The plasma membrane of epithelial cells is divided by tight junctions into two distinct domains, apical and basolateral, that differ markedly in their protein and lipid composition. A central question in cell biology is to determine how this asymmetry is generated and maintained. Viruses that cause influenza, measles, and other diseases depend on intracellular protein sorting pathways for their propagation. Understanding how intracellular sorting operates may provide new approaches to combating these diseases. Plasma membrane proteins and some membrane lipids are sorted in the trans Golgi network in certain cultured epithelial cells, and are packaged into transport vesicles destined for the apical or basolateral surface. Proteins anchored in membranes by glycosyl phosphatidyl inositol (GPI) are transported apically. GPI anchorage can cause this targeting. Glycosphingolipids are also delivered primarily to the apical surface. It has been proposed that GPI-anchored proteins may associate with glycosphingolipids inside the cell, and that the two may be sorted co-ordinatedly into the apical transport pathway. Our goal is to test this theory, and to isolate and characterize the lipid-protein complexes. Membrane complexes that contain both glycolipids and GPI-anchored proteins have been isolated, using their surprising insolubility in non-ionic detergents.
The aim of this application is to learn whether these complexes contain the domains of associated GPIanchored proteins and glycolipids from the Golgi that were proposed above. These domains would be likely to contain sorting proteins, since they form in the sorting compartment and contain two classes of molecules that are targeted to the same surface. Isolation of these sorting proteins is the long-range goal of this work. Biochemical and ultrastructural techniques will be used to characterize the complexes. The effects of altering the cellular lipid composition and the conditions of detergent lysis on complex formation will be determined, as will the behavior of GPI-anchored proteins reconstituted into vesicles of defined lipid composition. The intracellular site(s) of origin of the complexes will be determined by subcellular fractionation and by membrane labelling prior to isolation of complexes. The next aim is to determine the protein profile of the membrane complexes, to identify candidate proteins that may be important in sorting. Finally, the possible inter-relationship of glycolipid and GPI-anchored protein sorting will be determined using specific inhibitors.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Cellular Biology and Physiology Subcommittee 1 (CBY)
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State University New York Stony Brook
Schools of Arts and Sciences
Stony Brook
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Brown, Deborah A (2015) Preparation of detergent-resistant membranes (DRMs) from cultured mammalian cells. Methods Mol Biol 1232:55-64
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