Trafficking, stability and substrate specificity of glycosyltransferases (GTs) involved in xyloglucan biosynthesis in plant Golgi.
Date
2023-08
Authors
Zhang, Ning
Major Professor
Advisor
Zabotina, Olga A
Chen, Baoyu
Myers, Alan
Bassham, Diane
Yin, Yanhai
Committee Member
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Abstract
Xyloglucans (XyGs) are the predominant hemicellulosic polysaccharides in the primary cell walls of most flowering plants including the Arabidopsis thaliana (Arabidopsis) and directly affect cell wall mechanical strength, cell expansion, and plant ability to respond to environmental stresses. Glycosyltransferases (GTs) catalyze glycosylation of various molecules and the synthesis of numerous polysaccharides, including XyG, by transferring activated sugar residues from donors to acceptors in the Golgi and ER. The mechanism of transportation, stability, and assembly of XyG-synthesizing GTs are critical questions for understanding polysaccharide biosynthesis in plants and is the objective of our investigations. In my dissertation, I revealed the specific function of the three xylosyltransferases, XXT3, XXT4, and XXT5, in adding the xylosyl residue on the third glucosyl in the glucan backbone of XyG to finalize the synthesis of the XXXG type XyGs. The knock-out mutant plants xxt3xxt4xxt5 showed a reduced length of root hairs and siliques. I also investigated the ER-to-Golgi trafficking of the XyG synthesizing GTs. I demonstrated that all enzymes involved in XyG synthesis are delivered to Golgi independently from each other and involved in protein-protein interactions after being localized in Golgi. Next, I evaluated the stability of all XyG-synthesizing GTs by determining their half-lives using treatment with Cycloheximide (CHI) and two independent approaches measuring protein degradation. Obtained results demonstrated that the XyG-synthesizing GTs have different stabilities and can be divided into two main groups: (a) the long-living proteins, including MUR3, FUT1 and CSLC4, and (b) the short-living proteins, including XXT1, XXT2, XXT5 and XLT2. Lastly, I investigated the role of the N-terminal cytosolic tail and the transmembrane domain (TMDs) of two XyG xylosyltransferases (XXT2 and XXT5) in their ER to Golgi export and Golgi localization. Using the set of deletion mutants and confocal microscopy, I demonstrated that, the specific protein sequence “RALRQLK” and “LPTTTLTNGGGRGGR” in N-termini of XXT2 and XTT5, respectively, are critical for their Golgi-localization. The di-arginine motifs were found in both proteins’ N-termini and the mutation of these arginine residues to glutamines alter XXT2 and XXT5 localization, significantly reducing their Golgi-localization and increasing their retaining to ER or causing their aggregation and non-Golgi dot-like localization, indicating the critical function of di-arginine motifs in ER-Golgi transportation of XXTs. In summary, all obtained results broaden our knowledge about functions of the polysaccharide synthesizing GTs, including their ER-Golgi transport, turnover in Golgi, and potential formation of multiple complexes to synthesize polysaccharides efficiently and with high reproducibility of their structural patterns.
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dissertation