Understanding the molecular details of low density lipoprotein (LDL) receptor-mediated endocytosis and developing new insights into the mechanisms of membrane glycoprotein synthesis, structure and function, protein sorting, and the control of membrane fusions and fissions are the long term objectives of this proposal.
The specific aims are to isolate and characterize mutant Chinese hamster ovary (CHO) cells with defects in the endocytosis of LDL and to use these mutants to help define the gene products and functions required for LDL receptor activity. This information will be useful not only because of its direct relevance to LDL receptors and to cholesterol and LDL metabolism and thus hypercholesterolemia and atherosclerosis), but also because the LDL receptor provides a powerful model for many other surface receptors and membrane glycoproteins. The combination of somatic cell genetics and molecular cytological techniques should help provide answers to fundamental questions about mammalian cell function in general, and LDL receptors and endocytosis in particular. New types of mutants and revertants will be isolated from cells treated with a variety of mutagens using previously described (e.g., reconstituted LDL, MeLoCo) and newly developed (flow cytometry, glycoprotein toxicity) selections. New conditional- viable and conditional-lethal (temperature-sensitive) mutants will be isolated and studied along with previously isolated conditional mutants. Revertants defining extragenic suppressors (e.g., RevC- 13) will also be isolated and characterized. characterization will include genetic (e.g., complementation) and biochemical (e.g., 125- LDL and cholesterol metabolism) analyses. To define the function ldlC, a gene previously shown to be required for LDL receptor function, its human analogue will be cloned by a transfection/complementation technique. Human genomic DNA containing the ldlC gene and linked human-specific repetitive sequences will be purified by multiple rounds of transfection/selection and isolated from a genomic library from secondary or tertiary transfectants. Total human genomic DNA will be used as the probe for the library screening. Repeat-free genomic probes will then be used for cDNA cloning and the cDNA will be sequenced. Antibodies to this gene's product will be prepared for ultrastructural and biochemical analyses designed to elucidate the function of ldlC.
