Growth Of Yeast Strains Research Articles

Proteasomes: Isolation and Activity Assays.

In eukaryotes, damaged or unneeded proteins are typically degraded by the ubiquitin-proteasome system. In this system, the protein substrate is often first covalently modified with a chain of ubiquitin polypeptides. This chain serves as a signal for delivery to the 26S proteasome, a 2.5-MDa, ATP-dependent multisubunit protease complex. The proteasome consists of a barrel-shaped 20S core particle (CP) that is capped on one or both of its ends by a 19S regulatory particle (RP). The RP is responsible for recognizing the substrate, unfolding it, and translocating it into the CP for destruction. Here we describe simple, one-step purification schemes for isolating the 26S proteasome and its 19S RP and 20S CP subcomplexes from the yeast Saccharomyces cerevisiae. A gel filtration step can be added to further enhance purity. We also describe assays for measuring ubiquitin-dependent and ubiquitin-independent proteolytic activity in vitro. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Purification of active 26S proteasomes Support Protocol 1: Growth of yeast strains and production of yeast cell powder Support Protocol 2: Regeneration of anti-flag M2 affinity gel Basic Protocol 2: Purification of the 19S regulatory particle (RP) Basic Protocol 3: Purification of active 20S CP Basic Protocol 4: In-gel peptidase activity assay for 20S CP and 26S proteasomes Basic Protocol 5: In-solution peptidase activity assay for 20S and 26S proteasomes Basic Protocol 6: Measuring degradation of polyubiquitinated SIC1PY Basic Protocol 7: Gel filtration of purified proteasomes and subcomplexes.

Poacic acid, a β‐1,3‐glucan–binding antifungal agent, inhibits cell‐wall remodeling and activates transcriptional responses regulated by the cell‐wall integrity and high‐osmolarity glycerol pathways in yeast

As a result of the relatively few available antifungals and the increasing frequency of resistance to them, the development of novel antifungals is increasingly important. The plant natural product poacic acid (PA) inhibits β-1,3-glucan synthesis in Saccharomyces cerevisiae and has antifungal activity against a wide range of plant pathogens. However, the mode of action of PA is unclear. Here, we reveal that PA specifically binds to β-1,3-glucan, its affinity for which is ~30-fold that for chitin. Besides its effect on β-1,3-glucan synthase activity, PA inhibited the yeast glucan-elongating activity of Gas1 and Gas2 and the chitin-glucan transglycosylase activity of Crh1. Regarding the cellular response to PA, transcriptional co-regulation was mediated by parallel activation of the cell-wall integrity (CWI) and high-osmolarity glycerol signaling pathways. Despite targeting β-1,3-glucan remodeling, the transcriptional profiles and regulatory circuits activated by caspofungin, zymolyase, and PA differed, indicating that their effects on CWI have different mechanisms. The effects of PA on the growth of yeast strains indicated that it has a mode of action distinct from that of echinocandins, suggesting it is a unique antifungal agent.

Production of added-value microbial metabolites during growth of yeast strains on media composed of biodiesel-derived crude glycerol and glycerol/xylose blends.

A total of 11 yeast strains of Yarrowia lipolytica, Metschnikowia sp., Rhodotorula sp. and Rhodosporidium toruloides were grown under nitrogen-limited conditions with crude glycerol employed as substrate in shake flasks, presenting interesting dry cell weight (DCW) production. Three of these strains belonging to Metschnikowia sp. accumulated significant quantities of endopolysaccharides (i.e. the strain V.V.-D4 produced 11.0g/L of endopolysaccharides, with polysaccharides in DCW≈63% w/w). A total of six Y. lipolytica strains produced either citric acid or mannitol. Most of the screened yeasts presented somehow elevated lipid and polysaccharides in DCW values at the early steps of growth despite nitrogen appearance in the fermentation medium. Lipid in DCW values decreased as growth proceeded. R. toruloides DSM 4444 cultivated on media presenting higher glycerol concentrations presented interesting lipid-accumulating capacities (maximum lipid=12.5g/L, maximum lipid in DCW=43.0-46.0% w/w, conversion yield on glycerol=0.16g/g). Replacement of crude glycerol by xylose resulted in somehow decreased lipid accumulation. In xylose/glycerol mixtures, xylose was more rapidly assimilated from glycerol. R. toruloides total lipids were mainly composed of triacylglycerols. Total cellular fatty acid composition on xylose presented some differences compared with that on glycerol. Cellular lipids contained mainly oleic and palmitic acid.

Epistasis and mutational fitness landscape

Molecular Evolution A fitness landscape of a gene defines the molecular potential of evolution. This can help us understand the current state of evolution as well as predict unrealized potential. Using deep sequencing to examine mutations in nonessential genes that affect the growth of yeast strains, two studies have generated fitness landscapes and measured the effect of epistatic interactions (see the Perspective by He and Liu). Li et al. generated a library of mutants in a transfer RNA gene, including all single and many double and multiple mutants. The RNA secondary structure was generally predictive of bases under selection. Similarly, Puchta et al. assessed a small nucleolar RNA gene for the fitness effects of individual mutations, which correlated with evolutionary conservation and structural stability. Both studies suggest that epistasis—the combined functional effect—for double substitutions is more often negative than positive. Science , this issue pp. [837][1] and [840][2]; see also p. [769][3] [1]: /lookup/doi/10.1126/science.aae0568 [2]: /lookup/doi/10.1126/science.aaf0965 [3]: /lookup/doi/10.1126/science.aaf7543

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein’s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein’s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Visible light alters yeast metabolic rhythms by inhibiting respiration

Exposure of cells to visible light in nature or in fluorescence microscopy often is considered to be relatively innocuous. However, using the yeast respiratory oscillation (YRO) as a sensitive measurement of metabolism, we find that non-UV visible light has a significant impact on yeast metabolism. Blue/green wavelengths of visible light shorten the period and dampen the amplitude of the YRO, which is an ultradian rhythm of cell metabolism and transcription. The wavelengths of light that have the greatest effect coincide with the peak absorption regions of cytochromes. Moreover, treating yeast with the electron transport inhibitor sodium azide has similar effects on the YRO as visible light. Because impairment of respiration by light would change several state variables believed to play vital roles in the YRO (e.g., oxygen tension and ATP levels), we tested oxygen's role in YRO stability and found that externally induced oxygen depletion can reset the phase of the oscillation, demonstrating that respiratory capacity plays a role in the oscillation's period and phase. Light-induced damage to the cytochromes also produces reactive oxygen species that up-regulate the oxidative stress response gene TRX2 that is involved in pathways that enable sustained growth in bright visible light. Therefore, visible light can modulate cellular rhythmicity and metabolism through unexpectedly photosensitive pathways.

Biosynthesis of Crystalline Silver and Gold Nanoparticles by Extremophilic Yeasts

The biosynthesis of Ag and Au nanoparticles (NPs) was investigated using an extremophilic yeast strain isolated from acid mine drainage in Portugal. Three distinct studies were performed, namely, the growth of yeast strain in presence of metal ions, the use of yeast biomass for the metal nanoparticles synthesis, and of the supernatant obtained after 24-hour incubation of yeast biomass in water. The extremophilic strain under study was able to grow up to an Ag ion concentration of 1.5 mM whereas an increase of Au ion concentration over 0.09 mM caused a strong inhibitory effect. A successful route for the metal NPs synthesis was obtained using the yeast biomass. When the washed yeast cells were in contact with Ag or Au solutions, AgNPs smaller than 20 nm were produced, as for the AuNPs diameter ranged from 30 to 100 nm, as determined through transmission electron microscopy and confirmed by energy-dispersive X-ray spectra. The supernatant-based strategy provided evidence that proteins were released to the medium by the yeasts, which could be responsible for the formation and stabilisation of the Ag NPs, although the involvement of the cell wall seems fundamental for AuNPs synthesis.

Inactivation of PPX1 and PPN1 genes encoding exopolyphosphatases of Saccharomyces cerevisiae does not prevent utilization of polyphosphates as phosphate reserve

Cytosol polyphosphates (polyPs) are the main phosphate (P(i)) reserve in the yeast Saccharomyces cerevisiae. In this work, the participation of cytosol polyPs and exopolyphosphatases in maintenance of P(i) homeostasis under P(i) deficit in the cultivation medium has been studied in different strains of S. cerevisiae. The growth of yeast strains with inactivated genes PPX1 and PPN1 encoding the yeast exopolyphosphatases and a strain with double mutations in these genes in a P(i)-deficient medium is not disturbed. All the studied strains are able to maintain relatively constant P(i) levels in the cytosol. In P(i)-deficient medium, polyP hydrolysis in the cytosol of the parent and PPN1-deficient strains seems to be performed by exopolyphosphatase Ppx1 and proceeds without any change of the spectrum of polyP chain lengths. In the PPX1-deficient strain, long-chain polyPs are depleted first, and only then short-chain polyPs are hydrolyzed. In the double PPX1 and PPN1 mutant having low exopolyphosphatase activity, polyP hydrolysis in the cytosol starts with a notable delay, and about 20% of short-chain polyPs still remain after the polyP hydrolysis in other strains has almost been completed. This fact suggests that S. cerevisiae possesses a system, which makes it possible to compensate for inactivation of the PPX1 and PPN1 genes encoding exopolyphosphatases of the yeast cells.

Role of the yeast ABC transporter Yor1p in cadmium detoxification

Growth of yeast strains, either deleted for the vacuolar ABC transporter Ycf1 or deleted for the plasma membrane ABC transporter Yor1p or overexpressing Yor1p, were compared for their sensitivity to cadmium. On solid medium cell death (or growth inhibition) was observed at cadmium concentrations higher than 100 μM when yeasts were grown at 30 °C for 24 h. However, for all tested strains cell death (or growth inhibition) was already observed at 40 μM cadmium when incubated at 23 °C for 60 h. Thus cadmium is more toxic to yeast at 23 °C than at 30 °C. At 23 °C, the Δ yor1 strain grew more slowly than the wild-type strain and the double Δ yor1, Δ ycf1 deleted strain was much more sensitive to cadmium than each single Δ yor1 or Δ ycf1 deletant. Overexpression of Yor1p in a Δ ycf1 strain restores full growth. Cadmium uptake measurements show that Δ ycf1 yeast strains expressing or overexpressing Yor1p store less cadmium than the corresponding Δ ycf1, Δ yor1 strain. The strains expressing Yor1p display an energy-dependent efflux of cadmium estimated for the yeast overexpressing Yor1p to be about 0.02 nmol 109Cd/mg protein/min. Yeast cells loaded with radiolabeled glutathione and then with radioactive cadmium displayed a twice-higher efflux of glutathione than that of cadmium suggesting that Yor1p transports both compounds as a bis-glutathionato-cadmium complex. All together, these results suggest that in addition to being accumulated in the yeast vacuole by Ycf1p, cadmium is also effluxed out of the cell by Yor1p.

HIGH OXYGEN CONCENTRATION IN COMBINATION WITH ELEVATED CARBON DIOXIDE TO AFFECT GROWTH OF FRESH-CUT PRODUCE MICRO-ORGANISMS

High oxygen atmospheres (> 70 %) can be used as an alternative for the nowadays applied low O-2/elevated CO2 modifled atmosphere, to prolong shelf life of respiring fresh-cut vegetables and fruit. The effect of high oxygen atmosphere, applied alone or in combination with elevated CO2 concentrations, on the growth of spoilage micro-organisms (14 species) and pathogens (Listeria monocytogenes and Aeromonas caviae (HG4)), isolated from fresh-cut produce, has been studied. Of each species only one strain was considered, except for Candida lambica, from which 5 different strains were analyzed to check possible differences in sensitivity for high O-2 concentrations between strains of the same species. Lag phase (lambda), maximum specific growth rate (mu(max)), initial count (N-0) and maximum count (N-max) were determined applying the Baranyi model. The maximum specific growth rate of G(-) spoilage bacteria was slightly reduced by high O-2. Growth rate of the two tested pathogens was rather stimulated by high O-2. Lactic acid bacteria were much more sensitive to high O-2 concentrations, but no additive or synergistic effect was found when high O-2 was combined with high CO2. In most of the cases, growth of yeast strains was strongly inhibited by high O-2, and this effect was even more pronounced when applying 80 % O-2 + 20 % CO2 (additive effect). Only one strain of Candida lambica was not at all affected by high O-2 concentrations and/or elevated CO2. Growth of Candida humicola and of another strain of Candida lambica was significantly stimulated by high O-2. So although yeast are generally inhibited during storage experiments with fresh-cut produce under high O-2 atmosphere, this inhibitory effect is not true for every individual yeast strain in in vitro experiments.