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Adaptation to potassium starvation of wild-type and K(+)-transport mutant (trk1,2) of Saccharomyces cerevisiae: 2-dimensional gel electrophoresis-based proteomic approach.

ABSTRACT: Saccharomyces cerevisiae wild-type (BY4741) and the corresponding mutant lacking the plasma membrane main potassium uptake systems (trk1,trk2) were used to analyze the consequences of K(+) starvation following a proteomic approach. In order to trigger high-affinity mode of potassium transport, cells were transferred to potassium-free medium. Protein profile was followed by two-dimensional (2-D) gels in samples taken at 0, 30, 60, 120, 180, and 300 min during starvation. We observed a general decrease of protein content during starvation that was especially drastic in the mutant strain as it was the case of an important number of proteins involved in glycolysis. On the contrary, we identified proteins related to stress response and alternative energetic metabolism that remained clearly present. Neural network-based analysis indicated that wild type was able to adapt much faster than the mutant to the stress process. We conclude that complete potassium starvation is a stressful process for yeast cells, especially for potassium transport mutants, and we propose that less stressing conditions should be used in order to study potassium homeostasis in yeast.

SUBMITTER: Gelis S

PROVIDER: S-EPMC3426419 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Adaptation to potassium starvation of wild-type and K(+)-transport mutant (trk1,2) of Saccharomyces cerevisiae: 2-dimensional gel electrophoresis-based proteomic approach.

Gelis Samuel S Curto Miguel M Valledor Luis L González Asier A Ariño Joaquín J Jorrín Jesús J Ramos José J

MicrobiologyOpen 20120601 2

Saccharomyces cerevisiae wild-type (BY4741) and the corresponding mutant lacking the plasma membrane main potassium uptake systems (trk1,trk2) were used to analyze the consequences of K(+) starvation following a proteomic approach. In order to trigger high-affinity mode of potassium transport, cells were transferred to potassium-free medium. Protein profile was followed by two-dimensional (2-D) gels in samples taken at 0, 30, 60, 120, 180, and 300 min during starvation. We observed a general dec ...[more]

PMID: 22950024

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Project description:BackgroundTwo-dimensional gel electrophoresis (2DE) is one of the most popular methods in proteomics. Currently, most 2DE experiments are performed using immobilized pH gradient (IPG) in the first dimension; however, some laboratories still use carrier ampholytes-based isoelectric focusing technique. The aim of this study was to directly compare IPG-based and non-equilibrium pH gradient electrophoresis (NEPHGE)-based 2DE techniques by using the same samples and identical second dimension procedures. We have used commercially available Invitrogen ZOOM IPGRunner and WITAvision systems for IPG and NEPHGE, respectively. The effectiveness of IPG-based and NEPHGE-based 2DE methods was compared by analysing differential protein expression during cytosolic unfolded protein response (UPR-Cyto) in Saccharomyces cerevisiae.ResultsProtein loss during 2DE procedure was higher in IPG-based method, especially for basic (pI?>?7) proteins. Overall reproducibility of spots was slightly better in NEPHGE-based method; however, there was a marked difference when evaluating basic and acidic protein spots. Using Coomassie staining, about half of detected basic protein spots were not reproducible by IPG-based 2DE, whereas NEPHGE-based method showed excellent reproducibility in the basic gel zone. The reproducibility of acidic proteins was similar in both methods. Absolute and relative volume variability of separate protein spots was comparable in both 2DE techniques. Regarding proteomic analysis of UPR-Cyto, the results exemplified parameters of general comparison of the methods. New highly basic protein Sis1p, overexpressed during UPR-Cyto stress, was identified by NEPHGE-based 2DE method, whereas IPG-based method showed unreliable results in the basic pI range and did not provide any new information on basic UPR-Cyto proteins. In the acidic range, the main UPR-Cyto proteins were detected and quantified by both methods. The drawback of NEPHGE-based 2DE method is its failure to detect some highly acidic proteins. The advantage of NEPHGE is higher protein capacity with good reproducibility and quality of spots at high protein load.ConclusionsComparison of broad range (pH3-10) gradient-based 2DE methods suggests that NEPHGE-based method is preferable over IPG (Invitrogen) 2DE method for the analysis of basic proteins. Nevertheless, the narrow range (pH4-7) IPG technique is a method of choice for the analysis of acidic proteins.

Dataset Information

Adaptation to potassium starvation of wild-type and K(+)-transport mutant (trk1,2) of Saccharomyces cerevisiae: 2-dimensional gel electrophoresis-based proteomic approach.

Publications

Adaptation to potassium starvation of wild-type and K(+)-transport mutant (trk1,2) of Saccharomyces cerevisiae: 2-dimensional gel electrophoresis-based proteomic approach.

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