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Title: Dynamics of Golgi matrix proteins in planta
Author: Osterrieder, Anne
ISNI:       0000 0001 3460 891X
Awarding Body: Oxford Brookes University
Current Institution: Oxford Brookes University
Date of Award: 2008
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The plant Golgi apparatus plays a central role in the secretory pathway, being the site of protein glycosylation, sorting and the synthesis of cell wall polysaccharides and glycolipids. Golgi matrix proteins are involved in the formation and maintenance of the Golgi stack and putative plant homologues have been identified, but their general function and role in plant Golgi biogenesis still is unknown. To study Golgi biogenesis, the Golgi was disrupted using both the drug Brefeldin A (BFA) and a mutated GTP-Iocked form of the small GTPase Sarlp, blocking protein transport from the endoplasmic reticulum to the Golgi. As prolonged expression of Sarl-GTP is toxic to cells, the gene was inserted into a dexamethasone-inducible promoter system, allowing controlled expression and Golgi disruption. The system was characterised by live cell imaging of fluorescent Golgi and ER exit site markers and at the ultrastructural level by electron microscopy. A novel method to reverse the dexamethasone-inducib'e system in tobacco using the glucocorticoid antagonist RU486 was established. The subcellular location of the Golgi matrix proteins GFP-AtCASP, GFP-golgin-84, AtGRIP-GFP and GFP-TMF and other Golgi markers fused to fluorescent proteins was studied during Golgi disruption and reformation. After disruption of Golgi membranes, the trans-Golgi matrix proteins GFP-TMF and AtGRIP-GFP were redistributed into the cytoplasm. The cis-Golgi matrix proteins GFP-AtCASP and GFP-golgin-84 labelled not only the ER and cytoplasm respectively, but also remnant punctate structures. Those remnants were found to co-locate with the ER exit site marker Sarl-GTP-YFP, indicating a close relationship between the cis-Golgi matrix and ER exit sites. After BFA washout, cis-Golgi matrix proteins labelled the reforming Golgi before transGolgi matrix proteins. Interactions between Golgi matrix proteins and regulatory proteins were visualised in vivo in tobacco leaf epidermal cells using fluorescence lifetime Imagmg (FUM) combined with fluorescence resonance energy transfer (FRET).
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available