The general aim of this proposal is to understand how proteins are targeted to their correct intracellular compartments after synthesis. Specifically, we will use the pituitary cell line, AtT- 2O, to study molecular events underlying the assembly of secretory granules. Recent evidence suggests that neuroendocrine cells possess a constitutive and a regulated pathway for secretion, and that proteins destined for the cell surface are selectively sorted into different secretory vesicles for externalization. How proteins select the proper organelles, and how the machinery of these two types of secretory vesicles differs from each other are not understood. In this proposal, we will try to identify the sorting signals that direct peptide hormones into regulated granules by in vitro mutagenesis and DNA transfection. We will also purify the sorting compartment and characterize them biochemically. To elucidate the cellular sorting mechanisms, we will isolate specific sorting proteins from the sorting compartment and study their interactions with the transported proteins. We will ask if different sorting proteins are used by different types of cells with regulated secretory functions, or whether these cells share a common sorting machinery. To identify other components involved in sorting, we will set up an in vitro system to assay sorting in permeabilized cells. This system will be used to assess the role of clathrin in regulated secretion. Finally, we will devise systems to dissect functional components of the granule that are involved in the temporal and spatial regulation of secretory activities.
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