The assembly of iron-sulphur proteins in germinating seeds

点击翻译按钮获取中文摘要

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.