REPROGRAMMED EXPRESSION OF SUBUNIT-9 OF THE MITOCHONDRIAL ATPASE COMPLEX OF SACCHAROMYCES-CEREVISIAE - EXPRESSION INVITRO FROM A CHEMICALLY SYNTHESIZED GENE AND IMPORT INTO ISOLATED-MITOCHONDRIA
REPROGRAMMED EXPRESSION OF SUBUNIT-9 OF THE MITOCHONDRIAL ATPASE COMPLEX OF SACCHAROMYCES-CEREVISIAE - EXPRESSION INVITRO FROM A CHEMICALLY SYNTHESIZED GENE AND IMPORT INTO ISOLATED-MITOCHONDRIA
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DOI：
10.1111/j.1432-1033.1988.tb13976.x
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发表时间：
1988-04-05
期刊：
影响因子：
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通讯作者：
NAGLEY, P
中科院分区：
文献类型：
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作者：
FARRELL, LB;GEARING, DP;NAGLEY, P
合成基因已被设计和构建的全化学合成作为第一步，在功能搬迁的基因编码的亚基9的酵母线粒体ATP酶复合物的胞核。该基因（NAP 9）包含酿酒酵母核基因中常用的密码子，另外还包括一系列独特的限制性内切酶切割位点，以便于将来对该基因及其蛋白质产物进行系统操作。NAP 9基因通过体外转录和翻译表达后，产生了放射性标记的蛋白质，其显示出由线粒体oli 1基因在体内编码的真实亚基9蛋白脂质的凝胶电泳迁移率和在氯仿/甲醇中的溶解度特征。为了实现将酵母亚基9导入线粒体，在NAP 9基因和编码来自粗糙脉孢菌的亚基9的核编码前体的可裂解前序列的DNA之间进行融合。体外表达后，导入所得融合蛋白并通过分离的酵母线粒体进行适当加工。酵母亚基9的导入效率低于在平行导入实验中观察到的酵母亚基8连接到相同的前序列或与天然存在的完整的N。crassa亚基9前体。缺乏前导序列的酵母亚基9不能进入线粒体，但与亚基8不同的是，尽管它具有高度疏水性，但它不会将自己嵌入外膜。
A synthetic gene has been designed and constructed by total chemical synthesis as a first step in the functional relocation from the mitochondrion to the nucleus of a gene encoding subunit 9 of the yeast mitochondrial ATPase complex. This gene (NAP9) incorporates codons frequently used in nuclear genes of Saccharomyces cerevisiae and additionally includes a series of unique restriction enzyme cleavage sites to facilitate future systematic manipulations of the gene and its protein product. Following the expression of the NAP9 gene by transcription and translation in vitro, a radiolabelled protein was produced which displayed a gel electrophoretic mobility and solubility in chloroform/methanol characteristic of the authentic subunit 9 proteolipid encoded in vivo by the mitochondrial oli1 gene. In order to achieve import into mitochondria of yeast subunit 9, a fusion was made between the NAP9 gene and DNA encoding the cleavable presequence of the nuclearly encoded precursor to subunit 9 from Neurospora crassa. Following expression in vitro, the resultant fusion protein was imported and appropriately processed by isolated yeast mitochondria. The import ofyeast subunit 9 was less efficient than that observed in parallel import experiments with yeast subunit 8 attached to the same presequence or with the naturally occurring intact N. crassa subunit 9 precursor. Yeast subunit 9 lacking a leader sequence is not imported into mitochondria but, unlike subunit 8, it does not embed itself into the outer membrane, in spite of its highly hydrophobic character.