GENES CONTROLLING LDL RECEPTOR STRUCTURE AND FUNCTION

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The long term goal of the proposed study is to define the molecular details of low density lipoprotein (LDL) receptor- mediated endocytosis, which can help provide fundamental understanding of lipoprotein metabolism and atherosclerosis. In addition, this work may contribute new insights into the general mechanisms underlying the structure and function of the Golgi apparatus, which plays a central role in controlling the flow of many integral membrane and lumenal soluble proteins through eukaryotic cells and also participates in the posttranslational modifications of these proteins (glycosylation, sulfation) as well as in glycolipid synthesis. Two cytoplasmic proteins, ldlBp and ldlCp, which were identified by expression cloning from recessive LDL receptor (LDLR)-defective Chinese hamster ovary (CHO) cell mutants, ldlB and ldlC, play critical roles in controlling intralumenal Golgi processing reactions (e.g., glycoconjugate synthesis and remodeling) in mammals. The ldlB and ldlC null mutants exhibit pleiotropic defects in multiple lumenal Golgi reactions, which result in the abnormal synthesis the LDLR and many other glycoconjugates. Both ldlBp and ldlCp bind to the cytoplasmic surface of the Golgi and are components of a very large macromolecular complex (approximately 950 kD, 'ldlCp complex'). Preliminary studies indicate that ldlCp is essential for development in the worm C. elegans. The goal of this proposal is to elucidate the mechanism underlying the control of Golgi lumenal enzymatic activities by the ldlBp/ldlCp system from the cytoplasmic surface of the Golgi. We will 1) identify additional components of the system (additional complex components, Golgi receptor, intralumenal factors and conditions), 2) describe the effects of the ldlBp/ldlCp-complex system on the structure, composition, and function of the Golgi in normal and mutant cells, and 3) characterize the physical and functional interactions of the components of the ldlBp/ldlCp-Golgi system, using a variety of techniques [immunochemistry, protein purification, ligand blotting, biophysics (EM,centrifugation), cloning, carbohydrate analysis, in vitro organelle assays, somatic cell genetics, and others]. It is likely that the molecular characterization of this system will provide fundamental new insights into the synthesis and processing of the LDLR and other membrane and secreted proteins, as well as the structure and function of the Golgi in higher eukaryotes. This should further our understanding of the complex physiologic and pathophysiologic (e.g., familial hypercholesterolemia, atherosclerosis) processes which are based on the function and dysfunction of the LDLR.