A fundamental event in any intracellular trafficking pathway is the fusion of a cargo vesicle with its target membrane. In eukaryotes, this membrane fusion event requires members of the SNARE family (soluble N-ethylmaleimide-sensitive factor adaptor protein receptors). SNAREs are characterised by the presence of a C-terminal transmembrane domain, a cytosolic N-terminal regulatory motif, and a cytoplamically oriented SNARE motif essential for the fusion event. Three SNAREs (t-SNAREs) present on the target membrane and one (v-SNARE) on the transport vesicle specifically interact, thus bringing membranes into close proximity and driving the fusion between them. In the present research project, I studied protein transport routes to the vacuole within the plant secretory pathway and aimed to shed light on the role of SNAREs in the biogenesis and filling of of protein storage vacuoles (PSVs) in maturing seeds of the model plant Arabidopsis thaliana. This thesis deals with the preliminary study of the t-SNARE BET11, a potential candidate for a role in the protein transport to PSVs in seeds. The expression patterns and the hypothetical function of the Arabidopsis thaliana BET11 were analysed by fluorescent protein fusions and confocal laser scanning microscopy, GUS assays and biochemical approaches. Transient expression in Nicotiana tabacum demonstrated that, fused to fluorescent proteins, BET11 localises at the trans-Golgi-Network (TGN), a compartment with a central role in both biosynthetic and endocytic protein transport. This led to the more extended analysis of BET11 in the model plant to ascertain its localisation in the secretory pathway. In addition, a bet11 knockout was identified and characterised, and its phenotypes analysed. Also an Arabidopsis line expressing a reporter protein bearing the characterised C-terminal vacuolar sorting signal (ctVSS) (sp-RFP-AFVY) in the bet11 background was generated. Within the knockout background, the vacuolar reporter was secreted, especially in vegetative tissues, and surprisingly not in seeds. The missorting phenotype could be complemented by ectopic expression of BET11.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:560277 |
| Date | January 2011 |
| Creators | Di Benedetto, Sara |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/51382/ |
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