The surface membrane is a major site for signal transduction events and transport functions. A number of specific lipid components of this membrane may participate in aspects of these various activities including the recycling of receptors and transporters. Because of their special properties sphingolipids, phosphatidylinositides, phosphatidylethanolamine, and cholesterol are of particular interest.
The aims and methods of this proposal are as follows. The isolation of mutants of CHO and LM cells in sphingolipid synthesis and phosphatidylinositide turnover using colony replication and autoradiographic screening procedures. Biochemical characterization and genetic classification (dominance and complementation analysis) of the mutants to explore regulation of sphingolipid and phosphatidylinositide metabolism. Using the mutant cell lines as recipients in DNA- mediated gene transfer experiments, isolation of human genes and then the gene products associated with key aspects in the metabolism of these two membrane lipids. Exploration of the involvement of sphingolipids and phosphatidylinositides in surface membrane assembly and/or function. Currently, considerable interest centers around phosphatidylinositide metabolism because it has been implicated in a number of physiological processes including growth control. The cell lines selected for the proposed work are ideal for developing a biochemical genetic approach to elucidating the mechanisms for regulating sphingolipid and phosphatidylinositide metabolism and for establishing the participation of these two classes of surface membrane lipids in specific cellular events. In the long run, the proposed work could provide critical information for subsequent isolation of similar mutants in cells which have differentiated phenotypes and are more appropriate for investigating the involvement of these specific lipids in the mechanism(s) which regulate and sustain normal cell growth.
