Squalene lipotoxicity in a lipid droplet-less yeast mutant is linked to plasma membrane dysfunction.

Squalene is a naturally occurring triterpene with wide industrial applications. Due to limited natural resources, production of this valuable lipid in yeast is of high commercial relevance. Typically low levels of squalene in yeast can be significantly increased by specific cultivation conditions or genetic modifications. Under normal conditions, excess squalene is stored in lipid droplets (LD), while in a Saccharomyces cerevisiae mutant unable to form LD it is distributed to cellular membranes. We present here the evidence that squalene accumulation in this LD-less mutant treated with squalene monooxygenase inhibitor terbinafine induces growth defects and loss of viability. We show that plasma membrane malfunction is involved in squalene toxicity. We have found that subinhibitory concentrations of terbinafine increased the sensitivity of LD-less mutant to several membrane-active substances. Furthermore, squalene accumulation in terbinafine-treated LD-less cells disturbed the maintenance of membrane potential and increased plasma membrane permeability to rhodamine 6G. LD-less cells treated with terbinafine showed also high sensitivity to osmotic stress. To confirm the causal relationship between squalene accumulation, loss of viability and impaired plasma membrane functions we treated LD-less cells simultaneously with terbinafine and squalene synthase inhibitor zaragozic acid. Reduction of squalene levels by zaragozic acid improved cell growth and viability and decreased plasma membrane permeability to rhodamine 6G in terbinafine-treated LD-less cells. Our results support the hypothesis that plasma membrane malfunction is involved in the mechanisms of squalene lipotoxicity in yeast cells with defective lipid storage.

Mutations in the nucleotide-binding domain of putative sterol importers Aus1 and Pdr11 selectively affect utilization of exogenous sterol species in yeast.

Sterol uptake in the yeast Saccharomyces cerevisiae is mediated by two plasma membrane ATP-binding cassette transporters, Aus1 and Pdr11. Their expression is regulated by oxygen and is triggered by anaerobic growth conditions. Under these conditions, internal ergosterol synthesis is arrested and utilization of exogenous sterol is vital for yeast cells. Here, we demonstrate that Aus1 is the major importer of non-yeast sterols, mammalian cholesterol, and plant sterols under anaerobic conditions. In contrast, uptake of yeast native sterol, ergosterol, is relatively low. This uptake could not be enhanced by overexpression of either of the transporters. Interestingly, overexpression of the minor importer Pdr11 resulted in a substantial import of non-yeast sterols. We show that mutation of the conserved residue in one of the ABC characteristic motifs-the H-loop in Aus1 and Pdr11-lowered their ATPase activity. The residual activity was sufficient to import exogenous sterols and to preserve cell viability. Importantly, the reduction of sterol import was dramatic for mammalian cholesterol and plant sterols, whereas import of yeast ergosterol was decreased only slightly indicating substrate selectivity of the sterol utilization process.

The complete mitochondrial DNA sequence from Kazachstania sinensis reveals a general +1C frameshift mechanism in CTGY codons.

The complete mitochondrial DNA (mtDNA) sequence from Kazachstania sinensis was analysed and compared to mtDNA from related yeasts. It contained the same set of genes; however, it only contained 23 tRNAs, as the trnR2 gene was absent. Most of the 12 introns within cox1, cob and rnl genes were inserted in the same sites as in other yeasts; however, two introns in rnl were in unusual positions. Traits such as gene order and GC cluster number were more related to Saccharomyces than to the other Kazachstania or linked clades. The most exceptional feature was the +1 frameshift in cox3, atp6 and cob open reading frames that was also found in other Kazachstania, Nakaseomyces delphensis and Candida glabrata. Comparison of DNA and protein sequences revealed the universal sites of +1C frameshifts were either CTGT or CTGC sequences. Moreover, an A→G substitution was found at position 37 in the anticodon stem loop tRNA gene for cysteine in all species with frameshifts but not in other sibling yeasts. This substitution allowed strong Watson-Crick base-pairing between an unmodified G (ACG) and the skipped C in the CTGY, leading to this quadruplet being read as cysteine.

Squalene is lipotoxic to yeast cells defective in lipid droplet biogenesis.

The toxic effect of overloaded lipids on cell physiology and viability was described in various organisms. In this study we focused on the potential lipotoxicity of squalene, a linear triterpene synthesized in eukaryotic cells as an intermediate in sterol biosynthesis. Squalene toxicity was studied in the yeast Saccharomyces cerevisiae, a model unicellular eukaryote established in lipotoxicity studies. Squalene levels in yeast are typically low but its accumulation can be induced under specific conditions, e.g. by inhibition of squalene monooxygenase with the antimycotic terbinafine. At higher levels squalene is stored in lipid droplets. We demonstrated that low doses of terbinafine caused severe impairment of growth and loss of viability of the yeast mutant dga1Δ lro1Δ are1Δ are2Δ unable to form lipid droplets and that these defects were linked to squalene accumulation. The hypersensitivity of the lipid droplet-less mutant to terbinafine was alleviated by decreasing squalene accumulation with low doses of squalene synthase inhibitor zaragozic acid. Our results proved that accumulated squalene is lipotoxic to yeast cells if it cannot be efficiently sequestered in lipid droplets. This supports the hypothesis about the role of squalene in the fungicidal activity of terbinafine. Squalene toxicity may represent also a limiting factor for production of this high-value lipid in yeast.

Squalene epoxidase as a target for manipulation of squalene levels in the yeast Saccharomyces cerevisiae.

Squalene is a valuable natural substance with several biotechnological applications. In the yeast Saccharomyces cerevisiae, it is produced in the isoprenoid pathway as the first precursor dedicated to ergosterol biosynthesis. The aim of this study was to explore the potential of squalene epoxidase encoded by the ERG1 gene as the target for manipulating squalene levels in yeast. Highest squalene levels (over 1000 µg squalene per 10(9)  cells) were induced by specific point mutations in ERG1 gene that reduced activity of squalene epoxidase and caused hypersensitivity to terbinafine. This accumulation of squalene in erg1 mutants did not significantly disturb their growth. Treatment with squalene epoxidase inhibitor terbinafine revealed a limit in squalene accumulation at 700 µg squalene per 10(9)  cells which was associated with pronounced growth defects. Inhibition of squalene epoxidase activity by anaerobiosis or heme deficiency resulted in relatively low squalene levels. These levels were significantly increased by ergosterol depletion in anaerobic cells which indicated feedback inhibition of squalene production by ergosterol. Accumulation of squalene in erg1 mutants and terbinafine-treated cells were associated with increased cellular content and aggregation of lipid droplets. Our results prove that targeted genetic manipulation of the ERG1 gene is a promising tool for increasing squalene production in yeast.

Yeast perilipin Pet10p/Pln1p interacts with Erg6p in ergosterol metabolism.

Lipid droplets (LD) are highly dynamic organelles specialized for the regulation of energy storage and cellular homeostasis. LD consist of a neutral lipid core surrounded by a phospholipid monolayer membrane with embedded proteins, most of which are involved in lipid homeostasis. In this study, we focused on one of the major LD proteins, sterol C24-methyltransferase, encoded by ERG6. We found that the absence of Erg6p resulted in an increased accumulation of yeast perilipin Pet10p in LD, while the disruption of PET10 was accompanied by Erg6p LD over-accumulation. An observed reciprocal enrichment of Erg6p and Pet10p in pet10Δ and erg6Δ mutants in LD, respectively, was related to specific functional changes in the LD and was not due to regulation on the expression level. The involvement of Pet10p in neutral lipid homeostasis was observed in experiments that focused on the dynamics of neutral lipid mobilization as time-dependent changes in the triacylglycerols (TAG) and steryl esters (SE) content. We found that the kinetics of SE hydrolysis was reduced in erg6Δ cells and the mobilization of SE was completely lost in mutants that lacked both Erg6p and Pet10p. In addition, we observed that decreased levels of SE in erg6Δpet10Δ was linked to an overexpression of steryl ester hydrolase Yeh1p. Lipid analysis of erg6Δpet10Δ showed that PET10 deletion altered the composition of ergosterol intermediates which had accumulated in erg6Δ. In conclusion, yeast perilipin Pet10p functionally interacts with Erg6p during the metabolism of ergosterol.

The yeast Saccharomyces cerevisiae Pdr16p restricts changes in ergosterol biosynthesis caused by the presence of azole antifungals.

Pdr16p belongs to the family of phosphatidylinositol transfer proteins in yeast. The absence of Pdr16p results in enhanced susceptibility to azole antifungals in Saccharomyces cerevisiae. In the major fungal human pathogen Candida albicans, CaPDR16 is a contributing factor to clinical azole resistance. The current study was aimed at better understanding the function of Pdr16p, especially in relation to azole resistance in S. cerevisiae. We show that deletion of the PDR16 gene increased susceptibility of S. cerevisiae to azole antifungals that are used in clinical medicine and agriculture. Significant differences in the inhibition of the sterol biosynthetic pathway were observed between the pdr16Δ strain and its corresponding wild-type (wt) strain when yeast cells were challenged by sub-inhibitory concentrations of the azoles miconazole or fluconazole. The increased susceptibility to azoles, and enhanced changes in sterol biosynthesis upon exposure to azoles of the pdr16Δ strain compared to wt strain, are not the results of increased intracellular concentration of azoles in the pdr16Δ cells. We also show that overexpression of PDR17 complemented the azole susceptible phenotype of the pdr16Δ strain and corrected the enhanced sterol alterations in pdr16Δ cells in the presence of azoles. Pdr17p was found previously to be an essential part of a complex required for intermembrane transport of phosphatidylserine at regions of membrane apposition. Based on these observations, we propose a hypothesis that Pdr16p assists in shuttling sterols or their intermediates between membranes or, alternatively, between sterol biosynthetic enzymes or complexes.

Is fat so bad? Modulation of endoplasmic reticulum stress by lipid droplet formation.

LDs (lipid droplets) have long been considered as inert particles used by the cells to store fatty acids and sterols as esterified non-toxic lipid species (i.e. triacylglycerols and steryl esters). However, accumulating evidence suggests that LDs behave as a dynamic compartment, which is involved in the regulation of several aspects of the homoeostasis of their originating organelle, namely the ER (endoplasmic reticulum). The ER is particularly sensitive to physiological/pathological stimuli, which can ultimately induce ER stress. In the present review, after considering the basic mechanisms of LD formation and the signal cascades leading to ER stress, we focus on the connections between these two pathways. Taking into consideration recent data from the literature, we will try to draw possible mechanisms for the role of LDs in the regulation of ER homoeostasis and in ER-stress-related diseases.

Effect of lipid particle biogenesis on the subcellular distribution of squalene in the yeast Saccharomyces cerevisiae.

Squalene belongs to the group of isoprenoids and is a precursor for the synthesis of sterols, steroids, and ubiquinones. In the yeast Saccharomyces cerevisiae, the amount of squalene can be increased by variation of growth conditions or by genetic manipulation. In this report, we show that a hem1Delta mutant accumulated a large amount of squalene, which was stored almost exclusively in cytoplasmic lipid particles/droplets. Interestingly, a strain bearing a hem1Delta deletion in a dga1Delta lro1Delta are1Delta are2Delta quadruple mutant background (QMhem1Delta), which is devoid of the classical storage lipids, triacylglycerols and steryl esters, and lacks lipid particles, accumulated squalene at similar amounts as the hem1Delta mutant in a wild type background. In QMhem1Delta, however, increased amounts of squalene were found in cellular membranes, especially in microsomes. The fact that QMhem1Delta did not form lipid particles indicated that accumulation of squalene solely was not sufficient to initiate proliferation of lipid particles. Most importantly, these results also demonstrated that (i) squalene was not lipotoxic under the conditions tested, and (ii) organelle membranes in yeast can accommodate relatively large quantities of this non-polar lipid without compromising cellular functions. In summary, localization of squalene as described here can be regarded as an unconventional example of non-polar lipid storage in cellular membranes.

The role of ABC proteins Aus1p and Pdr11p in the uptake of external sterols in yeast: dehydroergosterol fluorescence study.

Uptake of external sterols in the yeast Saccharomyces cerevisiae is a multistep process limited to anaerobiosis or heme deficiency. It includes crossing the cell wall, insertion of sterol molecules into plasma membrane and their internalization and integration into intracellular membranes. We applied the fluorescent ergosterol analog dehydroergosterol (DHE) to monitor the initial steps of sterol uptake by three independent approaches: fluorescence spectroscopy, fluorescence microscopy and sterol quantification by HPLC. Using specific fluorescence characteristics of DHE we showed that the entry of sterol molecules into plasma membrane is not spontaneous but requires assistance of two ABC (ATP-binding cassette) pumps--Aus1p or Pdr11p. DHE taken up by uptake-competent hem1ΔAUS1PDR11 cells could be directly visualized by UV-sensitive wide field fluorescence microscopy. HPLC analysis of sterols revealed significant amounts of exogenous ergosterol and DHE (but not cholesterol) associated with uptake-deficient hem1Δaus1Δpdr11Δ cells. Fluorescent sterol associated with these cells did not show the characteristic emission spectrum of membrane-integrated DHE. The amount of cell-associated DHE was significantly reduced after enzymatic removal of the cell wall. Our results demonstrate that the yeast cell wall is actively involved in binding and uptake of ergosterol-like sterols.

Chemogenomic and transcriptome analysis identifies mode of action of the chemosensitizing agent CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine).

BACKGROUND: CTBT (7-chlorotetrazolo [5,1-c]benzo[1,2,4]triazine) increases efficacy of commonly used antifungal agents by an unknown mechanism. It increases the susceptibility of Saccharomyces cerevisiae, Candida albicans and Candida glabrata cells to cycloheximide, 5-fluorocytosine and azole antimycotic drugs. Here we elucidate CTBT mode of action with a combination of systematic genetic and transcriptome analysis. RESULTS: To identify the cellular processes affected by CTBT, we screened the systematic haploid deletion mutant collection for CTBT sensitive mutants. We identified 169 hypersensitive deletion mutants. The deleted genes encode proteins mainly involved in mitochondrial functions, DNA repair, transcription and chromatin remodeling, and oxidative stress response. We found that the susceptibility of yeast cells to CTBT depends on molecular oxygen. Transcriptome analysis of the immediate early response to CTBT revealed rapid induction of oxidant and stress response defense genes. Many of these genes depend on the transcription factors Yap1 and Cin5. Yap1 accumulates rapidly in the nucleus in CTBT treated cells suggesting acute oxidative stress. Moreover, molecular calculations supported a superoxide generating activity of CTBT. Superoxide production in vivo by CTBT was found associated to mitochondria as indicated by oxidation of MitoSOX Red. CONCLUSION: We conclude that CTBT causes intracellular superoxide production and oxidative stress in fungal cells and is thus enhancing antimycotic drug effects by a secondary stress.

Metabolism of Storage Lipids and the Role of Lipid Droplets in the Yeast Schizosaccharomyces pombe.

Storage lipids, triacylglycerols (TAG), and steryl esters (SE), are predominant constituents of lipid droplets (LD) in fungi. In several yeast species, metabolism of TAG and SE is linked to various cellular processes, including cell division, sporulation, apoptosis, response to stress, and lipotoxicity. In addition, TAG are an important source for the generation of value-added lipids for industrial and biomedical applications. The fission yeast Schizosaccharomyces pombe is a widely used unicellular eukaryotic model organism. It is a powerful tractable system used to study various aspects of eukaryotic cellular and molecular biology. However, the knowledge of S. pombe neutral lipids metabolism is quite limited. In this review, we summarize and discuss the current knowledge of the homeostasis of storage lipids and of the role of LD in the fission yeast S. pombe with the aim to stimulate research of lipid metabolism and its connection with other essential cellular processes. We also discuss the advantages and disadvantages of fission yeast in lipid biotechnology and recent achievements in the use of S. pombe in the biotechnological production of valuable lipid compounds.

Control of lipid accumulation in the yeast Yarrowia lipolytica.

A genomic comparison of Yarrowia lipolytica and Saccharomyces cerevisiae indicates that the metabolism of Y. lipolytica is oriented toward the glycerol pathway. To redirect carbon flux toward lipid synthesis, the GUT2 gene, which codes for the glycerol-3-phosphate dehydrogenase isomer, was deleted in Y. lipolytica in this study. This Delta gut2 mutant strain demonstrated a threefold increase in lipid accumulation compared to the wild-type strain. However, mobilization of lipid reserves occurred after the exit from the exponential phase due to beta-oxidation. Y. lipolytica contains six acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX6 genes, that catalyze the limiting step of peroxisomal beta-oxidation. Additional deletion of the POX1 to POX6 genes in the Delta gut2 strain led to a fourfold increase in lipid content. The lipid composition of all of the strains tested demonstrated high proportions of FFA. The size and number of the lipid bodies in these strains were shown to be dependent on the lipid composition and accumulation ratio.

Chemosensitisation of drug-resistant and drug-sensitive yeast cells to antifungals.

Multidrug resistance in yeast results from overexpression of genes encoding drug efflux transporters owing to gain-of-function mutations in transcription factors regulating their expression. We have screened a library of synthetic compounds for modulators of drug resistance using the multidrug-resistant Saccharomyces cerevisiae pdr3-9 mutant strain. One of the compounds, 7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine (CTBT), displayed weak antifungal activity and strongly inhibited the growth of yeast cells in combination with subinhibitory concentrations of other antifungals with a different mode of action. Biological activity of CTBT was demonstrated in Saccharomyces, Kluyveromyces and Candida yeast species grown on solid and in liquid media. The chemosensitising effect of CTBT, manifested as increased antifungal activity of fluconazole, was demonstrated in yeast mutant strains with deleted genes encoding the major multidrug resistance transcription factors Yap1p, Pdr1p and Pdr3p as well as the drug efflux pumps Pdr5p and Snq2p in S. cerevisiae or their counterparts in Candida albicans and Candida glabrata, named Cdr1p and Mdr1p, respectively. Importantly, CTBT also increased the sensitivity to fluconazole in multidrug-resistant cells overexpressing the efflux pumps. Yeast cells grown in the presence of subinhibitory concentrations of CTBT exhibited an altered sterol composition and a slightly enhanced accumulation of Rhodamine 6G, which suggests that the plasma membrane plays a role in sensitisation. This novel chemosensitisation by CTBT that can overcome multidrug resistance in yeast may prove useful in combined treatment of infections caused by drug-resistant fungal pathogens.

Biosynthetic Approaches to Squalene Production: The Case of Yeast.

Squalene is a precursor in the eukaryotic sterol biosynthesis. It is a valuable compound with several human health-related applications. Since the traditional natural resources of squalene are limited, alternatives for the production of squalene on industrial scale have been intensively explored during past years. The yeast Saccharomyces cerevisiae represents an attractive option due to elaborated techniques of genetic and metabolic engineering that can be applied to improve squalene yields. We discuss in this chapter some theoretical aspects of genetic manipulations of the ergosterol biosynthesis pathway aimed at increased squalene production and describe analytical methods for squalene purification and determination of its content in yeast cells.

Baker's Yeast Deficient in Storage Lipid Synthesis Uses cis-Vaccenic Acid to Reduce Unsaturated Fatty Acid Toxicity.

The role of cis-vaccenic acid (18:1n-7) in the reduction of unsaturated fatty acids toxicity was investigated in baker's yeast Saccharomyces cerevisiae. The quadruple mutant (QM, dga1Δ lro1Δ are1Δ are2Δ) deficient in enzymes responsible for triacylglycerol and steryl ester synthesis has been previously shown to be highly sensitive to exogenous unsaturated fatty acids. We have found that cis-vaccenic acid accumulated during cultivation in the QM cells but not in the corresponding wild type strain. This accumulation was accompanied by a reduction in palmitoleic acid (16:1n-7) content in the QM cells that is consistent with the proposed formation of cis-vaccenic acid by elongation of palmitoleic acid. Fatty acid analysis of individual lipid classes from the QM strain revealed that cis-vaccenic acid was highly enriched in the free fatty acid pool. Furthermore, production of cis-vaccenic acid was arrested if the mechanism of fatty acids release to the medium was activated. We also showed that exogenous cis-vaccenic acid did not affect viability of the QM strain at concentrations toxic for palmitoleic or oleic acids. Moreover, addition of cis-vaccenic acid to the growth medium provided partial protection against the lipotoxic effects of exogenous oleic acid. Transformation of palmitoleic acid to cis-vaccenic acid is thus a rescue mechanism enabling S. cerevisiae cells to survive in the absence of triacylglycerol synthesis as the major mechanism for unsaturated fatty acid detoxification.

Heme-regulated expression of two yeast acyl-CoA:sterol acyltransferases is involved in the specific response of sterol esterification to anaerobiosis.

Sterol esterification in Saccharomyces cerevisiae is catalyzed by two acyl-CoA:sterol acyltransferases encoded by the genes ARE1 and ARE2. Using double mutants in the HEM1 gene and individual ARE genes we demonstrated that the relative contribution of these two enzymes to sterol esterification was dependent on cellular heme status. Observed changes in sterol esterification could be explained by a different effect of heme on the transcription of both genes: while the ARE1 transcript level was elevated in heme-deficient and anaerobic cells, the ARE2 gene transcript was more abundant in aerobic cells competent for heme synthesis. Our results indicate that transcriptional regulation of ARE genes by heme and specific substrate preferences of Are1p and Are2p may be involved in the adaptation of yeast sterol metabolism to hypoxia.

Low concentrations of the non-ionic detergent Nonidet P-40 interfere with sterol biogenesis and viability of the yeast Saccharomyces cerevisiae.

Mild non-ionic detergents are used for solubilization of hydrophobic substrates in yeast growth media at concentrations 0.1-1%. Our data show that low concentrations of Nonidet P-40 may significantly affect lipid biogenesis in the yeast Saccharomyces cerevisiae. The uptake and esterification of external [4-14C]-cholesterol is strongly reduced in hem1 mutants treated with low concentrations of Nonidet P-40. Significant inhibitory effect of NP-40 on sterol uptake and esterification was evident both in non-growing and growing cells supplemented with external cholesterol. Increased levels of sterol precursors (squalene, lanosterol) in hem1 cells grown in complex medium with cholesterol indicated general interference of NP-40 with sterol biosynthesis. NP-40 in the growth medium affected also cell viability estimated as the colony forming ability. More attention should be therefore paid to possible effects of mild detergents at low concentrations generally considered to be harmless, especially in cells with disturbed lipid biogenesis.