TRANSLUCENT

SysMO is a European transnational funding and research initiative on "Systems Biology of Microorganisms".

The goal pursued by SysMO was to record and describe the dynamic molecular processes going on in unicellular microorganisms in a comprehensive way and to present these processes in the form of computerized mathematical models.

Systems biology will raise biomedical and biotechnological research to a new quality level and contribute markedly to progress in understanding. Pooling European research ...

Investigation of how the ENA1 gene is transcriptionally regulated.

Experimental approaches to evaluate cellular response to Potassium limitation. For this purpose, a series of isogenic strains derived from BY4741 and lacking one (TRK1 or TRK2) or both TRK genes encoding specific potassium uptake systems was constructed using the homologous recombination and Cre-lox system.

Cellular parameters of strains lacking both TRK1 and TRK2 genes.

Investigation of the role of 14-3-3 proteins in the S. cerevisiae cation homeostasis

Experimental approaches to study the mechanism and ions involved in potassium uptake after long term potassium starvation.

Properties of cells lacking the NHA1 gene.

Multi experimental approach to define the gene expression remodelling under potassium starvation conditions.

How does the Volume, internal pH, membrane potential and intracellular cation content change in a time dependent scale during potassium limitation.

How do the fluxes of rubidium (potassium) change during potassium starvation.

What is the proteome of starved cells. Main characteristics?

Are there one or several stationary states for physiological parameters dependent on external KCl? For which range of external potassium the cell is able to maintain a constant pH, potassium content, membrane potential and volume?

How does the current mediated by Trk1,2 depend on external and internal ion concentrations? How is the membrane potential shifted by the concentrations of various ions, especially ammonium?

Does the general proteome of yeast cells change in a strain lacking Trk1,2 transporter? Under which conditions?

Studies on the effect of deletion of genes encoding 14-3-3 proteins on the transcriptional response of yeast cells to potassium starvation.

Studies on genetic interactions between genes encoding 14-3-3 proteins and genes encoding transporters to elucidate which transporter(s) are regulated by 14-3-3 proteins.

Bioinformatic studies to elucidate the role of 14-3-3 proteins in cation homeostasis.

How does the flux of potassium, hydrogen change during potassium uptake? Which ions are involved in maintaining the required charge balance?

How does the transport of potassium and hydrogen ions effect the concentrations in the near surrounding of the cell.

How does the current mediated by Trk1,2 depend on external and internal ion concentrations? How is the membrane potential shifted by the concentrations of various ions, especially ammonium?

At external potassium concentrations in the range of 10uM to 1mM Trk is major cellular uptake system for potassium. This system responds to the activiy of the proton pump. Transmembrane fluxes of protons and potassium cations are analysed in a signal-response relationship.

Selected strains from the collection of GFP-tagged S. cerevisiae strains are cultivated at different extracellular caion concentrations and the localization of the GFP-tagged protein will be studied by confocal microscopy. The effect of deletion of the BMH1 gene, encoding the major 14-3-3 isoform, will be analyzed.

The main input is the ENA review paper (Function and Regulation of the Saccharomyces cerevisiae ENA Sodium ATPase System, Ruiz&Ariño 2007) and the papers referenced. Another source are the papers linked from the ENA page of SGD http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=ENA1

Is cell volume affected by potassium starvation?

The volume reduction as a response to the shift of cells from a medium of high potassium to potassium free medium will be studied in mutants lacking certain membrane transporters like Trk1,2 Nha1, etc. The conditions for the experiments follow Navarrete et al. (2010). Additionally knockouts of related regulation proteins (SAP155, SAP185) will be tested. For each mutant several time points will be measured to generate time courses.

Time courses of the internal pH changes under the conditions of Navarrete et al. (2010) will be obtained. The usage of different mutants (transport systems and regulation factors) will reveal the influence and key systems of pH regulation.

To estimate the changes of internal pH, the pH-dependent variant of GFP pHluorin is expressed in cells from a multicopy plasmid, and the changes in cell fluorescence are monitored during 5 hours of incubation in YNB-F growth media without added potassium.

How does internal potassium changes (decreases) during several hours of potassium starvation. Is there a limit for internal potassium decrease? Sampling potassium content in cells during starvation The relative membrane potential will be measured according to the conditions of Navarrete et al. (2010). Various mutants will be tested for their effect. To estimate the relative changes of plasma-membrane potential, the diSC3(3) fluorescent probe was used and the changes in cell fluorescence monitored ...

Determination of protein content at several times.

The potassium content measurements of Navarrete et al. (2010) are the basis for potassium content analysis in various mutants (Nha1, Trk1, Trk2).

External concentration changes under the conditions of Navarrete et al. (2010) will be estimated from the internal concentration changes and the volume ratio of cell sample to medium.

External pH changes for the conditions of Navarrete et al. (2010) will be estimated from the internal pH changes and the volume ratio of cell sample to medium.

The potassium fluxes will be estimated from the internal and external concentration changes.

How potassium starvation regulates the parameters of rubidium (potassium) transport. Analysis of transport characteristics during the starvation process. Kinetic characteristics of rubidium transport.

Related to the internal pH changes the proton efflux will be estimated from the internal and external concentration changes.

Based on Hess et al. (2006) ammonium is suspected to be transported via Trk1,2 under potassium shortage. The ammonium concentration in the medium will be determined for several time points under the conditions of Navarrete et al. (2010).

Is it possible to see changes in the proteome after starvation in 2D- Gels? Preparation of 2D Gels of cells incubated different periods of time in the absence of potassium.

What are the main proteins identified? Spots sampling and identification by MS

The potassium content of cells grown overnight in a certain amount (0.1, 0.2, 0.5, 1, 5, 20, 50, 200 mM) of KCl will be determined. Additionally the potassium content of cells grown overnight in high potassium and shifted to the amounts of potassium used in the former experiment. After growing overnight again in the lower potassium, the cells should contain finally a comparable potassium concentration than the cells grown in the respecting KCl in the first experiment.

The pH of cells grown overnight in a certain concentration of KCl will be determined, to reveal a functional relationship between external KCl and a stable pH. See also "2D-Gel Electrophoresis" Assay for further details.

Confocal microscopy of CIN5-GFP from the collection of GFP strains at standard growth conditions (50 mM KCl)

Confocal microscopy of HOG1-GFP from GFP-strain collection under standard growth cionditions (50 mM KCl).

Confocal microscopy of Pma1-GFP cells after 60 min at 0 or 50 mM KCl .

Confocal microscopy of Nha1-GFP cells after 60 min at 0 or 50 mM KCl or at 1 M NaCl.

Confocal microscopy of Arl1-GFP cells after 60 min at 0 or 50 mM KCl or at 1 M NaCl.

Confocal microscopy of Ena1-GFP cells after 60 min at 0 or 50 mM KCl or at 1 M NaCl.

TRK1, TRK2

Growth yeast with 50mM K+ and short timing collection

Growth yeast with 50mM K+ and long timing collection

Growth yeast with 0mM K+ and short timing collection

Growth yeast with 0mM K+ and short timing collection

Growth yeast with 50mM K+ and short timing collection

Growth yeast with 0mM K+ and short timing collection

Growth yeast with 0mM K+ and long timing collection

Growth yeast with 50mM K+ and short timing collection

Growth yeast with 50mM K+ and long timing collection

Growth yeast with 50mM K+ and long timing collection

MS005_10

Growth yeast with 0mM K+ and long timing collection

MS008_6

Purpose: PROTEIN EXTRACTION FOR 2-DE BASED PROTEOMIC EXPERIMENTS. 2. Membrane proteins

Purpose: PROTEIN EXTRACTION FOR 2-DE BASED PROTEOMIC EXPERIMENTS. 1. Citosoluble proteins

Purpose: Cation content analysis - protocol

Purpose: