The role of K+ and H+ transport systems during glucose‐ and H2O2‐induced cell death in Saccharomyces cerevisiae

Glucose, in the absence of additional nutrients, induces programmed cell death in yeast. This phenomenon is independent of yeast metacaspase (Mca1/Yca1) and of calcineurin, requires ROS production and it is concomitant with loss of cellular K⁺ and vacuolar collapse. K⁺ is a key nutrient protecting the cells and this effect depends on the Trk1 uptake system and is associated with reduced ROS production. Mutants with decreased activity of plasma membrane H⁺‐ATPase are more tolerant to glucose‐induced cell death and exhibit less ROS production. A triple mutant ena1‐4 tok1 nha1, devoid of K⁺ efflux systems, is more tolerant to both glucose‐ and H₂O₂‐induced cell death. We hypothesize that ROS production, activated by glucose and H⁺‐ATPase and inhibited by K⁺ uptake, triggers leakage of K⁺, a process favoured by K⁺ efflux systems. Loss of cytosolic K⁺ probably causes osmotic lysis of vacuoles. The nature of the ROS‐producing system sensitive to K⁺ and H⁺ transport is unknown. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of phosphatidic acid phosphatase activity in the oleaginous yeast Yarrowia lipolytica and its role in lipid biosynthesis

Phosphatidic acid phosphatase (PAP) catalyses the committed step of triacylglycerol (TAG) biosynthesis and thus regulates the amounts of TAG produced by the cell. TAG is the target of biotechnological processes developed for the production of food lipids or biofuels. These processes are using oleaginous microorganisms like the yeast Yarrowia lipolytica as the TAG producers. Thus manipulating key enzymatic activities like PAP in Y. lipolytica could drive lipid biosynthesis towards TAG production and increase TAG yields. In this study, PAP activity in Y. lipolytica was characterized in detail and its role in lipid biosynthesis was addressed. PAP activity increased 2.5‐fold with the addition of Mg²⁺ (1 mm ) in the assay mixture, which means that most of the PAP activity was due to Mg²⁺‐dependent PAP enzymes (e.g. Pah1, App1). In contrast, N‐ethylmaleimide (NEM) potently inhibited PAP activity, indicating the presence of NEM‐sensitive PAP enzymes (e.g. App1, Lpp1). Localization studies revealed that the majority of PAP activity resides in the membrane fraction, while the cytosolic fraction harbours only a small amount of activity. PAP activity was regulated in a growth‐dependent manner, being induced at the early exponential phase and declining thereafter. PAP activity did not correlate with TAG synthesis, which increased as cells progressed from the exponential phase to the early stationary phase. In stationary phase, TAG was mobilized with the concomitant synthesis of sterols and sterol esters. These results provide the first insights into the role of PAP in lipid biosynthesis by Y. lipolytica. Copyright © 2016 John Wiley & Sons, Ltd.     

Endocytosis and vacuolar morphology in Saccharomyces cerevisiae are altered in response to ethanol stress or heat shock

The vital lipophilic dye N‐(3‐triethylammoniumpropyl)‐4‐[6‐(4‐(diethylamino)phenyl]hexatrienyl) pyridinium dibromide (FM 4‐64) was used to study the effect of ethanol stress and heat shock on endocytosis in the yeast Saccharomyces cerevisiae. Yeast cells stained with FM 4‐64 were placed in a culture chamber and the internalization of the dye was monitored by fluorescence microscopy during perfusion of the cells with fresh growth medium. In the absence of ethanol in the perfusion medium, the internalization of FM 4‐64 from the plasma membrane to the vacuolar membrane by yeast cells harvested from the exponential phase of growth was completed in 30 min. The presence of 6% (v/v) ethanol in the perfusion medium had no obvious effect on the internalization of FM 4‐64 from the plasma membrane, but did lead to an accumulation of the dye in endocytic intermediates. Consequently, vacuolar membrane staining was delayed. Cells stained with FM 4‐64 and subjected to heat shock displayed a similar effect, with endocytic intermediates becoming more prominent with the severity of the heat shock. For both ethanol stress and heat shock, vacuolar morphology altered from segregated structures to a single, large organelle. The findings of this study reinforce previous observations that ethanol stress and heat shock induce similar responses in yeast. Copyright © 1999 John Wiley & Sons, Ltd.     

Transport of organic acid anions and guanosine into vacuoles of Saccharomyces pastorianus

Isolated vacuoles of the yeast Saccharomyces pastorianus accumulate citrate, α-ketoglutarate, malate and guanosine. This accumulation is Mg ATP-dependent and inhibited by protonophores. The ionophores monensin and A23187 (electroneutral Meⁿ⁺/nH⁺-exchange) inhibit guanosine accumulation but fail to block citrate uptake. Mg²⁺ ions (2 mM ) increase the values of both Δ$ \tilde \mu $H⁺ components and stimulate the uptake of all the above compounds. Ca²⁺ ions (1 mM ), hyperpolarizing the yeast vacuolar membrane and dissipating the pH gradient, inhibit guanosine uptake and stimulate that of citrate. It is concluded that guanosine is transported into yeast vacuoles by an H⁺/guanosine antiporter while citrate, malate and α-ketoglutarate are translocated by a uniporter(s) at the expense of the membrane potential (positive inside).     

Regulation of alcoholic fermentation in batch and chemostat cultures of Kluyveromyces lactis CBS 2359

Kluyveromyces lactis is an important industrial yeast, as well as a popular laboratory model. There is currently no consensus in the literature on the physiology of this yeast, in particular with respect to aerobic alcoholic fermentation (‘Crabtree effect’). This study deals with regulation of alcoholic fermentation in K. lactis CBS 2359, a proposed reference strain for molecular studies. In aerobic, glucose-limited chemostat cultures (D=0·05–0·40 h⁻¹) growth was entirely respiratory, without significant accumulation of ethanol or other metabolites. Alcoholic fermentation occurred in glucose-grown shake-flask cultures, but was absent during batch cultivation on glucose in fermenters under strictly aerobic conditions. This indicated that ethanol formation in the shake-flask cultures resulted from oxygen limitation. Indeed, when the oxygen feed to steady-state chemostat cultures (D=0·10 h⁻¹) was lowered, a mixed respirofermentative metabolism only occurred at very low dissolved oxygen concentrations (less than 1% of air saturation). The onset of respirofermentative metabolism as a result of oxygen limitation was accompanied by an increase of the levels of pyruvate decarboxylase and alcohol dehydrogenase. When aerobic, glucose-limited chemostat cultures (D=0·10 h⁻¹) were pulsed with excess glucose, ethanol production did not occur during the first 40 min after the pulse. However, a slow aerobic ethanol formation was invariably observed after this period. Since alcoholic fermentation did not occur in aerobic batch cultures this is probably a transient response, caused by an imbalanced adjustment of enzyme levels during the transition from steady-state growth at μ=0·10 h⁻¹ to growth at μ_max. It is concluded that in K. lactis, as in other Crabtree-negative yeasts, the primary environmental trigger for occurrence of alcoholic fermentation is oxygen limitation. © 1998 John Wiley & Sons, Ltd.     

Heterologous expression reveals unique properties of Trk K+ importers from nonconventional biotechnologically relevant yeast species together with their potential to support Saccharomyces cerevisiae growth

In the model yeast Saccharomyces cerevisiae, Trk1 is the main K⁺ importer. It is involved in many important physiological processes, such as the maintenance of ion homeostasis, cell volume, intracellular pH, and plasma-membrane potential. The ScTrk1 protein can be of great interest to industry, as it was shown that changes in its activity influence ethanol production and tolerance in S. cerevisiae and also cell performance in the presence of organic acids or high ammonium under low K⁺ conditions. Nonconventional yeast species are attracting attention due to their unique properties and as a potential source of genes that encode proteins with unusual characteristics. In this work, we aimed to study and compare Trk proteins from Debaryomyces hansenii, Hortaea werneckii, Kluyveromyces marxianus, and Yarrowia lipolytica, four biotechnologically relevant yeasts that tolerate various extreme environments. Heterologous expression in S. cerevisiae cells lacking the endogenous Trk importers revealed differences in the studied Trk proteins' abilities to support the growth of cells under various cultivation conditions such as low K⁺ or the presence of toxic cations, to reduce plasma-membrane potential or to take up Rb⁺. Examination of the potential of Trks to support the stress resistance of S. cerevisiae wild-type strains showed that Y. lipolytica Trk1 is a promising tool for improving cell tolerance to both low K⁺ and high salt and that the overproduction of S. cerevisiae's own Trk1 was the most efficient at improving the growth of cells in the presence of highly toxic Li⁺ ions.     

Distribution of the actin cytoskeleton during the cell cycle of Yarrowia lipolytica and the visualization of the tubulin cytoskeleton by immunofluorescence

Actin distribution was examined during the cell cycle of the dimorphic yeast Yarrowia lipolytica, showing the correlation between bud growth, nuclear migration and rearrangement of the actin cytoskeleton. The results correspond with observations made in cells of Saccharomyces cerevisiae, S. uvarum and Candida albicans. Localization of actin was also determined in hyphal cells, where actin is stained predominantly in the tip and also at the septum of hyphae. The standard methods used for tubulin immunostaining in S. cerevisiae and C. albicans cells were adapted for application in Y. lipolytica. Copyright © 1999 John Wiley & Sons, Ltd.     

Human NKCC2 cation–Cl– co‐transporter complements lack of Vhc1 transporter in yeast vacuolar membranes

Cation–chloride co‐transporters serve to transport Cl^– and alkali metal cations. Whereas a large family of these exists in higher eukaryotes, yeasts only possess one cation–chloride co‐transporter, Vhc1, localized to the vacuolar membrane. In this study, the human cation–chloride co‐transporter NKCC2 complemented the phenotype of VHC1 deletion in Saccharomyces cerevisiae and its activity controlled the growth of salt‐sensitive yeast cells in the presence of high KCl, NaCl and LiCl. A S. cerevisiae mutant lacking plasma‐membrane alkali–metal cation exporters Nha1 and Ena1‐5 and the vacuolar cation–chloride co‐transporter Vhc1 is highly sensitive to increased concentrations of alkali–metal cations, and it proved to be a suitable model for characterizing the substrate specificity and transport activity of human wild‐type and mutated cation–chloride co‐transporters. Copyright © 2013 John Wiley & Sons, Ltd.     

Optimization of cyclopropane fatty acids production in Yarrowia lipolytica

Cyclopropane fatty acids, which can be simply converted to methylated fatty acids, are good unusual fatty acid candidates for long-term resistance to oxidization and low-temperature fluidity useful for oleochemistry and biofuels. Cyclopropane fatty acids are present in low amounts in plants or bacteria. In order to develop a process for large-scale biolipid production, we expressed 10 cyclopropane fatty acid synthases from various organisms in the oleaginous yeast Yarrowia lipolytica, a model yeast for lipid metabolism and naturally capable of producing large amounts of lipids. The Escherichia coli cyclopropane fatty acid synthase expression in Y. lipolytica allows the production of two classes of cyclopropane fatty acids, a C17:0 cyclopropanated form and a C19:0 cyclopropanated form, whereas others produce only the C17:0 form. Expression optimization and fed-batch fermentation set-up enable us to reach a specific productivity of 0.032 g·L⁻¹·hr⁻¹ with a genetically modified strain containing cyclopropane fatty acid up to 45% of the total lipid content corresponding to a titre of 2.3 ± 0.2 g/L and a yield of 56.2 ± 4.4 mg/g.     

Growth of a tropical marine yeast Yarrowia lipolytica NCIM 3589 on bromoalkanes: relevance of cell size and cell surface properties

Yarrowia lipolytica 3589, a tropical marine yeast, grew aerobically on a broad range of bromoalkanes varying in carbon chain length and differing in degree and position of bromide group. Amongst the bromoalkanes studied, viz. 2‐bromopropane (2‐BP), 1‐bromobutane (1‐BB), 1,5‐dibromopentane (1,5‐DBP) and 1‐bromodecane (1‐BD), the best utilized was 1‐BD, with a maximal growth rate (μ_max) of 0.055 h^?1 and an affinity ratio (μ_max/K_s) of 0.022. Utilization of these bromoalkanes as growth substrates was associated with a concomitant release of bromide (8202.9 µm ) and cell mass (36 × 10⁹ cells/ml), occurring maximally on 1‐BD. Adherence of yeast cells to these hydrophobic bromoalkanes was observed microscopically, with an increase in cell size and surface hydrophobicity. The maximal cell diameter was for 1‐BD (4.66 µm), resulting in an increase in the calculated cell surface area (68.19 µm²) and sedimentation velocity (1.31 µm/s). Cell surface hydrophobicity values by microbial adhesion to solvents (MATS) analysis for yeasts grown on bromoalkanes and glucose were significantly high, i.e. >80%. Similarly, water contact angles also indicate that the cell surface of yeast cells grown in glucose possess a relatively more hydrophilic cell surface (θ = 49.1°), whereas cells grown in 1‐BD possess a more hydrophobic cell surface (θ = 90.7°). No significant change in emulsification activity or surface tension was detected in the cell‐free supernatant. Thus adherence to the bromoalkane droplets by an increase in cell size and surface hydrophobicity leading to debromination of the substrate might be the strategy employed in bromoalkane utilization and growth by Y. lipolytica 3589. Copyright © 2011 John Wiley & Sons, Ltd.     

Transport of L-leucine in the fission yeast Schizosaccharomyces pombe

Transport of L -leucine into Schizosaccharomyces pombe cells from the stationary phase of growth (after preincubation for 60 min with 1% glucose) proceeds uphill, practically unidirectionally, and is mediated by at least two systems: a high-affinity system with a K_T of 0·045 mmol 1^?1 and J_max of 3·3 nmol min^?1 (mg dry weight)^?1 and a low-affinity system with a K_T of 1·25 mmol 1^?1 and J_max of 16·0 nmol min^?1 (mg dry weight)^?1. The high-affinity system has a pH optimum at 3.2, the accumulation ratio is highest at a cell density of 2–4 mg dry weight per ml and decreases with increasing leucine concentration. Transport of leucine by the high-affinity system is strongly inhibited by proton conductors, ammonium ions and by most amino acids, but only L -phenylalanine, L -isoleucine, L -valine and L -cysteine behave as fully competitive inhibitors. Systems of L -leucine transport in S. pombe are not constitutive. Transport activity appears only after preincubation of cells with a suitable source of energy. If cycloheximide is added during preincubation with glucose, no transport systems for leucine are synthesized. After removal of glucose, the activity of transport systems decays with a half-time of about 20 min. The presence of cyclic AMP increases the initial rate of leucine uptake only in cells preincubated with glucose and in the absence of cycloheximide.     

Characterization of a glycerol/H+ symport in the halotolerant yeast Pichia sorbitophila

Pichia sorbitophila is a halotolerant yeast capable of surviving to extracellular NaCl concentrations up to 4 M in mineral medium when glucose or glycerol are the only carbon and energy sources. Evidence is presented here that glycerol, the main compatible solute this yeast accumulates so as to maintain osmotic balance, is actively co-transported with protons. This transport system was shown to be constitutive, not needing induction by either glycerol or salt, and was not repressible by glucose. In glucose- or glycerol-grown cells, a simple diffusion was detectable, and iterative calculations were performed to calculate kinetic parameters, in the presence and in the absence of NaCl. At 25°C, pH 5·0, in glucose-grown cells these were: K_m = 0·81 ± 0·11 mM and V_max = 634·2 ± 164·8 μmol h^?1 per g (glycerol); K_m = 1·28 ± 0·60 mM and V_max = 558·6 · 100·6 μmol h^?1 per g (protons). Correspondent stoichiometry was approximately 1, either for these conditions or in the presence of 1 M -NaCl. An increase in acumulation capacity was evident when different concentrations of NaCl were present. This capacity was shown to be dependent on ΔpH and membrane potential, consistently with an electrogenic character. We suggest that the main role of this system is in osmoregulation, by keeping glycerol accumulated inside the cells, compensating for leakage, due to its liposoluble character.     

Deletion of the N-terminal domain of the yeast vacuolar (Na+,K+)/H+ antiporter Vnx1p improves salt tolerance in yeast and transgenic Arabidopsis

Cation/proton antiporters play a major role in the control of cytosolic ion concentrations in prokaryotes and eukaryotes organisms. In yeast, we previously demonstrated that Vnx1p is a vacuolar monovalent cation/H⁺ exchanger showing Na⁺/H⁺ and K⁺/H⁺ antiporter activity. We have also shown that disruption of VNX1 results in an almost complete abolishment of vacuolar Na⁺/H⁺ exchange, but yeast cells overexpressing the complete protein do not show improved salinity tolerance. In this study, we have identified an autoinhibitory N-terminal domain and have engineered a constitutively activated version of Vnx1p, by removing this domain. Contrary to the wild type protein, the activated protein has a pronounced effect on yeast salt tolerance and vacuolar pH. Expression of this truncated VNX1 gene also improves Arabidopsis salt tolerance and increases Na⁺ and K⁺ accumulation of salt grown plants thus suggesting a biotechnological potential of activated Vnx1p to improve salt tolerance of crop plants.     

Saccharomyces cerevisiae mutants altered in vacuole function are defective in copper detoxification and iron-responsive gene transcription

The metal ions, Cu^2+/+ and Fe^3+/2+, are essential co-factors for a wide variety of enzymatic reactions. However, both metal ions are toxic when hyper-accumulated or maldistributed within cells due to their ability to generate damaging free radicals or through the displacement of other physiological metal ions from metalloproteins. Although copper transport into yeast cells is apparently independent of iron, the known dependence on Cu²⁺ for high affinity transport of Fe²⁺ into yeast cells has established a physiological link between these two trace metal ions. In this study we demonstrate that proteins encoded by genes previously demonstrated to play critical roles in vacuole assembly or acidification, PEP3, PEP5 and VMA3, are also required for normal copper and iron metal ion homeostasis. Yeast cells lacking a functional PEP3 or PEP5 gene are hypersensitive to copper and render the normally iron-repressible FET3 gene, encoding a multi-copper Fe(II) oxidase involved in Fe²⁺ transport, also repressible by exogenous copper ions. The inability of these same vacuolar mutant strains to repress FET3 mRNA levels in the presence of an iron-unresponsive allele of the AFT1 regulatory gene are consistent with alterations in the intracellular distribution or redox states of Fe^3+/2+ in the presence of elevated extracellular concentrations of copper ions. Therefore, the yeast vacuole is an important organelle for maintaining the homeostatic convergence of the essential yet toxic copper and iron ions. © 1997 John Wiley & Sons, Ltd.     

The VPH2 gene encodes a 25 kDa protein required for activity of the yeast vacuolar H+-ATPase

Strains bearing the vph2 mutation are defective in vacuolar acidification. The VPH2 gene was isolated from a genomic DNA library by complementation of the zinc-sensitive phenotype of the mutant. Deletion analysis localized the complementing activity to a 1·2 kb DNA fragment. Sequence analysis of this fragment revealed the presence of a single open reading frame that encoded a protein of 215 amino acids. Computer analysis indicated that the protein, which has a predicted molecular mass of 25 286 Daltons, has two distinct membrane-spanning domains. Biochemical studies indicated that strains bearing the vph2 mutation have greatly reduced levels of vacuolar proton pumping and ATPase activity and that the nucleotide binding subunits of the multimeric vacuolar H⁺-ATPase failed to be correctly targeted to the vacuolar membrane. The vph2 mutant fails to grow on YEP glycerol medium and on media containing 100 mM -CaCl₂ or 4 mM -ZnCl₂ or buffered to pH 7·5, a phenotype observed in strains carrying deletions in the genes encoding several vacuolar H⁺-ATPase subunits. The VPH2 gene is identical to the VMA12 gene (T. Stevens and Y. Anraku, personal communication).     

Possible involvement of GDI1 protein, a GDP dissociation inhibitor related to vesicle transport, in an amelioration of zinc toxicity in Saccharomyces cerevisiae

The GDI1 protein related vesicle transport system was studied to investigate the possibility that an exclusion of toxic zinc (Zn) from the cytoplasm ameliorates Zn toxicity in Saccharomyces cerevisiae (yeast). A temperature‐sensitive gdi1 mutant (originally called sec19), in which the GDP dissociation inhibitor becomes inactive at the non‐permissive temperature (37 °C), was more sensitive to Zn than its parental GDI1 strain at 32 °C (a moderately non‐permissive temperature). The relative efflux of cytoplasmic Zn in the gdi1 mutant was lower than that in the control strain. Treatment with a vesicle transport‐specific inhibitor, Brefeldin A, caused an increase of Zn sensitivity and a decrease of Zn efflux in these strains. It is therefore suggested that the GDI1‐related vesicle transport system contributes to Zn tolerance in yeast. Furthermore, changes in the number of Zn‐specific fluorescent granules (zincosomes) were observed by zinquin staining in the mutant cells under Zn treatment at 32 °C and 37 °C. We concluded that the GDI1 protein is implicated in control of vesicle numbers. Collectively, the results suggest that the GDI1protein is involved in Zn efflux via small vesicle trafficking and contributes to the control of cytoplasmic Zn content, allowing yeast to survive in the presence of toxic Zn. Copyright © 2011 John Wiley & Sons, Ltd.     

The population change of yeasts in commercial salami

The survival of yeast during a pilot scale production of commercial salami was investigated. A total of 108 distinctive yeast strains were isolated and identified during the processing of the salami. Initially, the number of yeasts remained below 10³cfu g⁻¹but their numbers increased after the 12^thday of maturation, reaching a maximum of 2·0×10⁵cfu g⁻¹at day 20. During maturation, the pH declined from 5·72 to 4·36, water content from 58% to 43%, while the salt content increased by 1%. The number of lactic acid bacteria remained above 10⁵cfu g⁻¹throughout processing and maturation. Of the 108 yeast strains isolated, 22 strains were identified as members of the species Debaryomyces hansenii, being present in all samples taken. In descending numbers, Rhodotorula mucilaginosa, Yarrowia lipolytica and Cryptococcus albidus, were also frequently isolated during processing and maturation.