INTEGRATED MODELING OF PROTEIN AND MEMBRANE TRAFFIC IN THE SECRETORY PATHWAY

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DESCRIPTION (provided by applicant): The eukaryotic secretory pathway consists of the endoplasmic reticulum, Golgi apparatus, secretory vesicles, endosomes, lysosomes, and the plasma membrane. This pathway is responsible for synthesis, processing, and secretion of all proteins, hormones, and neurotransmitters produced by a given cell type. It also uniquely defines the functions and responses of each cell by delivering to the cell surface all receptors, enzymes, ion channels and transporters.With the advent of green fluorescent protein (GFP) tags we and others have measured, using time-lapse confocal microscopy in living cells, the dynamics of protein transport n the secretory pathway. These measurements have allowed us to test current theories of Golgi function and to propose a new theory that accounts for data that are unexplained by current paradigms. While this information is useful, it fails to account for the prominent role of membrane lipids and cholesterol in powering and regulating the secretory pathway. The long term goal of this research is formulation and testing of a mechanistic kinetic model of the secretory pathway that integrates five key subsystems or modules: cargo protein, glycerophospholipids (GPL), sphingolipids, cholesterol, and resident Golgi protein. To advance this goal we propose three specific aims: 1) Develop a kinetic model that combines cargo, glycerophospholipid, sphingolipid and resident protein modules and test it and two competing models against both classical observations and our new data on Golgi exit of a GFP-tagged cargo protein, vesicular stomatitis virus G protein (VSVG-GFP), 2) Construct a kinetic model of intracellular cholesterol trafficking and its hypothesized regulation of cargo export, combine it with the model from Aim 1 and test it against secretory cargo and sterol kinetic data in cells treated with agents that decrease, increase, or chemically modify cellular cholesterol content, 3) Modify the model from Aim 2 to include hypothesized protein-cholesterol interactions and test the resulting model against cargo and sterol kinetics in cells overexpressing either cargo proteins or resident ER or Golgi proteins. Methods: Kinetic modeling using ProcessDB and Berkeley Madonna software, GFP fusion proteins, time-lapse fluorescence microscopy, live cell imaging. Derangements of cellular cholesterol processing are central aspects of major human diseases including heart disease and diabetes. Since the secretory pathway is vital to normal cellular function we need to know how cholesterol controls it.