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

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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.