MUTANTS IN INTRACELLULAR CHOLESTEROL TRANSPORT

哺乳动物细胞严格调节它们的胆固醇含量和它 细胞内的处理方式。胆固醇并不均匀地分布在 细胞膜、胆固醇生物合成率、脂蛋白 内化和胆固醇酯化对细胞敏感 游离胆固醇水平。无论是感测机制还是 胆固醇在细胞内被分割的机制很好 明白了。 我们的长期目标是识别与细胞内有关的基因产物 胆固醇的运输和调节。我们已经分离出一组 细胞内有缺陷的隐性体细胞突变体 低密度脂蛋白(LDL)衍生的胆固醇的运输。 互补分析表明，至少有两个基因控制着低密度脂蛋白- 胆固醇信号和运输。初步分析表明， 下文：第一个基因的突变会损害来自 溶酶体。这些L类突变体似乎是一个体细胞模型 经典型的Niemann-Pick病C型(NPC)。第二个基因的突变 损害低密度脂蛋白-胆固醇信号，但不影响运输。这些2类突变体 似乎是鼻咽癌变异表型的模型。我们建议： 具体目标#1：分析L班和班级的生化表型 2互补组。我们将彻底调查这两人是如何 基因缺陷改变了细胞内胆固醇运输的关键方面 细胞胆固醇代谢。 具体目标2：确定其他2类突变体和其他 互补性小组。我们的结论是基于对 整个变种人的集合。CHO突变体与鼻咽癌的融合 将进行成纤维细胞以鉴定鼻咽癌的突变系 基因分型。 具体目标3：分离纠正或抑制突变的cDNA L和2类突变体。正常产量的cpNAs的鉴定 表型将揭示有关基因缺陷的信息 这些细胞系。 特定目标4：分离和分析表达显性基因的CHO品系 细胞内胆固醇转运缺陷是由于a基因过表达所致 编码正常细胞蛋白的cDNA.产生型DNA的鉴定 突变表型将揭示控制细胞的新基因产品 胆固醇的分布。

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.