Spontaneous Mutations in Saccharomyces cerevisiae mtDNA Increase Cell-to-Cell Variation in mtDNA Amount.

In a eukaryotic cell, the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) is usually maintained within a specific range. This suggests the presence of a negative feedback loop mechanism preventing extensive mtDNA replication and depletion. However, the experimental data on this hypothetical mechanism are limited. In this study, we suggested that deletions in mtDNA, known to increase mtDNA abundance, can disrupt this mechanism, and thus, increase cell-to-cell variance in the mtDNA copy numbers. To test this, we generated Saccharomyces cerevisiae rho- strains with large deletions in the mtDNA and rho0 strains depleted of mtDNA. Given that mtDNA contributes to the total DNA content of exponentially growing yeast cells, we showed that it can be quantified in individual cells by flow cytometry using the DNA-intercalating fluorescent dye SYTOX green. We found that the rho- mutations increased both the levels and cell-to-cell heterogeneity in the total DNA content of G1 and G2/M yeast cells, with no association with the cell size. Furthermore, the depletion of mtDNA in both the rho+ and rho- strains significantly decreased the SYTOX green signal variance. The high cell-to-cell heterogeneity of the mtDNA amount in the rho- strains suggests that mtDNA copy number regulation relies on full-length mtDNA, whereas the rho- mtDNAs partially escape this regulation.

ORFans in Mitochondrial Genomes of Marine Polychaete Polydora.

Most characterized metazoan mitochondrial genomes are compact and encode a small set of proteins that are essential for oxidative phosphorylation, as well as rRNA and tRNA for their expression. However, in rare cases, invertebrate taxa have additional open reading frames (ORFs) in their mtDNA sequences. Here, we sequenced and analyzed the mitochondrial genome of a polychaete worm, Polydora cf. ciliata, part of whose life cycle takes place in low-oxygen conditions. In the mitogenome, we found three "ORFan" regions (544, 1,060, and 427 bp) that have no resemblance to any standard metazoan mtDNA gene but lack stop codons in one of the reading frames. Similar regions are found in the mitochondrial genomes of three other Polydora species and Bocardiella hamata. All five species share the same gene order in their mitogenomes, which differ from that of other known Spionidae mitogenomes. By analyzing the ORFan sequences, we found that they are under purifying selection pressure and contain conservative regions. The codon adaptation indices (CAIs) of the ORFan genes were in the same range of values as the CAI of conventional protein-coding genes in corresponding mitochondrial genomes. The analysis of the P. cf. ciliata mitochondrial transcriptome showed that ORFan-544, ORFan-427, and a portion of the ORFan-1060 are transcribed. Together, this suggests that ORFan-544 and ORFan-427 encode functional proteins. It is likely that the ORFans originated when the Polydora/Bocardiella species complex separated from the rest of the Spionidae, and this event coincided with massive gene rearrangements in their mitochondrial genomes and tRNA-Met duplication.

Tyrosol induces multiple drug resistance in yeast Saccharomyces cerevisiae.

In yeast, multiple (pleiotropic) drug resistance (MDR) transporters efflux xenobiotics from the cytoplasm to the environment. Additionally, upon the accumulation of xenobiotics in the cells, MDR genes are induced. At the same time, fungal cells can produce secondary metabolites with physico-chemical properties similar to MDR transporter substrates. Nitrogen limitation in yeast Saccharomyces cerevisiae leads to the accumulation of phenylethanol, tryptophol, and tyrosol, which are products of aromatic amino acid catabolism. In this study, we investigated whether these compounds could induce or inhibit MDR in yeast. Double deletion of PDR1 and PDR3 genes, which are transcription factors that upregulate the expression of PDR genes, reduced yeast resistance to high concentrations of tyrosol (4-6 g/L) but not to the other two tested aromatic alcohols. PDR5 gene, but not other tested MDR transporter genes (SNQ2, YOR1, PDR10, PDR15) contributed to yeast resistance to tyrosol. Tyrosol inhibited the efflux of rhodamine 6G (R6G), a substrate for MDR transporters. However, preincubating yeast cells with tyrosol induced MDR, as evidenced by increased Pdr5-GFP levels and reduced yeast ability to accumulate Nile red, another fluorescent MDR-transporter substrate. Moreover, tyrosol inhibited the cytostatic effect of clotrimazole, the azole antifungal. Our results demonstrate that a natural secondary metabolite can modulate yeast MDR. We speculate that intermediates of aromatic amino acid metabolites coordinate cell metabolism and defense mechanisms against xenobiotics.

Secondary structure of the human mitochondrial genome affects formation of deletions.

BACKGROUND: Aging in postmitotic tissues is associated with clonal expansion of somatic mitochondrial deletions, the origin of which is not well understood. Such deletions are often flanked by direct nucleotide repeats, but this alone does not fully explain their distribution. Here, we hypothesized that the close proximity of direct repeats on single-stranded mitochondrial DNA (mtDNA) might play a role in the formation of deletions. RESULTS: By analyzing human mtDNA deletions in the major arc of mtDNA, which is single-stranded during replication and is characterized by a high number of deletions, we found a non-uniform distribution with a "hot spot" where one deletion breakpoint occurred within the region of 6-9 kb and another within 13-16 kb of the mtDNA. This distribution was not explained by the presence of direct repeats, suggesting that other factors, such as the spatial proximity of these two regions, can be the cause. In silico analyses revealed that the single-stranded major arc may be organized as a large-scale hairpin-like loop with a center close to 11 kb and contacting regions between 6-9 kb and 13-16 kb, which would explain the high deletion activity in this contact zone. The direct repeats located within the contact zone, such as the well-known common repeat with a first arm at 8470-8482 bp (base pair) and a second arm at 13,447-13,459 bp, are three times more likely to cause deletions compared to direct repeats located outside of the contact zone. A comparison of age- and disease-associated deletions demonstrated that the contact zone plays a crucial role in explaining the age-associated deletions, emphasizing its importance in the rate of healthy aging. CONCLUSIONS: Overall, we provide topological insights into the mechanism of age-associated deletion formation in human mtDNA, which could be used to predict somatic deletion burden and maximum lifespan in different human haplogroups and mammalian species.

Role of Mitochondrial DNA in Yeast Replicative Aging.

Despite the diverse manifestations of aging across different species, some common aging features and underlying mechanisms are shared. In particular, mitochondria appear to be among the most vulnerable systems in both metazoa and fungi. In this review, we discuss how mitochondrial dysfunction is related to replicative aging in the simplest eukaryotic model, the baker's yeast Saccharomyces cerevisiae. We discuss a chain of events that starts from asymmetric distribution of mitochondria between mother and daughter cells. With age, yeast mother cells start to experience a decrease in mitochondrial transmembrane potential and, consequently, a decrease in mitochondrial protein import efficiency. This induces mitochondrial protein precursors in the cytoplasm, the loss of mitochondrial DNA (mtDNA), and at the later stages - cell death. Interestingly, yeast strains without mtDNA can have either increased or decreased lifespan compared to the parental strains with mtDNA. The direction of the effect depends on their ability to activate compensatory mechanisms preventing or mitigating negative consequences of mitochondrial dysfunction. The central role of mitochondria in yeast aging and death indicates that it is one of the most complex and, therefore, deregulation-prone systems in eukaryotic cells.

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.

Heterogeneity of Starved Yeast Cells in IF1 Levels Suggests the Role of This Protein in vivo.

In mitochondria, a small protein IF1 suppresses the hydrolytic activity of ATP synthase and presumably prevents excessive ATP hydrolysis under conditions of energy deprivation. In yeast Saccharomyces cerevisiae, IF1 homologs are encoded by two paralogous genes: INH1 and STF1. INH1 expression is known to aggravate the deleterious effects of mitochondrial DNA (mtDNA) depletion. Surprisingly, no beneficial effects of INH1 and STF1 were documented for yeast so far, and the functions of INH1 and STF1 in wild type cells are unclear. Here, we put forward a hypothesis that INH1 and STF1 bring advantage during the fast start of proliferation after reentry into exponential growth from post-diauxic or stationary phases. We found that yeast cells increase the concentration of both proteins in the post-diauxic phase. Post-diauxic phase yeast cells formed two subpopulations distinct in Inh1p and Stf1p concentrations. Upon exit from the post-diauxic phase cells with high level of Inh1-GFP started growing earlier than cells devoid of Inh1-GFP. However, double deletion of INH1 and STF1 did not increase the lag period necessary for stationary phase yeast cells to start growing after reinoculation into the fresh medium. These results point to a redundancy of the mechanisms preventing uncontrolled ATP hydrolysis during energy deprivation.

Attenuated ADP-inhibition of FOF1 ATPase mitigates manifestations of mitochondrial dysfunction in yeast.

Proton-translocating FOF1 ATP synthase (F-ATPase) couples ATP synthesis or hydrolysis to transmembrane proton transport in bacteria, chloroplasts, and mitochondria. The primary function of the mitochondrial FOF1 is ATP synthesis driven by protonmotive force (pmf) generated by the respiratory chain. However, when pmf is low or absent (e.g. during anoxia), FOF1 consumes ATP and functions as a proton-pumping ATPase. Several regulatory mechanisms suppress the ATPase activity of FOF1 at low pmf. In yeast mitochondria they include special inhibitory proteins Inh1p and Stf1p, and non-competitive inhibition of ATP hydrolysis by MgADP (ADP-inhibition). Presumably, these mechanisms help the cell to preserve the ATP pool upon membrane de-energization. However, no direct evidence was presented to support this hypothesis so far. Here we report that a point mutation Q263L in subunit beta of Saccharomyces cerevisiae ATP synthase significantly attenuated ADP-inhibition of the enzyme without major effect on the rate of ATP production by mitochondria. The mutation also decreased the sensitivity of the enzyme ATPase activity to azide. Similar effects of the corresponding mutations were observed in earlier studies in bacterial enzymes. This observation indicates that the molecular mechanism of ADP-inhibition is probably the same in mitochondrial and in bacterial FOF1. The mutant yeast strain had lower growth rate and had a longer lag period preceding exponential growth phase when starved cells were transferred to fresh growth medium. However, upon the loss of mitochondrial DNA (ρ0) the ßQ263L mutation effect was reversed: the ßQ263L ρ0 mutant grew faster than the wild-type ρ0 yeast. The results suggest that ADP-inhibition might play a role in prevention of wasteful ATP hydrolysis in the mitochondrial matrix.

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.

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.

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.

Deletion of CDR1 reveals redox regulation of pleiotropic drug resistance in Candida glabrata.

Microbial cells sense the presence of xenobiotics and, in response, upregulate genes involved in pleiotropic drug resistance (PDR). In yeast, PDR activation to a major extent relies on the transcription factor Pdr1. In addition, many xenobiotics induce oxidative stress, which may upregulate PDR independently of Pdr1 activity. Mitochondria are important sources of reactive oxygen species under stressful conditions. To evaluate the relevance of this redox pathway, we studied the activation of PDR in the yeast Candida glabrata, which we treated with a mitochondrially targeted antioxidant plastoquinonyl-decyl-triphenylphosphonium and dodecyltriphenylphosphonium (C12TPP) as a control. We found that both compounds induced activation of PDR genes and decreased the intracellular concentration of the PDR transporter substrate Nile red. Interestingly, the deletion of PDR transporter gene CDR1 inhibited the decrease in Nile red accumulation induced by antioxidant plastoquinonyl-decyl-triphenylphosphonium but not that by C12TPP. Moreover, antioxidant alpha-tocopherol inhibited C12TPP-mediated activation of PDR in Δcdr1 but not in the wild-type strain. Furthermore, pre-incubation of yeast cells with low concentrations of hydrogen peroxide induced a decrease in the intracellular concentration of Nile red in Δcdr1 and Δpdr1 as well as in control cells. Deletion of PDR1 inhibited the C12TPP-induced activation of CDR1 but not that of FLR1, which is a redox-regulated PDR transporter gene. It appears that disruption of the PDR1/CDR1 regulatory circuit makes auxiliary PDR regulation mechanisms crucial. Our data suggest that redox regulation of PDR is dispensable in wild-type cells because of redundancy in the activation pathways, but is manifested upon deletion of CDR1.

Intracellular quality control of mitochondrial DNA: evidence and limitations.

Eukaryotic cells can harbour mitochondria with markedly different transmembrane potentials. Intracellular mitochondrial quality-control mechanisms (e.g. mitophagy) rely on this intracellular variation to distinguish functional and damaged (depolarized) mitochondria. Given that intracellular mitochondrial DNA (mtDNA) genetic variation can induce mitochondrial heterogeneity, mitophagy could remove deleterious mtDNA variants in cells. However, the reliance of mitophagy on the mitochondrial transmembrane potential suggests that mtDNAs with deleterious mutations in ATP synthase can evade the control. This evasion is possible because inhibition of ATP synthase can increase the mitochondrial transmembrane potential. Moreover, the linkage of the mtDNA genotype to individual mitochondrial performance is expected to be weak owing to intracellular mitochondrial intercomplementation. Nonetheless, I reason that intracellular mtDNA quality control is possible and crucial at the zygote stage of the life cycle. Indeed, species with biparental mtDNA inheritance or frequent 'leakage' of paternal mtDNA can be vulnerable to invasion of selfish mtDNAs at the stage of gamete fusion. Here, I critically review recent findings on intracellular mtDNA quality control by mitophagy and discuss other mechanisms by which the nuclear genome can affect the competition of mtDNA variants in the cell. This article is part of the theme issue 'Linking the mitochondrial genotype to phenotype: a complex endeavour'.

Effects of Sterols on the Interaction of SDS, Benzalkonium Chloride, and A Novel Compound, Kor105, with Membranes.

Sterols change the biophysical properties of lipid membranes. Here, we analyzed how sterols affect the activity of widely used antimicrobial membrane-active compounds, sodium dodecyl sulfate (SDS) and benzalkonium chloride (BAC). We also tested a novel benzalkonium-like substance, Kor105. Our data suggest that benzalkonium and Kor105 disturb the ordering of the membrane lipid packaging, and this disturbance is dampened by cholesterol. The disturbance induced by Kor105 is stronger than that induced by BAC because of the higher rigidity of the Kor105 molecule due to a shorter linker between the phenyl group and quaternary nitrogen. On the contrary, individual SDS molecules do not cause the disturbance. Thus, in the tested range of concentrations, SDS-membrane interaction is not influenced by cholesterol. To study how sterols influence the biological effects of these chemicals, we used yeast strains lacking Lam1-4 proteins. These proteins transport sterols from the plasma membrane into the endoplasmic reticulum. We found that the mutants are resistant to BAC and Kor105 but hypersensitive to SDS. Together, our findings show that sterols influence the interaction of SDS versus benzalkonium chloride and Kor105 with the membranes in a completely different manner.

Coding palindromes in mitochondrial genes of Nematomorpha.

Inverted repeats are common DNA elements, but they rarely overlap with protein-coding sequences due to the ensuing conflict with the structure and function of the encoded protein. We discovered numerous perfect inverted repeats of considerable length (up to 284 bp) embedded within the protein-coding genes in mitochondrial genomes of four Nematomorpha species. Strikingly, both arms of the inverted repeats encode conserved regions of the amino acid sequence. We confirmed enzymatic activity of the respiratory complex I encoded by inverted repeat-containing genes. The nucleotide composition of inverted repeats suggests strong selection at the amino acid level in these regions. We conclude that the inverted repeat-containing genes are transcribed and translated into functional proteins. The survey of available mitochondrial genomes reveals that several other organisms possess similar albeit shorter embedded repeats. Mitochondrial genomes of Nematomorpha demonstrate an extraordinary evolutionary compromise where protein function and stringent secondary structure elements within the coding regions are preserved simultaneously.

Replicative aging as a source of cell heterogeneity in budding yeast.

While deviations from the optimal phenotype are deleterious, increased variation can prevent population extinction under severe stresses. Cell division asymmetry is an important source of microbial phenotypic heterogeneity. A consecutive set of asymmetric divisions can cause the gradual accumulation of deleterious factors and, at late stages, the death of old pole (mother) cells. This phenomenon is known as replicative aging. As the old cells are constantly being diluted by the progeny, the majority of a microbial population is represented by replicatively young cells. Therefore, early-age changes in yeast mother cells have a much greater impact on the integral performance of the microbial population than does functional deterioration at later ages. Here, we review the early manifestations of replicative aging in Saccharomyces cerevisiae mother cells that occur during the first ten cell cycles. Early age-dependent changes occur in stress resistance, genomic instability, protein aggregate levels, redox balance and metabolism. We speculate that some of these manifestations can be beneficial during stress exposure; therefore, early aging may be a bet-hedging mechanism. Together, the data suggest that the age component of variation in populations of asymmetrically dividing microorganisms is substantial and may play an important role in adaptations to changing environments.

Penetrating cations induce pleiotropic drug resistance in yeast.

Substrates of pleiotropic drug resistance (PDR) transporters can induce the expression of corresponding transporter genes by binding to their transcription factors. Penetrating cations are substrates of PDR transporters and theoretically may also activate the expression of transporter genes. However, the accumulation of penetrating cations inside mitochondria may prevent the sensing of these molecules. Thus, whether penetrating cations induce PDR is unclear. Using Saccharomyces cerevisiae as a model, we studied the effects of penetrating cations on the activation of PDR. We found that the lipophilic cation dodecyltriphenylphosphonium (C12TPP) induced the expression of the plasma membrane PDR transporter genes PDR5, SNQ2 and YOR1. Moreover, a 1-hour incubation with C12TPP increased the concentration of Pdr5p and Snq2p and prevented the accumulation of the PDR transporter substrate Nile red. The transcription factor PDR1 was required to mediate these effects, while PDR3 was dispensable. The deletion of the YAP1 or RTG2 genes encoding components of the mitochondria-to-nucleus signalling pathway did not prevent the C12TPP-induced increase in Pdr5-GFP. Taken together, our data suggest (i) that the sequestration of lipophilic cations inside mitochondria does not significantly inhibit sensing by PDR activators and (ii) that the activation mechanisms do not require mitochondria as a signalling module.