Róża Kucharczyk, PhD, DSc, Prof. of IBB PAS

Laboratory of Bioenergetics and Mitochondrial Disease Mechanisms

Research Scope

Our research focuses on mitochondrial adenosine triphosphate (ATP) synthase biogenesis, regulation, and deficiencies, including control of the expression of mitochondrially encoded subunits of ATP synthase, S. cerevisiae as a model organism to study the effects of mutations of mitochondrial ATP6 and ATP8 genes, and the biological role of Fmp40 (i.e., the only known yeast ampylase) in the regulation of ATP synthase and redox homeostasis.


Main Scientific Achievements

  • We discovered the biological role of Fmp40 ampylase in yeast cells and its involvement in redox homeostasis.
  • We described mechanism of the pathology of 11 mutations of the mitochondrial ATP6 gene, four of them at the molecular level.
  • We created a yeast strain that expresses the β1-10 non-fluorescent fragment of GFP from the mitochondrial genome, thus permitting the screening of matrix-localized pools of proteins that have dual localization in the cell.

Research Description

Adenosine triphosphate (ATP) synthase is an enzyme of the inner mitochondrial membrane that is responsible for ATP synthesis in the process of oxidative phosphorylation. ATP synthase consists of 17 structural subunits. Three subunits in yeast (a, 8, and c) and two in humans (a and 8) are encoded by mitochondrial DNA (mtDNA). The biogenesis of this enzyme is a sophisticated process that requires the coordination of gene expression in both nuclear and mitochondrial genomes with assembly of the enzyme. Activity of the enzyme is closely related to activity of the respiratory chain and controlled, for example, by the natural hydrolytic activity inhibitor peptide IF1. Many modifications have been identified in various subunits of ATP synthase, including phosphorylation, acetylation, trimethylation, nitration, S-nitrosylation, and tryptophan oxidation. Some modifications were reported to affect the enzymatic activity of ATP synthase, but in most cases remaining unknown are the signaling pathways that are responsible for these modifications, the tissues where or biological conditions under which these modifications occur, or the ways in which they impact biochemical activity of the target protein and holoenzyme. Mutations of genes that encode ATP synthase subunits or assembly factors lead to neurodegenerative diseases, which are currently untreatable.

Our research is divided into several interconnected axes:

1) The mitochondrial ATP synthase is composed of subunits of dual genetic origin, nuclear and mitochondrial. We are investigating the mechanisms that control the biogenesis of ATP synthase, including the expression of its mitochondrially encoded subunits and their assembly to the enzyme. We are looking for new proteins that are involved in these processes.

2) Cells adapt energy supplies to their demands. Deficits in energy production lead to metabolic diseases, such as mitochondrial disease, diabetes, heart failure, cancer, and neurodegenerative diseases. Our research seeks to identify molecular mechanisms of ATP synthase diseases that are caused by mutations of mitochondrial ATP6 and ATP8 genes, which encode ATP synthase subunits a and 8, in yeast as a model organism. We created yeast strains that bear 20 of 48 mutations of the ATP6 gene and three mutations of ATP8 that lead to diseases and deciphered their pathogenic mechanisms for five mutations at the molecular level. We extended our research to include mutations of conserved residues of the ATP8 gene to understand the function of subunit 8.

3) We discovered that human selenoprotein O (SelO) and its yeast (Fmp40) and E. coli (YdiU) homologues, classified by our collaborator as pseudokinases, have AMPylase activity. SelO proteins attach the AMP to the threonine, tyrosine, or serine residues in the protein substrates. One of the biological roles of these proteins is the regulation of protein S-glutathionylation levels by AMPylation of the grx family and other proteins during oxidative stress. This is the only AMPylase that is described in yeast to date and only the second in human cells. Our research, apart from understanding the significance of the ampylation of Grx2 for redox homeostasis, seeks to find Fmp40 substrates beyond redoxins and understand the role of this protein in the regulation of mitochondrial bioenergetics.

4) A growing number of proteins that have been studied in yeast have dual cytosolic and mitochondrial localization. We screened such mitochondrial proteins and explored a split-green fluorescent protein (GFP) method that was designed by Cabantous and co-workers such that the synthesis and localization of one of the two fragments of Split-GFP are restricted to the mitochondrial compartment. Split-GFP is based on the partition of 11β strand-composed GFP into two fragments: one long fragment that encompasses the first 10 β strands (β1-10) and one smaller fragment that consists of the remaining b strand (β11). We engineered a yeast strain that harbors the gene that encodes β1-10 fragments of Split-GFP in the mitochondrial genome and is thus translated inside the mitochondrial matrix. The second β11 Split-GFP fragment can be fused to any nuclear-encoded protein that will be translated by cytosolic translation machinery. The fluorescence signal in the mitochondrial network appears only from the mitochondrial pool of the protein that is fused to β11. We are working on engineering a yeast strain that expresses β1-10 from mitochondrial DNA, in which β1-10 would be located in the mitochondrial inter-membrane space (IMS). This strain will allow searching among non-mitochondrial proteins for those that transfer to the IMS under certain conditions.

  • Bibliography
  1. Carraro et al. Cell Rep. 2020. doi: 10.1016/j.celrep.2020.108095
  2. Kucharczyk et al. BBA-BIO. doi: 10 10.18388/pb.2018_144
  3. Carraro et al. Cell Physiol Biochem. 2018. doi: 10.1159/000494864
  4. Kucharczyk et al. BBA-BIO 2019. doi: 10.1016/j.bbabio.2018.11.005
  5. Niedzwiecka et al. BBA-MCR 2016. doi: 10.1016/j.bbamcr.2017.10.003
  6. Wen et al. Sci Rep 2016. doi: 10.1038/srep36313
  7. Żurawik et al. PLOS-One 2016. doi : 10.1371/journal.pone.0161353
  8. Niedzwiecka et al. Mitochondrion 2016. doi: 10.1016/j.mito.2016.04.003
  9. Lasserre et al. Dis Model Mech 2015. doi: 10.1242/dmm.020438
  10. Aiyar et al. Nat Commun. 2014. doi: 10.1038/ncomms6585


  • Molecular biology: polymerase chain reaction, DNA cloning, electrophoresis, etc.
  • Yeast genetics: site-directed mutagenesis of nuclear and mitochondrial DNA, transformation into mitochondria (Biolistics), drug screening (in collaboration with JP di Rago).
  • Biochemical methods for the characterization of mitochondrial activity: oxygraphy, Clark electrode, spectrofluorometry, enzymatic activity.
  • Protein methods: immunoprecipitation, affinity chromatography, in-gel activities, BN/CN-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, expression and purification from bacteria, in vitro activity.

Selected Publications

  • Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP. Bader G, Enkler L, Araiso Y, Hemmerle M, Binko K, Baranowska E, De Craene JO, Ruer-Laventie J, Pieters J, Tribouillard-Tanvier D, Senger B, di Rago JP, Friant S, Kucharczyk R*, Becker HD*. eLife. 2020, 9:e56649. doi: 10.7554/eLife.56649.
  • Protein AMPylation by an Evolutionarily Conserved Pseudokinase. Sreelatha A, Yee SS, Lopez VA, Park BC, Kinch LN, Pilch S, Servage KA, Zhang J, Jiou J, Karasiewicz-Urbańska M, Łobocka M, Grishin NV, Orth K, Kucharczyk R, Pawłowski K, Tomchick DR, Tagliabracci VS. Cell. 2018, 175(3):809-821.e19. doi: 10.1016/j.cell.2018.08.046.
  • ATP Synthase Subunit a Supports Permeability Transition in Yeast Lacking Dimerization Subunits and Modulates yPTP Conductance. Niedzwiecka K, Baranowska E, Panja C, Kucharczyk R. Cell Physiol Biochem. 2020, 54(2):211-229. doi: 10.33594/000000215.
  • ATP Synthase Diseases of Mitochondrial Genetic Origin. Front Physiol. Dautant A, Meier T, Hahn A, Tribouillard-Tanvier D, di Rago JP, Kucharczyk. 2018, 9:329. doi: 10.3389/fphys.2018.00329.
  • Two mutations in mitochondrial ATP6 gene of ATP synthase, related to human cancer, affect ROS, calcium homeostasis and mitochondrial permeability transition in yeast. Niedzwiecka K, Tisi R, Penna S, Lichocka M, Plochocka D, Kucharczyk R. Biochim Biophys Acta Mol Cell Res. 2018, 1865(1):117-131. doi: 10.1016/j.bbamcr.2017.10.003.


Publications (IBB PAS affiliated)

KABALA A., LASSERRE J.-P., ACKERMAN S.H., DI RAGO J.-P., KUCHARCZYK R., Defining the impact on yeast ATP synthase of two pathogenic human mitochondrial DNA mutations, T9185C and T9191C. Biochimie (2014) 100: 200-206 IF 3.123
WYSOCKA-KAPCIŃSKA M., TOROCSIK B., TURIAK L., TSAPRAILIS G., DAVID C.L., HUNT A., VEKEY K., ADAM-VIZI V., KUCHARCZYK R., CHINOPOULOS C., The suppressor of AAC2 lethality SAL1 modulates sensitivity of heterologously expressed artemia ADP/ATP carrier to bongkrekate in yeast. PLOS ONE (2013) 8(9): e74187 (11p.) IF 3.730
KUCHARCZYK R., GIRAUD M.-F., BRÈTHES D., WYSOCKA-KAPCIŃSKA M., EZKURDIA N., SALIN B., VELOURS J., CAMOUGRAND N., HARAUX F., DI RAGO J.-P., Defining the pathogenesis of human mtDNA mutations using a yeast model: The case of T8851C. The International Journal of Biochemistry & Cell Biology (2013) 45(1): 130–140 IF 4.152
KLIONSKY D.J., KUCHARCZYK R., SIRKO A., ZOLADEK T., ET AL. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy (2012) 8(4): 445-544 IF 7.453
COUPLAN E., AIYAR R.S., KUCHARCZYK R., KABALA A., EZKURDIA N., GAGNEUR J., ST.ONGE R.P., SALIN B., SOUBIGOU F., LE CANN M., STEINMETZ L.M., DI RAGO J.-P., BLONDEL M., A yeast-based assay identifies drugs active against human mitochondrial disorders. PNAS - Proceedings of the National Academy of Sciences of the United States of America (2011) 108(29): 11989-11994 IF 9.771
PIEKARSKA I., KUCHARCZYK R., MICKOWSKA B., RYTKA J., REMPOLA B., Mutants of the Saccharomyces cerevisiae VPS genes CCZ1 and YPT7 are blocked in different stages of sporulation. European Journal of Cell Biology (2010) 89: 780-787 IF 3,314
HOFFMAN-SOMMER M., KUCHARCZYK R., PIEKARSKA I., KOZLOWSKA E., RYTKA J., Mutations in the Saccharomyces cerevisiae vacuolar fusion proteins Ccz1, Mon1 and Ypt7 cause defects in cell cycle progression in a num1∆ background. European Journal of Cell Biology (2009) 88: 639-652 IF 3,955
KUCHARCZYK R., RAK M., DI RAGO J.-P., Biochemical consequences in yeast of the human mitochondrial DNA 8992T>C mutation in the ATPase6 gene found in NARP/MILS patients. Biochimica et Biophysica Acta - Molecular Cell Research (2009) 1793: 817-824 IF 4,893
KUCHARCZYK R., HOFFMAN-SOMMER M., PIEKARSKA I., FISCHER von MOLLARD G., RYTKA J., The Saccharomyces cerevisiae protein Ccz1p interacts with components of the endosomal fusion machinery. FEMS Yeast Research (2009) 9: 565-573 IF 2,579
ZENG X., KUCHARCZYK R., DI RAGO J.-P., TZAGOLOFF A., The Leader Peptide of Yeast Atp6p is required for efficient interaction with the Atp9p ring of the Mitochondrial ATPase. The Journal of Biological Chemistry (2007) 282(50): 36167-36176 IF 5,808