Characterization of end4+, a gene required for endocytosis in Schizosaccharomyces pombe

To understand endocytic trafficking in Schizosaccharomyces pombe, we constructed an end4 disruption mutant. The end4+ gene encodes a protein homologous to Sla2p/End4p, which is essential for the assembly and function of the cytoskeleton and endocytosis in Saccharomyces cerevisiae. We characterized the fission yeast mutant end4 Delta as well as ypt7 Delta, which is deficient in vacuolar fusion and, hence, endocytosis. The delivery of FM4-64 to the vacuolar membrane, accumulation of Lucifer yellow CH and internalization of plasma membrane protein Map3-GFP were inhibited in the end4 mutant. Deletion of end4 resulted in pleiotropic phenotypes consistent with F-actin depolarization, including high temperature sensitivity, abnormal morphology and mating defects. Extensive missorting of carboxypeptidase Y was detected in the ypt7 mutant; however, little missorting was detected in the end4 mutant. These results indicate that End4p is essential for the internalization process and Ypt7p affects endocytosis at a post-internalization step after the intersection of the endocytic and the vacuolar protein-sorting pathways in fission yeast.     

Identification and characterization of Saccharomyces cerevisiae mutants defective in fluid-phase endocytosis

A mutant library generated by the European Functional Analysis Network (EUROFAN) was screened for strains defective in fluid-phase endocytosis. Accumulation of Lucifer yellow in the vacuole was used as a marker for efficient endocytosis. Fourteen mutants, including ede1Delta, rcy1Delta, sys1Delta and tlg2Delta, previously described to be involved in membrane trafficking, were identified in this screen. alpha-Factor uptake, endocytosis of FM4-64, carboxypeptidase Y secretion, vacuolar morphology, and a vma2 synthetic growth defect were used as criteria to characterize the endocytic defect of the mutant strains obtained. Accordingly, eight mutant strains have endocytic phenotypes in addition to their defect in Lucifer yellow accumulation. These fluid-phase endocytosis mutants are defective at different steps of the endocytic pathway. Interestingly, only two mutants were defective for internalization, two for vacuolar protein sorting and four mutants had aberrant vacuolar morphologies. Some of the mutants identified in this screen that sort carboxypeptidase Y correctly may affect endocytosis at an early post-internalization step before the intersection of the endocytic with the vacuolar protein-sorting pathway.     

Vacuolar functions are involved in stress-protective effect of intracellular proline in Saccharomyces cerevisiae

Proline protects yeast cells from damage caused by various stresses. A yeast Saccharomyces cerevisiae mutant with high levels of intracellular proline grown in a minimal medium accumulated proline in its vacuole, but when grown in a nutrient medium, accumulated proline mainly in the cytosol. To understand the role of the proline pool in the vacuole, we examined the stress-protective effect of proline in proline-accumulating yeast cells deficient in vacuolar functions. The disruption of PEP3 encoding a vacuolar membrane protein required for vacuolar biogenesis caused hypersensitivity to heat shock and ethanol stresses, probably due to disappearance of normal vacuoles. The vph1-disrupted cells lacking vacuolar-ATPase activity showed resistance to heat shock without any change in proline localization, but showed severe growth defects in an ethanol-containing medium. These results indicate that vacuolar functions are involved in the stress-protective effect of proline in S. cerevisiae. Also, it appears that excess proline is transported to the vacuole in an ATP-independent manner.     

Characterization of vps33+, a gene required for vacuolar biogenesis and protein sorting in Schizosaccharomyces pombe

From the fission yeast Schizosaccharomyces pombe we have identified and deleted vps33, a gene encoding a homologue of VPS33, which is required for vacuolar biogenesis in S. cerevisiae cells. When the vps33(+) gene is disrupted, Sz. pombe strains are temperature-sensitive for growth and contain numerous small vesicular structures stained with FM4-64 in the cells. Deletion of the Sz. pombe vps33(+) gene results in pleiotropic phenotypes consistent with the absence of normal vacuoles, including missorting of vacuolar carboxypeptidase Y, various ion- and drug-sensitivities, and sporulation defects. These results are consistent with Vps33p being necessary for the morphogenesis of vacuoles and subsequent expression of vacuolar functions in Sz. pombe cells.     

Overexpression of vacuolar H+‐ATPase‐related genes in bottom‐fermenting yeast enhances ethanol tolerance and fermentation rates during high‐gravity fermentation

Vacuolar H⁺‐ATPase (V‐ATPase) is thought to play a role in stress tolerance. In this study it was found that bottom‐fermenting yeast strains, in which the V‐ATPase‐related genes DBF2, VMA41/CYS4/NHS5 and RAV2 were overexpressed, exhibited stronger ethanol tolerance than the parent strain and showed increased fermentation rates in a high‐sugar medium simulating high‐gravity fermentation. Among the strains examined, the DBF2‐overexpressing bottom‐fermenting yeast strain exhibited the highest ethanol tolerance and fermentation rate in YPM20 medium. Using this strain, high‐gravity fermentation was performed by adding sugar to the wort, which led to increased fermentation rates and yeast viability compared with the parent strain. These findings indicate that V‐ATPase is a stress target in high‐gravity fermentation and suggests that enhancing the V‐ATPase activity increases the ethanol tolerance of bottom‐fermenting yeast, thereby improving the fermentation rate and cell viability under high‐gravity conditions. Copyright © 2012 The Institute of Brewing & Distilling     

A fission yeast kinesin affects Golgi membrane recycling

We report here an in vivo study of kinesin heavy chain (KHC) functions in yeast. We have identified in Schizosaccharomyces pombe a kinesin motor gene, klp3(+), which has the highest homology to the Neurospora crassa KHC. Using indirect immunofluorescence, HA epitope-tagged Klp3 protein is cytoplasmic and appears as one to a few distinct patches that are coincident with microtubules. The klp3 null allele is viable. In klp3 deleted cells, ER, Golgi and mitochondrial distribution appear normal. Mitochondrial distribution in S. pombe is known to be microtubule-associated. We show that latrunculin A does not cause mitochondria to aggregate, suggesting that mitochondrial distribution in fission yeast, unlike budding yeast, is not dependent upon actin-based processes. Neither latrunculin A nor thiabendazole affects ER or Golgi distribution. We also used the vital dye FM4-64 to visualize the internalization of the dye and its transport to vacuoles in fission yeast in the presence and absence of Klp3. We observed no significant difference between the wild-type and Klp3 null cells in either the dynamics of endocytosis or the distribution and fusion of vacuoles. The drug brefeldin A causes Golgi-to-ER recycling in wild-type fission yeast cells. Although recycling of Golgi to ER after brefeldin A treatment occurs in klp3 null cells, recycling is defective and the distribution pattern we see is different from that observed in the wild-type strain. We conclude that Klp3 plays a role in BFA-induced membrane transport. The nucleotide sequence of S. pombe klp3(+) was submitted to GenBank under Accession No. AF154055.     

Cellular mechanism for the improvement of multiple stress tolerance in brewer's yeast by potassium ion supplementation

The ethanol fermentation efficiency was affected by multiple stress tolerance of yeast during brewing and bioethanol industry. The effect of KCl on the multiple stress tolerance of yeast cells was examined. Results showed that KCl addition significantly enhanced the tolerance of yeast cells to osmotic and ethanol stress, which correlated with the decreased membrane permeability, the increased intracellular ergosterol and ATP content, and the improved activity of complex II and complex III in yeast cells. Biomass and viability of yeast cells under osmotic and ethanol stress were increased significantly by KCl addition. Supplementation of 4 and 10 g L⁻¹ KCl exhibited the best promotion activity for yeast cells present in medium with 500 g L⁻¹ sucrose and 10% (v v⁻¹) ethanol, respectively. These results suggested that exogenous potassium addition might be an effective strategy to improve yeast tolerance and fermentation efficiency during industrial very-high-gravity (VHG) fermentation.     

Fluorescence microscopy of NaCl-stressed, elongated Salmonella and Listeria cells reveals the presence of septa in filaments

The cell morphology of Salmonella enteritidis and Listeria monocytogenes after the application of stress was examined. Cells were stressed by plating the bacteria on Tryptone Soya Agar (TSA) plates, with 5-10% NaCl. The plates were subsequently incubated for 6 days at 25 degrees C. Finally, the cells were harvested and subjected to different fluorescent probes in order to visualize the possible presence of septa in elongated cells. Use of the stain 4',6-Diamidino-2-phenylindole (DAPI), which is a blue fluorescent nucleic acid stain that preferentially stains double-stranded DNA, showed clearly the presence of dark spots, probably cellular partitions where no nucleic acids were present, in both Salmonella and Listeria cells. Another stain, FM 4-64, a lipophilic styryl dye for red staining of the inner membrane, showed the presence of highly fluorescent spots in Listeria cells, probably indicating the presence of membranes. For Salmonella, however, FM 4-64 was not successful in revealing septa in filaments. Double staining applied to elongated Listeria cells showed areas with high fluorescence in DAPI-staining (DNA-rich spots) which contained low fluorescence in FM 4-64-staining (membrane spots) and vice versa, which is a confirmation that the elongated cells are indeed composed of several normal size cells.     

Ultrastructural changes in yeast following heat shock and recovery

The effects of heat shock and heat stress on the ultrastructure of Saccharomyces cerevisiae is reported. Following a mild heat shock, referred to as an increase in temperature from 25°C to 37°C for 30 min, we observed contraction of the nucleolus, formation of electron-dense particles (90 nm) in mitochondria and heat-shock granules (30–40 nm) in the cytoplasm. The electron-dense particles in the mitochondria were similar in appearance to those previously reported in plant cells exposed to elevated temperatures. In a heat-sensitive yeast strain, the nucleolus was severely aggregated after a mild heat shock, a treatment which hardly affected relatively more heat-resistant strains. The nucleolus was aggregated in all strains after a more severe heat stress (50°C for 2 or 4 min). When cells were observed during a recovery period after heat stress it was found that nucleolar ultrastructure was regained more rapidly in cells that were previously heat shocked compared to cells that were stressed directly with no prior heat shock.     

The deletion of CaVPS34 in the human pathogenic yeast Candida albicans causes defects in vesicle-mediated protein sorting and nuclear segregation

A Candida albicans null mutant of the phosphatidylinositol (PI) 3-kinase gene (CaVPS34) involved in virulence was examined by different microscopical techniques. We observed that vacuoles of the Cavps34 null mutant were considerably enlarged and electron-transparent. An interesting result obtained by transmission electron microscopy analysis of Cavps34 mutant cells was the aberrant patch-like accumulation of vesicles, which were localized in the periplasm close to the plasma membrane. We assume that the vesicles result from missorted prevacuolar compartments. In contrast to the accumulations of the specific endocytic dye FM4-64 in the vacuole membrane in C. albicans wild-type strains (ring staining pattern), the Cavps34 mutant strain showed a staining of punctuate structures, possibly multivesicular bodies (MVB), that are scattered all over the cell. This defect indicates a late block in endocytic vesicle transport. Measurement of the total activity of carboxypeptidase Y revealed significantly lower activity in Cavps34 mutant cells. This may indicate that carboxypeptidase Y is not properly activated as a result of mislocalization due to the lack of Vps34p. The deletion of the CaVPS34 gene caused disturbance of normal nuclear migration, which suggests that in the Cavps34 mutant the cell-size mediated control process of cell division is affected.     

Severe ethanol stress induces assembly of stress granules in Saccharomyces cerevisiae

Stress granules (SGs) and processing bodies (P bodies) are cytoplasmic domains and play a role in the control of translation and mRNA turnover in mammalian cells subjected to environmental stress. Recent studies have revealed that SGs also form in the budding yeast Saccharomyces cerevisiae in response to glucose depletion and robust heat shock. However, information about the types of stress that cause budding yeast SGs is quite limited. Here we demonstrate that severe ethanol stress generates budding yeast SGs in a manner independent of the phosphorylation of eIF2α. The concentration that generated budding yeast SGs (>10%) was higher than that causing P bodies (>6%), and P bodies were assembled prior to SGs. As well as mammalian SGs, the assembly of budding yeast SGs under ethanol stress was blocked by cycloheximide. On the other hand, the budding yeast SGs caused by ethanol stress contained eIF3c but not eIF3a and eIF3b, although the eIF3 complex is a core constituent of mammalian SGs. Moreover, null mutants (pbp1Δ, pub1Δ and tif4632Δ) with a strong reduction in SG formation did not resume proliferation after the elimination of ethanol stress, indicating that the formation of budding yeast SGs might play a role in sufficient recovery from ethanol stress.     

Characteristics of yeast flora in Chinese strong‐flavoured liquor fermentation in the Yibin region of China

The yeast community in the Chinese strong‐flavoured liquor region of Yibin was investigated and the ethanol producing abilities and extracellular enzymes activities of the isolates were tested. A total of 110 yeast were isolated on Wallerstein Laboratory medium and through 26S rRNA D1/D2 region sequence analysis identified as 13 yeast species. These were Wickerhamomyces anomalus, Debaryomyces hansenii, Issatchenkia orientalis, Lodderomyces elongisporus, Clavispora lusitaniae, Saccharomyces cerevisiae, Pichia fermentans, Pichia manshurica, Pichia membranifaciens, Torulaspora delbrueckii, Trichosporon insectorum, Trichosporonoides megachiliensis, Zygosaccharomyces bailii, and one uncertain species. These yeast species, composed of various strains, formed the special yeast community in the Yibin region. Approximately 73.6% of the strains belong to the four dominant species: W. anomalus, D. hansenii, I. orientalis and L. elongisporus. The 110 yeast strains produced 0.6–9.0% (v/v) alcohol (average of 5.4%, v/v) in a grain medium, and 0.2–7.2% (v/v) alcohol (average value of 2.9%, v/v) in a yeast extract–peptone–dextrose medium. Furthermore, the 49 strains that produced pectinase, lipase, cellulase, amylase or protease generally showed better ethanol‐producing ability than those strains that do not produce extracellular enzymes. This work profiles the ethanol‐producing ability and the organic matter utilization of the yeast community in Chinese strong‐flavoured liquor produced in the Yibin region and provides a better understanding of Chinese strong‐flavoured liquor fermentation. Copyright © 2016 The Institute of Brewing & Distilling     

Outward electron transfer by Saccharomyces cerevisiae monitored with a bi‐cathodic microbial fuel cell‐type activity sensor

A Janus head‐like bi‐cathodic microbial fuel cell was constructed to monitor the electron transfer from Saccharomyces cerevisiae to a woven carbon anode. The experiments were conducted during an ethanol cultivation of 170 g/l glucose in the presence and absence of yeast‐peptone medium. First, using a basic fuel‐cell type activity sensor, it was shown that yeast‐peptone medium contains electroactive compounds. For this purpose, 1% solutions of soy peptone and yeast extract were subjected to oxidative conditions, using a microbial fuel cell set‐up corresponding to a typical galvanic cell, consisting of culture medium in the anodic half‐cell and 0.5 M K₃Fe(CN)₆ in the cathodic half‐cell. Second, using a bi‐cathodic microbial fuel cell, it was shown that electrons were transferred from yeast cells to the carbon anode. The participation of electroactive compounds in the electron transport was separated as background current. This result was verified by applying medium‐free conditions, where only glucose was fed, confirming that electrons are transferred from yeast cells to the woven carbon anode. Knowledge about the electron transfer through the cell membrane is of importance in amperometric online monitoring of yeast fermentations and for electricity production with microbial fuel cells. Copyright © 2009 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.     

Change in transport activities of vacuoles of the yeast Yarrowia lipolytica during its growth on glucose

Vacuoles were isolated from Yarrowia lipolytica yeast cells taken at various growth phases under carbon or nitrogen limitation. Vacuoles from the cells at the logarithmic growth phase showed a high activity of vacuolar H⁺-ATPase (0·9–1·1 U/mg protein) and efficiently generated chemical proton gradient and membrane potential across the tonoplast. Ca²⁺- and citrate transport were found to be maximal at this growth phase. At growth retardation and then in the stationary phase all the parameters studied decreased irrespective of the method of growth limitation. The citrate-transporting activity of vacuoles completely disappeared at growth retardation, also irrespective of the limitation method and irrespective of whether yeast cells overproduced citrate in the culture medium. The citrate-transporting system of Y. lipolytica vacuolar membrane is concluded not to be involved in citrate efflux and this efflux is probably performed by the plasmalemma transport system.     

The yeast enzyme Eht1 is an octanoyl‐CoA:ethanol acyltransferase that also functions as a thioesterase

Fatty acid ethyl esters are secondary metabolites that are produced during microbial fermentation, in fruiting plants and in higher organisms during ethanol stress. In particular, volatile medium‐chain fatty acid ethyl esters are important flavour compounds that impart desirable fruit aromas to fermented beverages, including beer and wine. The biochemical synthesis of medium‐chain fatty acid ethyl esters is poorly understood but likely involves acyl‐CoA:ethanol O‐acyltransferases. Here, we characterize the enzyme ethanol hexanoyl transferase 1 (Eht1) from the brewer's yeast Saccharomyces cerevisiae. Full‐length Eht1 was successfully overexpressed from a recombinant yeast plasmid and purified at the milligram scale after detergent solubilization of sedimenting membranes. Recombinant Eht1 was functional as an acyltransferase and, unexpectedly, was optimally active toward octanoyl‐CoA, with k_cat = 0.28 ± 0.02/s and K_M = 1.9 ± 0.6 μm . Eht1 was also revealed to be active as a thioesterase but was not able to hydrolyse p‐nitrophenyl acyl esters, in contrast to the findings of a previous study. Low‐resolution structural data and site‐directed mutagenesis provide experimental support for a predicted α/β‐hydrolase domain featuring a Ser–Asp–His catalytic triad. The S. cerevisiae gene YBR177C/EHT1 should thus be reannotated as coding for an octanoyl‐CoA:ethanol acyltransferase that can also function as a thioesterase. © 2014 The Authors. Yeast published by John Wiley & Sons, Ltd.     

A mutant for the yeast scERV1 gene displays a new defect in mitochondrial morphology and distribution

The yeast scERV1 gene is the best characterized representative of a new gene family found in different lower and higher eukaryotes. The gene product is essential for the yeast cell and has a complex influence on different aspects of mitochondrial biogenesis. The homologous mammalian ALR(A ugmenter of L iver R egeneration) genes from man, mouse and rat are important at different developmental stages of the organism as, for example, in spermatogenesis and liver regeneration. In this study the influence of scERV1 on the morphology of mitochondria and its submitochondrial localization are investigated. A temperature‐sensitive mutant of the gene was stained with a mitochondria‐specific dye and fluorescence was inspected at the permissive and restrictive temperature. A new phenotype for morphological defects of mitochondria was identified. Already at the permissive temperature mitochondrial vesicles accumulate at defined positions in the cell. After shift to the restrictive temperature, morphological changes, and finally complete loss of mitochondrial structures, are observed. Ultrastructural studies confirm these findings and demonstrate the loss of the mitochondrial inner membrane and at the final stage a drastic reduction or complete absence of mitochondria from the cell. GFP fusion experiments with the scERV1 gene and subcellular localization by fractionation experiments identify the gene product inside mitoplasts and the cytosol. Re‐investigation of the mutant phenotype demonstrates that after longer incubation of the mutant at the restrictive temperature an irreversible defect of the cells, even on glucose complete medium, is found that is in accordance with a complete loss or irreversible damage of mitochondria. Copyright © 1999 John Wiley & Sons, Ltd.     

Disruption of ubiquitin-related genes in laboratory yeast strains enhances ethanol production during sake brewing

Sake yeast can produce high levels of ethanol in concentrated rice mash. While both sake and laboratory yeast strains belong to the species Saccharomyces cerevisiae, the laboratory strains produce much less ethanol. This disparity in fermentation activity may be due to the strains' different responses to environmental stresses, including ethanol accumulation. To obtain more insight into the stress response of yeast cells under sake brewing conditions, we carried out small-scale sake brewing tests using laboratory yeast strains disrupted in specific stress-related genes. Surprisingly, yeast strains with disrupted ubiquitin-related genes produced more ethanol than the parental strain during sake brewing. The elevated fermentation ability conferred by disruption of the ubiquitin-coding gene UBI4 was confined to laboratory strains, and the ubi4 disruptant of a sake yeast strain did not demonstrate a comparable increase in ethanol production. These findings suggest different roles for ubiquitin in sake and laboratory yeast strains.     

Fermentative capacity of Kluyveromyces marxianus and Saccharomyces cerevisiae after oxidative stress

Volatile compound production during alcoholic fermentation has been studied in the production of many beverages. Temperature, yeast strain, nutrients and pH have been identified as important factors in the production of volatile compounds. In addition, other factors could influence this production during the fermentation process as well. Oxidative stress could occur during yeast biomass production because oxygen is an essential nutrient that is added to the growth medium. The fermentation parameters and the volatile compound production of one Saccharomyces cerevisiae strain (MC4) and two Kluyveromyces marxianus strains (OFF1 and SLP1) were evaluated in relation to fermentation parameters after oxidative stress induced by hydrogen peroxide or menadione. These yeasts were compared with S. cerevisiae W303–1A and showed significant differences in ethanol production, ethanol yield and maximum ethanol production rate. K. marxianus (OFF1) showed better fermentative capacity after oxidative stress. The higher alcohol production decreased after oxidative stress by >35% after 72 h fermentation time, and the amyl alcohol decreased at a higher level (>60%); however, the isobutanol production increased after oxidative stress between 1.5 and 4 times. The yeasts produced significant concentrations of esters however ethyl lactate, ethyl caprylate and the ethyl caproate were not detected in the control fermentation, while in the stress fermentation they accounted for up to 3 mg/L. These results demonstrate that oxidative stress can play an important role in the final aroma profile; but it is necessary to guarantee adequate yeast growth to obtain the volatile compounds desired. Copyright © 2017 The Institute of Brewing & Distilling