is endowed with two genes coding for the main plasma membrane potassium transporters. These proteins are essential for the cell to grow at limiting potassium concentrations and mutants lacking the corresponding genes (TRK1
) show defective growth and transport at low potassium. However, at high potassium (in the mM range) other no specific systems can transport enough amounts of the cation, thus allowing mutant cells to grow at rates similar to those in the wild type (Navarrete et al. 2010
). A first proteomic study of trk1,2
mutants was recently published (Curto et al. 2010
). It is important to notice that in that paper both wild-type and mutant cells were grown under nonlimiting potassium concentrations (50 mM KCl) and the authors concluded that most of the differences observed between parental and DM strains corresponded to proteins related to glycolysis and redox-homeostasis enzymes.
Considering that full activity of the so-called high-affinity/high-velocity potassium transport process dependent upon the Trk1/2 system is usually observed after obtaining K+
-starved cells by incubation in media without added potassium during 4–5 h (Rodríguez-Navarro and Ramos 1984
; Bertl et al. 2003
), we decided to study changes in the proteome of the wild type and DM during the starvation process. The most important observation was the extra-ordinary decay in protein content and number of spots, observed during the 5 h of starvation with special relevance in the case of the mutant strain. It has to be considered that in 2D gels, only a fraction of the total proteome can be observed, mainly proteins that are the most abundant such as the housekeeping proteins (Buxbaum 2010
). Previous work reported that K+
deprivation during 24 h produced, in S. cerevisiae
, a decrease in cell viability by inducing a programmed cell death process (Lauff and Santa-María 2010
). Although this is a very important observation, in our conditions and after 5-h starvation we observed high decrease in protein content but no changes in cell viability. From these results, we conclude that potassium starvation is a very stressing process for the cells and it looks like 5-h incubation without potassium is excessive since it provokes a very important and general decay in many cellular processes. As mentioned above, 4–5 h K+
-starvation is a general method used to induce full activity of the Trk1/2 system. However, we have recently shown that in the newly designed medium YNB translucent (Navarrete et al. 2010
), used also in this work, adaptation to the high-affinity/high-velocity state is much faster. In fact, the higher affinity for Rb+
) is observed after 30-min starvation and higher Vmax
is reached after 2-h starvation. Therefore, we propose that this would be a much more rational way to obtain yeast cells expressing the high-affinity/high-velocity mode of transport.
PCA analysis allowed a clear classification of samples. The plot of PC1 (50.7%) and PC2 (22.2%) shows differences between mutant and wild type and also between the different sampling times. This supports the above-indicated hypothesis in terms of the differences found between strains behavior during the adaptation to the lack of potassium. This idea was confirmed after the application of a SOM neural network, a methodology for the classification of the samples more powerful than PCA analyses. Wild-type strain adapted quicker to the new conditions, since samples taken at 0, 30, and 60 min were already separated. Adaptation seems to be already completed after 3 h since samples taken at 180 and 300 min group together. On the other hand, the DM needed more time to try to adapt to the new environmental condition, that is, samples at time 0, 30, and 60 min grouped together. In conclusion, the wild-type strain adapted and got stabilized faster to the stress condition while the mutant seems to have problems to sense or adapt to the absence of potassium.
It is relevant that in the 2D gels we have identified most of the enzymes involved in glycolysis. During the starvation process, most of them enzymes were present in the wild type but in the mutant there was a fast decay. Our biochemical results on hexokinase activities are in agreement with this observation. The strong reduction in Hxk2 protein levels during starvation was not completely reflected in drastic changes in hexokinase activity. This apparent discrepancy could be explained by the induction of HXK1 and GLK1. The corresponding Hxk1 and Glk1 proteins, which would keep the capacity to phosphorylate glucose, are less abundant than Hxk2 and we did not identify them by proteomics; however, our transcriptomic results support that possibility.
On the other hand enzymes involved in two other important energetic pathways were detected: pentose phosphate and methylglyoxal pathways; in general, proteins from both pathways remained present during the starvation process. In fact, the transcriptomic profile of GLO1
, the two genes involved in detoxification of methylglyoxal, shows induction during starvation (not shown). It is tempting to speculate that the glycolysis pathway is more sensitive to low K+
than the alternative pathways and for that reason it is more inhibited in the mutant. It has been reported that, on the one hand, potassium plays a crucial role in the activation of the glycolytic enzyme pyruvate kinase (see Page and Di Cera (2006)
for a review) and, on the other hand, the mutant shows defective potassium transport. These facts may be related to the higher sensitivity of the glycolysis in the mutant.
We have mentioned the importance of the stress induced by potassium starvation, especially in mutant cells. The fact that two ubiquitin enzymes related with DNA-repair system (Ubc2 and Mms2) were identified along the 5 h of the experiment is in agreement with this observation (Broomfield et al. 1998
; Game and Chernikova 2009
). Even more in the mutant, the amount of the two ubiquitin proteins was not only present, but significantly increased during potassium starvation. On the other hand, some important pathways seem to be unaffected by starvation. Two examples are the metabolism of some amino acids (methionine, lysine) and bases (pyrimidine ribonucleotides). The application of two different algorithms for sample classification, one of them based on recent algorithms based on neural networks, lead to the obtaining of complementary results increasing the discriminatory power of this analysis (Valledor and Jorrín 2011
). Cluster analysis allowed a distance-based classification of the samples and spots reinforcing that idea. Five major groups of spots could be distinguished in the plot being relevant that most of the glycolytic proteins appear in groups I and II and show a completely different behavior in wild-type and mutant strain.
It is conceivable to pose the question about how TRK mutation affects these metabolic processes. We have no definitive answer to this question but our results indicating a defective metabolic adaptation to the lack of potassium in the mutant may be taken as a clue on the relevant role of potassium fluxes and/or levels triggering adaptation. Unpublished results of our group show that wild-type and trk1,2 cells grown under nonlimiting KCl are able to adapt and reach a new internal K+ stationary state when suspended in lower K+ concentrations, requiring mutant cells higher external K+ to keep similar internal amounts of the cation. In conclusion, the DM trk1,2 is still able to sense a decrease in external potassium but lacks the mechanism to properly adapt to this stress. A similar behavior may explain the defective metabolic adaptation during starvation.
In summary, the decrease in protein content during potassium starvation experiments lead to a global decrease of the basic cellular functions such as the cell energy production pathways, with a radical decrease of the glycolytic proteins that was more evident in the mutant. In the context of a general decrease of proteins, it is relevant that some cellular processes such as the pentose phosphate and methylglyoxal pathways were kept. These results indicate that conditions commonly used in the past to characterize adaptation to potassium (4–5 h in the absence of the cation) are too stressful for the cells and this should be considered in future studies on potassium homeostasis. In fact, the study of the proteome under less extreme potassium limitation is under way. This would allow to analyze differences between parental and mutant strains under more physiological conditions.