The assembly of iron-sulphur proteins in germinating seeds

种子含有大量的碳和矿物质，帮助萌发的幼苗在获得自己的水分、营养和能量之前，扎下根并折叠出第一片叶子。这一引人入胜的幼苗建立过程是由肉眼看不见的戏剧性生化变化支撑的。储存脂肪被分解并转化为糖和蛋白质，而光合作用机制和绿色色素则迅速组装起来。在这项提案中，我们将研究一种重要的营养物质--铁(Fe)是如何从种子中的储存中被动员起来，并被结合到铁酶的催化部位的。铁是一种基本矿物质，在电子转移和催化反应中起主要作用。通过铁与酶的相互作用，例如在血红素中的环结构中，或在铁-S簇合物中与无机硫化物一起作用，它的化学多功能性得到了增强。后一种形式的铁在植物中最占优势，因此是人类铁营养的重要来源。由于游离铁和硫化物是有毒的，需要严格控制Fe-S簇的组装，涉及小心处理铁和S的蛋白质，并将其安全地输送到新产生的酶中，而不会无意中释放中间产物。到目前为止，至少有30种蛋白质被鉴定出来，这些蛋白质参与了植物中的铁-S簇组装。这些蛋白质被分配到三种不同的途径，位于细胞的不同部分：线粒体、叶绿体和胞浆。虽然前两种途径在细菌中的途径相似，因此研究得相当好，但我们对胞质途径的了解非常有限。然而，这一途径对于一系列关键的铁-S酶是必不可少的，例如幼苗脂质动员的关键酶(乌头酸酶)，以及参与DNA复制、修复和调控的20多种酶。在过去的几年里，我们已经开始鉴定一种蛋白质NBP35，它被认为在细胞质中的铁-S组装途径中发挥关键作用。此外，我们有证据表明硫化物是由线粒体提供的。在接下来的几年里，我们希望找到铁的来源以及它是如何被运送到NBP35的，并寻找与NBP35一起发挥作用的其他蛋白质。为了探索铁的来源，我们将扩大我们对铁转运蛋白突变体的初步研究，并分析它们如何影响不同细胞内Fe-S酶的活性。由于对发芽幼苗中铁的动员知之甚少，我们还将编制一个数据集，记录1-10天内铁分布的变化，并将其与在处理或调节铁的过程中起作用的基因的蛋白质和转录水平的变化进行比较。系统方法将与S和铁向NBP35输送的分子生化研究相结合。为了寻找NBP35的直接相互作用伙伴，我们将从细胞提取液中与特异性抗体沉淀蛋白。共沉淀的蛋白质将用质谱学鉴定，它们与NBP35的相互作用将在所谓的酵母-2-杂交试验中进一步表征。最后但并非最不重要的一点是，来自系统或蛋白质相互作用研究的任何感兴趣的基因都将使用突变分析进行进一步研究。这将揭示这些蛋白质是否真的在胞内Fe-S簇组装中发挥功能。了解种子中铁的活化及其如何进入辅因子生物合成途径对于提高种子发芽成功率和作物产量具有重要意义。这些知识对于在植物中表达像固氮酶这样的铁-S蛋白也是很重要的。该项目与改进种子作为人类铁营养来源的研究相结合。

Seeds contain large amount of carbon and minerals to help the germinating seedling put down a root and fold out its first leaves, before it can obtain its own water, nutrition and energy. This fascinating process of seedling establishment is underpinned by dramatic biochemical changes invisible to the naked eye. Storage lipids are broken down and converted to sugars and protein, while the photosynthetic machinery and green pigments are rapidly assembled. In this proposal we will investigate how one important nutrient, iron (Fe), is mobilized from its stores in seed and is incorporated into the catalytic sites of Fe enzymes. Iron is an essential mineral that functions predominantly in electron transfer and catalytic reactions. Its chemical versatility is enhanced by how the iron interacts with the enzyme, such as in a ring structure in haem, or together with inorganic sulphide in Fe-S clusters. The latter form of Fe is most dominant in plants, and therefore an important source of human iron nutrition. Because free Fe and sulphide are toxic, the assembly of Fe-S clusters needs to be tightly controlled, involving proteins that carefully handle the Fe and S, and deliver it safely to the newly produced enzymes without inadvertent release of the intermediates. At least 30 proteins have so far been identified that are involved in Fe-S cluster assembly in plants. The proteins have been assigned to three different pathways, located in different parts of the cell: the mitochondria, chloroplasts and cytosol. While the former two are fairly well studied because the pathways are similar in bacteria, our knowledge on the cytosolic pathway is very limited. This pathway is essential for a range of critical Fe-S enzymes, however, such as a key enzyme in seedling lipid mobilization (aconitase), and more than 20 enzymes involved in DNA replication, repair and regulation.In the past years, we have started to characterize a protein, NBP35, that is thought to play a key role in the Fe-S assembly pathway in the cytosol. In addition, we have evidence that the sulphide is provided by the mitochondria. In the next few years we would like to find the source of iron and how it is delivered to NBP35, and identify other proteins that function together with NBP35.To investigate the source of iron, we will extend our initial studies on iron transporter mutants, and analyse how they are affected in the activity of Fe-S enzymes in different cell compartments. Since little is known about the mobilization of Fe in germinating seedlings, we will also compile a data set with changes in Fe distribution over day 1 - 10, and compare this to changes in protein and transcript levels of genes that play a role in handling or regulating Fe. The systems approach will be combined with a molecular-biochemical study of S and Fe delivery to NBP35. To find direct interaction partners of NBP35, we will precipitate the protein from cell extract with specific antibodies. Proteins that co-precipitate will be identified using mass spectrometry, and their interaction with NBP35 will be further characterized in so called yeast-2-hybrid assays. Last but not least, any genes of interest that come out of the systems- or protein-interaction studies will be further investigated using mutant analyses. This will reveal whether the proteins indeed function in cytosolic Fe-S cluster assembly. Knowledge on the mobilization of Fe in seeds and how it enters cofactor biosynthesis pathways is important for improving germination success rates and crop yields. This knowledge will also be important for expressing Fe-S proteins like nitrogenase in plants. This project links in with research to improve seeds as a source for human Fe nutrition.