MECHANISMS OF REGULATED EXOCYTOSIS IN TETRAHYMENA

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DESCRIPTION: Dense core granules store hormones, digestive enzymes, and other proteins for stimulation-coupled exocytosis. Dense core granules are found in number of unicellular organisms, including several medically relevant parasites. Although basic features of dense core granule exocytosis are conserved throughout eukaryotes, our understanding of fundamental steps in this pathway is limited by the paucity of appropriate experimental systems with flexible genetic and molecular genetic tools. Molecular genetic and biochemical approaches will be used to analyze the functions of proteins involved in the biosynthesis and exocytosis of dense-core granules in the ciliated protist, Tetrahymena thermophila. Granule assembly involves the ordered condensation of a set of granule proteins beginning in the trans-Golgi network. Proteolytic processing of the granule proteins appears to regulate granule formation. The principal investigator's lab has cloned the major Ca++-binding granule protein and has obtained evidence for a series of confrontational transitions that occur at specific steps in granule secretion. In the first specific aim the PI will determine whether the conformational changes direct granule assembly by expressing variants of the major granule protein Grl1p. The genes encoding the other major granule proteins will be disrupted to identify novel and overlapping functions. Following stimulated exocytosis of stored granules, new granules are synthesized de novo in a synchronous fashion. A subtractive library approach will be used to identify genes involved in granule synthesis. The function of these gene products will be analyzed by blocking translation using expressed antisense expression will be used to identify genes whose translational inhibition causes defects in granule exocytosis.