Final Report Summary - EXHOMO (Glycosylation of peptide hormones and extensins from the model plant Arabidopsis thaliana and in-vitro synthesis of glycosylated peptides.) 1. FINAL PUBLISHABLE SUMMARY REPORTThis project investigated the pathways by which arabinose sugars are added to two groups of proteins, extensins and peptide hormones, the latter of which is known to have a profound effect on the growth and development of various plant species, including crop plants. Arabinosylation appear to be required for complete biological activity but is challenging to achieve using synthetic chemistry. Identifying the enzymes that modify peptide hormones could help synthesize active signalling peptides in yeast or bacteria systems in the future. This project also sought to understand how the enzymes thought to be responsible for modifying these proteins are spatially arranged in the secretory system and which proteins they interacted with. Synthesis of complex plant polysaccharides in yeast requires the presence of the correct interacting proteins. Detailed knowledge of the interactions and compartmentalization of these enzymes is therefore required in they are ever to be synthesized in heterologous systems. This study concentrated on two peptide hormones, PSY1 and CLV3. A group of proteins (HPAT1, HPAT2 and HPAT3) had previously been identified as being responsible for the addition of the first of three arabinose residues on to PSY1. However, the enzymes adding the second and third arabinose residues had not been discovered. Conversely, the enzymes adding the second, third and fourth arabinose residues on to extensin (RRA1 – 3, XEG113, EXAD) had been identified by the host group but the enzyme adding the first arabinose was not known. This study demonstrated that HPAT1 -3 were involved in extensin arabinosylation as well as arabinosylation of PSY1 as a different ratio of arabinose decoration was observed on extensins extracted from Arabidopsis plants mutated in either the HPAT1, 2 or 3 compared to plants not carrying any mutants (Figure 1). It was concluded to be extremely likely that RRA3 were involved in arabinosylation of PSY1 as protein interactions were observed between HPAT3-RRA3 in yeast two hybrid assays and between HPAT2-RRA3 (Figure 2a) and HPAT3-RRA3 (Figure 2b) in BiFC assays in-planta. No interactions were seen with XEG113, so it was not possible to conclude whether this enzyme was likely to be involved in the arabinosylation of PSY1. The arrangement of enzymes in the extensin arabinosylation pathway was determined using Free Flow Electrophoresis. This is a little-used technique which Dr. Parsons has applied very successfully to the separation of the secretory pathway, where much protein modification occurs. By monitoring the abundance of enzymes in compartments belonging to the early (ER, cis-Golgi), middle (medial-Golgi) and late (trans- and post-Golgi) zones of the secretory pathway it became apparent that the first enzyme thought to act in this pathway (P4H5, catalysing the hydroxylation of prolines) occurred in the ER/cis-Golgi, whilst HPAT2, 3, RRA3 and XEG113 occurred in the medial-Golgi. This suggests that hydroxylation of proline is physically separated from arabinosylation but that addition of up to three arabinose residues all occurs in the medial Golgi. This implies that efficient arabinosylation of peptide hormones could be achieved if hydroxylated substrates were supplied in-vitro without compartmentalization of arabinosyl transferase enzymes. EXAD catalyzes the addition of the fourth and, usually, terminal arabinose on extensins. Subcellular localization of fluorescently labelled EXAD revealed a trans-, or post-Golgi localization, in agreement with free-flow electrophoresis results. However, EXAD did interact with RRA3, at which point it showed a more typical Golgi localization, suggesting that this enzyme had a slightly different distribution in the secretory system from other extensin enzymes. The lipid composition of membranes in different compartments of the secretory system is likely to play a key role in determining the localization and activity of membrane-anchored proteins such as those investigated here. Therefore further to the objectives in Annex 1, in collaboration with other groups at Copenhagen University, lipid analysis of the different subcompartments of the secretory pathway was investigated. This revealed clear changes in the major lipid groups. This is a precedential result as the resolution between subcompartments required for this type of analysis was only possible using free flow eelctrohpresis. Sub-compartment analyses were also extended to other glycosylated proteins and polysaccharides, revealing in which subcompartments significant structural changes were occurring. Related documents final1-final-report-pictures.docx
