Biology subjects

Kucharczyk, R.

Publications and source records attributed to Kucharczyk, R..

4 recordsLinked to original sources

bioRxiv2024

Fmp40 ampylase regulates cell survival upon oxidative stress by controlling Prx1 and Trx3 oxidation

Reactive oxygen species (ROS), play important roles in cellular signaling, nonetheless are toxic at higher concentrations. Cells have many interconnected, overlapped or backup systems to neutralize ROS, but their regulatory mechanisms remain poorly understood. Here, we reveal an essential role for mitochondrial AMPylase Fmp40 from budding yeast in regulating the redox states of mitochondrial 1-Cys peroxiredoxin, Prx1, which is the only protein shown to neutralize H2O2 with the oxidation of the mitochondrial glutathione and Trx3, thioredoxin, directly involved in the reduction of Prx1. Deletion of FMP40 impacts a cellular response to H2O2 treatment that leads to programmed cell death (PCD) induction and an adaptive response involving up or down regulation of genes encoding, among others the catalase Cta1, PCD inducing factor Aif1, and mitochondrial redoxins Trx3 and Grx2. This ultimately perturbs the reduced glutathione and NADPH cellular pools. We further demonstrated that Fmp40 AMPylates Prx1, Trx3, and Grx2 in vitro and interacts with Trx3 in vivo. AMPylation of the threonine residue 66 in Trx3 is essential for this proteins proper endogenous level of and its precursor forms maturation under oxidative stress conditions. Additionally, we showed the Grx2 involvement in the reduction of Trx3 in vivo. Taken together, Fmp40, through control of the reduction of mitochondrial redoxins, regulates the hydrogen peroxide, GSH and NADPH signaling influencing the programmed cell death execution. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/590396v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@2bae9borg.highwire.dtl.DTLVardef@1d46514org.highwire.dtl.DTLVardef@1380c2aorg.highwire.dtl.DTLVardef@a6bad9_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

A systematic bi-genomic split-GFP assay illuminates the mitochondrial matrix proteome and protein targeting routes

The majority of mitochondrial proteins are encoded in the nuclear genome and often lack clear targeting signals. Therefore, what constitutes the entire mitochondrial proteome is still unclear. We here build on our previously developed bi-genomic (BiG) split-GFP assay (Bader et al. 2020) to solidify the list of matrix and inner membrane mitochondrial proteins. The assay relies on one fragment (GFP1-10) encoded in the mitochondrial DNA enabling specific visualization of only the proteins tagged with a smaller fragment, GFP11, and localized to the mitochondrial matrix or the inner membrane. We used the SWAp-Tag (SWAT) strategy to tag every protein with GFP11 and mated them with the BiG GFP strain. Imaging the collection in six different conditions allowed us to visualize almost 400 mitochondrial proteins, 50 of which were never visualized in mitochondria before, and many are poorly studied dually localized proteins. We use structure-function analysis to characterize the dually localized protein Gpp1, revealing an upstream start codon that generates a mitochondrial targeting signal and explore its unique function. We also show how this data can be applied to study mitochondrial inner membrane protein topology and sorting. This work brings us closer to finalizing the mitochondrial proteome and the freely distributed library of GFP11-tagged strains will be a useful resource to study protein localization, biogenesis and interactions.

cell biology↗

bioRxiv2022

Small Protein Interactome analysis of ATP synthase identifies the uncharacterized 'subunit' Mco10 - a new modulator of permeability transition pore in S. cerevisiae

In S. cerevisiae, the uncharacterized protein Mco10 (Mitochondrial class one protein of 10 kDa) was previously found to be associated with mitochondrial ATP synthase and referred to as a new subunit l. However, recent cryo-EM structures of S. cerevisiae ATP synthase could not ascertain Mco10 as a structural subunit of the enzyme, either monomers or dimers, making questionable its role as a structural subunit. The N-terminal part of Mco10 is very similar to Atp19 (subunit k) of ATP synthase. The subunit k/Atp19, along with the subunits g/Atp20 and e/Atp21 plays a major role in stabilization of the ATP synthase dimers. In our effort to confidently define the small protein interactome of ATP synthase we similarly found Mco10 associated with ATP synthase of S. cerevisiae. We herein investigated the impact of Mco10 on ATP synthase functioning. Biochemical analysis revealed in spite of similarity in sequence and evolutionary lineage, that Mco10 and Atp19 differ significantly in function. This is the first work to show Mco10 is an auxiliary ATP synthase subunit that only functions in permeability transition.

cell biology↗

bioRxiv2022

The small GTPase Arf1 regulates ATP synthesis and mitochondria homeostasis by modulating fatty acid metabolism

Lipid mobilization through fatty acid {beta}-oxidation is a central process essential for energy production during nutrient shortage. In yeast, this catabolic process starts in the peroxisome from where {beta}-oxidation products enter mitochondria and fuel the TCA cycle. Little is known about the physical and metabolic cooperation between these organelles. We found that expression of fatty acid transporters and of the rate-limiting enzyme involved in {beta}-oxidation are decreased in cells expressing a hyperactive mutant of the small GTPase Arf1, leading to an accumulation of fatty acids in lipid droplets. As a consequence, mitochondria became fragmented and ATP synthesis decreased. Genetic and pharmacological depletion of fatty acids phenocopied the arf1 mutant mitochondrial phenotype. Although {beta}-oxidation occurs mainly in mitochondria in mammals, Arf1s role in fatty acid metabolism is conserved. Together, our results indicate that Arf1 integrates metabolism into energy production by regulating fatty acid storage and utilization, and presumably organelle contact-sites.

cell biology↗