Dimerisation of the Yeast K Translocation Protein Trk1 Depends on the K Concentration.

In baker's yeast (), Trk1, a member of the superfamily of K-transporters (SKT), is the main K uptake system under conditions when its concentration in the environment is low. Structurally, Trk1 is made up of four domains, each similar and homologous to a K-channel α subunit. Because most K-channels are proteins containing four channel-building α subunits, Trk1 could be functional as a monomer.

An Improved Transwell Design for Microelectrode Ion-Flux Measurements.

The microelectrode ion flux estimation (MIFE) is a powerful, non-invasive electrophysiological method for cellular membrane transport studies. Usually, the MIFE measurements are performed in a tissue culture dish or directly with tissues (roots, parts of the plants, and cell tissues). Here, we present a transwell system that allows for MIFE measurements on a cell monolayer.

MPM motifs of the yeast SKT protein Trk1 can assemble to form a functional K-translocation system.

The yeast Trk1 polypeptide, like other members of the Superfamily of K Transporters (SKT proteins) consists of four Membrane-Pore-Membrane motifs (MPMs A-D) each of which is homologous to a single K-channel subunit. SKT proteins are thought to have evolved from ancestral K-channels via two gene duplications and thus single MPMs might be able to assemble when located on different polypeptides. To test this hypothesis experimentally we generated a set of partial gene deletions to create alleles encoding one, two, or three MPMs, and analysed the cellular localisation and interactions of these Trk1 fragments using GFP tags and Bimolecular Fluorescence Complementation (BiFC).

The S. cerevisiae cation translocation protein Trk1 is functional without its "long hydrophilic loop" but LHL regulates cation translocation activity and selectivity.

In Saccharomyces cerevisiae, K-uptake under K-limiting conditions is largely mediated by the cation translocation systems Trk1 and Trk2 belonging to the family of SKT proteins. They are related to two-transmembrane-domain (inward rectifying K-) channels but unlike the symmetrical tetrameric structure of K-channels, a single Trk contains four pore-forming domains (A-D) encoded on one polypeptide chain. Between domains A and B Trks contain large cytosolic regions dubbed "long hydrophilic loop" (LHL).

Crystal structure of a novel domain of the motor subunit of the Type I restriction enzyme EcoR124 involved in complex assembly and DNA binding.

Although EcoR124 is one of the better-studied Type I restriction-modification enzymes, it still presents many challenges to detailed analyses because of its structural and functional complexity and missing structural information. In all available structures of its motor subunit HsdR, responsible for DNA translocation and cleavage, a large part of the HsdR C terminus remains unresolved. The crystal structure of the C terminus of HsdR, obtained with a crystallization chaperone in the form of pHluorin fusion and refined to 2.

pHluorin-assisted expression, purification, crystallization and X-ray diffraction data analysis of the C-terminal domain of the HsdR subunit of the Escherichia coli type I restriction-modification system EcoR124I.

The HsdR subunit of the type I restriction-modification system EcoR124I is responsible for the translocation as well as the restriction activity of the whole complex consisting of the HsdR, HsdM and HsdS subunits, and while crystal structures are available for the wild type and several mutants, the C-terminal domain comprising approximately 150 residues was not resolved in any of these structures. Here, three fusion constructs with the GFP variant pHluorin developed to overexpress, purify and crystallize the C-terminal domain of HsdR are reported. The shortest of the three encompassed HsdR residues 887-1038 and yielded crystals that belonged to the orthorhombic space group C2221, with unit-cell parameters a = 83.

Systems biology of monovalent cation homeostasis in yeast: the translucent contribution.

Maintenance of monovalent cation homeostasis (mainly K(+) and Na(+)) is vital for cell survival, and cation toxicity is at the basis of a myriad of relevant phenomena, such as salt stress in crops and diverse human diseases. Full understanding of the importance of monovalent cations in the biology of the cell can only be achieved from a systemic perspective. Translucent is a multinational project developed within the context of the SysMO (System Biology of Microorganisms) initiative and focussed in the study of cation homeostasis using the well-known yeast Saccharomyces cerevisiae as a model.

Early changes in membrane potential of Saccharomyces cerevisiae induced by varying extracellular K(+), Na (+) or H (+) concentrations.

Recently we introduced a fluorescent probe technique that makes possible to convert changes of equilibrium fluorescence spectra of 3,3'-dipropylthiadicarbocyanine, diS-C3(3), measured in yeast cell suspensions under defined conditions into underlying membrane potential differences, scaled in millivolts (Plasek et al. in J Bioenerg Biomembr 44: 559-569, 2012). The results presented in this paper disclose measurements of real early changes of plasma membrane potential induced by the increase of extracellular K(+), Na(+) and H(+) concentration in S.

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-C(3)(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-C(3)(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-C(3)(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.

Graphical analysis and experimental evaluation of Saccharomyces cerevisiae PTRK1|2 and PBMH1|2 promoter region.

We designed a simple graphical presentation for the results of a transcription factor (TF) pattern matching analysis. The TF analysis algorithm utilized known sequence signature motifs from several databases. The graphical presentation enabled a quick overview of potential TF binding sites, their frequency and spacing on both DNA strands and thus straight forward identification of promising candidates for further experimental investigations.

Electrophysiological and fluorescence microscopy studies with HERG channel/EGFP fusion proteins.

HERG (human ether-a-go-go-related gene) encodes the Kv11.1 protein alpha-subunit that underlies the rapidly activating delayed rectifier K+ current (IKr) in the heart. Alterations in the functional properties or membrane incorporation of HERG channels, either by genetic mutations or by administration of drugs, play major roles in the development of life-threatening torsades de pointes cardiac arrhythmias.

Functional expression of the voltage-gated neuronal mammalian potassium channel rat ether à go-go1 in yeast.

It has been shown previously that heterologous expression of inwardly rectifying potassium channels (K+-channels) from plants and mammals in K+-transport defective yeast mutants can restore the ability of growth in media with low [K+]. In this study, the functional expression of an outward rectifying mammalian K+-channel in yeast is presented for the first time. The outward-rectifying mammalian neuronal K+-channel rat ether à go-go channel 1 (rEAG1, Kv 10.

Ring test assessment of the mKir2.1 growth based assay in Saccharomyces cerevisiae using parametric models and model-free fits.

Inward rectifying K+ (Kir) channels are a subfamily of the potassium channel superfamily. They mediate potassium influx into the cells, a process responding to the polarization state, a variety of intracellular messengers and specific auxiliary proteins, thereby they are involved in important physiological processes such as the pacemaker activity in the heart, insulin release, and potassium uptake in glial cells. The Saccharomyces cerevisiae mKir2.

Estrogenic effects of natural and synthetic compounds including tibolone assessed in Saccharomyces cerevisiae expressing the human estrogen alpha and beta receptors.

The human estrogen receptors (hER)alpha and hERbeta, 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, hERalpha and hERbeta were expressed in a genetically modified Saccharomyces cerevisiae strain, devoid of three endogenous xenobiotic transporters (PDR5, SNQ2, and YOR1).

Computational recognition of potassium channel sequences.

Motivation: Potassium channels are mainly known for their role in regulating and maintaining the membrane potential. Since this is one of the key mechanisms of signal transduction, malfunction of these potassium channels leads to a wide variety of severe diseases. Thus potassium channels are priority targets of research for new drugs, despite the fact that this protein family is highly variable and closely related to other channels, which makes it very difficult to identify new types of potassium channel sequences.

Use of PMA1 as a housekeeping biomarker for assessment of toxicant-induced stress in Saccharomyces cerevisiae.

The brewer's yeast Saccharomyces cerevisiae has emerged as a versatile and robust model system for laboratory use to study toxic effects of various substances. In this study, toxicant-induced stresses of pure compounds were investigated in Saccharomyces cerevisiae utilizing a destabilized version of the green fluorescent protein optimized for expression in yeast (yEGFP3) under control of the promoter of the housekeeping plasma membrane ATPase gene PMA1. The responses of the biomarker upon increasing test compound concentrations were monitored by determining the decrease in fluorescence.

New phenotypes of functional expression of the mKir2.1 channel in potassium efflux-deficient Saccharomyces cerevisiae strains.

The functional expression of the mouse Kir2.1 potassium channel in yeast cells lacking transport systems for potassium and sodium efflux (ena1-4delta nha1delta) resulted in increased cell sensitivity to high external concentrations of potassium. The phenotype depended on the level of Kir2.

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 (Deltatrk1,2 and Deltatrk1,2 Deltatok1) 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.

Cod106, a novel synaptic protein expressed in sensory hair cells of the inner ear and in CNS neurons.

The exquisite performance of the highly specialized mammalian inner ear requires a multitude of specific proteins. Yet, only a subset of these proteins has been identified and studied in detail. Here, we describe a novel gene expressed in the organ of Corti that encodes a membrane-associated protein of 106 kDa.

Phenotypic yeast growth analysis for chronic toxicity testing.

In Saccharomyces cerevisiae the pH-dependent growth inhibition of the heavy metals Cu(2+), Cr(6+), Zn(2+), Co(2+), and Cd(2+) was examined in comparison to that of organic solvents and pure compounds DMSO, MNNG, 4-NQO, MTBE, ethanol, and 2-AA. The assay was based on both S. cerevisiae wild-type and genetically modified cells deleted in the transporters Pdr5, Snq2, and Yor1 that facilitate pleiotropic drug resistance to explore the potential for short-term chronic aquatic toxicity tests.

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.

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 trk1delta, trk2delta and tok1delta 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.