School of BioSciences - Theses
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ItemFrom nucleotide sugars to polysaccharides: How do plants control the delivery of substrates for cell wall biosynthesis and protein glycosylation?( 2021)Plant cell walls constitute one of the most abundant raw biomaterials on Earth. The synthesis of long-chain olysaccharides, the main components of plant cell walls starts ab ovo in the cytoplasm where most of the building blocks for polysaccharide synthesis, so-called nucleotide sugars, are produced. The monosaccharide moieties of nucleotide sugars are incorporated into growing polysaccharide chains either directly at the plasma membrane by lycosyltransferases (GTs) that form cellulose synthase complexes or by those residing in the Golgi apparatus. In the latter case, nucleotide sugars have to pass the Golgi membranes with the help of nucleotide sugar transporters (NSTs). Once inside, they are used by Golgi GTs which assemble polysaccharide chains and decorate proteins and lipids with sugar residues. Recent evidence suggests that in plants nucleotide sugars can be guided to specific polysaccharides and/or glycan decorations, yet the molecular mechanisms of these processes are not fully understood. This PhD research attempts to explore the phenomenon of the targeted substrate delivery by investigating two possible hypotheses: the spatiotemporal distribution of proteins within the Golgi apparatus and the occurrence of direct interactions between NSTs and GTs. The author of this dissertation has tested both of those hypotheses by investigating protein-protein interactions, localising the individual components to their specific sub-compartments within the cell and tracking changes in mutant plants where these processes are modulated. The bifunctional UDP-RHAMNOSE/UDP-GALACTOSE TRANSPORTER (URGT) family from the model plant Arabidopsis thaliana was selected for this study. While this family has six members which in vitro are capable of transporting the same substrates, plant mutant studies indicate substrate preference and targeted delivery to specific cell wall components in vivo. This thesis presents the first evidence that members of the URGT family localise to specific sub-Golgi compartments. The colocalisation studies undertaken as part of this thesis place URGT1 and URGT5 in the cis-Golgi, URGT2, URGT4 and URGT6 – in the medial Golgi, while URGT3 seems to localise to trans-Golgi stacks. Protein-protein interactions studies have identified multiple interaction partners for the six URGTs. Notably, many of those are known galactosyltransferases, which aligns with the transport function of the URGTs. It is therefore highly likely that the identified candidates are true interactors, which use the proximity of the transporter to increase the process efficiency by substrate channelling. This finding is further supported by the fact that observed interactions between URGT family members and GTs often localise to the same sub-Golgi compartment. The study identified new potential galactosyl- and rhamnosyltransferases, including two putative arabinogalactan protein galactosyltransferases. The data obtained during this project suggest, that URGTs may determine the flow of substrate through both spatial separation within the sub-Golgi stacks and direct interactions with GTs. The study presents new insight into the sugar substrate delivery processes in plants, suggests that similar processes may take place in other NST families and that their specificity may be similarly tuned by localisation and interactions.