Hella Lichtenberg-Fraté

Characterization of potassium transport in wild‐type and isogenic yeast strains carrying all combinations of trk1, trk2 and tok1 null mutations

Saccharomyces cerevisiae cells express three defined potassium‐specific transport systems en‐coded by TRK1 , TRK2 and TOK1 . To gain a more complete understanding of the physiological function of these transport proteins, we have constructed a set of isogenic yeast strains carrying all combinations of trk1 Δ, trk2 Δ and tok1 Δ null mutations. The in vivo K + transport characteristics of each strain have been documented using growth‐based assays, and the in vitro biochemical and electrophysiological properties associated with K + transport have been determined. As has been reported previously, Trk1p and Trk2p facilitate high‐affinity potassium uptake and appear to be functionally redundant under a wide range of environmental conditions. In the absence of TRK1 and TRK2 , strains lack the ability specifically to take up K + , and trk1 Δ trk2 Δ double mutant cells depend upon poorly understood non‐specific cation uptake mechanisms for growth. Under conditions that impair the activity of the non‐specific uptake system, termed NSC1, we have found that the presence of functional Tok1p renders cells sensitive to Cs + . Based on this finding, we have established a growth‐based assay that monitors the in vivo activity of Tok1p.

A yeast-based method for the detection of cyto and genotoxicity

A miniaturized short-term in vivo genotoxicity screening assay based on genetically modified yeast (Saccharomyces cerevisiae) cells was performed to explore the capacity of this eukaryotic organism to detect the presence of genotoxic compounds. An increased general sensitivity of yeast cells to toxic compounds was obtained by using a strain being deleted in the prominent pleiotropic drug resistance mediating efflux transporters PDR5, SNQ2 and YOR1. In order to detect genotoxic effects, a yeast optimized version of the green fluorescent protein (GFP) was fused to the RAD54 promoter that is activated upon DNA damage. Various model substances including the oxygenated fuel additive methyl tertiary-butyl ether (MTBE) and the direct acting genotoxins methyl-N-nitro-N-nitrosoguanidine (MNNG) and 4-nitroquinoline-1-oxide (4-NQO) were tested. All model substances were in parallel examined for chronic cytotoxicity. The results point out the sufficiency of both the sensitivity of the yeast cells to detect chronic cytotoxicity and the intensity of the fluorescence signal for the assessment of genotoxic effects. Thus, the test enables simultaneous detection of cytotoxic and genotoxic effects. By partial automation and implementation of the test in the microtitre scale this bioassay allows parallel sensitive pre-screening of numerous samples.

The SpTRK Gene Encodes a Potassium-specific Transport Protein TKHp in Schizosaccharomyces pombe

A somewhat different — albeit equally worrisome point — is that the large capacitive currents in Fig. 8D and E, again uncommented by the authors, indicate severe technical problems with either data acquisition or processing and open to doubt all point plots which are not directly supported by displayed current traces. Thus, this paper is deeply flawed in its failure to acknowledge my substantial contribution to the actual work, in its significant unadmitted reuse of previously published descriptions of methods, and — in my judgment — in its improper handling of the data actually obtained. That is a pity, because a precise and clear demonstration in Saccharomyces of the properties of a novel membrane transporter from Schizosaccharomyces would have been important. As this paper stands, however, its very structure puts all of its conclusions in doubt.

Impact of Acute Metal Stress in Saccharomyces cerevisiae

Although considered as essential cofactors for a variety of enzymatic reactions and for important structural and functional roles in cell metabolism, metals at high concentrations are potent toxic pollutants and pose complex biochemical problems for cells. We report results of single dose acute toxicity testing in the model organism S. cerevisiae. The effects of moderate toxic concentrations of 10 different human health relevant metals, Ag+, Al3+, As3+, Cd2+, Co2+, Hg2+, Mn2+, Ni2+, V3+, and Zn2+, following short-term exposure were analyzed by transcription profiling to provide the identification of early-on target genes or pathways. In contrast to common acute toxicity tests where defined endpoints are monitored we focused on the entire genomic response. We provide evidence that the induction of central elements of the oxidative stress response by the majority of investigated metals is the basic detoxification process against short-term metal exposure. General detoxification mechanisms also comprised the induction of genes coding for chaperones and those for chelation of metal ions via siderophores and amino acids. Hierarchical clustering, transcription factor analyses, and gene ontology data further revealed activation of genes involved in metal-specific protein catabolism along with repression of growth-related processes such as protein synthesis. Metal ion group specific differences in the expression responses with shared transcriptional regulators for both, up-regulation and repression were also observed. Additionally, some processes unique for individual metals were evident as well. In view of current concerns regarding environmental pollution our results may support ongoing attempts to develop methods to monitor potentially hazardous areas or liquids and to establish standardized tests using suitable eukaryotic a model organism.

Estrogenic effects of natural and synthetic compounds including tibolone assessed in Saccharomyces cerevisiae expressing the human estrogen α and β receptors

The human estrogen receptors (hER)α and hERβ, differentially expressed and localized in various tissues and cell types, mediate transcriptional activation of target genes. These encode a variety of physiological reproductive and nonreproductive functions involved in energy metabolism, salt balance, immune system, development, and differentiation. As a step toward developing a screening assay for the use in applications where significant numbers of compounds or complex matrices need to be tested for (anti) estrogenic bioactivity, hERα and hERβ were expressed in a genetically modified Saccharomyces cerevisiae strain, devoid of three endogenous xenobiotic transporters (PDR5, SNQ2, and FOR1). By using receptor‐mediated transcriptional activation of the green fluorescent protein optimized for expression in yeast (yEGFP) as reporter 17 natural, comprising estrogens and phytoestrogens or synthetic compounds among which tibolone with its metabolites, gestagens, and antiestrogens were investigated. The reporter assay deployed a simple and robust protocol for the rapid detection of estrogenic effects within a 96‐well microplate format. Results were expressed as effective concentrations (EC50) and correlated to other yeast based and cell line assays. Tibolone and its metabolites exerted clear estrogenic effects, though considerably less potent than all other natural and synthetic compounds. For the blood serum of two volunteers, considerable higher total estrogenic bioactivity than single estradiol concentrations as determined by immunoassay was found. Visualization of a hERα/GFP fusion protein in yeast revealed a sub cellular cytosolic localization. This study demonstrates the versatility of (anti) estrogenic bioactivity determination using sensitized S. cerevisiae cells to assess estrogenic exposure and effects.—Hasenbrink, G., Sievernich, A., Wildt, L., Ludwig, J., and Lichtenberg‐Fraté, H. Estrogenic effects of natural and synthetic compunds including tibolone assessed in Saccharomyces cerevisiae expressing the human estrogen α and β receptors. FASEB J. 20, E861–E870 (2006)

Association of serum sex steroid receptor bioactivity and sex steroid hormones with breast cancer risk in postmenopausal women

Postmenopausal women with elevated serum sex steroids have an increased risk of breast cancer. Most of this risk is believed to be exerted through binding of the sex steroids to their receptors. For the first time, we investigate the association of estrogen receptor (ER) and androgen receptor (AR) serum bioactivity (SB) in addition to hormone levels in samples from women with breast cancer collected before diagnosis. Two hundred postmenopausal women participating in the UK Collaborative Trial of Ovarian Cancer Screening who developed ER-positive breast cancer 0.6–5 years after sample donation were identified and matched to 400 controls. ER and AR bioassays were used to measure ERα, ERβ, and AR SB. Androgen and estrogen levels were measured with immunoassays. Subjects were classified according to quintiles of the respective marker among controls and the associations between SB and hormones with breast cancer risk were determined by logistic regression analysis. ERα and ERβ SB were significantly higher before diagnosis compared with controls, while estrogens showed no difference. Women had a twofold increased breast cancer risk if ERα SB (odds ratio (OR), 2.114; 95% confidence interval (CI), 1.050–4.425; P=0.040) was in the top quintile >2 years before diagnosis or estrone (OR, 2.205; 95% CI, 1.104–4.586; P=0.029) was in the top quintile <2 years before diagnosis. AR showed no significant association with breast cancer while androstenedione (OR, 3.187; 95% CI, 1.738–6.044; P=0.0003) and testosterone (OR, 2.145; 95% CI, 1.256–3.712; P=0.006) were significantly higher compared with controls and showed a strong association with an almost threefold increased breast cancer risk independent of time to diagnosis. This study provides further evidence on the association of androgens and estrogens with breast cancer. In addition, it reports that high ER but not AR SB is associated with increased breast risk >2 years before diagnosis.

Analysis of the mKir2.1 channel activity in potassium influx defective Saccharomyces cerevisiae strains determined as changes in growth characteristics.

Potassium uptake defective Saccharomyces cerevisiae strains (Δtrk1,2 and Δtrk1,2 Δtok1) were used for the phenotypic analysis of the mouse inward rectifying Kir2.1 channel by growth analysis. Functional expression of both, multi‐copy plasmid and chromosomally expressed GFP‐mKir2.1 fusion constructs complemented the potassium uptake deficient phenotype in a pHout dependent manner. Upon application of Hygromycin B to chromosomally mKir2.1 expressing cells, significantly lower toxin sensitivity (EC50 15.4 μM) compared to Δtrk1,2 Δtok1 cells (EC50 2.6 μM) was observed. Growth determination of mKir2.1 expressing strains upon application of Ag+, Cs+ and Ba2+ as known blockers of mKir2.1 channels revealed significantly decreased channel function. Cells with mKir2.1 were about double sensitive to AgNO3, 350‐fold more sensitive to CsCl and 1500‐fold more sensitive to BaCl2 in comparison to the respective controls indicating functional expression and correct pharmacology.

Monitoring of real changes of plasma membrane potential by diS-C 3 (3) fluorescence in yeast cell suspensions

The fluorescent dye 3,3′-dipropylthiadicarbocyanine, diS-C3(3), is a suitable probe to monitor real changes of plasma membrane potential in yeast cells which are too small for direct membrane potential measurements with microelectrodes. A method presented in this paper makes it possible to convert changes of equilibrium diS-C3(3) fluorescence spectra, measured in yeast cell suspensions under certain defined conditions, into underlying membrane potential differences, scaled in the units of millivolts. Spectral analysis of synchronously scanned diS-C3(3) fluorescence allows to assess the amount of dye accumulated in cells without otherwise necessary sample taking and following separation of cells from the medium. Moreover, membrane potential changes can be quantified without demanding calibration protocols. The applicability of this approach was demonstrated on the depolarization of Rhodotorula glutinis yeast cells upon acidification of cell suspensions and/or by increasing extracellular K+ concentration.

Properties and Heterologous Expression of the Glucose Transporter GHT1 from Schizosaccharomyces pombe

Genomic DNA of the Schizosaccharomyces pombe glucose transporter, GHT1, was obtained by complementation of the glucose transport deficient Sz. pombe strain YGS‐5. Here we describe the GHT1 gene that encodes a protein of 565 amino acids with a corresponding molecular mass of 62·5 kDa. This eukaryotic glucose transporter contains 12 putative transmembrane segments and is homologous to the HXT multigene family of S. cerevisiae with several amino acid motifs of this sugar transporter family. It is also homologous to other sugar carriers from human, mouse and Escherichia coli. The function of the Ght1 protein as a glucose transporter was proved both by homologous and heterologous expression in the Sz. pombe mutant YGS‐5 and in the S. cerevisiae hxt mutant RE700A, respectively. Both transformed yeast strains transported d‐glucose with substrate specificity similar to that in Sz. pombe wild‐type cells. Moreover, the cells of the two transformed yeast strains accumulated 2‐deoxy‐d‐glucose, a non‐metabolizable d‐glucose analogue, with an efficiency similar to Sz. pombe wild‐type cells. The ability of the S. cerevisiae mutant RE700A to accumulate 2DG in an Δμ  H + dependent manner after transformation with GHT1 provides evidence that the Sz. pombe transporter catalyses an energy‐dependent uptake of glucose. The sequence of GHT1 was deposited at EMBL, Outstation EBI, Accession Number X91218. ©1997 John Wiley & Sons, Ltd.

A Thermodynamic Model of Monovalent Cation Homeostasis in the Yeast Saccharomyces cerevisiae.

Cationic and heavy metal toxicity is involved in a substantial number of diseases in mammals and crop plants. Therefore, the understanding of tightly regulated transporter activities, as well as conceiving the interplay of regulatory mechanisms, is of substantial interest. A generalized thermodynamic description is developed for the complex interplay of the plasma membrane ion transporters, membrane potential and the consumption of energy for maintaining and restoring specific intracellular cation concentrations. This concept is applied to the homeostasis of cation concentrations in the yeast cells of S. cerevisiae. The thermodynamic approach allows to model passive ion fluxes driven by the electrochemical potential differences, but also primary or secondary active transport processes driven by the inter- play of different ions (symport, antiport) or by ATP consumption (ATPases). The model—confronted with experimental data—reproduces the experimentally observed potassium and proton fluxes induced by the external stimuli KCl and glucose. The estimated phenomenological constants combine kinetic parameters and transport coefficients. These are in good agreement with the biological understanding of the transporters thus providing a better understanding of the control exerted by the coupled fluxes. The model predicts the flux of additional ion species, like e.g. chloride, as a potential candidate for counterbalancing positive charges. Furthermore, the effect of a second KCl stimulus is simulated, predicting a reduced cellular response for cells that were first exposed to a high KCl stimulus compared to cells pretreated with a mild KCl stimulus. By describing the generalized forces that are responsible for a given flow, the model provides information and suggestions for new experiments. Furthermore, it can be extended to other systems such as e.g. Candida albicans, or selected plant cells.