Molecular analysis of fatty acid export from plastids of oilseed plants and green microalgae - towards developing tools to improve plant biofuel production

脂肪酸（FAs）构建了大部分的细胞脂质，这不仅对膜功能至关重要，而且对生物体的生长和发育也至关重要。例如，油籽植物种子中的三酰基甘油脂（TAGs）是碳能量储存的主要形式，或者植物表面的蜡质限制了水分的流失并提供了病原体保护。由于在植物中，FA的新合成发生在质体中，因此需要运输到内质网进行进一步的脂质组装。尽管人们普遍认为游离脂肪酸是通过质体包膜穿梭的，但质体中游离脂肪酸的输出模式仍然是一个谜。我们分离了FAX1（脂肪酸输出1），这是一种在拟南芥质体内膜中的新型膜蛋白，它对生物量生产、雄性繁殖和脂肪酸衍生化合物（如脂质、酮蜡或花粉细胞壁）的合成至关重要。当FAX1缺失时，er来源的脂质会减少，而质体产生的脂质水平会增加。FAX1过表达系表现相反，包括叶片和花的TAG增加。由于在酵母中，FAX1可以补充fa -运输，我们得出结论，FAX1介导fa -通过质体内包膜输出。在拟南芥中，FAX1属于一个由7个蛋白组成的家族。预计有4个在质体中，3个最有可能整合到分泌途径的膜中。绿色微藻莱茵衣藻包含这些亚群中的每一个成员。在脊椎动物中，FAX亲缘蛋白是线粒体膜“TMEM14”蛋白，与血红素代谢和细胞凋亡有关。然而，它们的生物学功能尚不清楚。因此，该蛋白家族不仅是增加绿色谱系中脂质/生物燃料生产的有力工具，而且还可以探索影响脊椎动物线粒体动力学和/或细胞凋亡的新型运输系统。该项目旨在表征拟南芥和衣藻中的FAX蛋白。研究应阐明它们对细胞代谢的影响，特别是对脂肪酸、脂质和脂肪酸/脂质衍生化合物（蜡、角质或奥西利平激素）以及碳能量代谢物的影响。重点将放在种子储存油的合成上，即FAX在TAG生产中的意义。在衣藻中，FAX蛋白（FA-transport）的过量生产与FA/脂质生物合成酶的增强表达将导致生物燃料生产策略的发展。FAX1的结构/功能相关性是完全新颖的，与“经典”FA或脂质转运蛋白无关。因此，研究将使我们能够研究新的细胞内fa运输途径的植物特异性和一般方面。评估FAX的拓扑结构、FA/脂质结合、异源酵母系统中的功能特征以及相互作用伙伴的识别将有助于描述质体FA输出途径。

Fatty acids (FAs) build the majority of cellular lipids, which are essential not only for membrane function, but also for growth and development of organisms. For example, triacylglycerol lipids (TAGs) in seeds of oilseed plants represent the major form of carbon energy storage, or waxes on the plant surface restrict loss of water and provide pathogen protection. Since in plants de novo FA synthesis occurs in plastids, transport to the endoplasmic reticulum for further lipid assembly is required. Although it is generally agreed that free FAs are shuttled across plastid envelope membranes, the mode of plastid FA-export, however, still remains enigmatic. We isolated FAX1 (fatty acid export 1), a novel membrane protein in the plastid inner envelope, which in Arabidopsis thaliana is crucial for biomass production, male fertility, and synthesis of FA-derived compounds such as lipids, ketone waxes or pollen cell walls. Whereas ER-derived lipids decrease when FAX1 is missing, levels of plastid-produced lipids increase. FAX1 over-expressing lines show the opposite, including a TAG increase in leaves and flowers. Since in yeast, FAX1 can complement for FA-transport, we conclude that FAX1 mediates FA-export across the plastid inner envelope. In Arabidopsis, FAX1 belongs to a family of seven proteins. Four are predicted to be in plastids, three most likely integrate into membranes of the secretory pathway. The green microalga Chlamydomonas reinhardtii contains one member of each of these subgroups. In vertebrates, FAX relatives are mitochondrial membrane 'TMEM14' proteins, linked to heme metabolism and apoptosis. However, their biological function is unknown. Thus, this protein family represents a powerful tool not only to increase lipid/biofuel production in the green lineage, but also to explore novel transport systems with impact on mitochondrial dynamics and/or apoptosis in vertebrates.The project aims to characterise FAX proteins in Arabidopsis and Chlamydomonas. Research should clarify their impact on cellular metabolism, in particular on FAs, lipids and FA/lipid-derived compounds (waxes, cutin or oxilipin hormones) as well as on carbon energy metabolites. A spotlight will be on synthesis of storage oils in seeds, i.e. the significance of FAX in TAG production. In Chlamydomonas, over-production of FAX proteins (FA-transport) in combination with enhanced expression of FA/lipid biosynthesis enzymes will lead to development of strategies for biofuel production. The structure/function correlation of FAX1 is completely novel and not related to 'classical' FA or lipid transporters. Thus, research will enable us to study plant-specific but also general aspects of a new intracellular FA-transport pathway. Evaluation of FAX topology, structure, and FA/lipid binding, functional characteristics in a heterologous yeast system as well as identification of interaction partners will help to describe the plastid FA-export pathway.