The goal of the proposed study is to define gene products and functions required for intracellular protein trafficking and normal low density lipoprotein receptor (LDLR) activity. The work will be based on the molecular genetic analysis of three classes of temperature sensitive (ts) Chinese hamster ovary cell lethal mutants with defects in LDLR function. The genetic lesions responsible for these mutant phenotypes will be identified using expression cloning techniques with retroviral cDNA libraries. After cloning the cDNAs, I will examine the role of their gene products in protein processing and trafficking using the LDLR as a paradigm. This will involve immunochemical, cell biological, and biochemical analysis of intracellular membrane transport and endocytosis. In addition, it should be possible to use the genes initially identified as stepping stones for the identification of additional genes important for membrane transport, e.g. using immunochemical or two hybrid screening techniques. These studies will broaden our knowledge of the molecular mechanisms of intracellular protein trafficking and provide further insights into the basic biology of the LDLR. In addition, this work will contribute to our understanding of cholesterol metabolism, the mechanics of hypercholesterolemia, and the development and progression of atherosclerosis.
