Phosphorylation plays a crucial role in the regulation of many fundamental cellular processes. Phosphorylation levels are increased in many cancer cells where they may promote changes in mitochondrial homeostasis. Proteomic studies on various types of cancer identified 17 phosphorylation sites within the human ATP-dependent protease Lon, which degrades misfolded, unassembled and oxidatively damaged proteins in mitochondria. Most of these sites were found in Lon's N-terminal (NTD) and ATPase domains, though little is known about the effects on their function. By combining the biochemical and cryo-electron microscopy studies, we show the effect of Tyr186 and Tyr394 phosphorylations in Lon's NTD, which greatly reduce all Lon activities without affecting its ability to bind substrates or perturbing its tertiary structure. A substantial reduction in Lon's activities is also observed in the presence of polyphosphate, whose amount significantly increases in cancer cells. Our study thus provides an insight into the possible fine-tuning of Lon activities in human diseases, which highlights Lon's importance in maintaining proteostasis in mitochondria.

CEITEC Masaryk University in Brno Brno Czech Republic

Department of Biochemistry and Protein Structure Institute of Molecular Biology Slovak Academy of Sciences Dúbravská Cesta 21 845 51 Bratislava Slovakia

Department of Structural and Computational Biology Max Perutz Labs University of Vienna Campus Vienna Biocenter 5 1030 Vienna Austria

Institute of Biology and Medical Genetics 1st Faculty of Medicine Charles University Prague Prague Czech Republic

Institute of Chemistry Slovak Academy of Sciences Bratislava Slovakia

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo Namesti 542 2 16000 Prague Czech Republic

Laboratory of Clinical and Molecular Genetics National Institute of Children's Diseases Limbová 1 833 40 Bratislava Slovakia

Medirex Group Academy Nitra Slovakia

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