Many aspects of membrane protein synthesis, intracellular trafficking, and distribution are well understand. In contrast, comparatively little information exists concerning the cellular physiology of glycosphingolipids. Glycolipids function as blood group antigens, receptors for toxins and microbes, and modulators of growth factor receptor activity. In order to better understand how glycolipids perform these functions, knowledge of glycolipid metabolism, trafficking, and distribution must be increased. To achieve that end, the following specific aims will be addressed in this project. The first aim is to determine the rate and extent of transport of glycolipids to the plasma membrane following their synthesis. These data will be used to clarify the metabolic relationships between glycolipids; of particular interest will be the nature of the precursor product relationship between glucosyl ceramide and more complex glycolipids. The second aim is to determine if cell surface glycolipids can serve as substrates for the synthesis of more complex structures. These experiments will be accomplished by cell surface and metabolic labeling of glycolipids combined with the use of inhibitors of intracellular vesicular traffic and subcellular fractionation. Purified glycolipids will be analyzed by thin layer chromatography (TLC). Cells to be utilized will be L5178Y mouse lymphoma cells and a mutant of Chinese hamster ovary cells which is deficient in 4- epimerase and therefore can synthesize galactose containing glycolipids only after addition of exogenous galactose. The third aim is to determine the pathway(s) by which plasma membrane glycolipids are internalized. Plasma membranes and intracellular vesicles representing different pathways and stages of endocytic traffic will be isolated from HeP 02 human hepatoma cells by the binding of colloidal iron dextran protein conjugates followed by high gradient magnetic affinity chromatography (HIMAC). The results will indicate the relative importance for glycolipid uptake of the coated versus non-coated pit pathways. The fourth aim is to use the magnetic bead method described above to determine if there is polarity in the distribution of the glycolipids of the plasma membrane domains of polarized epithelial cells. Studies to date have been hampered by the lack of a good technique for purifying apical and basolateral membranes. Each domain can be directly sampled using this new method.
NIH ExPorter
NIH ExPorter