A major development in the last few years has been the gradual realization of the magnitude and diversity of intracellular membrane traffic connected with such diverse cellular processes as secretion of macromolecules, endocytosis, transcellular transport, and membrane recycling. It has also become clear that the Golgi complex is the site where all this membrane and product traffic converges and is sorted and directed to its correct intracellular destinations. Little is known, however, about where in this heterogeneous organelle these sorting operations take place. It has also become clear that coated vesicles play a key role in intracellular transport operatons (e.g., in receptor-mediated endocytosis, membrane recycling, transport of lysosomal enzymes from Golgi to lysosomes and of plasmalemma proteins from Golgi to plasmalemma), but little information is available on the nature of the membranes or contents of these vesicles. Our interest has been centered for some time on the Golgi complex--especially in attempting to define Golgi subcompartments, and in determining the pathways taken by membranes and secretory products to and through this organelle in secretory cells. During the previous grant period we have: a) obtained new information on the pathways and kinetics of transport of secretory products; b) obtained data which validates the existence of considerable membrane traffic from the plasmalemma to the Golgi complex in a number of secretory cell types (endocrine, exocrine and immunoglobulin-secreting cells; and c) developed a novel method for the isolation of purified coated vesicle fractions from crude microsomal fractions (by immunoaffinity adsorption onto anticlathrin-coated Staph A cells). During the next grant period we intend to pursue these problems further and have defined three major goals: 1) To determine where in the Golgi complex sorting of lysosomal enzymes and secretory products takes place; 2) To determine whether or not the plasmalemma to Golgi traffic detected in secretory cells truly recycles--i.e., returns to the cell surface, and, if so, to find out whether plasmalemmal components are modified in their passage through the Golgi complex; and 3) to isolate and characterize subpopulations of coated vesicles involved in specific intracellular transport operations. A number of experiments designed to gain insight into these problems are proposed.
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