The general goal of these studies is to further our understanding of the functional organization of the Golgi apparatus and of Golgi-related traffic patterns in plant cells. In particular, we will a) determine whether a given Golgi stack can simultaneously assemble glycoproteins and polysaccharides, or if the two types of molecules are processed by different types of Golgi stacks; b) determine whether different types of complex polysaccharides are made in different Golgi cisternae and that they can exit the Golgi from these cisternae without having to pass through the trans Golgi network; c) examine where and how sorting of secretory and vacuolar proteins occurs in the Golgi apparatus of plants. Carrot suspension culture cells will be used as experimental system both because of the ease with which they can be grown, and because individual cells can be induced to differentiate into somatic embryos. The proposal is divided into two parts, a morphological and immunocytochemical section designed to delineate both structural and functional aspects of the Golgi apparatus and related membrane systems, and a biochemical section devoted to the development of methods for fractionating Golgi stacks, cis and trans cisternae, and secretory and coated vesicles, and to the biochemical characterization of the fractions in terms of enzymatic activities and of traffic patterns based on pulse-chase kinetics of glycoproteins and polysaccharides. Of central importance will be the use of polyclonal (monoclonal) antibodies against two complex polysaccharides (rhamnogalacturonan I and xyloglucan), two related cell wall glycoproteins, extensins-1 and -2, an inducible vacuolar enzyme, chitinase, and a chemical indicator of acidic membrane compartments, DAMP. The experiments will also make use of drugs (monensin, cytochalasin D, fluorescein isothiocyanate) that disrupt the secretory pathway at different points.
