The focus of this Ph.D. study has primarily been to utilize and adapt the acceptor photobleaching technique for measuring of Förster resonance energy transfer (FRET) to tudy proteinprotein interactions (PPIs) among glycosyltranseferases (GTs) and nucleotide ugar transporters (NSTs) localized to the plant Golgi apparatus and involved mainly in arabinogalactan protein (AGP) biosynthesis. Co-expression analysis identified 4 GTs and 4 NSTs possibly involved in AGP biosynthesis. As part of the method development, the cytoskeleton-acting agent Cytochalasin D was tested as an inhibitor of the actinomyosin based movement of Golgi vesicles, and was proved to be superior to commonly used fixatives such as the cross-linking agent paraformaldehyde which causes quenching of the fluorophores.
According to FRET analysis, the results showed association between two galactosyltransferases, AtGALT29A and AtGALT31A, which remarkably enhanced the nzymatic activity specifically towards the β-(1→6)-galactan side chain elongation of AGP. No interaction was found between AtGALT31A and AtGlcAT14A. In addition, the interaction between the putative arabinosyltransferases, ARAD1 and ARAD2, involved in rhamnogalacturonan-I biosynthesis was proved and further supported by BiFC and non-reducing gel. Finally, association among four different NSTs (AtUTr5, AtUTr5B, At5g41760 and At4g35335) was shown as both homo- and heterodimeric complexes.
In conclusion, our findings point to the notion that enzymes and transporters involved in glycosylation process require interaction partners; hence the screening for PPI would ubstantially facilitate the biochemical characterization of putative enzymes involved in glycoprotein glycosylation and cell wall polysaccharide biosynthesis.