Dissecting the molecular interactions of mitochondrial glutaredoxin S15 in plants

植物的一些代谢途径和细胞过程依赖于铁硫（Fe-S）蛋白的功能，其辅助因子通过存在于细胞质、质体和线粒体中的专用组装机制进行组装。仅举几个例子，Fe-S蛋白存在于光合作用和呼吸电子传递链中，它们是硫和氮同化或辅酶合成（如生物素和硫辛酸）所必需的。在植物和其他生物中，Fe-S簇整合到蛋白质中首先需要铁硫簇（ISCs）重新组装到支架蛋白上，并通过几种成熟因子的作用将其转移到受体蛋白上，其中包括II类glutaredoxins （GRXs）。最近的研究表明，GRXS15利用谷胱甘肽分子协调一个[2Fe-2S]簇，该簇可以转移到一个受体蛋白上，并且在拟南芥中，线粒体GRXS15的零突变体具有胚胎致死性，这为GRXS15是ISC转移机制的重要组成部分提供了明确的证据。这一发现为线粒体Fe-S蛋白如何组装的分子理解开辟了一条新的途径。该项目主要旨在分析GRXS15在拟南芥中ISCs向靶蛋白转移中的作用。通过体外厌氧重组获得的GRXS15全型的生化、光谱和结构分析将允许确定组装的ISCs的寡聚化状态和性质。在结构比对的基础上，利用靶向诱变和组合方法，我们将对该GRX和同类蛋白的结构-功能关系产生新的认识。这些变体的特性将通过以下方式确定：(i)分析其结合ISC的能力及其不稳定性，（ii）通过精细检查与已知或新发现的伴侣蛋白的蛋白质-蛋白质相互作用，（iii）通过在线粒体Grx5缺陷的酵母突变体中进行异源表达，以及（iv）通过使用roGFP2和氧化敏感性测试进行体外活性分析来评估其可能的氧化还原特性。根据早期的研究，通过表达异源grx或突变grxs15部分获救的grxs15零突变体有望表现出不同的发育和生理表型。因此，我们将研究这些表型是否与特定的Fe-S酶缺陷有关，以及线粒体ISC转移的瓶颈是否仅限于线粒体靶蛋白，还是也影响细胞质金属酶。这可能是因为已知一种未知的线粒体含硫化合物被输出用于细胞质和核蛋白的成熟，并且因为在几种细胞质Fe-S酶中发现的钼辅助因子的合成需要线粒体Fe-S酶。

Several metabolic pathways and cellular processes in plants depend on the functioning of iron-sulfur (Fe-S) proteins, whose cofactors are assembled through dedicated assembly machineries present in the cytosol, plastids and mitochondria. To cite only a few examples, Fe-S proteins are present in the photosynthetic and respiratory electron transfer chains and they are needed for sulfur and nitrogen assimilation, or co-enzyme synthesis such as biotin and lipoic acid. In plants as in other organisms, the incorporation of Fe-S clusters into proteins requires first the de novo assembly of iron-sulfur clusters (ISCs) onto scaffold proteins and their transfer to acceptor proteins via the action of several maturation factors, and among those class II glutaredoxins (GRXs). The recent demonstration that GRXS15 coordinates an [2Fe-2S] cluster using glutathione molecules which can be transferred to an acceptor protein and that null mutants for the mitochondrial GRXS15 in Arabidopsis are embryo-lethal provided clear evidence that GRXS15 is an essential component of the ISC transfer machinery. This finding opens a new avenue towards molecular understanding of how mitochondrial Fe-S proteins are assembled. The proposed project aims primarily at dissecting the role of GRXS15 in the transfer of ISCs to target proteins in Arabidopsis. The biochemical, spectroscopic and structural analysis of GRXS15 holoforms obtained by in vitro anaerobic reconstitution will allow determining the oligomerisation status and nature of the assembled ISCs. On the basis of structural alignments and using targeted mutagenesis and combinatorial approaches, we will generate new knowledge on the structure-function relationship of this GRX and of proteins of the same class. The properties of these variants will be determined (i) by analysing their ability to bind an ISC and its lability, (ii) by finely examining protein-protein interactions with known or newly identified partner proteins, (iii) by performing heterologous expression in a yeast mutant deficient in mitochondrial Grx5 and (iv) by assessing their possible redox properties using in vitro activity assays with roGFP2 and oxidation sensitivity tests. Following earlier work, grxs15 null mutants partially rescued through expression of heterologous GRXs or mutated GRXS15 are expected to display distinct developmental and physiological phenotypes. Thus, it will be investigated whether these phenotypes are related to specific Fe-S enzyme defects and whether a bottleneck in mitochondrial ISC transfer is restricted to mitochondrial target proteins or whether it also affects cytosolic metalloenzymes. This could be because a yet unknown mitochondrial sulfur-containing compound is known to be exported for the maturation of cytosolic and nuclear proteins, and because synthesis of the molybdenum cofactor found in several cytosolic Fe-S enzymes requires a mitochondrial Fe-S enzyme.