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.

• Klíčová slova
• Kluyveromyces marxianu, K+–H+ symporter, affinity, potassium, transporter, uniporter,
• MeSH
• draslík * metabolismus   MeSH
• fungální proteiny genetika   metabolismus   MeSH
• genový knockout MeSH
• Kluyveromyces * genetika   metabolismus   růst a vývoj   MeSH
• koncentrace vodíkových iontů MeSH
• membránové potenciály MeSH
• proteiny přenášející kationty * genetika   metabolismus   MeSH
• regulace genové exprese u hub MeSH
• Saccharomyces cerevisiae * genetika   metabolismus   růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• draslík * MeSH
• fungální proteiny MeSH
• proteiny přenášející kationty * MeSH

Yeasts need a high intracellular concentration of potassium to grow. The main K+ uptake system in Saccharomyces cerevisiae is the Trk1 transporter, a complex protein with four MPM helical membrane motifs. Trk1 has been shown to exist in low- or high-affinity modes, which reflect the availability of potassium in the environment. However, when and how the affinity changes, and whether the potassium availability is the only signal for the affinity switch, remains unknown. Here, we characterize the Trk1 kinetic parameters under various conditions and find that Trk1's KT and Vmax change gradually. This gliding adjustment is rapid and precisely reflects the changes in the intracellular potassium content and membrane potential. A detailed characterization of the specific mutations in the P-helices of the MPM segments reveals that the presence of proline in the P-helix of the second and third MPM domain (F820P and L949P) does not affect the function of Trk1 in general, but rather specifically prevents the transporter's transition to a high-affinity state. The analogous mutations in the two remaining MPM domains (L81P and L1115P) result in a mislocalized and inactive protein, highlighting the importance of the first and fourth P-helices in proper Trk1 folding and activity at the plasma membrane.

• Klíčová slova
• Saccharomyces cerevisiae, cation homeostasis, membrane potential, potassium uptake,
• Publikační typ
• časopisecké články MeSH

There are only a few antifungal drugs used systemically in treatment, and invasive fungal infections that are resistant to these drugs are an emerging problem in health care. In this study, we performed a high-copy-number genomic DNA (gDNA) library screening to find and characterize genes that reduce susceptibility to amphotericin B, caspofungin, and voriconazole in Saccharomyces cerevisiae We identified the PDR16 and PMP3 genes for amphotericin B, the RMD9 and SWH1 genes for caspofungin, and the MRS3 and TRI1 genes for voriconazole. The deletion mutants for PDR16 and PMP3 were drug susceptible, but the other mutants had no apparent susceptibility. Quantitative-PCR analyses suggested that the corresponding drugs upregulated expression of the PDR16, PMP3, SWH1, and MRS3 genes. To further characterize these genes, we also profiled the global expression patterns of the cells after treatment with the antifungals and determined the genes and paths that were up- or downregulated. We also cloned Candida albicans homologs of the PDR16, PMP3, MRS3, and TRI1 genes and expressed them in S. cerevisiae Heterologous expression of Candida homologs also provided reduced drug susceptibility to the budding yeast cells. Our analyses suggest the involvement of new genes in antifungal drug resistance.

• Klíčová slova
• amphotericin B, antifungal agents, caspofungin, drug resistance, genomics, multidrug resistance, voriconazole,
• MeSH
• amfotericin B farmakologie   MeSH
• antifungální látky farmakologie   MeSH
• Candida albicans účinky léků   genetika   metabolismus   MeSH
• fungální léková rezistence genetika   MeSH
• kaspofungin farmakologie   MeSH
• mikrobiální testy citlivosti MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae účinky léků   genetika   metabolismus   MeSH
• Saccharomycetales účinky léků   genetika   metabolismus   MeSH
• vorikonazol farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amfotericin B MeSH
• antifungální látky MeSH
• kaspofungin MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• vorikonazol 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.

• Klíčová slova
• Candida glabrata, Membrane potential, Potassium transport, Saccharomyces cerevisiae, Salt tolerance, Trk1,
• MeSH
• buněčná membrána metabolismus   MeSH
• Candida glabrata genetika   metabolismus   MeSH
• draslík metabolismus   MeSH
• fungální proteiny genetika   metabolismus   MeSH
• homeostáza MeSH
• koncentrace vodíkových iontů MeSH
• proteiny přenášející kationty genetika   metabolismus   MeSH
• regulace genové exprese u hub MeSH
• sodík metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• draslík MeSH
• fungální proteiny MeSH
• proteiny přenášející kationty MeSH
• sodík MeSH

Saccharomyces species, which are mostly used in the food and beverage industries, are known to differ in their fermentation efficiency and tolerance of adverse fermentation conditions. However, the basis of their difference has not been fully elucidated, although their genomes have been sequenced and analyzed. Five strains of four Saccharomyces species (S. cerevisiae, S. kudriavzevii, S. bayanus, and S. paradoxus), when grown in parallel in laboratory conditions, exhibit very similar basic physiological parameters such as membrane potential, intracellular pH, and the degree to which they are able to quickly activate their Pma1 H+-ATPase upon glucose addition. On the other hand, they differ in their ability to proliferate in media with a very low concentration of potassium, in their osmotolerance and tolerance to toxic cations and cationic drugs in a growth-medium specific manner, and in their capacity to survive anhydrobiosis. Overall, S. cerevisiae (T73 more than FL100) and S. paradoxus are the most robust, and S. kudriavzevii the most sensitive species. Our results suggest that the difference in stress survival is based on their ability to quickly accommodate their cell size and metabolism to changing environmental conditions and to adjust their portfolio of available detoxifying transporters.

• Klíčová slova
• Intracellular pH, Membrane potential, Saccharomyces, Stress tolerance,
• MeSH
• fermentace MeSH
• fungální proteiny genetika   metabolismus   MeSH
• fyziologický stres MeSH
• glukosa metabolismus   MeSH
• protonové ATPasy genetika   metabolismus   MeSH
• Saccharomyces klasifikace   genetika   růst a vývoj   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• fungální proteiny MeSH
• glukosa MeSH
• protonové ATPasy 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
• buněčná membrána metabolismus   MeSH
• Candida glabrata metabolismus   fyziologie   MeSH
• draslík metabolismus   MeSH
• homeostáza fyziologie   MeSH
• iontový transport fyziologie   MeSH
• kationty metabolismus   MeSH
• membránové potenciály fyziologie   MeSH
• proteiny přenášející kationty metabolismus   MeSH
• regulace genové exprese u hub fyziologie   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• draslík MeSH
• kationty MeSH
• proteiny přenášející kationty MeSH
• Trk1 protein, Candida albicans MeSH Prohlížeč