Longevity of U cells of differentiated yeast colonies grown on respiratory medium depends on active glycolysis

<p>Colonies of <i>Saccharomyces cerevisiae</i> laboratory strains pass through specific developmental phases when growing on solid respiratory medium. During entry into the so-called alkali phase, in which ammonia signaling is initiated, 2 prominent cell types are formed within the colonies: U cells in upper colony regions, which have a longevity phenotype and activate the expression of a large number of metabolic genes, and L cells in lower regions, which die more quickly and exhibit a starvation phenotype. Here, we performed a detailed analysis of the activities of enzymes of central carbon metabolism in lysates of both cell types and determined several fermentation end products, showing that previously reported expression differences are reflected in the different enzymatic capabilities of each cell type. Hence, U cells, despite being grown on respiratory medium, behave as fermenting cells, whereas L cells rely on respiratory metabolism and possess active gluconeogenesis. Using a spectrum of different inhibitors, we showed that glycolysis is essential for the formation, and particularly, the survival of U cells. We also showed that β-1,3-glucans that are released from the cell walls of L cells are the most likely source of carbohydrates for U cells.</p

Confocal wavelength titrations.

<p>Emission spectra (nm) at different pH from peripheral ROIs within Ato1p-pHluorin (A) and Ato1p-mCherry (B) yeast cells suspended in P-buffers of pH 4–11. Emission intensities were normalized to better show peak shifts. Emission wavelength versus pH titration curves from Ato1p-pHluorin (C) or Ato1p-mCherry (D) peripheral ROIs. Both fluorophores exhibited pH-dependent wavelength shifts indicative of ratiometry by emission at the given pH ranges. Ecliptic yEGFP1 maintained a constant wavelength as expected (C). Representative values were averaged from independent subsets of 10 cells with 20 subcellular ROIs each. Error bars indicate the standard error. The darker areas within the sigmoidal titration curves represent their quantitation limits.</p

Confocal wavelength stability tests.

<p>Emission spectra (nm) of sequential scans from peripheral ROIs within Ato1p-pHluorin (A) and Ato1p-mCherry (B) yeast cells under constant pH conditions. Emission intensities were normalized to better show peak shifts. Wavelength stability curves (solid lines) track peak wavelength maxima throughout 10 successive confocal spectral scans, evidencing an absence of statistically significant changes in Ato1p-pHluorin peripheral wavelength (C) and in the case of Ato1p-mCherry (D), modest fluctuations after the seventh scan. CFM wavelength titrations (dashed lines) projected over the additional top x-axis (pH) are included for comparison to illustrate the higher extent of the pH-induced emission shifts for both ratiometric FPs.</p

Confocal dual ratio stability tests.

<p>(A) Emission intensity (unitless) of sequential scans from peripheral ROIs within Ato1p-pHluorin yeast cells under excitation at 405 and 476 nm and constant pH conditions. (B) Dual ratio stability curves (solid line) track the intensity ratio (R_405/476) throughout 10 successive confocal spectral scans, evidencing statistically significant changes in Ato1p-pHluorin peripheral ratio after the third scan. Dual ratio titrations (dashed line) projected over the additional top x-axis (pH) illustrate that pH-unrelated ratio fluctuations rapidly exceeded the extent of the pH-induced shifts.</p

Model of cellular pH gradients.

<p>Schematic diagram illustrating intracellular pH gradients within yeast cells from alkali-phase colonies. Measurements with Ato1p-pHluorin, Ato1p-mCherry, Jen1p-mCherry and Met17p-pHluorin fusion proteins provided evidence for the existence of sharp intracellular pH gradients between peripheral (∼8.8) and internal (∼6.6) cytoplasmic regions during the ammonia-producing alkali phase. In contrast to protonated ammonium (NH₄⁺), volatile ammonia (NH₃) enters the cell by simple diffusion through the plasma membrane. High cytosolic pH values near the plasma membrane allow for the maintenance of high transmembrane electrochemical potential (Δψ) based on H⁺ gradient (ΔpH) under conditions of relatively high extracellular pH (∼7.3).</p

Confocal wavelength-based pH quantifications.

<p>Developmental phases of yeast giant colonies growing on GMA plates with BKP pH indicator (A) and extracellular pH changes in the colony surroundings (B). Timings of <i>ATO1</i>, <i>MET17</i> and <i>JEN1</i> gene expression (black, violet and yellow bars) and the ammonia-producing alkali phase (purple bar) are indicated above. Emission wavelength box plots from peripheral ROIs within Ato1p-pHluorin (C) and Ato1p-mCherry cells (D) suspended in N-buffer of pH 6 under near-native conditions. CFM scans were performed during 1^st acidic, alkali and 2^nd acidic phases. 2^nd acidic cells treated with 100 mM NH₃ served as an alkalinization positive control. Each sample subset was selected to represent cell variability within the colony and contained a total of 30 subcellular ROIs from 3 independent biological repeats. Box plots indicate median, quartiles, extremes and individual values within each subset. Titration-based pH box plots of Ato1p-pHluorin (E) Ato1p-mCherry (F) Met17p-pHluorin (G) and Jen1p-mCherry (H) extrapolated from wavelength data. The effective range of the extrapolations was strictly defined by the pKa and quantitation limits of the titrations. Extrapolations from wavelength populations displaying out-of-range interquartile or medians (yellow box plots) must therefore be treated as non-quantitative and are included for comparison.</p

Spectrofluorometer and confocal intensity titrations.

<p>Emission intensity (unitless) versus pH titration curves from Ato1p-yEGFP1 fresh cells (A) or individual organelles within fresh cells (B) suspended in N or P-buffers of pH 4–8. Spectrofluorometric analyses and confocal scans were respectively completed within 5 and 3 min after buffer suspension. Treatments with high-dose ethanol (60%) for 2 min before N-buffer suspension were included as invasive controls. Examination of the titration curves (A and B) implied a connection between the sigmoid slope and the degree of cell permeation, but only confocal titration curves (B) evidenced the altered response of ethanol-treated cells. Fluorescent signal for the confocal titrations was acquired from discrete ROIs in the confocal stacks (C and D). Confocal stack images of P-buffer cells with peripheral and vacuolar ROIs (C). Confocal stack images of ethanol-treated cells with whole-cell ROIs (D) revealing the effects of high ethanol doses on cell and organelle integrity. Images were digitally colored to depict ROI sampling.</p

SUN Family Proteins Sun4p, Uth1p and Sim1p Are Secreted from <i>Saccharomyces cerevisiae</i> and Produced Dependently on Oxygen Level

<div><p>The SUN family is comprised of proteins that are conserved among various yeasts and fungi, but that are absent in mammals and plants. Although the function(s) of these proteins are mostly unknown, they have been linked to various, often unrelated cellular processes such as those connected to mitochondrial and cell wall functions. Here we show that three of the four <i>Saccharomyces cerevisiae</i> SUN family proteins, Uth1p, Sim1p and Sun4p, are efficiently secreted out of the cells in different growth phases and their production is affected by the level of oxygen. The Uth1p, Sim1p, Sun4p and Nca3p are mostly synthesized during the growth phase of both yeast liquid cultures and colonies. Culture transition to slow-growing or stationary phases is linked with a decreased cellular concentration of Sim1p and Sun4p and with their efficient release from the cells. In contrast, Uth1p is released mainly from growing cells. The synthesis of Uth1p and Sim1p, but not of Sun4p, is repressed by anoxia. All four proteins confer cell sensitivity to zymolyase. In addition, Uth1p affects cell sensitivity to compounds influencing cell wall composition and integrity (such as Calcofluor white and Congo red) differently when growing on fermentative versus respiratory carbon sources. In contrast, Uth1p is essential for cell resistance to boric acids irrespective of carbon source. In summary, our novel findings support the hypothesis that SUN family proteins are involved in the remodeling of the yeast cell wall during the various phases of yeast culture development and under various environmental conditions. The finding that Uth1p is involved in cell sensitivity to boric acid, i.e. to a compound that is commonly used as an important antifungal in mycoses, opens up new possibilities of investigating the mechanisms of boric acid’s action.</p></div

Strains deficient in SUN proteins have different sensitivity to zymolyase and various drugs when grown on respiratory GMA agar.

<p>A–C, drop assays of cells of BY4742 (wt), BY-<i>uth1</i>Δ, BY-p_TEF-<i>UTH1</i>, BY-<i>sun4</i>Δ, BY-<i>sim1</i>Δ and BY-<i>nca3</i>Δ strains on GMA plates supplemented with various drugs. Representative experiments of at least three biological replicates are presented. Significant drug effects on particular strains are marked by grey boxes; the quantification of the drug effect is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073882#pone.0073882.s004" target="_blank">Figure S4A</a>. <b>A</b>, effect of Congo red (800 µg/ml) or Calcofluor white (1 mg/ml); cells were grown at 37°C for 4 days. <b>B</b>, effect of SDS (0.012%), cells were grown at 28°C for 4 or 7 days as indicated. <b>C</b>, effect of boric acid (0.4%), cells were grown at 28°C for 7 days. <b>D</b>, cell resistance to zymolyase (0.2 U/µl) presented as decrease in density of cell suspension. S.D. values were calculated from four independent biological replicates for the mutant strains and 10 replicates for the wt; the significance of the difference between BY4742 and the other four strains was determined using two-way ANOVA with p≤0.0001.</p

O₂ concentration affects SUN protein production.

<p><b>A</b>, Protein amounts in cell lysates of unwashed colonies of BY-Uth1p-HA, BY-Sun4p-HA and BY-Sim1p-HA strains. Strains were either grown on YEPDA-erg or incubated on GMA under normoxic, hypoxic or anoxic atmosphere. The representative Western blot of the 2 independent biological replicates is shown. <b>B</b>, Sun4p-HA amounts in cell lysates (CELL) and in extracellular extracts (EXTRA) from colonies. BY-Sun4p-HA strain was grown on YEPDA-erg under normoxic, hypoxic or anoxic conditions for 3 days. Two film-expositions are shown to better visualize differences in protein concentrations in individual samples. The value of Mw at the right side of immunoblots represents Mw of particular SUN protein linked to the HA tag. The standardized amounts of lysate-proteins or the corresponding amounts of extracellular extracts were loaded onto the gel (loading controls, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073882#pone.0073882.s003" target="_blank">Figure S3 D</a>).</p