAsH-TC. In addition, we carry out pulse-chase experiments using dual labelling FlAsH-TC/ ReAsH-TC or viceversa, in order to differentiate sub-populations of GEP during their biosynthesis on the secretory pathway. Now, we are imaging the expression of GEP not only at confocal microscopy but also at EM level using photo-oxidable ReAsH reagent. Using this multiple approaches, we expect to address interesting questions related with the subcellular localization of the HRGPs together with a deeper understanding of their post-translational modifications. Keywords. Arabinogalactans, cell wall glycoproteins, extensins, FlAsH-ReAsH tags, GEP genetically enconded peptides, O-glycosylation, proline-hydroxilation, subcelular localization.