Objective
- To understand the mechanisms of wheat gluten protein systhesis, transport, folding and assembly in relation to the formation of protein bodies and the structure and functional properties of gluten.
Results so far
- The production of suitable levels of in vitro synthesised gamma-gliadin in a cell free system; this has been facilitated by cloning the coding sequence of the gamma-gliadin cDNA into the pGEM3 transcription vector. Subsequent transcription and translation using this template resulted in easily detectable levels of radiolabelled protein.
- The gamma-gliadin pGEM template has been used to set up protein translocation assays in a cell free system containing pancreatic canine microsomal membranes. The translocation of the gamma-gliadin was observed, as determined by signal peptide cleavage and protease-protection assays. The use of stalled translocation-intermediates was attempted to examine the interaction between the gamma-gliadin-translocating nascent chain and chaperones such as BiP and PDI. Initial attempts utilised long nascent chains of greater than 20 kD in length. However these polypeptides have failed to produce crosslinks, indicating that such protein-protein interactions occur when the nascent chain initially enters the ER lumen. Therefore, shorter nascent chains have been generated and are at present being examined in vitro.
- Transgenic tobacco lines expressing gamma-gliadins with modified targeting information are being used for electron microscopic studies. The ultrastructure (in particular of the endomembrane system) is being examined. Immunogold labelling is being optimised for this particular system to allow the precise determination of the subcellular locations of the transgene products.
- Expression of an epitope-tagged version of the gamma-gliadin in transgenic wheat is also being carried out to determine the deposition of the protein in a homologous background. The c-myc epitope tag has been introduced, in frame, into the C-terminus of the gamma-gliadin, and this will be expressed in wheat under the control of the HMW-glutenin promoter. Transgene expression will be detected by the use of the 9E10 monoclonal against the c-myc tag.
- To determine the pathway and mechanism of gamma-gliadin translocation across the ER membrane, including interactions with proteins of the translocation machinery;
- To determine the stage at which the chaperone BiP interacts with the nascent gamma-gliadin chain to aid protein folding;
- To determine the effects of ER-localised enzymes and molecular chaperones on the folding of wheat storage proteins and their assembly into protein bodies using an in vitro system with proteins expressed in E. coli;
- To determine the assembly into protein bodies of natural and modified storage proteins in transgenic tobacco plants, and the effects of over-expression of ER localised enzymes and molecular chaperones on these processes.
Topic(s)
Data not availableCall for proposal
Data not availableFunding Scheme
CSC - Cost-sharing contractsCoordinator
BS41 9AF BRISTOL
United Kingdom