Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Průmyslová Vestec, Czech Republic.
Laboratory of Microbiology, Wageningen University and Research, Wageningen, The Netherlands.
PLoS Pathog. 2023 Oct 4;19(10):e1010773. doi: 10.1371/journal.ppat.1010773. eCollection 2023 Oct.
Mitochondrial metabolism is entirely dependent on the biosynthesis of the [4Fe-4S] clusters, which are part of the subunits of the respiratory chain. The mitochondrial late ISC pathway mediates the formation of these clusters from simpler [2Fe-2S] molecules and transfers them to client proteins. Here, we characterized the late ISC pathway in one of the simplest mitochondria, mitosomes, of the anaerobic protist Giardia intestinalis that lost the respiratory chain and other hallmarks of mitochondria. In addition to IscA2, Nfu1 and Grx5 we identified a novel BolA1 homologue in G. intestinalis mitosomes. It specifically interacts with Grx5 and according to the high-affinity pulldown also with other core mitosomal components. Using CRISPR/Cas9 we were able to establish full bolA1 knock out, the first cell line lacking a mitosomal protein. Despite the ISC pathway being the only metabolic role of the mitosome no significant changes in the mitosome biology could be observed as neither the number of the mitosomes or their capability to form [2Fe-2S] clusters in vitro was affected. We failed to identify natural client proteins that would require the [2Fe-2S] or [4Fe-4S] cluster within the mitosomes, with the exception of [2Fe-2S] ferredoxin, which is itself part of the ISC pathway. The overall uptake of iron into the cellular proteins remained unchanged as also observed for the grx5 knock out cell line. The pull-downs of all late ISC components were used to build the interactome of the pathway showing specific position of IscA2 due to its interaction with the outer mitosomal membrane proteins. Finally, the comparative analysis across Metamonada species suggested that the adaptation of the late ISC pathway identified in G. intestinalis occurred early in the evolution of this supergroup of eukaryotes.
线粒体代谢完全依赖于[4Fe-4S]簇的生物合成，而[4Fe-4S]簇是呼吸链亚基的一部分。线粒体晚期ISC 途径介导这些簇从更简单的[2Fe-2S]分子的形成，并将它们转移到客户蛋白上。在这里，我们在厌氧原生动物贾第虫的最简单的线粒体——mitosomes 中对晚期 ISC 途径进行了表征，该虫失去了呼吸链和其他线粒体的特征。除了 IscA2、Nfu1 和 Grx5 外，我们还在 G. intestinalis mitosomes 中鉴定到了一种新的 BolA1 同源物。它与 Grx5 特异性相互作用，并且根据高亲和力下拉实验也与其他核心 mitosomal 成分相互作用。使用 CRISPR/Cas9，我们能够建立完全的 bolA1 敲除，这是第一个缺乏 mitosomal 蛋白的细胞系。尽管 ISC 途径是 mitosome 的唯一代谢作用，但 mitosome 生物学没有发生显著变化，因为 mitosomes 的数量或它们在体外形成[2Fe-2S]簇的能力都没有受到影响。我们未能鉴定需要 mitosomes 内的[2Fe-2S]或[4Fe-4S]簇的天然客户蛋白，除了[2Fe-2S]铁氧还蛋白，它本身就是 ISC 途径的一部分。进入细胞蛋白的铁的总体摄取量保持不变，就像 grx5 敲除细胞系一样。所有晚期 ISC 成分的下拉实验被用来构建途径的互作组，显示出由于与外 mitosomal 膜蛋白的相互作用，IscA2 的特定位置。最后，对 Metamonada 物种的比较分析表明，在该超群真核生物的早期进化过程中，在 G. intestinalis 中发现的晚期 ISC 途径的适应就已经发生了。
