TGNPROTEOMICS · Proteomics of the trans-Golgi network in Arabidopsis thaliana using immunoisolation as a means of organelle purification

The aim of this proposal is to identify novel proteins in the Arabidopsis thaliana trans-Golgi network (TGN). This organelle is poorly characterised, yet is known to play an important role in sorting and forward transport of proteins leaving the Golgi apparatus, but also in retrograde transport and endocytosis. The knowledge generated in this project will help understanding cellular transport in plants, which, in the long term, will facilitate crop improvement in Europe. This proposal involves the development of a purification strategy for TGN membranes that can also be applied to isolate other compartments from plants. The TGN membranes will be purified from a line expressing a GFP fusion of the TGN-specific H+-ATPase subunit VHA-a1 (VHA-a1-GFP) throughout the plant. A TGN-enriched fraction will be obtained by density centrifugation of a complete lysate and this fraction will be used to immuno-purify TGN membranes using GFP antibodies immobilised on magnetic beads. The peptide content of the purified TGN will be determined by LC-MS/MS. The most interesting proteins will be selected for further characterisation using confocal microscopy, yeast two-hybrid analysis and reverse genetics approaches. The proposed work will be carried out in the laboratory of Professor Møller at the University of Stavanger in Norway. It will also form part of a recently established Centre for Organelle Research (CORE), which is an international research centre with seven research groups focusing on organelle biology. The centre includes a protein laboratory with highly advanced equipment operated by experienced staff. The applicant is a plant molecular biologist with four years of experience as post-doctoral researcher who has worked on Golgi and TGN proteins over the last three years. Using a multi-disciplinary approach this project with accompanying training programme ensures that she will be able to acquire a wide range of new technical and complementary skills.