Leber R, Landl K, Zinser E, Ahorn H, Spök A, Kohlwein S D, Turnowsky F, Daum G
Institut für Biochemie und Lebensmittelchemie and SFB Biomembrane Research Center, Technische Universität Graz, 1121 Vienna, Austria.
Mol Biol Cell. 1998 Feb;9(2):375-86. doi: 10.1091/mbc.9.2.375.
Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 x g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmic-phase cells. In contrast, sterol Delta24-methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.
角鲨烯环氧酶由酵母中的ERG1基因编码，是甾醇生物合成的关键酶。亚细胞组分分析表明，角鲨烯环氧酶存在于微粒体组分（30,000×g）中，并且也与脂质颗粒共分级分离。通过免疫荧光显微镜证实了Erg1p的双重定位。根据标记蛋白的分布，在对数后期细胞中，62%的细胞Erg1p可归属于内质网，38%归属于脂质颗粒。相比之下，甾醇Δ24-甲基转移酶（Erg6p）是催化甾醇生物合成后期步骤的一种酶，主要存在于与三酰甘油和甾醇酯共分级分离的脂质颗粒中。Erg1p在内质网和脂质颗粒之间的相对分布在生长过程中会发生变化。在erg1缺失菌株中不存在角鲨烯环氧酶（Erg1p），当从多拷贝质粒中过表达ERG1基因时，其在脂质颗粒和内质网中被诱导增加了五倍。用特比萘芬（一种真菌角鲨烯环氧酶抑制剂）处理酵母细胞也可使两个区室中的角鲨烯环氧酶量增加约五倍。与蛋白质的分布不同，角鲨烯环氧酶的酶活性仅在内质网中可检测到，而在分离的脂质颗粒中不存在。当野生型菌株的脂质颗粒与erg1缺失菌株的微粒体混合时，角鲨烯环氧酶活性部分恢复。这些发现表明内质网中存在的因子是角鲨烯环氧酶活性所必需的。脂质颗粒和内质网之间的紧密接触对于这两个区室在甾醇生物合成中的协同作用可能是必要的。
