The general goal of these studies is to analyze the functional organization of the plar Golgi apparatus produces both glycoproteins and proteoglycans as well as large quantities of complex polysaccharides has in the past caused many technical and experimental problems. Recent findings, however, suggest that the dual synthetic capabilities may also offer unique opportunities to gain new insights into its functional organization and its dynamic properties. Before this can happen, several technical hurdles have to be overcome. Thus, there are still no defined biochemical markers for cis, medial and trans Golgi cisternae and for the trans Golgi network (TGN), and subfractionation of plant Golgi stacks has not been achieved to date. For this reason, the first section of this proposal focuses on the development of novel marker enzyme systems for monitoring the presence of specific Golgi compartments based on results of our recent immunolocalization studies. The second section describes novel approaches for isolating purer Golgi fractions, as well as Golgi stacks with different ratios of cis, medial and trans cisternae, using 90% physiologically synchronized tobacco BY-2 cells. The third section builds on recent observations on the effects of brefeldin A (BFA) on the secretory apparatus of plant cells. Since the Golgi stacks in BFA- treated cells do not disappear, but secretion of most products is inhibited, we will determine if intercisternal transport within Golgi stacks can still occur. In addition, we will compare the membranes of secretion-incompetent secretory vesicles of BFA-treated cells with secretion competent vesicles of control cells. The fourth section will evaluate TGN38 as protein marker of the TGN of plant cells. Finally, in a collaborative study we will employ antibodies to immunolocalize at the electron microscopical level specific small GTP-binding proteins associated with the secretory apparatus of plant cells, both in the presence and absence of GTPyS.
