The non-conventional yeast Kluyveromyces marxianus has recently emerged as a promising candidate for many food, environment, and biotechnology applications. This yeast is thermotolerant and has robust growth under many adverse conditions. Here, we show that its ability to grow under potassium-limiting conditions is much better than that of Saccharomyces cerevisiae, suggesting a very efficient and high-affinity potassium uptake system(s) in this species. The K. marxianus genome contains two genes for putative potassium transporters: KmHAK1 and KmTRK1. To characterize the products of the two genes, we constructed single and double knock-out mutants in K. marxianus and also expressed both genes in an S. cerevisiae mutant, that lacks potassium importers. Our results in K. marxianus and S. cerevisiae revealed that both genes encode efficient high-affinity potassium transporters, contributing to potassium homeostasis and maintaining plasma-membrane potential and cytosolic pH. In K. marxianus, the presence of HAK1 supports growth at low K+ much better than that of TRK1, probably because the substrate affinity of KmHak1 is about 10-fold higher than that of KmTrk1, and its expression is induced ~80-fold upon potassium starvation. KmHak1 is crucial for salt stress survival in both K. marxianus and S. cerevisiae. In co-expression experiments with ScTrk1 and ScTrk2, its robustness contributes to an increased tolerance of S. cerevisiae cells to sodium and lithium salt stress.

• Keywords
• Kluyveromyces marxianu, K+–H+ symporter, affinity, potassium, transporter, uniporter,
• MeSH
• Potassium * metabolism   MeSH
• Fungal Proteins genetics   metabolism   MeSH
• Gene Knockout Techniques MeSH
• Kluyveromyces * genetics   metabolism   growth & development   MeSH
• Hydrogen-Ion Concentration MeSH
• Membrane Potentials MeSH
• Cation Transport Proteins * genetics   metabolism   MeSH
• Gene Expression Regulation, Fungal MeSH
• Saccharomyces cerevisiae * genetics   metabolism   growth & development   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Potassium * MeSH
• Fungal Proteins MeSH
• Cation Transport Proteins * MeSH

Acetic acid-induced stress is a common challenge in natural environments and industrial bioprocesses, significantly affecting the growth and metabolic performance of Saccharomyces cerevisiae. The adaptive response and tolerance to this stress involves the activation of a complex network of molecular pathways. This study aims to delve deeper into these mechanisms in S. cerevisiae, particularly focusing on the role of the Hrk1 kinase. Hrk1 is a key determinant of acetic acid tolerance, belonging to the NPR/Hal family, whose members are implicated in the modulation of the activity of plasma membrane transporters that orchestrate nutrient uptake and ion homeostasis. The influence of Hrk1 on S. cerevisiae adaptation to acetic acid-induced stress was explored by employing a physiological approach based on previous phosphoproteomics analyses. The results from this study reflect the multifunctional roles of Hrk1 in maintaining proton and potassium homeostasis during different phases of acetic acid-stressed cultivation. Hrk1 is shown to play a role in the activation of plasma membrane H+-ATPase, maintaining pH homeostasis, and in the modulation of plasma membrane potential under acetic acid stressed cultivation. Potassium (K+) supplementation of the growth medium, particularly when provided at limiting concentrations, led to a notable improvement in acetic acid stress tolerance of the hrk1Δ strain. Moreover, abrogation of this kinase expression is shown to confer a physiological advantage to growth under K+ limitation also in the absence of acetic acid stress. The involvement of the alkali metal cation/H+ exchanger Nha1, another proposed molecular target of Hrk1, in improving yeast growth under K+ limitation or acetic acid stress, is proposed.

• Keywords
• NPR/Hal family, Nha1, Pma1 activity, Saccharomyces cerevisiae, acetic acid tolerance, plasma membrane H+-ATPase, yeast kinases,
• Publication type
• Journal Article MeSH

The alteration of the fine-tuned balance of phospho/dephosphorylation reactions in the cell often results in functional disturbance. In the yeast Saccharomyces cerevisiae, the overexpression of Ser/Thr phosphatase Ppz1 drastically blocks cell proliferation, with a profound change in the transcriptomic and phosphoproteomic profiles. While the deleterious effect on growth likely derives from the alteration of multiple targets, the precise mechanisms are still obscure. Ppz1 is a negative effector of potassium influx. However, we show that the toxic effect of Ppz1 overexpression is unrelated to the Trk1/2 high-affinity potassium importers. Cells overexpressing Ppz1 exhibit decreased K+ content, increased cytosolic acidification, and fail to properly acidify the medium. These effects, as well as the growth defect, are counteracted by the deletion of NHA1 gene, which encodes a plasma membrane Na+, K+/H+ antiporter. The beneficial effect of a lack of Nha1 on the growth vanishes as the pH of the medium approaches neutrality, is not eliminated by the expression of two non-functional Nha1 variants (D145N or D177N), and is exacerbated by a hyperactive Nha1 version (S481A). All our results show that high levels of Ppz1 overactivate Nha1, leading to an excessive entry of H+ and efflux of K+, which is detrimental for growth.

• Keywords
• K+ transport, Nha1, Ppz1 phosphatase, Saccharomyces cerevisiae, cation homeostasis, intracellular pH,
• Publication type
• Journal Article MeSH

The gradual acidification of the secretory pathway is conserved and extremely important for eukaryotic cells, but until now there was no pH sensor available to monitor the pH of the early Golgi apparatus in Saccharomyces cerevisiae. Therefore, we developed a pHluorin-based sensor for in vivo measurements in the lumen of the Golgi. By using this new tool we show that the cis- and medial-Golgi pH is equal to 6.6-6.7 in wild type cells during exponential phase. As expected, V-ATPase inactivation results in a near neutral Golgi pH. We also uncover that surprisingly Vph1p isoform of the V-ATPase is prevalent to Stv1p for Golgi acidification. Additionally, we observe that during changes of the cytosolic pH, the Golgi pH is kept relatively stable, mainly thanks to the V-ATPase. Eventually, this new probe will allow to better understand the mechanisms involved in the acidification and the pH control within the secretory pathway.

• MeSH
• Biosensing Techniques instrumentation   MeSH
• Chemical Engineering MeSH
• Golgi Apparatus chemistry   MeSH
• Isoenzymes chemistry   MeSH
• Hydrogen-Ion Concentration MeSH
• Saccharomyces cerevisiae Proteins chemistry   MeSH
• Saccharomyces cerevisiae chemistry   enzymology   MeSH
• Vacuolar Proton-Translocating ATPases chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Isoenzymes MeSH
• Saccharomyces cerevisiae Proteins MeSH
• Vacuolar Proton-Translocating ATPases MeSH

Candida glabrata is a haploid yeast that is considered to be an emergent pathogen since it is the second most prevalent cause of candidiasis. Contrary to most yeasts, this species carries only one plasma membrane potassium transporter named CgTrk1. We show in this work that the activity of this transporter is regulated at the posttranslational level, and thus Trk1 contributes to potassium uptake under very different external cation concentrations. In addition to its function in potassium uptake, we report a diversity of physiological effects related to this transporter. CgTRK1 contributes to proper cell size, intracellular pH and membrane-potential homeostasis when expressed in Saccharomyces cerevisiae. Moreover, lithium influx experiments performed both in C. glabrata and S. cerevisiae indicate that the salt tolerance phenotype linked to CgTrk1 can be related to a high capacity to discriminate between potassium and lithium (or sodium) during the transport process. In summary, we show that CgTRK1 exerts a diversity of pleiotropic physiological roles and we propose that the corresponding protein may be an attractive pharmacological target for the development of new antifungal drugs.

• Keywords
• Candida glabrata, Membrane potential, Potassium transport, Saccharomyces cerevisiae, Salt tolerance, Trk1,
• MeSH
• Cell Membrane metabolism   MeSH
• Candida glabrata genetics   metabolism   MeSH
• Potassium metabolism   MeSH
• Fungal Proteins genetics   metabolism   MeSH
• Homeostasis MeSH
• Hydrogen-Ion Concentration MeSH
• Cation Transport Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Fungal MeSH
• Sodium metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Potassium MeSH
• Fungal Proteins MeSH
• Cation Transport Proteins MeSH
• Sodium MeSH

The maintenance of potassium homeostasis is crucial for all types of cells, including Candida glabrata. Three types of plasma-membrane systems mediating potassium influx with different transport mechanisms have been described in yeasts: the Trk1 uniporter, the Hak cation-proton symporter and the Acu ATPase. The C. glabrata genome contains only one gene encoding putative system for potassium uptake, the Trk1 uniporter. Therefore, its importance in maintaining adequate levels of intracellular potassium appears to be critical for C. glabrata cells. In this study, we first confirmed the potassium-uptake activity of the identified gene's product by heterologous expression in a suitable S. cerevisiae mutant, further we generated a corresponding deletion mutant in C. glabrata and analysed its phenotype in detail. The obtained results show a pleiotropic effect on the cell physiology when CgTRK1 is deleted, affecting not only the ability of trk1Δ to grow at low potassium concentrations, but also the tolerance to toxic alkali-metal cations and cationic drugs, as well as the membrane potential and intracellular pH. Taken together, our results find the sole potassium uptake system in C. glabrata cells to be a promising target in the search for its specific inhibitors and in developing new antifungal drugs.

• MeSH
• Cell Membrane metabolism   MeSH
• Candida glabrata metabolism   physiology   MeSH
• Potassium metabolism   MeSH
• Homeostasis physiology   MeSH
• Ion Transport physiology   MeSH
• Cations metabolism   MeSH
• Membrane Potentials physiology   MeSH
• Cation Transport Proteins metabolism   MeSH
• Gene Expression Regulation, Fungal physiology   MeSH
• Saccharomyces cerevisiae metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Potassium MeSH
• Cations MeSH
• Cation Transport Proteins MeSH
• Trk1 protein, Candida albicans MeSH Browser