The regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase requires a short-lived protein and occurs in the endoplasmic reticulum.
The regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase requires a short-lived protein and occurs in the endoplasmic reticulum.
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DOI：
10.1016/s0021-9258(18)45839-5
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发表时间：
1990-12
期刊：
影响因子：
--
通讯作者：
K. Chun;S. Bar-Nun;R. D. Simoni
中科院分区：
文献类型：
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作者：
K. Chun;S. Bar-Nun;R. D. Simoni
先前已经构建了一个由内质网蛋白膜结构域3-羟基-3-甲基戊二酰辅酶A （HMG-CoA）还原酶编码序列与大肠杆菌可溶性酶-半乳糖苷酶编码序列融合而成的嵌合基因。这种融合蛋白HMGal已经定位于转染了这种嵌合基因的中国仓鼠卵巢细胞的内质网膜上，并且其-半乳糖苷酶活性在低密度脂蛋白存在下下降（Skalnik， D. G., Narita， H., Kent， C., and Simoni， R. D. (1988) J. Biol.）。化学，263,6836-6841)。在本报告中，我们证明β -半乳糖苷酶活性的丧失是由于HMGal蛋白的加速降解造成的。利用荧光激活细胞分选技术，我们选择了表达足够水平HMGal的转染细胞，以提高其免疫检测。脉冲追踪实验表明，HMGal的半衰期为6.0 h，在20 mM甲羟戊酸的存在下，半衰期下降1.7倍。在这些条件下，甲羟戊酸加速HMG-CoA还原酶在这些细胞中的降解1.6倍，从8.4 h到5.3 h，很可能是通过相同的机制。甲羟戊酸调节的HMGal降解不是由于HMGal与还原酶的异聚结合，因为在缺乏还原酶蛋白的细胞中观察到同样的效果。此外，我们证明了用环己亚胺抑制蛋白质合成可以消除甲羟戊酸依赖的HMGal加速降解，这与先前的研究一致，这些研究提出了间接证据，证明短寿命蛋白质对于介导HMG-CoA还原酶活性的丧失是必不可少的。最后，使用brefeldin A，我们表明甲基戊酸依赖的HMGal加速降解可能发生在内质网中。
A chimeric gene consisting of the coding sequence for the membrane domain of the endoplasmic reticulum protein, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, fused to the coding sequence for the soluble enzyme, beta-galactosidase of Escherichia coli, has been previously constructed. This fusion protein, HMGal, has been localized to the membrane of the endoplasmic reticulum of Chinese hamster ovary cells transfected with this chimeric gene, and its beta-galactosidase activity has declined in the presence of low density lipoprotein (Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). In this report, we demonstrate that the loss of beta-galactosidase activity results from the accelerated degradation of the HMGal protein. Taking advantage of a fluorescence-activated cell sorter technique, we have selected transfected cells which express sufficient levels of HMGal to improve its immunodetection. Based on pulse-chase experiments, the half-life of HMGal is 6.0 h, and, in the presence of 20 mM mevalonate, the half-life declines 1.7-fold. Under these conditions, mevalonate accelerates the degradation of HMG-CoA reductase in these cells 1.6-fold, from 8.4 h to 5.3 h, most probably by the same mechanism. This mevalonate-regulated degradation of HMGal is not due to a heteromeric association of HMGal with reductase, since the same effect has been observed in cells lacking the reductase protein. In addition, we demonstrate that inhibition of protein synthesis with cycloheximide abolishes the mevalonate-dependent accelerated degradation of HMGal, in agreement with previous studies which have presented indirect evidence that a short-lived protein is essential for mediating the loss of HMG-CoA reductase activity. Finally, using brefeldin A, we show that the mevalonate-dependent accelerated degradation of HMGal may occur in the endoplasmic reticulum.