The essential protein Mak16 binds an Fe/S center: Implications for its function in ribosomal assembly

Mak 16是核糖体组装过程中60 S rRNA成熟所需的必需蛋白。然而，其确切的分子作用是未知的。Mak 16与其他核糖体生物合成因子相互作用，正如我们发现的那样，与负责细胞溶质铁硫（Fe/S）蛋白组装（CIA机器）的蛋白质相互作用。在此之前，我们对CIA因子的研究发现，所有真核生物的DNA聚合酶都含有Fe/S簇，这些簇对于与辅助亚基的组装和DNA复制至关重要。现在，我们的初步工作表明，在真核生物Mak 16，包括人Mak 16中存在一个必要的Fe/S簇。在这个提议中，我们想解开Mak 16的Fe/S簇在核糖体组装中的作用。我们计划Mak 16蛋白在酿酒酵母体内的结构-功能研究和Mak 16蛋白在体外的生化特性。方法包括EPR，穆斯堡尔和顺磁NMR光谱。为了解决Mak 16的Fe/S簇与相互作用伴侣形成复合物的重要性，将使用创新的非放射性脉冲追踪表位标记方法对野生型和Fe/S缺陷型Mak 16突变体进行广泛的相互作用组分析。Mak 16的独特特征包括与核糖体L28 e蛋白家族的相似性和高度碱性的N-末端结构域，表明rRNA结合。这个问题将通过使用NMR光谱的蛋白质/RNA结合研究来研究。我们对Mak 16的研究对于在分子水平上理解Fe/S簇在核糖体生物发生中的作用具有重要意义。

Mak16 is an essential protein that is required for the maturation of 60S rRNA during assembly of ribosomes. However, its precise molecular role is unknown. Mak16 interacts with other ribosome biogenesis factors and, as we found, with proteins responsible for cytosolic iron-sulfur (Fe/S) protein assembly (CIA machinery). Previously, our study of CIA factors led to the discovery that all eukaryotic DNA polymerases contain Fe/S clusters, which are crucial for the assembly with accessory subunits and DNA replication. Now, our preliminary work demonstrates the presence of an essential Fe/S cluster in eukaryotic Mak16, including human Mak16. In this proposal we want to unravel the role of the Fe/S cluster of Mak16 for its function in ribosomal assembly. We plan structure-function studies of Mak16 proteins in vivo in Saccharomyces cerevisiae and biochemical characterization of Mak16 proteins in vitro. The methods include EPR, Mössbauer and paramagnetic NMR spectroscopy. To address the essentiality of the Fe/S cluster of Mak16 for complex formation with interactions partners, extensive interactome analysis of wild-type and Fe/S-defective Mak16 mutants will be performed using an innovative, non-radioactive, pulse-chase epitope labeling approach. Unique features of Mak16 include similarity to the ribosomal L28e protein family and a highly basic N-terminal domain, suggesting rRNA binding. This issue will be investigated by protein/RNA binding studies using NMR spectroscopy. Our research on Mak16 is pivotal for understanding of the role of Fe/S clusters in ribosome biogenesis at molecular level.