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

Mak16是核糖体组装过程中60rRNA成熟所必需的蛋白质。然而，其确切的分子作用尚不清楚。Mak16与其他核糖体生物发生因子相互作用，并且如我们所发现的，与负责胞浆铁-硫(Fe/S)蛋白组装(CIA机制)的蛋白相互作用。以前，我们对CIA因子的研究发现，所有真核细胞的DNA聚合酶都含有Fe/S簇，这对辅助亚基的组装和DNA的复制至关重要。现在，我们的初步工作表明，在真核细胞Mak16，包括人Mak16中，存在一个必需的Fe/S簇。在这项研究中，我们想要揭示Mak16的Fe/S簇在核糖体组装中的作用。我们计划在酿酒酵母中进行Mak16蛋白的体内结构和功能研究，并在体外进行Mak16蛋白的生化特性研究。方法包括电子顺磁共振、穆斯堡尔谱和顺磁核磁共振波谱。为了说明Mak16的Fe/S簇对于与相互作用伙伴形成复杂的相互作用伙伴的重要性，将使用一种创新的、非放射性的脉冲追逐表位标记方法对野生型和Fe/S缺陷的Mak16突变体进行广泛的交互作用组分析。Mak16的独特功能包括与核糖体L28e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核磁共振光谱进行蛋白质/RNA结合研究来研究。我们对Mak16的研究对于从分子水平上理解Fe/S簇在核糖体生物发生中的作用是至关重要的。

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.