Dosage rescue by UBC4 restores cell wall integrity in Saccharomyces cerevisiae lacking the myosin type II gene MYO1

Myosin II is important for normal cytokinesis and cell wall maintenance in yeast cells. Myosin II-deficient (myo1) strains of the budding yeast Saccharomyces cerevisiae are hypersensitive to nikkomycin Z (NZ), a competitive inhibitor of chitin synthase III (Chs3p), a phenotype that is consistent with compromised cell wall integrity in this mutant. To explain this observation, we hypothesized that the absence of myosin type II will alter the normal levels of proteins that regulate cell wall integrity and that this deficiency can be overcome by the overexpression of their corresponding genes. We further hypothesized that such genes would restore normal (wild-type) NZ resistance. A haploid myo1 strain was transformed with a yeast pRS316-GAL1-cDNA expression library and the cells were positively selected with an inhibitory dose of NZ. We found that high expression of the ubiquitin-conjugating protein cDNA, UBC4, restores NZ resistance to myo1 cells. Downregulation of the cell wall stress pathway and changes in cell wall properties in these cells suggested that changes in cell wall architecture were induced by overexpression of UBC4. UBC4-dependent resistance to NZ in myo1 cells was not prevented by the proteasome inhibitor clasto-lactacystin-beta-lactone and required the expression of the vacuolar protein sorting gene VPS4, suggesting that rescue of cell wall integrity involves sorting of ubiquitinated proteins to the PVC/LE-vacuole pathway. These results point to Ubc4p as an important enzyme in the process of cell wall remodelling in myo1 cells.     

DETERMINATION OF SOME LIPID CONSTITUENTS OF BAKER'S YEAST

Lipid was extracted from whole yeast cells with difficulty because of the limited porosity of the cell wall and its sensitivity to dehydrating solvents. Extraction from mechanically broken cells was rapid and complete. Metabolic alteration of lipid when cells were disintegrated was minimized by first heating a yeast suspension briefly at 90°C. Details are given for the routine analysis of yeast phospholipids by quantitative paper chromatography and of neutral lipids by column chromatography. Lysophosphatides were found in some types of yeast after drying, but not in pressed commercial yeast or in yeast grown on synthetic media. They were formed when yeast was suspended in methanol-water, as yeast phospholipase A was activated and split palmitoleic acid from lecithin and phosphatidyl ethanolamine. Phosphatidyl inositol in baker's yeast contained a higher proportion of saturated fatty acids than other phospholipids. Sterols were present free and as esters with palmitoleic and oleic acids. Phospholipid content was affected by growth conditions. A high level of inositol in the growth medium reduced the formation of neutral lipid but did not affect the content of yeast phospholipid.     

Production of phytase in a low phosphate medium by a novel yeast Candida krusei

A yeast strain producing high levels of phytase was isolated from soil and identified as Candida krusei. The phytase was located on the yeast cell wall and was a glucanase-extractable protein. The phytase production was controlled by the phosphate concentration in the medium used. The maximum production of phytase occurred in a medium containing 0.5 mg of phosphorus per 100 ml, and most of the cells were ellipsoid-shaped and did not exhibit budding. Increasing the concentration of phosphorus in the medium to more than 5 mg of phosphorus per 100 ml caused inhibition of phytase production and 90% of the cells exhibited budding. On the other hand, transferring cells grown in the high-phosphate medium into a phosphate-free one derepressed the phytase production. For example, transferring cells grown in 2 mg of phosphorus per 100 ml into the phosphate-free medium, enhanced the total phytase activity up to 5.5-fold that in the medium containing 0.5 mg of phosphorus per 100 ml. The phytase showed two optimum pHs of 2.5 and 5.5, an optimum temperature of 40 degrees C and the K(m) value for Na-phytate was 0.03 mM. Using in vitro experiments that simulated the conditions of the digestive tract, 50-80% phosphorus was liberated from different plant samples (wheat bran, rice bran and feeds) by the strain.     

Binding of heterocyclic amines by yeast cells and their fractions

The binding of mutagenic pyrolysis products to cells of 50 yeast strains and their cell fractions was investigated. Cells of all yeast strains effectively bound 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). Cell walls (CW), and cell wall glucan and mannan (5 mg in each case) showed the highest binding of Trp-P-1 (50 μg ml^?1); glucan adsorbed virtually all of the Trp-P-1. Cytoplasm also showed some binding but was much less effective. Glucans also bound well with 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo 4,5-quinoxaline (MeIQX) much more than CW, but 2-amino-5-phenylpyridine (Phe-P-1) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MelQ) were not effectively bound. The quantity of IQ, MeIQ, Phe-P-1 and MeIQX bound was dependent on the strain of yeast. The mutagenic pyrolysis products bound to cells were effectively extracted by aqueous methanol, ammonia (50 g litre^?1) and alcohol, but not by water. The binding was pH dependent and inhibited by metal salts. When yeast cells were heated to 100° for 15 min, the binding of Trp-P-1 decreased by about 30% but Saccharomyces cerevisiae 50 heated to 100° did not differ much from untreated cells in its binding ability.     

Surfome analysis of a wild-type wine Saccharomyces cerevisiae strain

The yeast Saccharomyces cerevisiae, besides being an eukaryotic cell model, plays a fundamental role in the production of fermented foods. In the winemaking industry, yeast cell walls may be involved in numerous processes and contribute substantially to the final chemical and sensorial profiles of wines. Nonetheless, apart from mannoproteins, little is known on the protein components of the yeast cell wall and their changes during the fermentation of must into wine. In this work, we performed a dynamic analysis of the cell surface proteome (surfome) of an autochthonous wine yeast strain (previously selected as a wine fermentation starter) by shaving intact cells with trypsin and identifying tryptic peptides by means of nLC-ESI-LIT-MS/MS. Out of the 42 identified proteins, 16 and 14 were found to be specifically expressed in wine yeast surfome at the beginning and at the end of fermentation, respectively. The molecular functions of these specifically expressed proteins might help in explaining their roles in the cell wall as a response to the alcoholic fermentation-related stresses. Additionally, we provided the identification of 20 new potential cell wall related proteins. Globally, our results might provide new useful data for the selection and characterization of yeast strains to be used in the winemaking industry.     

COMPARISON OF METHODS FOR THE DETERMINATION OF CELL VIABILITY IN STORED BAKER'S YEAST

The classical plate method for discriminating between viable and nonviable yeast cells in stored baker's yeast was compared with dead cell staining techniques using methylene blue and three fluorochrome stains. The increase of dead yeast cells during storage of baker's yeast for up to 16 days at 5°C, 20°C and 35°C was determined. During prolonged storage, especially at 35°C, the death rate increased rapidly and the yeast cake began to soften because of autolysis. In these conditions the choice of method for determining the proportion of dead cells proved to be of great importance. Useful results for yeast stored for some time at 35°C could be obtained only by the fluorochrome technique using primuline, acridine orange or acriflavine as fluorochromes.     

Phenotypic analysis of gene deletant strains for sensitivity to oxidative stress

Ascertaining the impact of inhibitors on the growth phenotype of yeast mutants can be useful in elucidating the function of genes within the cell. Microtitre plates and robotics have been used to screen over 600 deletions from EUROSCARF, constructed in an FY1679 strain background, for sensitivity to various oxidants. These included the inorganic hydroperoxide, H(2)O(2), an organic peroxide (cumene hydroperoxide) and a lipid hydroperoxide (linoleic acid hydroperoxide). These produce within the cell several different reactive oxygen species that can cause damage to DNA, proteins and lipids. Approximately 14% of deletants displayed sensitivity to at least one of the oxidants and there was also a distribution of deletants that showed sensitivity to all or different combinations of the oxidants. Deletants included genes encoding proteins involved in stress responses, heavy metal homeostasis and putative cell wall proteins. Although global mechanisms have been identified that provide general stress responses, these results imply that there are also distinct mechanisms involved in the protection of the cell against specific damage caused by different oxidants. Further analysis of these genes may reveal unknown mechanisms protecting the cell against reactive oxygen species.     

APPLICABILITY OF ALGINATE ENTRAPPED YEAST CELLS FOR THE PRODUCTION OF LACTOSE-HYDROLYZED MILK

To overcome the problem of enzyme extraction and poor permeability of cell membrane to lactose, permeabilized Kluyveromyces marxianus NCIM 3465 cells as a source of β‐D‐galactosidase were employed for the production of lactose‐hydrolyzed milk. In view of the advantages of an immobilized cell system over a free cell system, the yeast cells were entrapped in alginate gel for their subsequent use in lactose hydrolysis. Different process parameters (alginate concentration, bead size, biomass load, temperature, agitation and incubation time) were monitored to enhance lactose hydrolysis in milk. Maximum lactose hydrolysis (87.9%) was observed with yeast cells immobilized in 2% (w/v) alginate concentration with a bead size of 2.90 mm at 30C under agitation (80 rpm) after 150 min of incubation. The developed system was highly stable and the alginate entrapped yeast cells can be recycled up to the eight cycle without any marked change in their ability to carry out the lactose hydrolysis.     

Use of Residual Yeast Cell Wall for New Biobased Materials Production: Effect of Plasticization on Film Properties

The use of renewable resources to develop food contact materials, such as proteins or polysaccharides, and the use of industrial residues for alternative applications are trending topics for researchers and the industry. Yeast cell wall (YCW) is a very rich waste from the yeast extract industry. Due to this, the aim of this work is to develop new biodegradable films based on residual YCW and the study of the effect of plasticization on films properties. Residual YCW was used as base matrix and different concentrations of glycerol (0, 15, 25 and 35 wt%) were tested to obtain casted films. Homogeneous and yellow-brownish films, which allow seeing through them, were obtained from the YCW. Total soluble matter demonstrated that glycerol enhanced solubility of films but glycerol was retained in the polymer matrix. TGA studies indicated that YCW films exhibited substantial degradation at temperatures above 180 °C. FTIR spectra of the casted films were representative of yeast cell wall material and SEM photographs showed that cell wall maintained their shape after film formation. As expected, Young’s modulus and tensile strength values were decreased with the increasing amount of glycerol. However, elongation at break was not increased further with higher concentration of plasticizer and the addition of 15 wt% of glycerol seemed to be enough to improve mechanical properties. The linear increment of water vapour permeability with glycerol concentration was produced by the increase in water solubility in the film. Therefore, based on solubility in water, mechanical, and barrier properties, it is possible to propose yeast cells residues as film-forming material for biodegradable film developments.     

Translational regulator RpL10p/Grc5p interacts physically and functionally with Sed1p,a dynamic component of the yeast cell surface

Biogenesis of an active ribosome complement and a dynamic cell surface complement are two major determinants of cellular growth. In yeast, the 60S ribosomal subunit protein RpL10p/Grc5p functions during successive stages in ribosome biogenesis, specifically rRNA processing, nucle(ol)ar preribosomal subunit assembly, nucleo-cytoplasmic transport and cytoplasmic maturation of ribosomes. Here, we report that a two-hybrid screen identified yeast genes SED1, ACS2 and PLB3 as encoding proteins physically interacting with both ribosomal RpL10p/Grc5p and its human homologue hRpL10p/QMp. SED1 encodes a differentially expressed cell wall protein which is proposed to be first transiently secreted to the plasma membrane as a GPI (glycosylated derivative of phosphoinositol)-anchored form and to be then transferred to the glucan layer of the cell wall. Ectopic expression of SED1 rescues both the aberrant growth phenotype and the translation defect of grc5-1(ts) temperature-sensitive cells. Furthermore, we report that Sed1p associates with translating ribosomes suggesting a novel, cytoplasmic role for Sed1p. ACS2 encodes one of the two yeast acetyl-CoA synthases and represents a key enzyme in one of several metabolic routes to produce acetyl-CoA, which in turn is indispensable for lipid biosynthesis. PLB3 encodes a phospholipase, which is active in the breakdown of membrane lipids. Our results support the view that Grc5p/RpL10p links ribosome function to membrane turnover and cell surface biogenesis.     

ON THE MEASUREMENT OF THE FLOCCULATION CHARACTERISTICS OF BREWERS' YEAST

The capacity of certain yeast strains to flocculate is important to the brewing industry. So is the determination of the flocculation characteristics of a yeast strain. In this study we subdivided the flocculation characteristics into three phenomena. A proposal for the most suitable method to quantify each phenomenon is given. For this, four parameters (bond strength, floc size, settling rate and number of single cells) that serve as a measure to these phenomena have been studied. Next to this, attention is payed to the influence of environmental conditions (temperature, calcium concentration, pH and the hydrodynamic conditions during the test) on the result of the test. During this part of the study the flocculence of the yeast cells was constant, so the effect of the yeast on the results of the test is excluded. It turned out that the temperature of the medium and the hydrodynamic conditions during the test most strongly influence floc formation. Next to this, medium viscosity is important if the flocculation characteristics are quantified via settling experiments.     

SOME CONSIDERATIONS OF THE FLOCCULATION CHARACTERISTICS OF ALE AND LAGER YEAST STRAINS*

While some ale yeast strains are able to flocculate when cultured in a defined medium of glucose, ammonium salts, vitamins and ions, others require the presence of a nitrogen-containing inducer in the growth medium. On the other hand, all flocculent lager strains examined to date are able to flocculate after being cultured in a defined medium and do not appear to require the addition of inducer material to the growth medium. The inducer material present in wort has been identified as peptide. By the use of ion exchange chromatography the peptide fraction that induces flocculation has been found to contain a high level of acidic amino acid residues with a very similar structure to that reported for the α-factor involved in sexual agglutination of haploid α and a cells of Sacch. cerevisiae. Studies on the adsorption of Ca⁺⁺ ion by the cell wall failed to reveal any significant differences in total uptake between flocculent and non-flocculent cultures. It would appear that Ca⁺⁺ ions are bound less tightly by non-flocculent cells than by flocculent cells. The contribution of calcium to flocculation is not the absolute amount of this ion adsorbed by the yeast cell wall but rather the stereo-specific manner by which it is bound, i.e., its position relative to the three-dimensional structure of the yeast cell wall.     

Use of FT-NIR and XPS techniques to distinguish cell hull fractions prepared by autolysis or HPH from Saccharomyces cerevisiae and Brettanomyces bruxellensis strains

Yeast hulls can be used to adsorb undesirable compounds such as volatile phenols that may be present in wine. To understand this adsorption process, the properties of the cell walls and their chemical composition need to be better understood. A study was conducted using four different yeast fractions of either autolysed or high-pressure homogeniser (HPH)-crushed yeast biomasses of Saccharomyces cerevisiae and Brettanomyces bruxellensis. Near-infrared spectroscopy (FT-NIR) coupled with X-ray photoelectron spectroscopy (XPS) was used and analysed by principal component analysis (PCA). The FT-NIR spectral region of Saccharomyces and Brettanomyces statistically analysed by PCA showed a clear discrimination accounting for 76% of the variation in the data for PC1; moreover, yeast hulls prepared from the same strain and subjected to two different treatments were also separated. These methods classify yeast cell hulls (YCH) according to strain, composition and treatment applied. Our results indicate that yeast hulls obtained by autolysis are less rich in proteins than those resulting from HPH treatment due to the high pressure that releases more proteins and exposes them on the surface of the cell wall. The composition of YCH at the extreme surface is similar to that found deeper in the wall.     

THE STRUCTURE,SYNTHESIS AND FUNCTIONS OF THE YEAST CELL WALL—A REVIEW

Glucan, mannan, chitin, protein, phosphate and lipid are found in the walls of all brewing yeasts so far examined although the contents and composition of each varies, as does the content of wall in the cell. Both glucan and mannan have branched structures and are linked to protein. Chitin is probably also bound to protein and phosphate to mannan. Some of the enzymic activities present in the wall are associated with the mannan-protein fraction. This fraction is located on the outer surface of the wall and appears to play a part in the appearance of flocculence in brewing yeast during fermentation.     

Specific Labelling of Cell Wall Proteins by Biotinylation. Identification of Four Covalently Linked O-mannosylated Proteins of Saccharomyces cerevisiae

Intact Saccharomyces cerevisiae cells were biotinylated with the non-permeable sulfosuccinimidyl-6-(biotinamido)hexanoate reagent. Twenty specifically labelled cell wall proteins could be extracted and visualized on SDS gels via streptavidin/horseradish peroxidase. Nine cell wall proteins were released by SDS extraction under reducing conditions and were designated Scw1–9p for (soluble cell wall proteins); five proteins were released from SDS-extracted cell walls by laminarinase (Ccw1–5p for covalently linked cell wall proteins) and six with mild (30 mm-NaOH, 4°C, 14 h) alkali treatment (Ccw6–11p). N-terminal sequences of the Ccw proteins 6, 7, 8 and 11 showed that these cell wall proteins are members of the PIR gene family (predicted proteins with internal repeats), CCW6 being identical to PIR1 and CCW8 to PIR3. Single gene disruptions of all four genes did not yield a phenotype. In the CCW11 disruption the Ccw11p as well as the laminarinase-extracted Ccw5 protein was missing. The new cell wall proteins are O-mannosylated, contain a Kex2 processing site, but no C-terminal GPI anchor sequence. © 1997 John Wiley & Sons, Ltd.     

EFFECT OF ENVIRONMENTAL CONDITIONS ON FLOCCULATION AND IMMOBILISATION OF BREWER'S YEAST DURING PRODUCTION OF ALCOHOL-FREE BEER

A method using immobilised yeasts has been developed and successfully applied for production of alcohol-free beer. The influence of environmental conditions present during alcohol-free beer production on the flocculation and immobilisation of the yeast Saccharomyces cerevisiae var. uvarum was investigated in the present study. In wort, the cells developed flocculation at the end of exponential growth, according to the NewFlo phenotype. In defined medium, the flocculation capacity appeared to be temporary and was lost rapidly during the stationary phase. No increase in cell wall hydrophobicity at the onset of flocculation was observed in either medium. Low growth temperatures increased flocculation capacity approximately four-fold, compared to growth at high temperatures. The optimum temperature for flocculation was at 25°C with cells grown at low or high temperature . A novel method using carboxyfluorescein-stained cells was developed to analyse the initial adhesion of cells to carrier. This method also allowed rapid analysis of the effects of immobilisation to DEAE-cellulose carrier during alcohol-free beer production process. It appeared that a high flocculation capacity stimulated adhesion to the DEAE-carrier .     

SYNTHESIS OF LIPIDS BY CERTAIN YEAST STRAINS GROWN ON WHEY PERMEATE

Four yeast strains, Apiotrichum curvatum ATCC 10567, Cryptococcus albidus ATCC 56297, Lipomyces starkeyi ATCC 12659, and Rhodosporidium toruloides ATCC 10788, were grown on whey permeate in order to synthesize lipid. Lipomyces starkeyii ATCC 12659 was identified as a high producer of lipid. It synthesized 36.9% lipids based on dry cell mass at a carbon:nitrogen ratio of 30:1 in shake-flask experiments. The significant (p < 0.05) effect of both carbon:nitrogen ratio and yeast strain was observed on dry cell mass yield and lipid biosynthesis. All yeast strains except Rhodosporidium toruloides ATCC 10788 produced more triacylglycerols than phospholipids.     

THE RESPONSE OF BREWING YEASTS TO ACID WASHING

Brewing yeasts are inherently resistant to acid washing treatments but, under some conditions this resistance is diminished. Sixteen yeast strains, including both ale and lager strains, have been successfully washed using phosphoric acid (pH 2.1) and, in some cases, sulphuric acid (pH 2.0) or acidified 0.75% w/v ammonium persulphate (pH 2.8). Cell viability, fermentation performance and flocculation/fining behaviour were unaffected. However, changes could be observed in the yeast resulting from the washing treatment. These included alterations of the cell surface, as shown by scanning electron microscopy and leakage of adenosine triphosphate from the cells during the wash. Acid washing is successful provided that, i) food grade acid is used, ii) the acid is chilled before use, iii) the yeast and acid are well mixed, iv) the temperature of the yeast does not exceed 5°C during washing and, v) the yeast is pitched immediately after washing. ‘Unhealthy’ yeast (yeast which has been stored for long periods, heavily contaminated yeast, or yeast from slow fermentations) may respond poorly to acid washing and a shorter washing time, or higher wash pH value should be employed.     

Finnish Fermented Milk “Viili”: Involvement of Two Cell Surface Proteins in Production of Slime by Streptococcus lactis ssp. cremoris

The cell surface protein profile of the slime-forming, encapsulated (mucoid) Streptococcus lactis ssp. cremoris T₅ from “viili” is compared with two variants of the same strain that do not produce slime: strain T₅/30 was obtained by using an elevated growth temperature of 30°C and the nonmucoid strain T₅/NS spontaneously at the growth temperature of 17°C.The profiles of cell surface proteins extractable by Triton X-100 were studied by the immunoblotting method. The spontaneous change of T₅ phenotype to T₅/NS brought about appreciable diminution in four and a loss in two cell surface polypeptides. Four of these, with molecular weights of 70,000, 54,000, 47,000, and 40,000, have previously been found to be located in the cell wall of two slime-forming, encapsulated strains. The polypeptides with molecular weights of 42,000 and 26,000 are present in the slime-forming mucoid variant T₅ and also in T₅/30 cells but absent from the nonmucoid T₅/NS-cells. These two polypeptides may have some role in slime production and mucoidness and remain to be studied further. These analyses provide useful information for the purification and further characterization of surface antigens connected with slime production or excretion by the mucoid phenotype S. lactis ssp. cremoris and facilitate the selection of slime-forming variants for dairy starters.     

INVESTIGATION OF THE MECHANICS OF SINGLE TOMATO FRUIT CELLS

The mechanical behavior of single tomato fruit cells has been characterized using high strain‐rate microcompression testing. Single cells isolated by gentle washing from inner pericarp tissue were compressed to a wide range of deformations at a speed of 1500 µm/s, and then released. The cells were larger than any tested previously by microcompression, and had very low initial turgor. Force‐deformation data were modeled to find cell wall material properties, assuming water loss during compression could be neglected because of fast compression. Repeat compression‐release experiments were conducted to discover when cell deformation was no longer recoverable upon release. Cells from three commercially grown tomatoes were elastic to deformations of just over 11%. The elastic moduli of the cell walls were found by modeling to be 30 to 80 MPa, significantly lower than suspension‐cultured cell walls. The cell walls yielded at about 2% wall strain. High‐speed compression testing is a powerful tool for studying low turgor cells, such as those found during ripening.