Skalnik D G, Narita H, Kent C, Simoni R D
Department of Biological Sciences, Stanford University, California 94305.
J Biol Chem. 1988 May 15;263(14):6836-41.
A hybrid gene has been constructed consisting of coding sequence for the membrane domain of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase linked to the coding sequence for the soluble enzyme Escherichia coli beta-galactosidase. Expression of the hybrid gene in transfected Chinese hamster ovary cells results in the production of a fusion protein (HMGal) which is localized in the endoplasmic reticulum. The fusion protein contains the high-mannose oligosaccharides characteristic of HMG-CoA reductase. Importantly the beta-galactosidase activity of HMGal decreases when low density lipoprotein is added to the culture media. Therefore, the membrane domain of HMG-CoA reductase is sufficient to determine both correct intracellular localization and sterol-regulation of degradation. Mutant fusion proteins which lack 64, 85, or 98 amino acid residues from within the membrane domain of HMG-CoA reductase are found to be localized in the endoplasmic reticulum and to retain beta-galactosidase activity. However, sterol-regulation of degradation is abolished.
构建了一种杂合基因，其由内质网蛋白3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）还原酶的膜结构域编码序列与可溶性酶大肠杆菌β-半乳糖苷酶的编码序列相连组成。该杂合基因在转染的中国仓鼠卵巢细胞中的表达导致产生一种定位于内质网的融合蛋白（HMGal）。融合蛋白含有HMG-CoA还原酶特有的高甘露糖寡糖。重要的是，当向培养基中添加低密度脂蛋白时，HMGal的β-半乳糖苷酶活性降低。因此，HMG-CoA还原酶的膜结构域足以决定正确的细胞内定位和降解的固醇调节。发现从HMG-CoA还原酶膜结构域内缺失64、85或98个氨基酸残基的突变融合蛋白定位于内质网并保留β-半乳糖苷酶活性。然而，降解的固醇调节被消除。
