Mammalian cells tightly regulate their cholesterol content and its intracellular disposition. Cholesterol is not uniformly distributed among cell membranes, and rates of cholesterol biosynthesis, lipoprotein internalization, and cholesterol esterification are sensitive to cellular levels of free cholesterol. Neither the sensing mechanism nor the mechanism by which cholesterol is compartmentalized within cells are well understood. Our long term goal is to identify gene products involved in intracellular cholesterol transport and regulation. We have isolated a battery of recessive somatic cell mutants that are defective in the intracellular transport of low density lipoprotein (LDL)-derived cholesterol. Complementation analysis reveals that at least two genes control LDL- cholesterol signaling and transport. Preliminary analysis suggests the following: Mutations in the first gene impair LDL-cholesterol egress from lysosomes. These Class l mutants appear to be a somatic cell model for classical Niemann-Pick disease type C (NPC). Mutation in the second gene impairs LDL-cholesterol signaling but not transport. These Class 2 mutants appear to be a model for a variant phenotype of NPC. We propose:
Specific Aim #1 : To analyze the biochemical phenotype of Class l and Class 2 complementation groups. We will thoroughly investigate how these two gene defects alter key aspects of intracellular cholesterol transport and cellular cholesterol metabolism.
Specific Aim #2 : To identify other Class 2 mutants and additional complementation groups. Our conclusions are based on a partial analysis of the entire collection of mutants. Fusion of CHO mutants with NPC fibroblasts will be performed to identify the mutant line with the NPC genotype.
Specific Aim #3 : To isolate cDNAs that correct or suppress the mutations in Class l and Class 2 mutants. Identification of cpNAs yielding normal phenotypes will reveal information on the genes that are defective in these cell lines.
Specific Aim #4 : To isolate and analyze CHO lines expressing dominant defects in intracellular cholesterol transport due to overexpression of a cDNA encoding a normal cellular protein. Identification of cDNAs yielding mutant phenotypes will reveal novel gene products that control cellular cholesterol distribution.
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