Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

The biogenesis of mammalian ATP synthase is complex process believed to proceed via several modules. It starts with the formation of F1 catalytic part, which is in the later steps connected with the membranous subcomplex. The final phase is represented by incorporation of the two mtDNA-encoded subunits Fo-a and A6L. However, little is known about the position of two newly described Fo accessory subunits DAPIT (also termed Usmg5) and MLQ (also known as c14orf2) in the assembly scheme and about their role in regulation of ATP synthase biogenesis. To resolve this, we have utilised several model systems, namely rho0 cells lacking mtDNA and thus both subunits Fo-a and A6L, cells harbouring 9205delTA microdeletion, which results in the absence of the subunit Fo-a, HEK293 cells with knockdown of DAPIT protein and HEK293 cells with knockout of MLQ protein and followed the assembly state of ATP synthase among them. Contrary to previously reported data, we observed normal levels of assembled ATP synthase in DAPIT knockdown and MLQ knockout cells. Our results indicate that lack of DAPIT protein leads to the assembly of more labile, but complete and functional holoenzyme. Absence of either Fo-a alone or Fo-a and A6L results into the normal levels of structurally altered, labile, and ~60 kDa smaller vestigial enzyme complex, which also lacks DAPIT and MLQ. This complex retains the ATP hydrolytic activity but is unable to synthesize ATP. Cells with the MLQ knockout presented with the phenotype similar to the lack of Fo-a: normal content of smaller and labile complex. In the absence of MLQ, vestigial ATP synthase did not contain also subunits Fo-a and A6L. This complex also retained ATP hydrolytic activity, while its phosphorylating capacity was affected. In all the cell lines tested, the individual subunits seemed to be associated only with assembled ATP synthase complex, indicating that once subunits dissociate from the complex, they are degraded in the cell. This hypothesis is supported by the fact, that in the cells lacking subunit MLQ the biosynthesis of both mtDNA-encoded subunits Fo-a and A6L is normal, but they are degraded at faster pace than the rest of the complex. Based on our data, we conclude that MLQ and Fo-a closely associate and their incorporation into the enzyme complex depends on each another. On the contrary, DAPIT protein seems to be incorporated at the very last step and its presence stabilises the holoenzyme.

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.

Mitochondria dysfunction is involved in the pathomechanism of many illnesses including Parkinsons disease. PINK1, which is mutated in some cases of familiar Parkinsonism, is a key component in the degradation of damaged mitochondria by mitophagy. The accumulation of PINK1 on the mitochondrial outer membrane (MOM) of compromised organelles is crucial for the induction of mitophagy, but the molecular mechanism of this process is still unresolved. Here, we investigate the association of PINK1 with the TOM complex. We demonstrate that PINK1 heavily relies on the import receptor TOM70 for its association with mitochondria and directly interacts with this receptor. The structural protein TOM7 appears to play only a moderate role in PINK1 association with the TOM complex, probably due to its role in stabilizing this complex. PINK1 requires the TOM40 pore lumen for its stable interaction with the TOM complex and apparently remains there during its further association with the MOM. Overall, this study provides new insights on the role of the individual TOM subunits in the association of PINK1 with the MOM of depolarized mitochondria.

To date knowledge about plant RAC/ROP-GTPase effectors and downstream targets is still limited. This work aims on elucidation of related signal transduction networks involved in pollen tube growth. Yeast two-hybrid and Pull Down methodology were used to identify and characterize hitherto unknown components of RAC-related protein complexes from Nicotiana tabacum. Nt-RIC11pt specifically interacts with diverse active tobacco RAC-GTPases, and it is particularly significant, that their binding affinity is differential, thus implicating a multifaceted role in an interconnected RIC-RAC network. Moreover, Y2H-screening for Nt-RIC11pt targets identified Nt-CAR4, which is phylogenetically assigned to a multifaceted family of novel unusual GTPase activating proteins (GAP). It is argued that scaffold Nt-RIC11pt connects active Nt-RAC3 with membrane-bound Nt-CAR4, thus relaying GAP-activity. Quantitative RT-PCR demonstrates Nt-RIC11pt is primarily expressed in pollen and YFP-fusion proteins show homogeneous cytoplasmic localization in growing tubes, what builds the prerequisite for a proposed role in broader signal transduction. By synoptically integrating experimental data, bioinformatic sequence comparison, phylogenetic analyses, and detailed literature review, this study hypothesizes a concept in which RIC-effectors collectively constitute a multifaceted network hub linking diverse GTPase-dependent processes.

The Twin-arginine translocation (Tat) machinery, which is found in most cellular membranes containing a respiratory or photosynthetic electron transport chain, is characterized by its unique ability to catalyze membrane transport of folded proteins without impairing the membrane potential. In plant thylakoids, Tat machinery consists of three subunits, TatA, TatB, and TatC, with the latter two, TatB and TatC, forming membrane-integral multimeric TatBC receptor complexes. Here we have analyzed the stability and the subunit composition of these complexes after solubilization of thylakoids with the mild detergent digitonin as well as after additional affinity-purification. Employing different detergent combinations and/or heat treatment (40{degrees}C) followed by BN-PAGE and Western analysis we could identify four distinct Tat complexes with apparent molecular masses ranging from approximately 230 kDa to 620 kDa. Treatment of the largest Tat complex with either heat or detergents like DDM or Triton X-114 led to its stepwise breakdown into the three smaller complexes resulting from the successive release of TatB subunits from a relatively stable TatC core complex. From these data we conclude that the fully assembled, physiologically active TatBC receptor complex consists of a stable, trimeric TatC core to which three TatB subunits are bound independently from each other.

Mitochondrial integrity is a crucial determinant of overall cellular health. Mitochondrial dysfunction and impediments in regulating organellar homeostasis contribute majorly to the pathophysiological manifestation of several neurological disorders. Mutations in human DJ-1 (PARK7) have been implicated in the deregulation of mitochondrial homeostasis, a critical cellular etiology observed in Parkinsons disease progression. DJ-1 is a multifunctional protein belonging to the DJ-1/ThiJ/PfpI superfamily, conserved across the phylogeny. Although the pathophysiological significance of DJ-1 has been well-established, the underlying molecular mechanism(s) by which DJ-1 paralogs modulate mitochondrial maintenance and other cellular processes remains elusive. Using Saccharomyces cerevisiae as the model organism, we unravel the intricate mechanism by which yeast DJ-1 paralogs (collectively called Hsp31 paralogs) modulate mitochondrial homeostasis. Our study establishes a genetic synthetic interaction between Ubp2, a cysteine-dependent deubiquitinase, and DJ-1 paralogs. In the absence of DJ-1 paralogs, mitochondria adapt to a highly tubular network due to enhanced expression of Fzo1. Intriguingly, the loss of Ubp2 restores the mitochondrial integrity in the DJ-1 deletion background by modulating the ubiquitination status of Fzo1. Besides, the loss of Ubp2 in the absence of DJ-1 restores mitochondrial respiration and functionality by regulating the mitophagic flux. Further, Ubp2 deletion makes cells resistant to oxidative stress without DJ-1 paralogs. For the first time, our study deciphers functional crosstalk between Ubp2 and DJ-1 in regulating mitochondrial homeostasis and cellular health. Author SummaryMitochondria are dynamic organelles essential for generating the energy required to maintain cellular viability and drive biological processes. Mitochondrial structures undergo continuous remodeling, modulating their function in response to cellular cues. The plasticity of mitochondrial structures is due to conserved fusion-fission proteins, thus enabling cells to adapt to metabolic changes. Mutations in PARK7, encoding for DJ-1, lead to an imbalance in mitochondrial dynamics and culminate in the progression of neurodegenerative disorders such as Parkinsons disease (PD). DJ-1 belongs to the highly conserved DJ-1/ThiJ/Pfp superfamily of multifunctional proteins. Saccharomyces cerevisiae encodes for four paralogs, which belong to the DJ-1 superfamily. Recent studies demonstrate the role of yeast DJ-1 members in regulating mitochondrial integrity and oxidative stress response. However, the mechanism(s) by which the paralogs mediate cytoprotective action remains elusive. The current study addresses the mechanistic lacuna by delineating cross-talk between Ubp2, a deubiquitinase, and redox-sensitive DJ-1 paralogs in regulating mitochondrial health. Our results suggest that elevated expression of Ubp2 in cells lacking DJ-1 paralogs promotes hyperfused mitochondrial structures. At the same time, in the absence of DJ-1 paralogs, the levels of Fzo1 expression are enhanced significantly due to its altered ubiquitination status. Intriguingly, mitochondrial dynamics and cellular health were reinstated upon deletion of Ubp2, particularly in cells with combinatorial deletion of DJ-1 paralogs in yeast. The study thus provides evidence linking the role of DJ-1 and deubiquitinase in the maintenance of mitochondrial dynamics, which can further aid in understanding the mechanism causing PD progression.

AIF1 overexpression is intimately linked to the sensitivity of the yeast cells towards hydrogen peroxide or acetic acid. Therefore, studying the mechanism of its regulation in the cell would provide a significant understanding of the factors ultimately guiding yeast apoptosis. In this report, we establish the time-dependent induction of AIF1 in hydrogen peroxide stress. Additionally, the AIF1 expression in hydrogen peroxide is mediated by two transcription factors, Yap5 (DNA binding) and Cdc73 (non-DNA binding). Furthermore, substituting the H3K36 residue with another significantly abrogates the AIF1 expression. However, substituting H3K4 or H3K79 with A does not affect the AIF1 expression level under hydrogen peroxide stress. Altogether, the significant reduction of AIF1 expression in cdc73{Delta} cells plausibly reflects the reduced H3K36me3 modification and is independent of the H3K4me3 modification.

In previous papers, using the eGFP-RBD3 probe, which binds Ras-GTP with high affinity, we showed that activated Ras proteins are localized to the plasma membrane and in the nucleus in wild-type Saccharomyces cerevisiae cells growing exponentially on glucose, while an aberrant accumulation of activated Ras in mitochondria correlates to mitochondrial dysfunction, accumulation of ROS and an increase of apoptosis. In this paper, we show that lack of TPS1, which is known to trigger apoptosis in S. cerevisiae, induces localization of active Ras proteins in mitochondria, confirming the above-mentioned correlation. Next, by characterizing the ras1{Delta} and ras2{Delta} mutants concerning localization of active Ras proteins and propensity to undergo cell death, we show that active Ras2 proteins, which accumulate in the mitochondria following addition of acetic acid, a well-known pro-apoptotic stimulus, might be the GTPases involved in regulated cell death, while active Ras1 proteins, constitutively localized in mitochondria, might be involved in a pro-survival molecular machinery. Finally, by characterizing the gpa2{Delta} and cyr1{Delta} mutants concerning the propensity to undergo cell death, we show that active mitochondrial Ras proteins promote apoptosis through the cAMP/PKA pathway.

Degradation of short-lived and abnormal proteins are essential for normal cellular homeostasis. In eukaryotes, such unstable cellular proteins are selectively degraded by the ubiquitin proteasome system (UPS). Furthermore, abnormalities in protein degradation by the UPS have been linked to several human diseases. Ccr4 protein is a known component of the Ccr4-Not complex, which has established roles in transcription, mRNA de-adenylation and RNA degradation etc. Excitingly in this study, we show that Ccr4 protein has a novel function as a shuttle factor that promotes ubiquitin-dependent degradation of short-lived proteins by the 26S proteasome. Using a substrate of the well-studied ubiquitin fusion degradation (UFD) pathway, we found that its UPS-mediated degradation was severely impaired upon deletion of CCR4 in Saccharomyces cerevisiae. Additionally, we show that Ccr4 binds to cellular ubiquitin conjugates and the proteasome. In contrast to Ccr4, most other subunits of the Ccr4-Not complex proteins are dispensable for UFD substrate degradation. From our findings we conclude that Ccr4 functions in the UPS as a shuttle factor targeting ubiquitylated substrates for proteasomal degradation.

Two multisubunit protein complexes for membrane protein insertion were recently identified in the endoplasmic reticulum (ER): The guided entry of tail anchor proteins (GET) complex and ER membrane complex (EMC). The structures of both of their hydrophobic core subunits, that are required for the insertion reaction, revealed an overall similarity to the YidC/Oxa1/Alb3 family members found in bacteria, mitochondria and chloroplasts. This suggests that these membrane insertion machineries all share a common ancestry. To test whether these ER proteins can functionally replace Oxa1 in yeast mitochondria, we generated strains that express mitochondria-targeted Get2-Get1 and Emc6-Emc3 fusion proteins in Oxa1 deletion mutants. Interestingly, the Emc6-Emc3 fusion was able to complement an{Delta} oxa1 mutant and restored its respiratory competence. The Emc6-Emc3 fusion promoted the insertion of the mitochondrially encoded protein Cox2 as well as of nuclear encoded inner membrane proteins though was not able to facilitate the assembly of the Atp9 ring. Our observations indicate that protein insertion into the ER is functionally conserved to the insertion mechanism in bacteria and mitochondria and adheres to similar topological principles.

The mitochondrial F1F0-ATP synthase is crucial for maintaining the ATP/ADP balance which is critical for cell metabolism, ion homeostasis, cell division, proliferation and motility. This enzyme, conserved across evolution, is found in the mitochondria or chloroplasts of eukaryotic cells and the plasma membrane of bacteria. In vitro studies have shown that the mitochondrial F1F0-ATP synthase is reversible, capable of hydrolyzing instead of synthesizing ATP. In vivo, its reversibility is inhibited by the endogenous peptide If1 (Inhibitory Factor 1), which specifically prevents ATP hydrolysis in a pH-dependent manner. Despite its presumed importance, the loss of If1 in various model organisms does not cause severe phenotypes, suggesting its role may be confined to specific stress or metabolic conditions yet to be discovered. In this study, we explored the structural and physiological importances of If1 inhibitory peptides in Saccharomyces cerevisiae. Our analyses indicate that inhibitory peptides are crucial in mitigating metabolic adverse outcomes caused by mitochondrial depolarizing stress under glyco-oxidative metabolic conditions. Under glyco-oxidative metabolic state, the energy maintenance relies both on glycolysis and oxidative phosphorylation. Additionally, we found that the absence of If1 destabilizes the nuclear-encoded free F1 subcomplex. This novel mechanism of action highlights the role of If1 in preventing harmful ATP wastage, offering new insights into its function under physiological and pathological conditions.

Cyclophilin A (CyPA) is an abundant and ubiquitously expressed protein belonging to the immunophilin family that has intrinsic peptidyl-prolyl-(cis/trans)-isomerase enzymatic activity. In addition to mediating the immunosuppressive effects of the drug cyclosporine A, CyPA is involved in multiple cellular processes such as protein folding, intracellular trafficking, signal transduction, and transcriptional regulation. Because CyPA is also a molecular chaperone, its expression is induced by several stressor agents and is a highly abundant protein in cancer cells. In this study, it is demonstrated that in several cell types and at least in murine liver, a significant pool of this immunophilin is primarily an intramitochondrial factor that migrates to the nucleus upon the onset of stress. It is also shown that CyPA has antiapoptotic action. Importantly, the capability of CyPA to form complexes with the small acidic cochaperone p23 is proven, this interaction being independent of the usual association of p23 with the heat-shock protein of 90-kDa, Hsp90. Furthermore, it is demonstrated that the CyPA*p23 complex enhances the antiapoptotic response of the cell, suggesting that both proteins form a functional unit whose high level of expression plays a significant role in cell survival.

Cells can regulate the abundance and composition of peroxisomes to adapt to environmental changes. In the bakers yeast, S. cerevisiae, peroxisomes represent the only site for degradation of fatty acids. Hence, it is not surprising that growth of yeast cells on oleic acid results in a massive proliferation of peroxisomes. New peroxisomes can form either by division of pre-existing peroxisomes or de novo in a Pex25p-dependent process with the involvement of the Endoplasmic Reticulum (ER). In search for further factors involved in de novo formation of peroxisomes, we screened ~6,000 yeast mutants that were depleted of peroxisomes by conditional inhibition of PEX19 expression. Screening the mutants for the reappearance of peroxisomes upon expression of PEX19 identified Pex34p, in addition to the well-known component Pex25p, as crucial determinants for de novo biogenesis. Pex34p interacts with Pex19p and with different Peroxisomal Membrane Proteins (PMPs) in a PEX19-dependent manner. Depletion of Pex34p results in reduced numbers of import-competent peroxisomes formed de novo and Pex3p is partly retained and distributed in ER-like structures. We suggest that Pex25p and Pex34p are both required to maintain peroxisome number in a cell and that they perform non-redundant roles in the de novo formation of peroxisomes.

The mTOR complexes play a fundamental role in mitochondrial biogenesis and cellular homeostasis. Wat1, an ortholog of mammalian Lst8 is an important component of TOR complex and is essential for the regulation of downstream signaling. Earlier we reported the role of Wat1 in oxidative stress response. Here, we show that the inactivation of wat1 leads to respiratory defects and mitochondrial depolarization leading to decrease in ATP production. The confocal and electron microscopy in wat1{Delta} cells revealed the fragmented mitochondrial morphology implying its role in mitochondrial fission. Furthermore, we also showed its role in autophagy and the maintenance of calcium ion homeostasis. Additionally, tor2-287 mutant cells also exhibit defects in mitochondrial integrity indicating the TORC1-dependent involvement of Wat1 in the maintenance of mitochondrial homeostasis. The interaction studies of Wat1 and Tor2 with Por1 and Mmm1 proteins revealed a cross-talk between mitochondria and endoplasmic reticulum through the Mitochondria-associated membranes (MAM) and endoplasmic reticulum-mitochondria encounter structure (ERMES) complex, involving TORC1. Taken together, this study demonstrates involvement of Wat1/mLst8 in harmonizing various mitochondrial functions, redox status, and Ca2+ homeostasis.

O-GlcNAcylation is a reversible post-translational modification involved the regulation of cytosolic, nuclear and mitochondrial proteins. Only two enzymes, OGT and OGA, control attachment and removal of O-GlcNAc on proteins, respectively. Whereas a variant OGT (mOGT) has been proposed as the main isoform that O-GlcNAcylates proteins in mitochondria, identification of a mitochondrial OGA has not been performed yet. Two splice variants of OGA (short and long isoforms) have been described previously. In this work, using cell fractionation experiments, we show that short-OGA is preferentially recovered in mitochondria-enriched fractions from HEK-293T cells as well as mouse embryonic fibroblasts. Moreover, fluorescent microscopy imaging confirmed that GFP-tagged short-OGA is addressed to mitochondria. In addition, using a BRET-based mitochondrial O-GlcNAcylation biosensor, we show that co-transfection of short-OGA markedly reduced O-GlcNAcylation of the biosensor, whereas long-OGA had no significant effect. Finally, using genetically encoded or chemical fluorescent mitochondrial probes, we showed that short-OGA overexpression increases mitochondrial ROS levels, whereas long-OGA had no significant effect. Together, our work reveals that the short-OGA isoform is targeted to the mitochondria where it regulates ROS homoeostasis.

Signaling by formyl peptide receptor 1 (FPR1), the prototype G protein-coupled receptor (GPCR) expressed in neutrophil leukocytes, is initiated by an activation of a G protein containing a Gi subunit. FPR1 activation results in an increase in the cytosolic concentration of free calcium ions ([Ca2+]i), and an activation of the superoxide anion producing NADPH oxidase. Receptor downstream signals generated by the danger molecule ATP recognized by the purinergic receptor P2Y2 are transduced by a G protein containing a Gq subunit. The neutrophil response induced by ATP also includes a transient rise in [Ca2+]i, but the downstream signals do not activate the NADPH oxidase. ATP can, however, activate this enzyme system through a receptor transactivation mechanism dependent not only on the ATP receptor but also on the free fatty acid receptor FFA2R, provided that this receptor is allosterically modulated. This occurs through a novel mechanism whereby FFA2R is activated from the cytosolic side of the plasma membrane by Gq transduced signals generated by the ATP receptor. Furthermore, in neutrophils with a disrupted actin cytoskeleton, ATP (as well as platelet activating factor; recognized by the Gq-coupled PAFR) becomes a potent NADPH oxidase activating agonist. At high concentrations of the actin cytoskeleton disrupting drug latrunculin A the activation was only partly reduced by Gq inhibition. More importantly, this response was also partly inhibited by pertussis toxin. The effects on the ATP-induced NADPH oxidase activity, of the Gq inhibitor and pertussis toxin were more and less pronounced, respectively, when the concentration of latrunculin A was reduced. Taken together, we show that in primary human neutrophils the actin cytoskeleton is part of the regulatory machinery that determines the activation of NADPH oxidase activation and the G protein recruitment profile downstream of activated of Gq-coupled GPCRs. HighlightsO_LIATP is a biased signaling agonist unable to activate the neutrophil NADPH oxidase C_LIO_LIATP activates the NADPH oxidase through P2Y2R mediated transactivation of FFA2R C_LIO_LIActin cytoskeleton disruption enables ATP to activate the NADPH oxidase C_LIO_LICytoskeleton regulated NADPH oxidase activation depends on Gi and Gq signaling C_LIO_LIThe actin cytoskeleton regulates the G protein recruitment profile of P2Y2R C_LI

Treatment of endothelial cells with bacterial lipopolysaccharide (LPS) evokes a number of metabolic and functional consequences which built a multifaceted physiological response of endothelium to bacterial infection. Here effects of LPS on human aortic endothelial cells (HAEC) have been investigated. Among the spectrum of biochemical changes substantially elevated N-nicotinamide methyltransferase (NNMT) protein level was particularly intriguing. This important enzyme may potentially affect cellular metabolism by two means: direct regulation of methylnicotinamide level and availability of nicotinamide, that at least potentially may influence NAD+ synthesis, and regulation of S-adenosylmethionine concentration and therefore controlling methylation of many proteins including chromatin. This may have epigenetic consequences. This paper is focused on NNMT, despite the fact that in the presence of LPS additional effects of this compound mask pure (canonical) consequences of the elevated NNMT protein which are an increased MNA synthesis or reduced NAD+ level. On the other hand, however, it has been shown that silencing of the NNMT-encoding gene prevents several changes which are observed in control HAECs treated with LPS. They include significantly increased calcium response to thapsigargin (store-operated calcium entry), altered energy metabolism which is switched to anaerobic glycolysis and rearrangement of the mitochondrial network. However, a biochemical mechanism behind the protective consequences of the NNMT deficiency in cells treated with LPS remains unexplained.

Monomers, dimers, and individual FOF1-ATP synthase subunits are, presumably, involved in the formation of the mitochondrial permeability transition pore (PTP), which molecular structure, however, is still unknown. We hypothesized that upon the Ca2+-dependent assembly of PTP complex, F-ATP synthase (subunits) recruits mitochondrial proteins that do not interact or weakly interact with F-ATP synthase under normal conditions. Therefore, we examined whether the PTP opening in mitochondria before the separation of supercomplexes by BN-PAGE will increases the channel stability and channel-forming capacity of isolated F-ATP synthase dimers and monomers in planar lipid membranes. Besides, we studied the specific activity and protein composition of F-ATP synthase dimers and monomers from rat liver and heart mitochondria before and after PTP opening. By contrast to our expectations, preliminary PTP opening dramatically suppressed the high-conductance channel activity of F-ATP synthase dimers and monomers and decreased their specific "in gel" activity. The decline in the channel-forming activity correlated with the reduced levels of as few as two proteins in the bands: methylmalonate-semialdehyde dehydrogenase and prohibitin 2. These data indicate that proteins accompanying F-ATP synthase may be important players in the PTP formation and stabilization.

The biological effects of paraquat (PQ), an herbicide linked to Parkinsons disease (PD) risk in humans can be studied in Saccharomyces cerevisiae due to its evolutionary conservation with mammals. To understand how genetic background influences PQ toxicity, we treated four yeast strains (NA, SA, WA, and WE) with PQ and assessed physiological (growth curves), molecular (superoxide and peroxide levels) and cellular consequences (vacuolar disaggregation) on each genetic background. PQ significantly reduced specific growth rates (Max) in WE and WA, while SA and NA remained unaffected. Furthermore, PQ increased superoxide and peroxide levels across the strains, but to different extents, being the SA and WE the most affected. PQ also influenced vacuolar morphologies strain-dependently, shifting from one large organelle to small disaggregate vacuoles, with WE being the most susceptible. Interestingly, we found an inverse association between superoxide levels and Max. Given the known involvement of lysosomal dysfunction in pesticide-induced PD, we investigated correlations between predicted missense variants in vacuolar genes and PQ responses across the yeast strains. We identified associations between fen2 variants, the human orthologue of SLC17A5, and vacuolar disaggregation. To validate this, we exposed a fen2-deleted strain to PQ, which exhibited increased vacuolar disaggregation. In conclusion, our findings demonstrate that the strain-specific susceptibility to PQ is associated with superoxide levels and that fen2 likely plays a role in the vacuolar adaptive response to PQ exposure. We speculate that the most resistant strains may facilitate the development of novel therapeutics for humans exposed to PQ.

Exposure to cigarette smoke is one of the major risk factors for developing various diseases such as chronic obstructive pulmonary disease (COPD), cardiovascular disorders, and cancer mediated via cellular oxidative stress and organelle dysfunction. To this end, the current study investigated how cigarette smoke extract (CSE) affects vacuole structure and function in Saccharomyces cerevisiae, as vacuole plays a crucial role in handling oxidative stress-induced misfolded proteins. Our results showed that CSE exposure causes transient vacuolar fragmentation up to 1 h to increase its surface area to facilitate microautophagy in clearing CSE-mediated misfolded protein and promoting cell survival. However, excessive fragmentation or vacuolar fusion sensitizes cells towards CSE-mediated cellular toxicity. Towards understanding the underlying mechanism, the current study demonstrated the involvement of PI3P and PI (3,5) P2-mediated signaling and phospholipase-driven remodeling of lipid moieties. Moreover, the current study also showed the importance of mitochondrial activity in CSE-mediated vacuolar fragmentation. Prolonged exposure to CSE impairs mitochondrial function and thus disrupts fragmentation, the adaptive survival strategy against CS. It results in proteostasis collapse, which is a characteristic shared by many inflammatory and degenerative disorders. Taken together, the current study reveals a previously unrecognized cellular protection mechanism induced by cigarette smoke and highlights potential therapeutic targets for mitigating CS-mediated diseases