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SummarySaccharomyces cerevisiae Hal3 and Vhs3 are moonlighting proteins, forming an atypical heterotrimeric decarboxylase (PPCDC) required for CoA biosynthesis, and regulating cation homeostasis by inhibition of the Ppz1 phosphatase. The Schizosaccharomyces pombe ORF SPAC15E1.04 (renamed as Sp hal3) encodes a protein whose amino-terminal half is similar to Sc Hal3 whereas its carboxyl-terminal half is related to thymidylate synthase (TS). We show that Sp Hal3 and/or its N-terminal domain retain the ability to bind to and modestly inhibit in vitro S. cerevisiae Ppz1 as well as its S. pombe homolog Pzh1, and also exhibit PPCDC activity in vitro and provide PPCDC function in vivo, indicating that Sp Hal3 is a monogenic PPCDC in fission yeast. Whereas the Sp Hal3 N-terminal domain partially mimics Sc Hal3 functions, the entire protein and its carboxyl-terminal domain rescue the S. cerevisiae cdc21 mutant, thus proving TS function. Additionally, we show that the 70 kDa Sp Hal3 protein is not proteolytically processed under diverse forms of stress and that, as predicted, Sp hal3 is an essential gene. Therefore, Sp hal3 represents a fusion event that joined three different functional activities in the same gene. The possible advantage derived from this surprising combination of essential proteins is discussed.

Living cells accumulate potassium (K⁺) to fulfil multiple functions. It is well documented that the model yeast Saccharomyces cerevisiae grows at very different concentrations of external alkali cations and keeps high and low intracellular concentrations of K⁺ and sodium (Na⁺) respectively. However less attention has been paid to the study of the intracellular distribution of these cations. The most widely used experimental approach, plasma membrane permeabilization, produces incomplete results, since it usually considers only cytoplasm and vacuoles as compartments where the cations are present in significant amounts. By isolating and analysing the main yeast organelles, we have determined the subcellular location of K⁺ and Na⁺ in S. cerevisiae. We show that while vacuoles accumulate most of the intracellular K⁺ and Na⁺, the cytosol contains relatively low amounts, which is especially relevant in the case of Na⁺. However K⁺ concentrations in the cytosol are kept rather constant during the K⁺-starvation process and we conclude that, for that purpose, vacuolar K⁺ has to be rapidly mobilized. We also show that this intracellular distribution is altered in four different mutants with impaired vacuolar physiology. Finally, we show that both in wild-type and vacuolar mutants, nuclei contain and keep a relatively constant and important percentage of total intracellular K⁺ and Na⁺, which most probably is involved in the neutralization of negative charges.

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Saccharomyces cerevisiae cells are able to grow at very different potassium concentrations adapting its intracellular cation levels to changes in the external milieu. Potassium homeostasis in wild type cells resuspended in media with low potassium is an example of non-perfect adaptation since the same intracellular concentration is not approached irrespective of the extracellular levels of the cation. By using yeasts lacking the Trk1,2 system or expressing different versions of the mutated main plasma membrane potassium transporter (Trk1), we show that Trk1 is not essential for adaptation to potassium changes but the dynamics of potassium loss is very different in the wild type and in trk1,2 mutant or in yeasts expressing Trk1 versions with highly impaired transport characteristics. We also show that the pattern here described can be also fulfilled by heterologous expression of NcHAK1, a potassium transporter not belonging to the TRK family. Hyperpolarization and cationic drugs sensitivity in mutants with defective transport capacity provide additional support to the hypothesis of connections between the activity of the Trk system and the plasma membrane H(+) ATPase (Pma1) in the adaptive process.

Saccharomyces cerevisiae mutants lacking both isoforms of the main plasma membrane potassium transporter display impaired potassium transport and defective growth at limiting concentrations of the cation. Moreover, they are hyperpolarized and have a lower intracellular pH than wild-type. In order to unravel global physiological processes altered in trk1,2 mutants, we have established conditions at which both wild-type and mutants can grow at different rates. Using a combination of physiological, biochemical and proteomic approaches, we show that during growth at suboptimal potassium concentrations, double trk1,2 mutants accumulate less potassium and reach lower yields. In contrast, the mutants maintain increased viability in the stationary phase and retain more potassium. Moreover, the mutants show increased expression of stress-related proteins such as catalase T, thioredoxin peroxidase or hexokinase 2, suggesting that they are better adapted to the additional stress factors associated with entry into stationary growth phase.

Ypi1 was discovered as an essential protein able to act as a regulatory subunit of the Saccharomyces cerevisiae type 1 protein phosphatase Glc7 and play a key role in mitosis. We show here that partial depletion of Ypi1 causes lithium sensitivity and that high levels of this protein confer a lithium-tolerant phenotype to yeast cells. Remarkably, this phenotype was independent of the role of Ypi1 as a Glc7 regulatory subunit. Lithium tolerance in cells overexpressing Ypi1 was caused by a combination of increased efflux of lithium, mediated by augmented expression of the alkaline cation ATPase ENA1, and decreased lithium influx through the Trk1,2 high-affinity potassium transporters. Deletion of CNB1, encoding the regulatory subunit of the calcineurin phosphatase, blocked Ypi1-induced expression of ENA1, normalized Li + fluxes, and abolished the Li + hypertolerant phenotype of Ypi1-overexpressing cells. These results point to a complex role of Ypi1 on the regulation of cation homeostasis, largely mediated by the calcineurin phosphatase.

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.