Grohmann L, Rasmusson A G, Heiser V, Thieck O, Brennicke A
Institut für Genbiologische Forschung Berlin, Germany.
Plant J. 1996 Nov;10(5):793-803. doi: 10.1046/j.1365-313x.1996.10050793.x.
In higher plants, genes for subunits of respiratory chain complex I (NADH:ubiquinone oxidoreductase) have so far been identified solely in organellar genomes. At least nine subunits are encoded by the mitochondrial DNA and 11 homologues by the plastid DNA. One of the 'key' components of complex I is the subunit binding the substrate NADH. The corresponding gene for the mitochondrial subunit has now been cloned and identified in the nuclear genome from potato (Solanum tuberosum). The mature protein consists of 457 amino acids and is preceded by a mitochondrial targeting sequence of 30 amino acids. The protein is evolutionarily related to the NADH-binding subunits of complex I from other eukaryotes and is well conserved in the structural domains predicted for binding the substrate NADH, the FMN and one iron-sulphur cluster. Expression examined in different potato tissues by Northern blot analysis shows the highest steady-state mRNA levels in flowers. Precursor proteins translated in vitro from the cDNA are imported into isolated potato mitochondria in a delta psi-dependent manner. The processed translation product has an apparent molecular mass of 55 kDa, identical to the mature protein present in the purified plant mitochondrial complex I. However, the in-vitro translated protein is not imported into isolated chloroplasts. To further investigate whether the complex I-like enzyme in chloroplasts contains an analogous subunit for binding of NAD(P)H, different plastid protein fractions were tested with a polyclonal antiserum directed against the bovine 51 kDa NADH-binding subunit. In none of the different thylakoid or stroma protein fractions analysed were specific cross-reactive polypeptides detected. These results are discussed particularly with respect to the structure of a potential complex I in chloroplasts and the nature of its acceptor site.
在高等植物中，呼吸链复合体I（NADH：泛醌氧化还原酶）亚基的基因迄今仅在细胞器基因组中被鉴定出来。线粒体DNA编码至少9个亚基，质体DNA编码11个同源亚基。复合体I的“关键”组分之一是结合底物NADH的亚基。现在已经从马铃薯（Solanum tuberosum）的核基因组中克隆并鉴定出了线粒体亚基的相应基因。成熟蛋白由457个氨基酸组成，前面有一个30个氨基酸的线粒体靶向序列。该蛋白在进化上与其他真核生物复合体I的NADH结合亚基相关，并且在预测用于结合底物NADH、FMN和一个铁硫簇的结构域中高度保守。通过Northern印迹分析在不同马铃薯组织中检测的表达显示，花中的稳态mRNA水平最高。从cDNA体外翻译的前体蛋白以依赖膜电位差（Δψ）的方式导入分离的马铃薯线粒体。加工后的翻译产物的表观分子量为55 kDa，与纯化的植物线粒体复合体I中存在的成熟蛋白相同。然而，体外翻译的蛋白不能导入分离的叶绿体。为了进一步研究叶绿体中类似复合体I的酶是否含有用于结合NAD(P)H的类似亚基，用针对牛51 kDa NADH结合亚基的多克隆抗血清检测了不同的质体蛋白组分。在所分析的不同类囊体或基质蛋白组分中均未检测到特异性交叉反应多肽。特别针对叶绿体中潜在复合体I的结构及其受体位点的性质对这些结果进行了讨论。
