Cupp J R, McAlister-Henn L
Department of Biological Chemistry, University of California, Irvine 92717.
J Biol Chem. 1992 Aug 15;267(23):16417-23.
NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is composed of two nonidentical subunits, designated IDH1 and IDH2. The gene encoding IDH2 was previously cloned and sequenced (Cupp, J.R., and McAlister-Henn, L. (1991) J. Biol. Chem. 266, 22199-22205), and in this paper we describe the isolation of a yeast genomic clone containing the IDH1 gene. A fragment of the IDH1 gene was amplified by the polymerase chain reaction method utilizing degenerate oligonucleotides based on tryptic peptide sequences of the purified subunit; this fragment was used to isolate a full length IDH1 clone. The nucleotide sequence of the IDH1 coding region was determined and encodes a 360-residue polypeptide including an 11-residue mitochondrial targeting presequence. Amino acid sequence comparison between IDH1 and IDH2 reveals a 42% sequence identity, and both IDH1 and IDH2 show approximately 32% identity to Escherichia coli NAD(P)(+)-dependent isocitrate dehydrogenase. To examine the function of the IDH1 subunit and to determine the metabolic role of NAD(+)-dependent isocitrate dehydrogenase the IDH1 gene was disrupted in a wild type haploid yeast strain and in a haploid strain lacking IDH2. The IDH1 disruption strains expressed no detectable IDH1 as determined by Western blot analysis, and these strains were found to lack NAD(+)-dependent isocitrate dehydrogenase activity indicating that IDH1 is essential for a functional enzyme. Over-expression of IDH1 in a strain containing IDH2 restored wild type activity but did not result in increased levels of activity, suggesting that both IDH1 and IDH2 are required for a functional enzyme. Growth phenotype analysis of the IDH1 disruption strains revealed that they grew at a reduced rate on the nonfermentable carbon sources examined (glycerol, lactate, and acetate), consistent with NAD(+)-dependent isocitrate dehydrogenase performing a critical role in oxidative function of the citric acid cycle. In addition, the IDH1 disruption strains grew at wild type rates in the absence of glutamate, indicating that these strains are not glutamate auxotrophs.
来自酿酒酵母的NAD(+)依赖性异柠檬酸脱氢酶由两个不同的亚基组成，分别命名为IDH1和IDH2。编码IDH2的基因先前已被克隆和测序（Cupp, J.R., and McAlister-Henn, L. (1991) J. Biol. Chem. 266, 22199 - 22205），在本文中，我们描述了包含IDH1基因的酵母基因组克隆的分离。利用基于纯化亚基胰蛋白酶肽序列的简并寡核苷酸，通过聚合酶链反应方法扩增IDH1基因的一个片段；该片段用于分离全长IDH1克隆。测定了IDH1编码区的核苷酸序列，其编码一个360个残基的多肽，包括一个11个残基的线粒体靶向前序列。IDH1和IDH2之间的氨基酸序列比较显示有42%的序列同一性，并且IDH1和IDH2与大肠杆菌NAD(P)(+)依赖性异柠檬酸脱氢酶均显示约32%的同一性。为了研究IDH1亚基的功能并确定NAD(+)依赖性异柠檬酸脱氢酶的代谢作用，在野生型单倍体酵母菌株和缺乏IDH2的单倍体菌株中破坏了IDH1基因。通过蛋白质免疫印迹分析确定，IDH1破坏菌株未表达可检测到的IDH1，并且发现这些菌株缺乏NAD(+)依赖性异柠檬酸脱氢酶活性，表明IDH1对于功能性酶是必需的。在含有IDH2的菌株中过表达IDH1恢复了野生型活性，但未导致活性水平增加，这表明功能性酶需要IDH1和IDH2两者。IDH1破坏菌株的生长表型分析表明，它们在所检测的非发酵碳源（甘油、乳酸和乙酸盐）上生长速率降低，这与NAD(+)依赖性异柠檬酸脱氢酶在柠檬酸循环的氧化功能中起关键作用一致。此外，IDH1破坏菌株在没有谷氨酸的情况下以野生型速率生长，表明这些菌株不是谷氨酸营养缺陷型。
