Molecular Dissection of the Plant Golgi: A Heterologous Approach

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The focus of this project is on the Golgi apparatus, which is the subcellular site of the biosynthesis, modification, and sorting of glycoproteins and complex polysaccharides of the plant cell surface. Very little is known about the molecular composition of the Golgi in plants. Few of the enzymes involved in the synthesis of cell wall matrix polysaccharides have been identified and characterized. Nothing is known about the organization of these enzymes within the Golgi, or how such organization is maintained, or how polysaccharide synthesis is initiated, or how complex polysaccharides are assembled and targeted to specific locations in the cell wall. Part of the difficulty has been an inability to subfractionate plant Golgi into cis-, medial, and trans-cisternal elements. A novel approach is proposed to achieve such separation, in which a chimaeric protein (translational fusion between the E1 glycoprotein of avian Infectious Bronchitis Virus, IBV-E1, and GUS) will be used to target the manipulable GUS marker to the Golgi. This idea is based on observations in animal systems that IBV-E1 is targeted to and accumulates in the membranes of the cis-Golgi cisternae. In this project, the ultrastructural location of chimaeric markers containing all or part of the IBV-E1 coding sequence will be determined by EM colloidal gold immunolocalization (to be done in collaboration with experts in this technique). These studies will establish the feasibility of more extensive biochemical work exploiting the IBV-E1 fusion expressed in plant cells. %%% A large fraction of the dry weight of plants is in the cell walls. Plant cell walls are critical to the survival of the plants, since they provide the rigidity needed to maintain morphology and turgor pressure. There are indications that cell walls are also critical components of defense mechanisms against plant pathogens. They are the biological sources of significant commercial products, such as cellulose and cellulose-based products, wood products, and dietary fiber. This novel approach to understanding the cell biology of plant wall biosynthesis may open up the possibility of genetic engineering of plant walls for specific purposes. In addition, the research should provide important information about the parameters that affect expression of animal glycoproteins within genetically engineered plants. This should be relevant for applications involving the recovery of commercially useful animal glycoproteins from transgenic plants.