Tiny metal building block determines whether protein factories in cells work smoothly
A single iron-sulphur building block directly determines whether ribosomes - the protein factories of our cells - work smoothly or not. This is the conclusion of a current research project led by the RPTU University Kaiserslautern-Landau (RPTU). The findings significantly expand our understanding of the roles of metal ions in protein production - and have been published in the renowned scientific journal PNAS (Proceedings of the National Academy of Sciences of the United States of America).
Metal ions are important building blocks of life: iron ions in so-called iron-sulphur clusters play a central role in proteins, i.e. in protein molecules that are involved in various essential processes in biology: These include metabolic pathways such as the mitochondrial respiratory chain or the citrate cycle.
Researchers at RPTU have now been able to show that a tiny metal building block is just as crucial for protein production itself. PD Dr. Daili Netz from the Department of Chemistry at RPTU and doctoral student Nadine Duppe have taken a closer look at the protein Mak16. You should know this: Mak16 plays a key role in the production of ribosomes, i.e. in the production of the "protein factories" in our cells.
Daili Netz's team discovered that Mak16 is only stable and works correctly with its important partner protein Rpf1 when it contains the iron-sulphur building block [4Fe-4S]. The [4Fe-4S] cluster has a structure in which four iron and four sulphur ions are alternately located at the corners of the "cube" in a roughly cubic arrangement. If this building block is missing, then - to put it simply - ribosome production stops and the cell cannot produce any new proteins.
If the cluster is missing, the ribosomes cannot be assembled properly
"Mak16 carries an iron-sulphur building block in a pocket in the protein," explains Daili Netz on the detailed structure of the compound. "This pocket consists of four amino acids, the cysteine residues, which hold the cluster in place and help it to bind stably in the protein."
To show how important this cluster is for cooperation with Rpf1, the research team specifically produced Mak16 in two variants: once in its "normal" form with intact pocket and cluster. And once modified so that the pocket can no longer hold the cluster. Using immunoprecipitation, which can be thought of as a kind of "protein fishing", the researchers were able to show that only Mak16 with an intact pocket and cluster can reliably retain the Rpf1 protein. "If the cluster is missing, the binding does not work at all, no complex is formed," explains Daili Netz - and adds, with a view to the fact that ribosomes consist of proteins and ribosomal RNA (rRNA): "We also looked at whether the ribosomes are assembled correctly in yeast cells. And we were able to see that the production of rRNA and the maturation of ribosomes strongly depend on whether Mak16 carries the cluster. If the cluster is missing, the ribosomes cannot be assembled correctly."
Details of the iron-sulphur building block elucidated
Under the direction of Professor Antonio Pierik, Department of Chemistry at RPTU, the metallic nature of the cluster was detected using electron spin resonance (EPR) spectroscopy, supplemented by Mössbauer analyses by Professor Volker Schünemann, Department of Physics at RPTU, and his doctoral student Lukas Knauer. Mössbauer spectroscopy can be understood as a kind of super-precise iron scanner - because using this highly specialized method, the researchers were able to analyze, among other things, how the iron building blocks are bound in the protein structure. Antonio Pierik explains the background to the investigations: "With EPR spectroscopy, we can see the iron ions because they have unpaired electrons that create a fingerprint in the magnetic field at very low temperatures. The sulphur ions themselves cannot be seen directly, but they influence the iron ions so strongly that their presence and arrangement can also be recognized." EPR and Mössbauer analyses show in summary that Mak16 contains a [4Fe-4S] cluster that occurs in two stable states. Daili Netz: "This enabled us to understand how the metal ions are organized in the protein and how Mak16 can thus fulfill its tasks in the cell."
Another finding of the researchers: The [4Fe-4S] cluster reacts very sensitively to oxidative stress. If the cluster breaks down as a result, ribosome production stops. The cluster thus not only acts as an essential building block, but also as a sensor that signals to the cell when protein production should be reduced.
The work was funded by the German Research Foundation (DFG). "Iron-sulphur clusters control central cell processes such as metabolism, DNA synthesis and repair, signal transmission and enzymatic functions and help the cells to react to stress. The fact that a single [4Fe-4S] cluster now directly influences the assembly of ribosomes gives us new insights into the mechanisms of protein production, expands our understanding of cell biology and explains how disruptions in these processes can lead to problems in protein production or cellular stress responses," says Daili Netz, highlighting the special features of the current research findings.
The publication:
Nadine Duppe, Lukas Knauer, Marc Hagebölling, Lena Langner, Martin Stümpfig, Volker Schünemann, Antonio J Pierik, Daili J Netz (2025): The function of Mak16 in ribosome biogenesis depends on its [4Fe-4S] cluster; PNAS; https://doi.org/10.1073/pnas.2513844122
Scientific contact person:
PD Dr. Daili J.A. Netz
Department of Chemistry at RPTU Kaiserslautern-Landau
T.: +49-(0)631-205-2968