Activation of H+-ATPase of the Plasma Membrane of Saccharomyces cerevisiae by Glucose: The Role of Sphingolipid and Lateral Enzyme Mobility

Activation of the plasma membrane H+-ATPase of the yeast Saccharomyces cerevisiae by glucose is a complex process that has not yet been completely elucidated. This study aimed to shed light on the role of lipids and the lateral mobility of the enzyme complex during its activation by glucose. The significance of H+-ATPase oligomerization for the activation of H+-ATPase by glucose was shown using the strains lcb1-100 and erg6, with the disturbed synthesis of sphyngolipid and ergosterol, respectively. Experiments with GFP-fused H+-ATPase showed a decrease in fluorescence anisotropy during the course of glucose activation, suggesting structural reorganization of the molecular domains. An immunogold assay showed that the incubation with glucose results in the spatial redistribution of ATPase complexes in the plasma membrane. The data suggest that (1) to be activated by glucose, H+-ATPase is supposed to be in an oligomeric state, and (2) glucose activation is accompanied by the spatial movements of H+-ATPase clusters in the PM

Phosphate efflux as a test of plasma membrane leakage in Saccharomyces cerevisiae cells

Plasma membrane integrity is a key to cell viability. Currently, the main approach to assessing plasma membrane integrity is the detection of penetration of special dyes, such as trypan blue and propidium iodide, into the cells. However, this method needs expensive equipment: a fluorescent microscope or a flow cytometer. Besides, staining with propidium iodide occasionally gives false-positive results. Here, we suggest the phosphate (Pi) leakage assay as an approach to assess the increase in permeability of the plasma membrane of yeast cells. We studied the dependence of phosphate efflux and uptake into Saccharomyces cerevisiae cells on the composition of the incubation medium, time, and ambient pH. The difference in optimal conditions for these processes suggests that Pi efflux is not conducted by the Pi uptake system. The Pi efflux in water correlated with the proportion of cells stained with propidium iodide. This indicated that Pi efflux is associated with cytoplasmic membrane disruption in a portion of the yeast cell population. The assay of Pi efflux was used to evaluate membrane disruption in S. cerevisiae cells treated with some heavy metal ions and detergents.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Sugar-Induced Cell Death in the Yeast <i>S. cerevisiae</i> Is Accompanied by the Release of Octanoic Acid, Which Does Not Originate from the Fatty Acid Synthesis Type II Mitochondrial System

Incubation of the yeast S. cerevisiae with glucose, in the absence of other nutrients, leads to Sugar-Induced Cell Death (SICD), accompanied by the accumulation of Reactive Oxygen Species (ROS). Yeast acidifies the environment during glucose metabolism not only as a result of the activity of the H+-ATPase of the plasma membrane but also due to the release of carboxylic acids. Acetic acid is known to induce apoptosis in growing yeast. We analyzed the composition of the incubation medium and found octanoic acid (OA) but no other carboxylic acids. Its concentration (0.675 µM) was significantly lower than the one at which OA had a toxic effect on the cell. However, the theoretically calculated concentration of OA inside the cell (about 200 μM) was found to be high enough to lead to cell necrosis. To test the hypothesis that OA might cause SICD, we used a ΔACP1 strain incapable of synthesizing OA in the yeast mitochondrial Fatty Acid Synthesis type II system (FAS-II). The deletion of the ACP1 gene did not affect the OA content in the medium. But, on the other hand, OA is a precursor of lipoic acid, which has antioxidant properties. However, strains with deleted genes for lipoic acid biosynthesis from OA (ΔPPT2, ΔLIP2, ΔLIP5, and ΔSGV3) showed no change in ROS and SICD levels. Thus, lipoic acid synthesized in FAS-II does not protect cells from ROS accumulated during SICD. We conclude that OA synthesized in the mitochondrial FAS-II system and its derivative lipoic acid are not involved in SICD in yeast S. cerevisiae

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

Immunogold labeling of Pma1 in the plasma membrane of <i>S. cerevisiae erg6</i> and <i>lcb1-100</i>.

<p>(A) – glucose-starved cells of the <i>erg6</i> strain, Pma1 was distributed in the membrane as single structures; (B) – <i>erg6</i> cells that had metabolized glucose for 15 min, Pma1 formed complexes; (C) - glucose-starved cells of the <i>lcb1-100</i> strain, Pma1 was distributed in the membrane as single structures; (D) – <i>lcb1-100</i> cells that had metabolized glucose for 15 min, Pma1 was distributed in the membrane as single structures. CW = cell wall; PM = plasma membrane.</p

Immunogold labeling of Pma1 in the plasma membrane of <i>S. cerevisiae SEY6210</i>.

<p>(A) – glucose-starved cells, Pma1 was distributed in the membrane as single structures; (B) – cells that had metabolized glucose for 15 min, Pma1 formed large bunch-like complexes; (C) – enlarged fragment of photograph (B) CW = cell wall; PM = plasma membrane.</p

Pma1 activity in situ (nmol P_i/min/mg total cell protein, <i>n</i> = 3±SD) after 15-min incubation of <i>S. cerevisiae</i> whole cells with 100 mM glucose or 100 mM deoxyglucose. The change of activity in % of initial activity is given in parenthesis.

<p>Pma1 activity in situ (nmol P_i/min/mg total cell protein, <i>n</i> = 3±SD) after 15-min incubation of <i>S. cerevisiae</i> whole cells with 100 mM glucose or 100 mM deoxyglucose. The change of activity in % of initial activity is given in parenthesis.</p

Fluorescence depolarization (anisotropy) <b>r</b> of PMA1-GFP in whole cells after 15 min incubation with 100 mM glucose or deoxyglucose.

<p>Fluorescence depolarization (anisotropy) <b>r</b> of PMA1-GFP in whole cells after 15 min incubation with 100 mM glucose or deoxyglucose.</p

The Reduced Level of Inorganic Polyphosphate Mobilizes Antioxidant and Manganese-Resistance Systems in <i>Saccharomyces cerevisiae</i>

Inorganic polyphosphate (polyP) is crucial for adaptive reactions and stress response in microorganisms. A convenient model to study the role of polyP in yeast is the Saccharomyces cerevisiae strain CRN/PPN1 that overexpresses polyphosphatase Ppn1 with stably decreased polyphosphate level. In this study, we combined the whole-transcriptome sequencing, fluorescence microscopy, and polyP quantification to characterize the CRN/PPN1 response to manganese and oxidative stresses. CRN/PPN1 exhibits enhanced resistance to manganese and peroxide due to its pre-adaptive state observed in normal conditions. The pre-adaptive state is characterized by up-regulated genes involved in response to an external stimulus, plasma membrane organization, and oxidation/reduction. The transcriptome-wide data allowed the identification of particular genes crucial for overcoming the manganese excess. The key gene responsible for manganese resistance is PHO84 encoding a low-affinity manganese transporter: Strong PHO84 down-regulation in CRN/PPN1 increases manganese resistance by reduced manganese uptake. On the contrary, PHM7, the top up-regulated gene in CRN/PPN1, is also strongly up-regulated in the manganese-adapted parent strain. Phm7 is an unannotated protein, but manganese adaptation is significantly impaired in &#916;phm7, thus suggesting its essential function in manganese or phosphate transport