Mutations in SNF1 complex genes affect yeast cell wall strength

The trimeric SNF1 complex from Saccharomyces cerevisiae, a homolog of mammalian AMP-activated kinase, has been primarily implicated in signaling for the utilization of alternative carbon sources to glucose. We here find that snf1 deletion mutants are hypersensitive to different cell wall stresses, such as the presence of Calcofluor white, Congo red, Zymolyase or the glucan synthase inhibitor Caspofungin in the growth medium. They also have a thinner cell wall. Caspofungin treatment triggers the phosphorylation of the catalytic Snf1 kinase subunit at Thr210 and removal of this phosphorylation site by mutagenesis (Snf1-T210A) abolishes the function of Snf1 in cell wall integrity. Deletion of the PFK1 gene encoding the α-subunit of the heterooctameric yeast phosphofructokinase suppresses the cell wall phenotypes of a snf1 deletion, which suggests a compensatory effect of central carbohydrate metabolism. Epistasis analyses with mutants in cell wall integrity (CWI) signaling confirm that the SNF1 complex and the CWI pathway independently affect yeast cell integrity.     

Alleviating monoterpene toxicity using a two-phase extractive fermentation for the bioproduction of jet fuel mixtures in Saccharomyces cerevisiae

Monoterpenes are a diverse class of compounds with applications as flavors and fragrances, pharmaceuticals and more recently, jet fuels. Engineering biosynthetic pathways for monoterpene production in microbial hosts has received increasing attention. However, monoterpenes are highly toxic to many microorganisms including Saccharomyces cerevisiae, a widely used industrial biocatalyst. In this work, the minimum inhibitory concentration (MIC) for S. cerevisiae was determined for five monoterpenes: β‐pinene, limonene, myrcene, γ‐terpinene, and terpinolene (1.52, 0.44, 2.12, 0.70, 0.53 mM, respectively). Given the low MIC for all compounds tested, a liquid two‐phase solvent extraction system to alleviate toxicity during fermentation was evaluated. Ten solvents were tested for biocompatibility, monoterpene distribution, phase separation, and price. The solvents dioctyl phthalate, dibutyl phthalate, isopropyl myristate, and farnesene showed greater than 100‐fold increase in the MIC compared to the monoterpenes in a solvent‐free system. In particular, the MIC for limonene in dibutyl phthalate showed a 702‐fold (308 mM, 42.1 g L^?1 of limonene) improvement while cell viability was maintained above 90%, demonstrating that extractive fermentation is a suitable tool for the reduction of monoterpene toxicity. Finally, we estimated that a limonane to farnesane ratio of 1:9 has physicochemical properties similar to traditional Jet‐A aviation fuel. Since farnesene is currently produced in S. cerevisiae, its use as a co‐product and extractant for microbial terpene‐based jet fuel production in a two‐phase system offers an attractive bioprocessing option. Biotechnol. Bioeng. 2012; 109: 2513–2522. © 2012 Wiley Periodicals, Inc.     

Kin1 is a plasma membrane‐associated kinase that regulates the cell surface in fission yeast

Cell morphogenesis is a complex process that depends on cytoskeleton and membrane organization, intracellular signalling and vesicular trafficking. The rod shape of the fission yeast Schizosaccharomyces pombe and the availability of powerful genetic tools make this species an excellent model to study cell morphology. Here we have investigated the function of the conserved Kin1 kinase. Kin1‐GFP associates dynamically with the plasma membrane at sites of active cell surface remodelling and is present in the membrane fraction. Kin1Δ null cells show severe defects in cell wall structure and are unable to maintain a rod shape. To explore Kin1 primary function, we constructed an ATP analogue‐sensitive allele kin1‐as1. Kin1 inhibition primarily promotes delocalization of plasma membrane‐associated markers of actively growing cell surface regions. Kin1 itself is depolarized and its mobility is strongly reduced. Subsequently, amorphous cell wall material accumulates at the cell surface, a phenotype that is dependent on vesicular trafficking, and the cell wall integrity mitogen‐activated protein kinase pathway is activated. Deletion of cell wall integrity mitogen‐activated protein kinase components reduces kin1Δ hypersensitivity to stresses such as those induced by Calcofluor white and SDS. We propose that Kin1 is required for a tight link between the plasma membrane and the cell wall.     

Structural and immunological studies on the protoplast membrane of the yeast Candida utilis

Cell membranes of the yeast Candida utilis isolated by lysis of protoplasts have been shown to be lipoprotein in nature. Electron microscopy shows that Mg⁺⁺ is responsible for maintaining the integrity of the membrane. A close serological relationship was found between membranes and cell walls isolated from the yeast. This relationship was exhibited not only by membranes obtained by strepzyme treatment but also by those obtained from the action of helicase enzyme. No such relationship existed between membranes and whole cells. Related data have been obtained by treatment of yeasts with different digestive enzymes. All of the results suggest that the protoplast membrane possesses traces of structural cell wall material. This material is detectable by serological tests, but not by electron microscopy.     

Evaluating the influence of inhibitors present in lignocellulosic hydrolysates on the cell membrane integrity of oleaginous yeast Trichosporon fermentans by flow cytometry

The effect of ten typical organic acids and five aldehydes present in lignocellulosic hydrolysates on the cell membrane integrity of oleaginous yeast Trichosporon fermentans was evaluated by flow cytometry. Overall, organic acids affected the cell membrane integrity of T. fermentans more significantly than that of aldehydes albeit aldehydes are more toxic to T. fermentans. The PI (Propidium Iodide) uptake rate of T. fermentans’ cells gradually decreased as fermentation going on, indicating that T. fermentans could overcome the inhibition of organic acids or aldehydes by adaption. Interestingly, in some cases, the effect of organic acids or aldehydes on the cell membrane integrity of T. fermentans was well related to their hydrophobicity. However, for the outliers, no obvious similar phenomena were observed. Thus, the attack on hydrophobic sites of cell membrane was not the only determinant for the damage of organic acids or aldehydes on cell membrane integrity of T. fermentans.     

Cell wall integrity maintenance in plants: Lessons to be learned from yeast?

The plant cell wall is involved in different biological processes like cell morphogenesis and response to biotic/abiotic stress. Functional integrity of the wall is apparently being maintained during these processes by changing structure/composition and coordinating cell wall with cellular metabolism. In S.cerevisiae a well-characterized mechanism exists that is maintaining functional integrity of yeast the cell wall during similar processes. During the last years it has become obvious that plants have evolved a mechanism to monitor and maintain functional integrity of their cell walls. However, our understanding of the mechanism is rather limited. The available evidence suggests that similar signaling cascades may be involved and particular protein activities may be conserved between plants and yeast. Here we review the available evidence briefly and highlight similarities between yeast and plants that could help us to understand the mode of action of the signaling cascades maintaining plant cell wall integrity.     

Limonene Synthase, the Enzyme Responsible for Monoterpene Biosynthesis in Peppermint, Is Localized to Leucoplasts of Oil Gland Secretory Cells

Circumstantial evidence based on ultrastructural correlation, specific labeling, and subcellular fractionation studies indicates that at least the early steps of monoterpene biosynthesis occur in plastids. (4S)-Limonene synthase, which is responsible for the first dedicated step of monoterpene biosynthesis in mint species, appears to be translated as a preprotein bearing a long plastidial transit peptide. Immunogold labeling using polyclonal antibodies raised to the native enzyme demonstrated the specific localization of limonene synthase to the leucoplasts of peppermint (Mentha × piperita) oil gland secretory cells during the period of essential oil production. Labeling was shown to be absent from all other plastid types examined, including the basal and stalk cell plastids of the secretory phase glandular trichomes. Furthermore, in vitro translation of the preprotein and import experiments with isolated pea chloroplasts were consistent in demonstrating import of the nascent protein to the plastid stroma and proteolytic processing to the mature enzyme at this site. These experiments confirm that the leucoplastidome of the oil gland secretory cells is the exclusive location of limonene synthase, and almost certainly the preceding steps of monoterpene biosynthesis, in peppermint leaves. However, succeeding steps of monoterpene metabolism in mint appear to occur outside the leucoplasts of oil gland cells.     

Changes of Saccharomyces cerevisiae cell membrane components and promotion to ethanol tolerance during the bioethanol fermentation

During bioethanol fermentation process, Saccharomyces cerevisiae cell membrane might provide main protection to tolerate accumulated ethanol, and S. cerevisiae cells might also remodel their membrane compositions or structure to try to adapt to or tolerate the ethanol stress. However, the exact changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation still remains poorly understood. This study was performed to clarify changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation. Both cell diameter and membrane integrity decreased as fermentation time lasting. Moreover, compared with cells at lag phase, cells at exponential and stationary phases had higher contents of ergosterol and oleic acid (C_18:1) but lower levels of hexadecanoic (C_16:0) and palmitelaidic (C_16:1) acids. Contents of most detected phospholipids presented an increase tendency during fermentation process. Increased contents of oleic acid and phospholipids containing unsaturated fatty acids might indicate enhanced cell membrane fluidity. Compared with cells at lag phase, cells at exponential and stationary phases had higher expressions of ACC1 and HFA1. However, OLE1 expression underwent an evident increase at exponential phase but a decrease at following stationary phase. These results indicated that during bioethanol fermentation process, yeast cells remodeled membrane and more changeable cell membrane contributed to acquiring higher ethanol tolerance of S. cerevisiae cells. These results highlighted our knowledge about relationship between the variation of cell membrane structure and compositions and ethanol tolerance, and would contribute to a better understanding of bioethanol fermentation process and construction of industrial ethanologenic strains with higher ethanol tolerance.     

Alkamides from Echinacea disrupt the fungal cell wall-membrane complex

We tested the hypothesis that alkamides from Echinacea exert antifungal activity by disrupting the fungal cell wall/membrane complex. Saccharomyces cerevisiae cells were treated separately with each of seven synthetic alkamides found in Echinacea extracts. The resulting cell wall damage and cell viability were assessed by fluorescence microscopy after mild sonication. Membrane disrupting properties of test compounds were studied using liposomes encapsulating carboxyfluorescein. Negative controls included hygromycin and nourseothricin (aminoglycosides that inhibit protein synthesis), and the positive control used was caspofungin (an echinocandin that disrupts fungal cell walls). The results show that yeast cells exposed to sub-inhibitory concentrations of each of the seven alkamides and Echinacea extract exhibit increased frequencies of cell wall damage and death that were comparable to caspofungin and significantly greater than negative controls. Consistent with effects of cell wall damaging agents, the growth inhibition by three representative alkamides tested and caspofungin, but not hygromycin B, were partially reversed in sorbitol protection assays. Membrane disruption assays showed that the Echinacea extract and alkamides have pronounced membrane disruption activity, in contrast to caspofungin and other controls that all had little effect on membrane stability. A Quantitative Structure-Activity Relationship (QSAR) analysis was performed to study the effect of structural substituents on the antifungal activity of the alkamides. Among the set studied, diynoic alkamides showed the greatest antifungal and cell wall disruption activities while an opposite trend was observed in the membrane disruption assay where the dienoic group was more effective. We propose that alkamides found in Echinacea act synergistically to disrupt the fungal cell wall/membrane complex, an excellent target for specific inhibition of fungal pathogens. Structure-function relationships provide opportunities for synthesis of alkamide analogs with improved antifungal activities.     

Rho Signaling Participates in Membrane Fluidity Homeostasis

Preservation of both the integrity and fluidity of biological membranes is a critical cellular homeostatic function. Signaling pathways that govern lipid bilayer fluidity have long been known in bacteria, yet no such pathways have been identified in eukaryotes. Here we identify mutants of the yeast Saccharomyces cerevisiae whose growth is differentially influenced by its two principal unsaturated fatty acids, oleic and palmitoleic acid. Strains deficient in the core components of the cell wall integrity (CWI) pathway, a MAP kinase pathway dependent on both Pkc1 (yeast''s sole protein kinase C) and Rho1 (the yeast RhoA-like small GTPase), were among those inhibited by palmitoleate yet stimulated by oleate. A single GEF (Tus1) and a single GAP (Sac7) of Rho1 were also identified, neither of which participate in the CWI pathway. In contrast, key components of the CWI pathway, such as Rom2, Bem2 and Rlm1, failed to influence fatty acid sensitivity. The differential influence of palmitoleate and oleate on growth of key mutants correlated with changes in membrane fluidity measured by fluorescence anisotropy of TMA-DPH, a plasma membrane-bound dye. This work provides the first evidence for the existence of a signaling pathway that enables eukaryotic cells to control membrane fluidity, a requirement for division, differentiation and environmental adaptation.     

An investigation into the feasibility of using yeast protoplasts to study the ion transport properties of the plasma membrane

A method for studying ion uptake in enzymatically isolated protoplasts from the yeast, Saccharomyces cerevisiae, is described. The kinetics of K⁺ and Rb⁺ uptake, metabolic proton extrusion and cell electrophoretic mobility bave been determined. Enzymic removal of the cell wall does not significantly alter the above-mentioned properties of the yeast cells. It is concluded that studies of these properties can be performed equally well with intact yeast cells or protoplasts. However, in studies aimed at determining effects of complex organic substances, e.g., antibiotics, on plasma membrane function the use of protoplasts is recommended. The effectiveness of the antibiotic, Dio-9, for example, in reversing the metabolic proton extrusion into a net proton influx is at least 50 times higher after enzymic removal of the yeast cell wall.     

Chemosensitization prevents tolerance of Aspergillus fumigatus to antimycotic drugs

Tolerance of human pathogenic fungi to antifungal drugs is an emerging medical problem. We show how strains of the causative agent of human aspergillosis, Aspergillus fumigatus, tolerant to cell wall-interfering antimycotic drugs become susceptible through chemosensitization by natural compounds. Tolerance of the A. fumigatus mitogen-activated protein kinase (MAPK) mutant, sakAΔ, to these drugs indicates the osmotic/oxidative stress MAPK pathway is involved in maintaining cell wall integrity. Using deletion mutants of the yeast, Saccharomyces cerevisiae, we first identified thymol and 2,3-dihydroxybenzaldehyde (2,3-D) as potent chemosensitizing agents that target the cell wall. We then used these chemosensitizing agents to act as synergists to commercial antifungal drugs against tolerant strains of A. fumigatus. Thymol was an especially potent chemosensitizing agent for amphotericin B, fluconazole or ketoconazole. The potential use of natural, safe chemosensitizing agents in antifungal chemotherapy of human mycoses as an alternative to combination therapy is discussed.