Assays

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.

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

The volume of cells grown overnight in a certain concentration of KCl will be determined, to reveal a relation between the osmotic effects of external KCl and the cell volume. See also 1.4.1. for further details.

What is the relationship between the activity of Trk1,2 system and cell volume? Is cell volume affected by lack of the main potassium uptake systems? Comparison of cell volume in wild type and TRK mutants.

To estimate the differnces of internal pH between the wild type, single trk1 and trk2 or double trk1 trk2 mutants, the pH-dependent variant of GFP pHluorin was expressed in cells from a multicopy plasmid, cells were grown under various conditions, and the cell fluorescence was monitored.

To estimate the relative level of plasma-membrane potential in the wild type, single trk1 and trk2 or double trk1 trk2 mutants, the diSC3(3) fluorescent probe was added to cells grown under various conditions and the levels of cell fluorescence were monitored.

To estimate the relative level of plasma-membrane potential in the wild type, single trk1 and trk2 or double trk1 trk2 mutants, the diSC3(3) fluorescent probe was added to cells grown under various conditions and the levels of cell fluorescence were monitored.

Is protein content of trk1,2 mutants affected? Determination of proteins in wild type and TRK mutants

Is internal potassium affected by mutations in the Trk1,2 system? Under which conditions? Potassium measurements in wild type and TRK mutants grown and/or incubated under several external conditions.

The current-voltage relation for Trk1,2 will be determined according to the conditions of Kuroda et al.

2D Gels in wild type and mutants grown or incubated under several conditions: starvation, potassium limiting or different growth phases.