Chun K T, Simoni R D
Department of Biological Sciences, Stanford University, California 94305-5020.
J Biol Chem. 1992 Feb 25;267(6):4236-46.
We have constructed a series of mutations in the membrane and linker domains of Syrian hamster 3-hydroxy-3-methylglutaryl-(HMG) CoA reductase in order to determine the regions critical for the regulated degradation of the enzyme. In transfected Chinese hamster ovary cells, we have expressed a fusion protein, HMGal, which consists of the membrane and linker domains of the Syrian hamster HMG-CoA reductase fused to beta-galactosidase. Using this fusion protein, we have determined that a deletion of 64 amino acids from the central region of the membrane domain causes the protein to be degraded extremely rapidly. In addition, deletion of PEST sequences has little effect on degradation, but deletion of the linker domain makes the protein's degradation insensitive to sterols and mevalonate. In addition to deletion mutations, we have systematically replaced each hydrophobic, putative membrane spanning region of the membrane domain with the first transmembrane sequence from bacteriorhodopsin. Replacement of span 4 has no effect on degradation. Replacements of spans 5 or 6 result in a protein which has a normal basal rate of degradation, but this rate of degradation is not accelerated by mevalonate, low density lipoprotein, or 25-hydroxycholesterol. Replacement of span 3 results in a protein whose degradation is similarly not accelerated by sterols or mevalonate, but since this protein might be mislocalized, these results are inconclusive. Replacement of span 7 yields a short-lived protein which is degraded more rapidly in response to mevalonate but not in response to exogenous sterols. Replacement of span 8 extends both the basal and mevalonate-accelerated half-life about 5-fold. This work begins to define the critical regions for regulated degradation within the membrane domain of HMG-CoA reductase.
为了确定对叙利亚仓鼠3-羟基-3-甲基戊二酰辅酶A（HMG）还原酶调节性降解至关重要的区域，我们构建了一系列该酶膜结构域和连接结构域的突变体。在转染的中国仓鼠卵巢细胞中，我们表达了一种融合蛋白HMGal，它由叙利亚仓鼠HMG-CoA还原酶的膜结构域和连接结构域与β-半乳糖苷酶融合而成。利用这种融合蛋白，我们确定从膜结构域中心区域缺失64个氨基酸会导致该蛋白极快速地降解。此外，缺失PEST序列对降解影响不大，但缺失连接结构域会使该蛋白的降解对固醇和甲羟戊酸不敏感。除了缺失突变，我们还将膜结构域中每个疏水的、推测的跨膜区域系统地替换为细菌视紫红质的第一个跨膜序列。替换第4跨膜区对降解没有影响。替换第5或第6跨膜区会产生一种蛋白，其基础降解速率正常，但甲羟戊酸、低密度脂蛋白或25-羟基胆固醇不会加速其降解速率。替换第3跨膜区会产生一种蛋白，其降解同样不会被固醇或甲羟戊酸加速，但由于这种蛋白可能定位错误，这些结果尚无定论。替换第7跨膜区会产生一种半衰期短的蛋白，它对甲羟戊酸有反应而降解更快，但对外源固醇无反应。替换第8跨膜区会使基础半衰期和甲羟戊酸加速后的半衰期都延长约5倍。这项工作开始确定HMG-CoA还原酶膜结构域内调节性降解的关键区域。
