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Identification of yeast-derived emulsification proteins through analyses of proteins distributed into the emulsified phase
Abstract
Emulsifiers are widely used in food manufacturing, and cell wall mannoproteins from Saccharomyces cerevisiae, a representative food yeast, have been proposed as potential new emulsifiers. However, their use has not become widespread. One reason is that the protein responsible for emulsification has not been identified so no efficient preparation method has been established. In this study, the emulsification substances were identified. Many mannoproteins are fixed to the cell wall via a glycosylphosphatidylinositol (GPI) anchor. Because emulsifying activity was found in the culture supernatant (CS) in gup1Δ with a mutation of GPI anchor synthesis, a protein distributed from the CS into the emulsified phase by emulsification treatment was analyzed. Using mass spectrometry, it was identified to be Gas1, a mannoprotein localized to the cell wall via a GPI anchor. Further, recombinant Gas1 without a C-terminal GPI anchor binding signal was purified, and it showed activity similar to bovine serum albumin, a well-known protein emulsifier. Gas1 also retained its activity under a wide range of pH and high salt conditions. Saccharomyces cerevisiae has Gas3 and Gas5, proteins homologous to Gas1, and recombinant Gas3 and Gas5 without the C-terminal signal were also purified and found to have emulsifying activity similar to that of recombinant Gas1. These results strongly suggest that Gas proteins take part in the emulsifying activity of yeast mannoproteins. It was also demonstrated that emulsification proteins could be identified by analyzing proteins distributed in an emulsified phase, so this method will be effective in finding new emulsification proteins.
... Although the first evidence of yeast cells stabilising Pickering droplets dates back from 1970s from oil sector [39] and many studies thereafter involved using kerosene [40,41] and hexadecane [21,35,36] to showcase Pickering effects of yeast cells (Table 1) (2) and (3) results in conventional molecularly adsorbed emulsions. The proteins that are metabolically engineered using yeast cells as host organisms (cell factories) for protein production [23] are out of scope for this review as indicated in the red box. ...
... The BSY extracts pp 4M and pp 200 • C also presented higher stability at acidic pH (pH 3) and ultraturrax stirring when compared to the reference emulsifiers, possibly due to the lower solubility of xanthan gum (pKa of 3.1) and poor stability of lecithin, the main emulsifying agent in egg yolk [3]. The stability of pp 4M and pp 200 • C, together with the neutral charge of β-glucans, also suggested that the proteins contributing to their emulsification properties belong to the mannose-rich glycosylphosphatidylinositol (GPI)anchored proteins family of yeast, proteins that yield emulsions resilient to pH variations (3-7) [22]. The improvement of the stability of pp 4M emulsion could be explained by the high energy associated to ultraturrax, which promotes the incorporation of short chain mannans and β-glucans into the emulsion. ...
Brewer's spent yeast (BSY) mannoproteins have been reported to possess thickening and emulsifying properties. The commercial interest in yeast mannoproteins might be boosted considering the consolidation of their properties supported by structure/function relationships. This work aimed to attest the use of extracted BSY mannoproteins as a clean label and vegan source of ingredients for the replacement of food additives and protein from animal sources. To achieve this, structure/function relationships were performed by isolating polysaccharides with distinct structural features from BSY, either by using alkaline extraction (mild treatment) or subcritical water extraction (SWE) using microwave technology (hard treatment), and assessment of their emulsifying properties. Alkaline extractions solubilized mostly highly branched mannoproteins (N-linked type; 75%) and glycogen (25%), while SWE solubilized mannoproteins with short mannan chains (O-linked type; 55%) and (1→4)- and (β1→3)-linked glucans, 33 and 12%, respectively. Extracts with high protein content yielded the most stable emulsions obtained by hand shaking, while the extracts composed of short chain mannans and β-glucans yielded the best emulsions by using ultraturrax stirring. β-Glucans and O-linked mannoproteins were found to contribute to emulsion stability by preventing Ostwald ripening. When applied in mayonnaise model emulsions, BSY extracts presented higher stability and yet similar texture properties as the reference emulsifiers. When used in a mayonnaise formulation, the BSY extracts were also able to replace egg yolk and modified starch (E1422) at 1/3 of their concentration. This shows that BSY alkali soluble mannoproteins and subcritical water extracted β-glucans can be used as replacers of animal protein and additives in sauces.
... Due to their molecular structure being highly mannosylated, YCW MNPs are integrated as additives in the food industry. Besides their use as bioemulsifiers 10,11 and food stabilizers, 12 they are mainly known for their enological application, 13 thanks to their complexation with phenolic compounds, 14,15 the inhibition of tartrate salt crystallization, and the influence of wine aspect both by prevention of haze formation and promotion of yeast flocculation. 16 Moreover, YCW MNPs have shown interesting health-promoting features by various mechanisms, such as their antioxidant 17 and immunomodulatory roles through interactions with the host immune system, 18 as well as a reported antitumor action. ...
Saccharomyces cerevisiae yeast is a fungus presenting a peripheral organelle called the cell wall. The cell wall protects the yeast cell from stress and provides means for communication with the surrounding environment. It has a complex molecular structure, composed of an internal part of cross-linked polysaccharides and an external part of mannoproteins. These latter are very interesting owing to their functional properties, dependent on their molecular features with massive mannosylations. Therefore, the molecular characterization of mannoproteins is a must relying on the optimal isolation and preparation of the cell wall fraction. Multiple methods are well reported for yeast cell wall isolation. The most applied one consists of yeast cell lysis by mechanical disruption. However, applying this classical approach to S288C yeast cells showed considerable contamination with noncell wall proteins, mainly comprising mitochondrial proteins. Herein, we tried to further purify the yeast cell wall preparation by two means: ultracentrifugation and Triton X-100 addition. While the first strategy showed limited outcomes in mitochondrial protein removal, the second strategy showed optimal results when Triton X-100 was added at 5%, allowing the identification of more mannoproteins and significantly enriching their amounts. This promising method could be reliably implemented on the lab scale for identification of mannoproteins and molecular characterization and industrial processes for "pure" cell wall isolation.
Introduction:
A previous study demonstrated a strong emulsification ability of the culture supernatant obtained by cultivation of Candida albicans in a medium containing a β-1,3-glucan synthesis inhibitor and proposed a novel screening method using emulsification as an indicator for β-1,3-glucan synthesis inhibition (Nerome et al., 2021. Evaluating β-1,3-glucan synthesis inhibition using emulsion formation as an indicator. J Microbiol Methods. 190:106327). The emulsification was presumed to be caused by the proteins released from the cells; however, which proteins have a strong emulsification ability was unclear. Furthermore, as many cell wall proteins are connected to β-1,3-glucan via the carbohydrate moiety of the glycosylphosphatidylinositol (GPI)-anchor, which remains when detached from the cell membrane, emulsification might be detected by inhibiting GPI-anchor synthesis.
Objective:
This study aimed to confirm whether emulsification could be detected by inhibiting GPI-anchor synthesis and identifying emulsification proteins released by inhibiting the synthesis of GPI-anchor or β-1,3-glucan.
Methods:
C. albicans was cultured in a medium containing a GPI-anchor synthesis inhibitor, and the emulsification by the culture supernatant was evaluated. We identified cell wall proteins released from the cells upon inhibition of β-1,3-glucan or GPI-anchor synthesis by mass spectrometry, their recombinant proteins were prepared, and their emulsification efficacy was evaluated.
Results:
In GPI-anchor synthesis inhibition, a weak emulsification phenomenon was observed compared to the β-1,3-glucan synthesis inhibition. Phr2 protein was released from the cells upon GPI-anchor synthesis inhibition, and recombinant Phr2 showed a strong emulsification activity. Phr2 and Fba1 proteins were released upon β-1,3-glucan synthesis inhibition, and recombinant Fba1 showed a strong emulsification activity.
Conclusions:
We concluded that the emulsion phenomenon could be used to screen β-1,3-glucan and GPI-anchor synthesis inhibitors. Also, the two kinds of inhibitors could be distinguished by differences in the growth recovery by osmotic support and strength of emulsification. In addition, we identified the proteins involved in emulsification.
Introduction
The cell wall β-1,3-glucan of fungal pathogen C. albicans is an attractive antifungal target. β-1,3-Glucan is the skeletal structure in the cell wall and the major scaffold for cell wall proteins. In previous studies using Saccharomyces cerevisiae, strong emulsification was detected by mixing cell wall proteins with oil. To date, there have been no reports of applying an emulsification phenomenon to assessing β-1,3-glucan synthesis inhibition.
Objective
The aim of this study was to clarify that emulsification is useful as an indicator for evaluating β-1,3-glucan synthesis inhibition in C. albicans.
Methods
At first, whether cell wall proteins released from cells by β-1,3-glucanase treatment worked as an effective emulsifier in C. albicans was examined. Next, whether emulsification occurred even in the culture supernatant brought about by treating with bioactive compounds, including β-1,3-glucan synthesis inhibitors, under osmotic protection was investigated. In addition, the release of cell wall proteins into the culture medium by treating with those compounds was examined. Finally, a simpler evaluation method using emulsion formation was examined for application to screening of inhibitors.
Results
Emulsification occurred by cell wall proteins obtained by treating with β-1,3-glucanase in C. albicans. In addition, cell wall proteins were released into the culture medium by treating with β-1,3-glucan synthesis inhibitors, resulting in emulsification. However, such phenomena were not observed in the case of other bioactive compounds. Furthermore, emulsification could be detected in the culture broth obtained by static culture on a small scale.
Conclusions
The obtained results strongly implied that emulsification results from decreased β-1,3-glucan levels in the cell wall. As emulsification can be simply evaluated by mixing the culture broth with oil, in the future application to the initial assessment and screening of β-1,3-glucan synthesis inhibitors is expected.
BACKGROUND
Chlorella protothecoides is one of the most widely commercialized and studied microalgae species. Recent research reported improved emulsifying properties of the insoluble protein fraction from C. protothecoides after thermal–acid treatment.
RESULTS
In this research, we studied the influence of ionic strength (sodium chloride 50–500 mmol L⁻¹ or calcium chloride 5–50 m mol L⁻¹) and pH (2–9) on the stability of oil‐in‐water emulsions prepared by 3% (w/w) of the untreated insoluble microalgae protein fraction or hydrolysates obtained after treatment with hydrochloric acid at 65 °C (Hydrolysates 65) or 85 °C (Hydrolysates 85) for 4 h. The emulsions were prepared by mixing 10% (w/w) oil and homogenized at 68.9 MPa. The ionic strength and pH were, subsequently, adjusted. The mean particle diameter of emulsions remained constant despite extensive variations in ionic strength or pH. Emulsion droplets stabilized by Hydrolysates 85 were stable against coalescence at all ionic strengths or pH values tested.
CONCLUSION
The results indicate a high potential to use acid‐hydrolyzed insoluble microalgae protein fractions for the formulation of various emulsion‐based food systems. © 2020 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
BACKGROUND
Glycosylated compounds are one of the main fractions of the yeast cell wall. Thanks to their amphiphilic structure, they have been studied as stabilizers in food emulsions over a broad range of pH conditions with encouraging results. Nevertheless, extraction costs still represent an important limit for their application in the food industry.
RESULTS
In this research, four extraction methods were applied to yeast cells exploiting both physical (heating and sonication) and enzymatic approaches (use of three industrial enzyme preparations, namely Glucanex®, Sur Lies and Elevage). A fifth method involving a pure β‐glucanase enzyme (Zymolyase) was taken as reference. These extraction methods were applied to the oenological strain Saccharomyces cerevisiae EC1118, and their extraction yields and chemical properties (quantitative and qualitative determination of sugars and proteins) were studied. Emulsifying activities were determined at three different pH values (3, 5 and 7). Extractions with Physical, Glucanex and Sur Lies methods were the most successful approaches to obtain relevant amounts of yeast compounds with good emulsifying activities for 2:1 oil‐in‐water emulsions at pH 3 and 7 over 48 h.
CONCLUSIONS
These results indicate that there is the potential for the extraction approaches here proposed to become viable tools for the recovery of yeast compounds to be used as emulsifiers in foods. This approach can be considered as the starting point to explore the possibility to exploit yeast by‐products from the fermentation processes (e.g. fermentation lees from wine and beer making) as valuable compounds for food applications. © 2019 Society of Chemical Industry
As the most abundant protein in chicken eggs, ovalbumin plays an important role in the processing of high value-added poultry products. The present study investigated the effects of hydroxyl radical-induced early stage oxidation on the physicochemical and interfacial properties of chicken egg white ovalbumin. Protein carbonyl content of ovalbumin increased (from 0.78 to 1.13 nmol/mg) with the oxidation time (0–5 h), while free sulfhydryl content (from 0.43 to 0.09 nmol/mg) and free amino group content (from 0.49 to 0.43 nmol/mg) decreased. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that the exposure of ovalbumin to hydroxyl radicals caused self-cross-linking and resulted in the formation of dimers and trimers. Accompanied by these changes, the surface hydrophobicity of ovalbumin was enhanced about 1.5-fold with the deepening of oxidation, and the value of zeta potential became more negative (from –7.15 to –20.51 mv). About 2 h of moderate oxidation improved the foaming and emulsifying properties of ovalbumin (1.2-fold to 1.8-fold), while excessive oxidation (3 h) decreased these interface properties. Hydroxyl radical-induced oxidation changed the surface chemical groups and structures of ovalbumin, thereby affecting the surface properties. The foaming and emulsifying properties of ovalbumin could be improved by oxidation, increasing the application possibilities of ovalbumin in the food interface system.
During downstream operations involved in the purification of hydrophobic biofuels produced by microorganisms, undesired stable emulsions may be formed. Understanding the mechanisms behind this stability is a pre-requisite for designing cost-effective strategies to break these emulsions. In this work, we aimed at increasing our knowledge on the mechanisms responsible for stabilizing yeast-containing oil-in-water emulsions. For this purpose, emulsions containing hexadecane and different yeast-based aqueous phases were prepared and analyzed for phase separation, surface charge density, particle size, and rheology. First, we observed that compounds present in fresh tablet baker’s yeast contribute to emulsion stability. In order to eliminate this effect, we generated stocks with this yeast in the laboratory, and compared its performance with an industrial fuel ethanol strain, namely Saccharomyces cerevisiae PE-2. We confirmed that the presence of yeast cells enhances emulsion stability. The cultivation medium (complex or defined) in which cells are grown, as well as the physiological state of the cells (pre- or post-diauxic), prior to emulsion preparation, influenced emulsion stability. The smaller cell size of tablet yeast probably also contributes to more stable emulsions, when compared to those prepared with yeast cells grown in the laboratory. Baker’s and fuel ethanol yeast cells in post-diauxic phase promote the formation of more stable emulsions than those with cells in the pre-diauxic physiological state. Finally, we propose a mechanism to explain the enhanced emulsion stability due to the presence of yeast cells, with electrostatic repulsion between emulsion droplets having the prevailing effect.
Saccharomyces cerevisiae KA01 was isolated from palm wine obtained from a local brewer in Songkhla province, Thailand. Mannoproteins with emulsification properties were extracted from the cell walls of S. cerevisiae KA01 cultivated in YM medium by autoclaving in a pH 7.0 citrate buffer for 60 min with a yield of 0.32 g/g wet cells. The mannoprotein extract obtained was evaluated for its chemical and physical stability to establish its potential use as a natural emulsifier in processed foods. The mannoprotein extract exhibited emulsion with the vegetable oils tested and showed emulsion activity of 65% towards palm oil as oil-in-water with a critical emulsifier concentration of 20 g/l. The mannoprotein extract had similar emulsifying properties to the commonly used food emulsifiers gum arabic and lecithin. Palm oil-in-water emulsions were stabilized over a broad range of conditions from pH 5 to 8 with up to 3% (w/v) sodium chloride and up to 0.1% (w/v) CaCl2 and MgCl2 in the aqueous phase. Temperature did not affect the emulsion activity of the mannoprotein extract. Preliminary trials showed that mannoproteins from S. cerevisiae KA01 had potential for use in salad dressing.
The GAS multigene family of Saccharomyces cerevisiae is composed of five paralogs (GAS1 to GAS5). GAS1 is the only one of these genes that has been characterized to date. It encodes a glycosylphosphatidylinositol-anchored protein functioning as a beta(1,3)-glucan elongase and required for proper cell wall assembly during vegetative growth. In this study, we characterize the roles of the GAS2 and GAS4 genes. These genes are expressed exclusively during sporulation. Their mRNA levels showed a peak at 7 h from induction of sporulation and then decreased. Gas2 and Gas4 proteins were detected and reached maximum levels between 8 and 10 h from induction of sporulation, a time roughly coincident with spore wall assembly. The double null gas2 gas4 diploid mutant showed a severe reduction in the efficiency of sporulation, an increased permeability of the spores to exogenous substances, and production of inviable spores, whereas the single gas2 and gas4 null diploids were similar to the parental strain. An analysis of spore ultrastructure indicated that the loss of Gas2 and Gas4 proteins affected the proper attachment of the glucan to the chitosan layer, probably as a consequence of the lack of coherence of the glucan layer. The ectopic expression of GAS2 and GAS4 genes in a gas1 null mutant revealed that these proteins are redundant versions of Gas1p specialized to function in a compartment at a pH value close to neutral.
A previous study revealed that Saccharomyces cerevisiae mcd4Δ, a cell wall mutant with a defect in the synthesis of the glycosylphosphatidylinositol anchor, has a strong macrophage activation ability. In this study, remarkable emulsion formation after cell suspensions of mcd4Δ and anp1Δ (which exhibit an extreme reduction of mannan) were mixed with oil was found. Moreover, the relationship between cell wall mutation and emulsion formation was investigated, suggesting that och1Δ with a defect in the formation of N-linked glycans also had a strong emulsification ability and that high molecular weight materials released from the cells were involved in emulsion formation. Furthermore, two strains (asc1Δ and scp160Δ) with a strong emulsification ability without a large decrease in mannan content were also found from the wide screening of strains that exhibit an emulsifying activity using more than 5000 gene-deficient strains. These results provide valuable information for the development of a yeast-derived emulsifier.
Yam soluble protein (YSP) has been reported to have many physiological activities, such as scavenging free radicals, immune activation, and anti-hypertensive activities. Protein solubility and emulsifying activity are important protein-associated functional properties for the application of proteins in food systems. During this study, the factors of protein concentration, pH, temperature and salt concentration that influenced the solubility of YSP were investigated. As a result, the solubility was minimal near its isoelectric point (pH 3.5) and was highest at 45 °C in a temperature range of 40–60 °C. With an increase of protein concentration, the solubility decreased. According to the results of response surface methodology analysis, the interaction between pH and temperature on the solubility of YSP was significant, and the maximum solubility (87.5%) was obtained when the temperature was close to 40 °C, the pH was approximately 7 and the NaCl concentration approached 0.5 mol/L. As the protein concentration increased, the average particle size of the YSP emulsion decreased, and the particle size distribution gradually became balanced. Additionally, the microphotograph of the YSP emulsion reflected its distribution. The results of this study will provide data and a theoretical basis for the understanding of YSP’s physicochemical properties and its application in the food industry.
Broad molecular weight (MW) distribution and variability of mannan to protein ratio of purified mannoproteins (MP), isolated from yeast cell walls upon the enzymatic treatment, revealed their multiplicity. The main fraction of high-MW Agrimos®-MP1 and YCW-b-MP1′ contained mannoproteins with a mannan to protein ratio of 3.5 and 6.9, respectively. Low-MW YCW-b-MP2′ was mainly comprised of mannan, with a ratio of 181, whereas low-MW Agrimos®-MP2 was characterized by a ratio of 12.2. The solubility of MP1/MP2 was higher than that of MP1′/MP2′. Mannoproteins showed similar or lower solubility than mannan, and they exhibited a Newtonian behaviour. Sonication was the appropriate method for the formation of mannoproteins-based emulsions. Contrary to MP1/MP1′-based emulsions, MP2/MP2′-based ones showed higher affinity towards soybean oil than glyceryl-trioleate. pH affected the emulsifying ability of MP1/MP1′. MP1/MP1′ showed similar or slightly inferior emulsifying properties than lecithin. This study is expected to broaden the applications of mannoproteins as value-added ingredients.
Sustainability driven production of food ingredients is in the center of discussion the past years, with plants being a promising source, since they are widely available and have smaller environmental impact compared to animals. However, plant material consists of a sturdy configuration comprising many components, like proteins, which cannot be readily liberated. Thus, downstream processing of plants often involves intensive physicochemical and thermal processing, which might be accompanied by alteration of protein properties, like emulsification ability. Here, the aim was to investigate the emulsification ability of the native mixtures derived from sunflower seeds, obtained via simple separation steps and link their properties with their molecular composition. The investigated molecular mixtures were the cold-pressed sunflower cake, a protein-based and a fibre-based mixture. It was demonstrated that the residual oil in both the Sf cake and the protein-based mixture was present in the form of naturally emulsified oil droplets, so-called oil bodies. Oil bodies did not have a notable impact on the interfacial activity of the samples in contrast with the destabilization effect of polysaccharides. Despite their complex composition all mixtures could efficiently stabilize oil/water interfaces, showing similar properties compared to isolated proteins. This is an intriguing bottom line regarding the necessity for using pure emulsifiers. The findings prove that molecular mixtures which contain even minor amounts of proteins, can be used as ingredients for efficient emulsion stabilization.
Consumer concern about human and environmental health is encouraging food manufacturers to use more natural and sustainable food ingredients. In particular, there is interest in replacing synthetic ingredients with natural ones, and in replacing animal-based ingredients with plant-based ones. This article provides a review of the various types of natural emulsifiers with potential application in the food industry, including phospholipids, biosurfactants, proteins, polysaccharides, and natural colloidal particles. Increased utilization of natural emulsifiers in food products may lead to a healthier and more sustainable food supply. However, more research is needed to identify, isolate, and characterize new sources of commercially viable natural emulsifiers suitable for food use. Expected final online publication date for the Annual Review of Food Science and Technology Volume 8 is February 28, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
In this work, ovalbumin (OVA) was phosphorylated by dry-heating at three different pH values (5.0, 7.0 and 9.0) and 45 °C for 12 hours in the presence of sodium tripolyphosphate (STP). The results indicated that, when compared with native OVA (N-OVA), the zeta potential absolute value of phosphorylated OVA (PP-OVA) increased from 2.13 mv (N-OVA) to 13.57(PP-OVA-pH5.0), 16.00(PP-OVA-pH 7.0) and 11.20 mv (PP-OVA-pH 9.0); the volume-weighted mean diameter(D[4,3])of emulsions stabilized by the N- and PP-OVA decreased from 70.22 μm(N-OVA) to 37.16(PP-OVA-pH 5.0), 16.21(PP-OVA-pH 7.0) and 43.52μm(PP-OVA-pH 9.0); and PP-OVA emulsions exhibited a more narrow monomodal size distribution and a smaller particle size than those of N-OVA, with the corresponding emulsifying activities index (EAI) increased from 92.71(N-OVA) to 142.51(PP-OVA-pH5.0), 224.88(PP-OVA-pH 7.0), and 150.97(PP-OVA-pH 9.0), respectively. The microscopic observation showed that the emulsion droplets of PP-OVA were more densely and uniformly distributed than those of N-OVA. Furthermore, characterization of PP-OVA by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and 31P nuclear magnetic resonance ( 31P NMR) revealed that phosphate groups were bound to the -OH on serine and threonine and the -NH2 on lysine and arginine, with covalent interactions (C-O-P and C-N-P bounds) formed between OVA and STP. This study demonstrated that the introduction of phosphate groups could increase the amount of negative charge and intermolecular repulsion, decrease the particle size of droplets and improve the emulsifying ability of PP-OVA.
The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches
are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third
of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of
more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects
in either rich or minimal medium.
In budding yeast, the neck that connects the mother and daughter cell is the site of essential functions such as organelle trafficking, septum formation and cytokinesis. Therefore, the morphology of this region, which depends on the surrounding cell wall, must be maintained throughout the cell cycle. Growth at the neck is prevented, redundantly, by a septin ring inside the cell membrane and a chitin ring in the cell wall. Here, we describe recent work supporting the hypothesis that attachment of the chitin ring, which forms at the mother-bud neck during budding, to β-1,3-glucan in the cell wall is necessary to stop growth at the neck. Thus, in this scenario, chemistry controls morphogenesis.
The wall gives a Saccharomyces cerevisiae cell its osmotic integrity; defines cell shape during budding growth, mating, sporulation, and pseudohypha formation; and presents adhesive glycoproteins to other yeast cells. The wall consists of β1,3- and β1,6-glucans, a small amount of chitin, and many different proteins that may bear N- and O-linked glycans and a glycolipid anchor. These components become cross-linked in various ways to form higher-order complexes. Wall composition and degree of cross-linking vary during growth and development and change in response to cell wall stress. This article reviews wall biogenesis in vegetative cells, covering the structure of wall components and how they are cross-linked; the biosynthesis of N- and O-linked glycans, glycosylphosphatidylinositol membrane anchors, β1,3- and β1,6-linked glucans, and chitin; the reactions that cross-link wall components; and the possible functions of enzymatic and nonenzymatic cell wall proteins.
The GAS multigene family of Saccharomyces cerevisiae is constituted by five genes (GAS1-GAS5), but GAS1 was the only one to have been characterized to date. Gas1 is a glycosylphosphatidylinositol-anchored protein predominantly localized in the plasma membrane and is also a representative of family GH72 of glycosidase/transglycosidases, a wide group of yeast and fungal enzymes involved in cell wall assembly. Gas1-Gas5 proteins share a common N-terminal domain but exhibit different C-terminal extensions, in which a domain named Cys-Box is located. This domain is similar to the carbohydrate binding module 43 and is present only in Gas1p and Gas2p. Here we report the expression in P. pastoris of soluble forms of Gas proteins. Gas1, 2, 4 and 5 proteins were secreted with a yield of about 30-40 mg/l of medium, whereas the yield for Gas3p was about three times lower. Gas proteins proved to be N-glycosylated. Purified Gas proteins were tested for enzymatic activity. Gas2, Gas4 and Gas5p showed a beta-(1,3)-glucanosyltransferase activity similar to Gas1p. A phylogenetic tree of the N-terminal regions of family GH72 members was constructed. Two subfamilies of N-terminal regions were distinguished: one subfamily, GH72(+), contains proteins that possess a Cys-box in the C-terminal region, whereas family GH72(-) comprises proteins that lack a Cys-box. On the basis of this net distinction, we speculate that the type of C-tail region imposed constraints to the evolution of the N-terminal portion.
The multigene GAS family of Saccharomyces cerevisiae is constituted by five genes encoding GPI-anchored proteins required for cell wall or spore wall assembly. GAS1 and GAS5 are expressed in vegetative growth and repressed during sporulation, whereas GAS2 and GAS4 exhibit the opposite expression pattern. This study focuses on GAS3, a still poorly characterized member of the family. To date, attempts to reveal the glucan elongase activity typical of Gas proteins have been unsuccessful, suggesting that Gas3p is the only inactive member of the family. Here, we compared the mRNA levels of GAS1, GAS3 and GAS5 and demonstrate that GAS3 is the weakest-expressed paralogue in vegetative growth. Moreover, GAS3 mRNA increased during sporulation, showing a bimodal profile typical of the early-middle meiotic genes. GAS3 product was identified as a low-abundance, polydisperse mannoprotein. Loss of Gas3p did not affect growth and sporulation. The overexpression of GAS3, driven by the GAS1 promoter, slightly reduced growth rate in a wild-type strain and led to hyperaccumulation of Gas3p in the membranes and in the cell wall. To determine whether GAS3 could replace GAS1 function in vivo, GAS3 was also overexpressed in a gas1Delta mutant. Increased amounts of Gas3p were not only unable to complement the defects of the gas1Delta cells but exacerbated them. A mutated Gas3p-E283Q, where one of the catalytic glutamate residues essential for GH72 enzyme activity was replaced by glutamine, was also noxious to gas1Delta cells, indicating that the increased expression of Gas3p, rather than a potential activity, is deleterious for gas1Delta cells.
The widely used pESC vector series (Stratagene, La Jolla, CA, USA) with the bidirectional GAL1/GAL10 promoter provides the possibility of simultaneously expressing two different genes from a single vector in Saccharomyces cerevisiae. This system can be induced by galactose and is repressed by glucose. Since S. cerevisiae prefers glucose as a carbon source, and since its growth rate is higher in glucose than in galactose-containing media, we compared and evaluated seven different promoters expressed during growth on glucose (pTEF1, pADH1, pTPI1, pHXT7, pTDH3, pPGK1 and pPYK1) with two strong galactose-induced promoters (pGAL1 and pGAL10), using lacZ as a reporter gene and measuring LacZ activity in batch and continuous cultivation. TEF1 and PGK1 promoters showed the most constant activity pattern at different glucose concentrations. Based on these results, we designed and constructed two new expression vectors which contain the two constitutive promoters, TEF1 and PGK1, in opposite orientation to each other. These new vectors retain all the features from the pESC-URA plasmid except that gene expression is mediated by constitutive promoters.
A fast yeast-transformation technique has been developed by adding thio compounds to alkali-ion based protocols and incubating at 45 degrees C. This procedure is especially recommended for cells from stationary phase at a density up to 2.5 x 10(8) cells/ml. It involves only one step for the preparation and transformation of competent cells within 30 min. The yield was more than 10(4) transformants/microgram plasmid DNA. This protocol is easy to scale up for many DNA samples and is also applicable for yeast cells from different types of storages.
The mannoprotein which is a major component of the cell wall of Saccharomyces cerevisiae is an effective bioemulsifier. Mannoprotein emulsifier was extracted in a high yield from whole cells of fresh bakers' yeast by two methods, by autoclaving in neutral citrate buffer and by digestion with Zymolase (Miles Laboratories; Toronto, Ontario, Canada), a beta-1,3-glucanase. Heat-extracted emulsifier was purified by ultrafiltration and contained approximately 44% carbohydrate (mannose) and 17% protein. Treatment of the emulsifier with protease eliminated emulsification. Kerosene-in-water emulsions were stabilized over a broad range of conditions, from pH 2 to 11, with up to 5% sodium chloride or up to 50% ethanol in the aqueous phase. In the presence of a low concentration of various solutes, emulsions were stable to three cycles of freezing and thawing. An emulsifying agent was extracted from each species or strain of yeast tested, including 13 species of genera other than Saccharomyces. Spent yeast from the manufacture of beer and wine was demonstrated to be a possible source for the large-scale production of this bioemulsifier.
A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes.
The yeast cell wall, which for years has been regarded as a static cellular component, has been revealed to be dynamic in its structure and composition and complex in its enzymatic activity. The S. cerevisiae cell wall is composed of beta-1,3/beta-1,6-glucans, mannoproteins, and chitin, which are assembled into an extracellular matrix essential for maintenance of cell integrity. Gas1p, a glycoprotein anchored to the outer leaflet of the plasma membrane through a glycosylphosphatidylinositol, plays a key role in cell wall assembly. Loss of Gas1p leads to several morphogenetic defects and to a decrease in the amount of cross-links between the cell wall glucans. These defects in turn trigger a compensatory response that guarantees cell viability. Several Gas1p homologs have been isolated from Candida species and S. pombe. The Gas1p family also includes two plant proteins with endo-beta-1,3-glucanase activity. Sequence comparisons reveal that Gas1p family proteins have a modular organization of domains. The genetic and molecular analyses reviewed here suggest that Gas1p could play a role as a polymer cross-linker, presumably by catalyzing a transglycosylation reaction.
Glycosylphosphatidylinositol (GPI)-anchored proteins are cell surface-localized proteins that serve many important cellular functions. The pathway mediating synthesis and attachment of the GPI anchor to these proteins in eukaryotic cells is complex, highly conserved, and plays a critical role in the proper targeting, transport, and function of all GPI-anchored protein family members. In this article, we demonstrate that MCD4, an essential gene that was initially identified in a genetic screen to isolate Saccharomyces cerevisiae mutants defective for bud emergence, encodes a previously unidentified component of the GPI anchor synthesis pathway. Mcd4p is a multimembrane-spanning protein that localizes to the endoplasmic reticulum (ER) and contains a large NH2-terminal ER lumenal domain. We have also cloned the human MCD4 gene and found that Mcd4p is both highly conserved throughout eukaryotes and has two yeast homologues. Mcd4p's lumenal domain contains three conserved motifs found in mammalian phosphodiesterases and nucleotide pyrophosphases; notably, the temperature-conditional MCD4 allele used for our studies (mcd4-174) harbors a single amino acid change in motif 2. The mcd4-174 mutant (1) is defective in ER-to-Golgi transport of GPI-anchored proteins (i.e., Gas1p) while other proteins (i.e., CPY) are unaffected; (2) secretes and releases (potentially up-regulated cell wall) proteins into the medium, suggesting a defect in cell wall integrity; and (3) exhibits marked morphological defects, most notably the accumulation of distorted, ER- and vesicle-like membranes. mcd4-174 cells synthesize all classes of inositolphosphoceramides, indicating that the GPI protein transport block is not due to deficient ceramide synthesis. However, mcd4-174 cells have a severe defect in incorporation of [3H]inositol into proteins and accumulate several previously uncharacterized [3H]inositol-labeled lipids whose properties are consistent with their being GPI precursors. Together, these studies demonstrate that MCD4 encodes a new, conserved component of the GPI anchor synthesis pathway and highlight the intimate connections between GPI anchoring, bud emergence, cell wall function, and feedback mechanisms likely to be involved in regulating each of these essential processes. A putative role for Mcd4p as participating in the modification of GPI anchors with side chain phosphoethanolamine is also discussed.
Most proteins involved in the synthesis of the GPI core structure of Saccharomyces cerevisiae are essential for growth. To explore the relationship between the GPI anchor structure and beta-1,6-glucan synthesis, we screened deletion mutants in genes involved in GPI synthesis for osmotic remedial growth. Heterozygous diploid strains were dissected on medium with osmotic support and slow growth of the mcd 4 deletion mutant was observed. The mcd 4 mutant showed abnormal morphology and cell aggregation, and was hypersensitive to SDS, hygromycin B and K1 killer toxin. Incorporation of GPI cell wall proteins was examined using a GPI-Flo 1 fusion protein. The result suggested that the mcd 4 deletion causes a decrease in GPI cell wall proteins levels. The mutation also caused a decrease in mannan levels and an increase in alkali-insoluble beta-1,6-glucan and chitin levels in the cell wall.
In this review, we discuss new insights in cell wall architecture and cell wall construction in the ascomycetous yeast Saccharomyces cerevisiae. Transcriptional profiling studies combined with biochemical work have provided ample evidence that the cell wall is a highly adaptable organelle. In particular, the protein population that is anchored to the stress-bearing polysaccharides of the cell wall, and forms the interface with the outside world, is highly diverse. This diversity is believed to play an important role in adaptation of the cell to environmental conditions, in growth mode and in survival. Cell wall construction is tightly controlled and strictly coordinated with progression of the cell cycle. This is reflected in the usage of specific cell wall proteins during consecutive phases of the cell cycle and in the recent discovery of a cell wall integrity checkpoint. When the cell is challenged with stress conditions that affect the cell wall, a specific transcriptional response is observed that includes the general stress response, the cell wall integrity pathway and the calcineurin pathway. This salvage mechanism includes increased expression of putative cell wall assemblases and some potential cross-linking cell wall proteins, and crucial changes in cell wall architecture. We discuss some more enzymes involved in cell wall construction and also potential inhibitors of these enzymes. Finally, we use both biochemical and genomic data to infer that the architectural principles used by S. cerevisiae to build its cell wall are also used by many other ascomycetous yeasts and also by some mycelial ascomycetous fungi.
The anchors of mature glycosylphosphatidylinositol (GPI)-anchored proteins of Saccharomyces cerevisiae contain either ceramide or diacylglycerol with a C26:0 fatty acid in the sn2 position. The primary GPI lipid added to newly synthesized proteins in the ER consists of diacylglycerol with conventional C16 and C18 fatty acids. Here we show that GUP1 is essential for the synthesis of the C26:0-containing diacylglycerol anchors. Gup1p is an ER membrane protein with multiple membrane-spanning domains harboring a motif that is characteristic of membrane-bound O-acyl-transferases (MBOAT). Gup1Delta cells make normal amounts of GPI proteins but most mature GPI anchors contain lyso-phosphatidylinositol, and others possess phosphatidylinositol with conventional C16 and C18 fatty acids. The incorporation of the normal ceramides into the anchors is also disturbed. As a consequence, the ER-to-Golgi transport of the GPI protein Gas1p is slow, and mature Gas1p is lost from the plasma membrane into the medium. Gup1Delta cells have fragile cell walls and a defect in bipolar bud site selection. GUP1 function depends on the active site histidine of the MBOAT motif. GUP1 is highly conserved among fungi and protozoa and the gup1Delta phenotype is partially corrected by GUP1 homologues of Aspergillus fumigatus and Trypanosoma cruzi.
An extracellular matrix composed of a layered meshwork of beta-glucans, chitin, and mannoproteins encapsulates cells of the yeast Saccharomyces cerevisiae. This organelle determines cellular morphology and plays a critical role in maintaining cell integrity during cell growth and division, under stress conditions, upon cell fusion in mating, and in the durable ascospore cell wall. Here we assess recent progress in understanding the molecular biology and biochemistry of cell wall synthesis and its remodeling in S. cerevisiae. We then review the regulatory dynamics of cell wall assembly, an area where functional genomics offers new insights into the integration of cell wall growth and morphogenesis with a polarized secretory system that is under cell cycle and cell type program controls.
The deletion of MCD4 leads to an increase in beta-1,6-glucan level and a decrease in glycosylphosphatidylinositol-anchored protein and mannan levels in the cell wall of Saccharomyces cerevisiae, suggesting that mcd4 deletion mutant (mcd4Delta) displays beta-glucans on the cell surface without a mannan cover. An observation of the cell surface of mcd4Delta cells and an examination of the effect of contact between mcd4Delta cells and mouse macrophages indicated that macrophages were activated by contact with mcd4Delta cells displaying beta-glucans on the cell surface. We further examined the effect of intraperitoneal ethanol-fixed mcd4Delta cells on the survival period of mice infected with Candida albicans. mcd4Delta cells prolonged the survival period, implying that mcd4Delta cells may enhance the immune function of mice via macrophage activation. Moreover, we examined the structures of beta-glucans (i.e., alkali- and acetic acid-insoluble beta-glucans) extracted from mcd4Delta with (13)C-NMR and the effect of extracted beta-glucans on TNF-alpha secretion from macrophages. The structures of the beta-glucans from mcd4Delta differed from those of wild type (WT); however, there was no difference in tumor necrosis factor-alpha (TNF-alpha) secretion level between beta-glucans from mcd4Delta and those from WT. The yield of purified beta-glucans obtained from dry cells of mcd4Delta was higher than that obtained from dry cells of WT. mcd4Delta may be a superior strain for the preparation of beta-glucans.
Methods in yeast genetics
- F Sherman
- G Fink
- J B Hicks
Sherman, F., Fink, G., & Hicks, J. B. (1982). Methods in yeast genetics. Cold Spring Harbor,
NY: Cold Spring Harbor Laboratory.