Hasslacher M, Ivessa A S, Paltauf F, Kohlwein S D
Institut für Biochemie und Lebensmittelchemie, Technische Universität Graz, Austria.
J Biol Chem. 1993 May 25;268(15):10946-52.
We have isolated a 1.2-kilobase pair cDNA fragment in a screening for yeast genes regulated at the level of transcription by soluble lipid precursors, inositol and choline. Sequence analysis and comparison of the deduced amino acid sequence to protein databases unveiled 68% similarity of a 374-amino acid peptide fragment to published C termini of chicken and rat acetyl-CoA carboxylase and almost 100% identity to the product of the FAS3 gene from yeast. Several lines of evidence confirm that the cloned gene represents the yeast structural gene ACC1 encoding acetyl-CoA carboxylase. Overexpression of the ACC1 gene from a high copy number plasmid resulted in overexpression of a 250-kDa biotin-enzyme and enzymatic activity of acetyl-CoA carboxylase. Disruption of one ACC1 allele in a diploid wild-type strain resulted in 50% reduction of ACC1-specific mRNA and acetyl-CoA carboxylase specific activity and a marked decrease of biotin associated with a 250-kDa protein, compared to wild-type. After sporulation of diploid disruptants, spores containing the disrupted acc1 allele failed to enter vegetative growth, despite fatty acid supplementation, suggesting that acetyl-CoA carboxylase activity is essential for a process other than de novo fatty acid synthesis and that only a single functional copy of the ACC1 gene exists. ACC1 transcription was repressed 3-fold by lipid precursors, inositol and choline, and was also controlled by regulatory factors Ino2p, Ino4p, and Opi1p, providing evidence that the key step of fatty acid synthesis is regulated in conjunction with phospholipid synthesis at the level of gene expression. The 5'-untranslated region of the ACC1 gene contains a sequence reminiscent of an inositol/choline-responsive element identified in genes encoding phospholipid biosynthetic enzymes.
我们在一项针对受可溶性脂质前体、肌醇和胆碱转录水平调控的酵母基因的筛选中，分离出了一个1.2千碱基对的cDNA片段。对推导的氨基酸序列进行序列分析并与蛋白质数据库进行比较后发现，一个374个氨基酸的肽片段与已发表的鸡和大鼠乙酰辅酶A羧化酶的C末端有68%的相似性，与酵母FAS3基因的产物几乎100%相同。多条证据证实，克隆的基因代表编码乙酰辅酶A羧化酶的酵母结构基因ACC1。从高拷贝数质粒中过表达ACC1基因导致一种250 kDa生物素酶的过表达以及乙酰辅酶A羧化酶的酶活性。与野生型相比, 在二倍体野生型菌株中破坏一个ACC1等位基因导致ACC1特异性mRNA和乙酰辅酶A羧化酶比活性降低50%，与250 kDa蛋白质相关的生物素显著减少。二倍体破坏株形成孢子后，尽管补充了脂肪酸，但含有被破坏的acc1等位基因的孢子仍无法进入营养生长阶段，这表明乙酰辅酶A羧化酶活性对于从头合成脂肪酸以外的过程至关重要，并且ACC1基因仅存在一个功能拷贝。脂质前体、肌醇和胆碱可使ACC1转录受到3倍的抑制，并且还受调控因子Ino2p、Ino4p和Opi1p的控制，这证明脂肪酸合成的关键步骤在基因表达水平上与磷脂合成一起受到调控。ACC1基因的5'-非翻译区包含一个序列，该序列让人联想到在编码磷脂生物合成酶的基因中鉴定出的肌醇/胆碱反应元件。
