Two Types of Survival Curves of Different Lines of Progeric Mice.

For most of their lifespan, the probability of death for many animal species increases with age. Gompertz law states that this increase is exponential. In this work, we have compared previously published data on the survival kinetics of different lines of progeric mice. Visual analysis showed that in six lines of these rapidly aging mutants, the probability of death did not strictly depend on age. In contrast, ten lines of progeric mice have survival curves similar to those of the control animals, that is, in agreement with Gompertz law, similar to the shape of an exponential curve upside down. Interestingly, these ten mutations cause completely different cell malfunctions. We speculate that what these mutations have in common is a reduction in the lifespan of cells and/or an acceleration of the transition to the state of cell senescence. Thus, our analysis, similar to the conclusions of many previously published works, indicates that the aging of an organism is a consequence of the aging of individual cells.

The Imidazolium Ionic Liquids Toxicity is Due to Their Effect on the Plasma Membrane.

Ionic liquids (ILs) are organic salts with a low melting point. This is due to the fact that their alkyl side chains, which are covalently connected to the ion, hinder the crystallization of ILs. The low melting point of ILs has led to their widespread use as relatively harmless solvents. However, ILs do have toxic properties, the mechanism of which is largely unknown, so identifying the cellular targets of ILs is of practical importance. In our work, we showed that imidazolium ILs are not able to penetrate model membranes without damaging them. We also found that inactivation of multidrug resistance (MDR) pumps in yeast cells does not increase their sensitivity to imidazolium ILs. The latter indicates that the target of toxicity of imidazolium ILs is not in the cytoplasm. Thus, it can be assumed that the disruption of the barrier properties of the plasma membrane is the main reason for the toxicity of low concentrations of imidazolium ILs. We also showed that supplementation with imidazolium ILs restores the growth of cells with kinetically blocked glycolysis. Apparently, a slight disruption of the plasma membrane caused by ILs can, in some cases, be beneficial for the cell.

Structural Role of Plasma Membrane Sterols in Osmotic Stress Tolerance of Yeast Saccharomyces cerevisiae.

Yeast S. cerevisiae has been shown to suppress a sterol biosynthesis as a response to hyperosmotic stress. In the case of sodium stress, the failure to suppress biosynthesis leads to an increase in cytosolic sodium. The major yeast sterol, ergosterol, is known to regulate functioning of plasma membrane proteins. Therefore, it has been suggested that the suppression of its biosynthesis is needed to adjust the activity of the plasma membrane sodium pumps and channels. However, as the sterol concentration is in the range of thirty to forty percent of total plasma membrane lipids, it is believed that its primary biological role is not regulatory but structural. Here we studied how lowering the sterol content affects the response of a lipid bilayer to an osmotic stress. In accordance with previous observations, we found that a decrease of the sterol fraction increases a water permeability of the liposomal membranes. Yet, we also found that sterol-free giant unilamellar vesicles reduced their volume during transient application of the hyperosmotic stress to a greater extent than the sterol-rich ones. Furthermore, our data suggest that lowering the sterol content in yeast cells allows the shrinkage to prevent the osmotic pressure-induced plasma membrane rupture. We also found that mutant yeast cells with the elevated level of sterol accumulated propidium iodide when exposed to mild hyperosmotic conditions followed by hypoosmotic stress. It is likely that the decrease in a plasma membrane sterol content stimulates a drop in cell volume under hyperosmotic stress, which is beneficial in the case of a subsequent hypo-osmotic one.

Cytostatic effects of structurally different ginsenosides on yeast cells with altered sterol biosynthesis and transport.

Triterpene glycosides are a diverse group of plant secondary metabolites, consisting of a sterol-like aglycon and one or several sugar groups. A number of triterpene glycosides show membranolytic activity, and, therefore, are considered to be promising antimicrobial drugs. However, the interrelation between their structure, biological activities, and target membrane lipid composition remains elusive. Here we studied the antifungal effects of four Panax triterpene glycosides (ginsenosides) with sugar moieties at the C-3 (ginsenosides Rg3, Rh2), C-20 (compound K), and both (ginsenoside F2) positions in Saccharomyces cerevisiae mutants with altered sterol plasma membrane composition. We observed reduced cytostatic activity of the Rg3 and compound K in the UPC2-1 strain with high membrane sterol content. Moreover, LAM gene deletion reduced yeast resistance to Rg3 and digitonin, another saponin with glycosylated aglycon in the C-3 position. LAM genes encode plasma membrane-anchored StARkin superfamily-member sterol transporters. We also showed that the deletion of the ERG6 gene that inhibits ergosterol biosynthesis at the stage of zymosterol increased the cytostatic effects of Rg3 and Rh2, but not the other two tested ginsenosides. At the same time, in silico simulation revealed that the substitution of ergosterol with zymosterol in the membrane changes the spatial orientation of Rg3 and Rh2 in the membranes. These results imply that the plasma membrane sterol composition defines its interaction with triterpene glycoside depending on their glycoside group position. Our results also suggest that the biological role of membrane-anchored StARkin family protein is to protect eukaryotic cells from triterpenes glycosylated at the C-3 position.

Lipophilic Cations Rescue the Growth of Yeast under the Conditions of Glycolysis Overflow.

Chemicals inducing a mild decrease in the ATP/ADP ratio are considered as caloric restriction mimetics as well as treatments against obesity. Screening for such chemicals in animal model systems requires a lot of time and labor. Here, we present a system for the rapid screening of non-toxic substances causing such a de-energization of cells. We looked for chemicals allowing the growth of yeast lacking trehalose phosphate synthase on a non-fermentable carbon source in the presence of glucose. Under such conditions, the cells cannot grow because the cellular phosphate is mostly being used to phosphorylate the sugars in upper glycolysis, while the biosynthesis of bisphosphoglycerate is blocked. We reasoned that by decreasing the ATP/ADP ratio, one might prevent the phosphorylation of the sugars and also boost bisphosphoglycerate synthesis by providing the substrate, i.e., inorganic phosphate. We confirmed that a complete inhibition of oxidative phosphorylation alleviates the block. As our system includes a non-fermentable carbon source, only the chemicals that did not cause a complete block of mitochondrial ATP synthesis allowed the initial depletion of glucose followed by respiratory growth. Using this system, we found two novel compounds, dodecylmethyl diphenylamine (FS1) and diethyl (tetradecyl) phenyl ammonium bromide (Kor105), which possess a mild membrane-depolarizing activity.

Protonophore FCCP provides fitness advantage to PDR-deficient yeast cells.

Pleiotropic drug resistance (PDR) plasma membrane transporters mediate xenobiotic efflux from the cells and thereby help pathogenic microorganisms to withstand antimicrobial therapies. Given that xenobiotic efflux is an energy-consuming process, cells with upregulated PDR can be sensitive to perturbations in cellular energetics. Protonophores dissipate proton gradient across the cellular membranes and thus increase ATP spendings to their maintenance. We hypothesised that chronic exposure of yeast cells to the protonophores can favour the selection of cells with inactive PDR. To test this, we measured growth rates of the wild type Saccharomyces cerevisiae and PDR-deficient Δpdr1Δpdr3 strains in the presence of protonophores carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), pentachlorophenol (PCP) and niclosamide (NCA). Although the protonophore-induced respiration rates of these two strains were similar, the PDR-deficient strain outperformed the control one in the growth rate on non-fermentable carbon source supplemented with low concentrations of FCCP. Thus, active PDR can be deleterious under conditions of partially uncoupled oxidative-phosphorylation. Furthermore, our results suggest that tested anionic protonophores are poor substrates of PDR-transporters. At the same time, protonophores imparted azole tolerance to yeasts, pointing that they are potent PDR inducers. Interestingly, protonophore PCP led to a persistent increase in the levels of a major ABC-transporter Pdr5p, while azole clotrimazole induced only a temporary increase. Together, our data provides an insight into the effects of the protonophores in the eukaryotes at the cellular level and support the idea that cells with activated PDR can be selected out upon conditions of energy limitations.

The GAR domain integrates functions that are necessary for the proper localization of fibrillarin (FBL) inside eukaryotic cells.

Fibrillarin (FBL) is an essential nucleolar protein that participates in pre-rRNA methylation and processing. The methyltransferase domain of FBL is an example of an extremely well-conserved protein domain in which the amino acid sequence was not substantially modified during the evolution from Archaea to Eukaryota. An additional N-terminal glycine-arginine-rich (GAR) domain is present in the FBL of eukaryotes. Here, we demonstrate that the GAR domain is involved in FBL functioning and integrates the functions of the nuclear localization signal and the nucleolar localization signal (NoLS). The methylation of the arginine residues in the GAR domain is necessary for nuclear import but decreases the efficiency of nucleolar retention via the NoLS. The presented data indicate that the GAR domain can be considered an evolutionary innovation that integrates several functional activities and thereby adapts FBL to the highly compartmentalized content of the eukaryotic cell.

LAM Genes Contribute to Environmental Stress Tolerance but Sensibilize Yeast Cells to Azoles.

Lam proteins transport sterols between the membranes of different cellular compartments. In Saccharomyces cerevisiae, the LAM gene family consists of three pairs of paralogs. Because the function of paralogous genes can be redundant, the phenotypes of only a small number of LAM gene deletions have been reported; thus, the role of these genes in yeast physiology is still unclear. Here, we surveyed the phenotypes of double and quadruple deletants of paralogous LAM2(YSP2)/LAM4 and LAM1(YSP1)/LAM3(SIP3) genes that encode proteins localized in the junctions of the plasma membrane and endoplasmic reticulum. The quadruple deletant showed increased sterol content and a strong decrease in ethanol, heat shock and high osmolarity resistance. Surprisingly, the quadruple deletant and LAM2/LAM4 double deletion strain showed increased tolerance to the azole antifungals clotrimazole and miconazole. This effect was not associated with an increased rate of ABC-transporter substrate efflux. Possibly, increased sterol pool in the LAM deletion strains postpones the effect of azoles on cell growth. Alternatively, LAM deletions might alleviate the toxic effect of sterols as Lam proteins can transport toxic sterol biosynthesis intermediates into membrane compartments that are sensitive to these compounds. Our findings reveal novel biological roles of LAM genes in stress tolerance and suggest that mutations in these genes may confer upregulation of a mechanism that provides resistance to azole antifungals in pathogenic fungi.

Mitochondrial dynamics in yeast with repressed adenine nucleotide translocator AAC2.

The mitochondrial network structure dynamically adapts to cellular metabolic challenges. Mitochondrial depolarisation, particularly, induces fragmentation of the network. This fragmentation may be a result of either a direct regulation of the mitochondrial fusion machinery by transmembrane potential or an indirect effect of metabolic remodelling. Activities of ATP synthase and adenine nucleotide translocator (ANT) link the mitochondrial transmembrane potential with the cytosolic NTP/NDP ratio. Given that mitochondrial fusion requires cytosolic GTP, a decrease in the NTP/NDP ratio might also account for protonophore-induced mitochondrial fragmentation. For evaluating the contributions of direct and indirect mechanisms to mitochondrial remodelling, we assessed the morphology of the mitochondrial network in yeast cells with inhibited ANT. We showed that the repression of AAC2 (PET9), a major ANT gene in yeast, increases mitochondrial transmembrane potential. However, the mitochondrial network in this strain was fragmented. Meanwhile, AAC2 repression did not prevent mitochondrial fusion in zygotes; nor did it inhibit mitochondrial hyperfusion induced by Dnm1p inhibitor mdivi-1. These results suggest that the inhibition of ANT, rather than preventing mitochondrial fusion, facilitates mitochondrial fission. The protonophores were not able to induce additional mitochondrial fragmentation in an AAC2-repressed strain and in yeast cells with inhibited ATP synthase. Importantly, treatment with the ATP synthase inhibitor oligomycin A also induced mitochondrial fragmentation and hyperpolarization. Taken together, our data suggest that ATP/ADP translocation plays a crucial role in shaping of the mitochondrial network and exemplify that an increase in mitochondrial membrane potential does not necessarily oppose mitochondrial fragmentation.

Catalytically important flavin linked through a phosphoester bond in a eukaryotic fumarate reductase.

One of the three domains of kinetoplastid NADH:fumarate oxidoreductase (FRD) is homologous to bacterial flavin transferase that catalyzes transfer of FMN residue from FAD to threonine in flavoproteins. Leptomonas pyrrhocoris FRD produced in yeast cells, which lack flavin transferase gene in their proteome, reduces fumarate in the presence of NADH and contains an FMN residue covalently linked to a Ser9 residue. The conserved flavinylation motif of FRD, D3(g/s)x(s/t)(s/g)AS9, is similar to the Dxx(s/t)gAT motif recognized by flavin transferase in prokaryotic proteins. Ser9 replacement abolished the flavinylation and fumarate reductase activity of FRD. These findings suggest that the flavinylation is important for the activity of FRD and that this post-translational modification is carried out by the own flavin transferase domain.

Mitochondrial depolarization in yeast zygotes inhibits clonal expansion of selfish mtDNA.

Non-identical copies of mitochondrial DNA (mtDNA) compete with each other within a cell and the ultimate variant of mtDNA present depends on their relative replication rates. Using yeast Saccharomyces cerevisiae cells as a model, we studied the effects of mitochondrial inhibitors on the competition between wild-type mtDNA and mutant selfish mtDNA in heteroplasmic zygotes. We found that decreasing mitochondrial transmembrane potential by adding uncouplers or valinomycin changes the competition outcomes in favor of the wild-type mtDNA. This effect was significantly lower in cells with disrupted mitochondria fission or repression of the autophagy-related genes ATG8, ATG32 or ATG33, implying that heteroplasmic zygotes activate mitochondrial degradation in response to the depolarization. Moreover, the rate of mitochondrially targeted GFP turnover was higher in zygotes treated with uncoupler than in haploid cells or untreated zygotes. Finally, we showed that vacuoles of zygotes with uncoupler-activated autophagy contained DNA. Taken together, our data demonstrate that mitochondrial depolarization inhibits clonal expansion of selfish mtDNA and this effect depends on mitochondrial fission and autophagy. These observations suggest an activation of mitochondria quality control mechanisms in heteroplasmic yeast zygotes.

How do yeast sense mitochondrial dysfunction?

Apart from energy transformation, mitochondria play important signaling roles. In yeast, mitochondrial signaling relies on several molecular cascades. However, it is not clear how a cell detects a particular mitochondrial malfunction. The problem is that there are many possible manifestations of mitochondrial dysfunction. For example, exposure to the specific antibiotics can either decrease (inhibitors of respiratory chain) or increase (inhibitors of ATP-synthase) mitochondrial transmembrane potential. Moreover, even in the absence of the dysfunctions, a cell needs feedback from mitochondria to coordinate mitochondrial biogenesis and/or removal by mitophagy during the division cycle. To cope with the complexity, only a limited set of compounds is monitored by yeast cells to estimate mitochondrial functionality. The known examples of such compounds are ATP, reactive oxygen species, intermediates of amino acids synthesis, short peptides, Fe-S clusters and heme, and also the precursor proteins which fail to be imported by mitochondria. On one hand, the levels of these molecules depend not only on mitochondria. On the other hand, these substances are recognized by the cytosolic sensors which transmit the signals to the nucleus leading to general, as opposed to mitochondria-specific, transcriptional response. Therefore, we argue that both ways of mitochondria-to-nucleus communication in yeast are mostly (if not completely) unspecific, are mediated by the cytosolic signaling machinery and strongly depend on cellular metabolic state.

Dodecyltriphenylphosphonium inhibits multiple drug resistance in the yeast Saccharomyces cerevisiae.

Multiple drug resistance pumps are potential drug targets. Here we asked whether the lipophilic cation dodecyltriphenylphosphonium (C12TPP) can interfere with their functioning. First, we found that suppression of ABC transporter gene PDR5 increases the toxicity of C12TPP in yeast. Second, C12TPP appeared to prevent the efflux of rhodamine 6G - a fluorescent substrate of Pdr5p. Moreover, C12TPP increased the cytostatic effects of some other known Pdr5p substrates. The chemical nature of C12TPP suggests that after Pdr5p-driven extrusion the molecules return to the plasma membrane and then into the cytosol, thus effectively competing with other substrates of the pump.

Roles of mitochondrial dynamics under stressful and normal conditions in yeast cells.

Eukaryotic cells contain dynamic mitochondrial filaments: they fuse and divide. Here we summarize data on the protein machinery driving mitochondrial dynamics in yeast and also discuss the factors that affect the fusion-fission balance. Fission is a general stress response of cells, and in the case of yeast this response appears to be prosurvival. At the same time, even under normal conditions yeast mitochondria undergo continuous cycles of fusion and fission. This seems to be a futile cycle and also expensive from the energy point of view. Why does it exist? Benefits might be the same as in the case of sexual reproduction. Indeed, mixing and separating of mitochondrial content allows mitochondrial DNA to segregate and recombine randomly, leading to high variation in the numbers of mutations per individual mitochondrion. This opens a possibility for effective purifying selection-elimination of mitochondria highly contaminated by deleterious mutations. The beneficial action presumes a mechanism for removal of defective mitochondria. We argue that selective mitochondrial autophagy or asymmetrical distribution of mitochondria during cell division could be at the core of such mechanism.

Mitochondrially-encoded protein Var1 promotes loss of respiratory function in Saccharomyces cerevisiae under stressful conditions.

Stressed Saccharomyces cerevisiae cells easily lose respiratory function due to deletions in mitochondrial DNA, and this increases their general stress resistance. Is the loss active? We found that erythromycin (an inhibitor of mitochondrial translation) prevents the loss in control cells but not in the ones expressing mitochondrially-encoded protein Var1 in the nucleus. Var1 is a component of mitochondrial ribosomes; it is hydrophilic, positively charged, and prone to aggregation. Addition of DNase altered Var1 content in a preparation of mitochondrial nucleoids. Our data indicate that Var1 physically interacts with mitochondrial DNA and under stress negatively regulates its maintenance.

Mitochondrial signaling in Saccharomyces cerevisiae pseudohyphae formation induced by butanol.

Yeasts growing limited for nitrogen source or treated with fusel alcohols form elongated cells--pseudohyphae. Absence of mitochondrial DNA or anaerobic conditions inhibits this process, but the precise role of mitochondria is not clear. We found that a significant percentage of pseudohyphal cells contained mitochondria with different levels of membrane potential within one cell. An uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), but not the ATP-synthase inhibitor oligomycin D, prevented pseudohyphal growth. Interestingly, repression of the MIH1 gene encoding phosphatase activator of the G2/M transition partially restores the ability of yeast to form pseudohyphal cells in the presence of FCCP or in the absence of mitochondrial DNA. At the same time, retrograde signaling (the one triggered by dysfunctional mitochondria) appeared to be a positive regulator of butanol-induced pseudohyphae formation: the deletion of any of the retrograde signaling genes (RTG1, RTG2, or RTG3) partially suppressed pseudohyphal growth. Together, our data suggest that two subpopulations of mitochondria are required for filamentous growth: one with high and another with low transmembrane potential. These mitochondria-activated signaling pathways appear to converge at Mih1p level.

Inactivation of Pmc1 vacuolar Ca(2+) ATPase causes G(2) cell cycle delay in Hansenula polymorpha.

The vacuolar Ca(2+) ATPase Pmc1 is involved in maintenance of a low Ca(2+) concentration in cytosol in yeast cells. Here we observed that increase of Ca(2+) cytosolic concentration in yeast Hansenula polymorpha due to inactivation of Pmc1 resulted in sensitivity to sodium dodecyl sulfate (SDS). To elucidate the mechanisms of the observed effect, a screening for mutations suppressing SDS sensitivity of the H. polymorpha pmc1 mutant was performed. As a result, three genes were identified. Two of them, designated as their Saccharomyces cerevisiae orthologs CCH1 and HOG1 encoded the plasma membrane voltage-gated high-affinity calcium channel and the MAP kinase involved in osmoregulation, respectively. The third gene, designated as WEE1, coded for the ortholog of Wee1/Swe1 kinase involved in cell cycle regulation by inhibiting of the G(2)/M transition. Detailed analysis of this mutant demonstrated that suppression of pmc1 SDS sensitivity by the wee1 mutation depended on an accompanying chromosomal rearrangement, whereas inactivation of WEE1 in the absence of this rearrangement caused SDS sensitivity. Expression of a chimeric protein containing an N-terminal portion of Wee1 in the pmc1 mutant led to abnormal morphology characteristic of G(2) delay. Our data indicate that cytosolic Ca(2+) rise causes SDS sensitivity in H. polymorpha through the activation of the Wee1 kinase, which is mediated by the Hog1 kinase. Wee1 has a dual role in the manifestation of SDS sensitivity in the H. polymorpha pmc1 mutant. Mechanisms of influence of the obtained mutations on the G(2)/M transition are discussed.

Unexpected link between anaphase promoting complex and the toxicity of expanded polyglutamines expressed in yeast.

Protein aggregation is intimately linked to a number of neurodegenerative diseases. Expansion of the huntingtin polyglutamine-rich domain causes protein aggregation and neuronal degeneration. Recently we found that, similar to neurons, yeast expressing the expanded domain show markers of programmed cell death. Here we showed that deletion of yeast metacaspase gene YCA1 partly rescues the toxic effect of the domain overexpression. We also performed genetic screen for other genes deletions alleviating the toxic effect and found ASE1. Ase1 is a substrate of the Cdh1 form of anaphase promoting complex, APC/Cdh1. We tested Cdh1 overexpression and the deletion of CLB2 (mitotic cyclin, substrate of APC/Cdh1) and found that both mutations had a rescuing effect on the expanded polyglutamine toxicity. Our data suggest that the toxic effect of aggregated proteins is partly indirect. We speculate that cellular attempt to degrade the aggregates overloads the proteasome, and this leads to pathological accumulation of APC substrates.