MUTANTS IN INTRACELLULAR CHOLESTEROL TRANSPORT

哺乳动物细胞严格调节其胆固醇含量， 胞内处置胆固醇并不是均匀分布在 细胞膜和胆固醇生物合成率，脂蛋白 内化和胆固醇酯化是敏感的细胞 游离胆固醇水平。无论是传感机构还是 胆固醇在细胞内的区室化机制是很好的 明白 我们的长期目标是鉴定参与细胞内 胆固醇的运输和调节。我们分离出了一组 隐性体细胞突变体，其在细胞内 低密度脂蛋白（LDL）衍生胆固醇的转运。 互补分析表明，至少有两个基因控制LDL- 胆固醇信号传导和运输。初步分析表明， 以下：第一个基因的突变损害LDL-胆固醇从 溶酶体这些I类突变体似乎是用于以下的体细胞模型： 典型的C型尼曼-匹克病（NPC）。第二个基因突变 损害LDL-胆固醇信号传导，但不损害转运。这些二级变异体 似乎是NPC变异表型的模型。我们建议： 具体目标#1：分析I类和II类的生化表型 2个互补组。我们会彻底调查这两个人 基因缺陷改变细胞内胆固醇转运的关键方面， 细胞胆固醇代谢 具体目标#2：鉴定其他2类突变体和额外的 互补群我们的结论是基于部分分析， 所有变种人CHO突变体与NPC的融合 将进行成纤维细胞鉴定NPC突变株系 基因型 具体目标#3：分离纠正或抑制突变的cDNA 在I类和II类突变体中。鉴定产生正常的cpNA 表型将揭示基因缺陷的信息， 这些细胞系。 具体目标#4：分离和分析表达显性的CHO细胞系 细胞内胆固醇转运的缺陷，由于过度表达的一个 编码正常细胞蛋白质的cDNA。鉴定cDNA产生 突变表型将揭示新的基因产物， 胆固醇分布

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.