In Saccharomyces cerevisiae, TRK1 and TRK2 genes encode partially redundant K(+) transporters. Direct involvement in K(+) uptake has been shown for Trk1p since cells growing under limiting environmental K(+) concentrations demand its presence. The biological role of Trk2p is less understood. In our experiments, TRK2 overexpression improved the ability of trk1 cells to grow in low K(+) and led to a higher accumulation of K(+). Using diS-C(3)(3) as a potentiometric probe, we revealed a higher hyperpolarization of trk2 cells compared to the wild type. In addition, the deletion of TRK2 in the trk1 genetic background increased the cell sensitivity to hygromycin B, spermine, and TMA. Our studies reinforced the conclusion that Trk1p is the prominent K(+) uptake transporter and for the first time revealed that though Trk2p is much less effective, its activity contributes significantly to K(+) supply and the maintenance of plasma-membrane potential.

• MeSH
• biologický transport MeSH
• buněčná membrána fyziologie   MeSH
• draslík metabolismus   MeSH
• membránové potenciály * MeSH
• proteiny přenášející kationty genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• draslík MeSH
• proteiny přenášející kationty MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• TRK1 protein, S cerevisiae MeSH Prohlížeč
• TRK2 protein, S cerevisiae MeSH Prohlížeč

The maintenance of K+ and Ca2+ homeostasis is crucial for many cellular functions. Potassium is accumulated in cells at high concentrations, while the cytosolic level of calcium, to ensure its signalling function, is kept at low levels and transiently increases in response to stresses. We examined Ca2+ homeostasis and Ca2+ signalling in Saccharomyces cerevisiae strains lacking plasma-membrane K+ influx (Trk1 and Trk2) or efflux (Tok1, Nha1 and Ena1-5) systems. The lack of K+ exporters slightly increased the cytosolic Ca2+, but did not alter the Ca2+ tolerance or Ca2+-stress response. In contrast, the K+-importers Trk1 and Trk2 play important and distinct roles in the maintenance of Ca2+ homeostasis. The presence of Trk1 was vital mainly for the growth of cells in the presence of high extracellular Ca2+, whilst the lack of Trk2 doubled steady-state intracellular Ca2+ levels. The absence of both K+ importers highly increased the Ca2+ response to osmotic or CaCl2 stresses and altered the balance between Ca2+ flux from external media and intracellular compartments. In addition, we found Trk2 to be important for the tolerance to high KCl and hygromycin B in cells growing on minimal media. All the data describe new interconnections between potassium and calcium homeostasis in S. cerevisiae.

• Klíčová slova
• K+-transporter, Trk1, Trk2, calcium, osmotic shock, potassium, yeast,
• MeSH
• chlorid draselný farmakologie   MeSH
• cinnamáty farmakologie   MeSH
• draslík metabolismus   MeSH
• homeostáza * MeSH
• hygromycin B analogy a deriváty   farmakologie   MeSH
• proteiny přenášející kationty genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae účinky léků   genetika   metabolismus   MeSH
• signální transdukce * MeSH
• vápník metabolismus   farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chlorid draselný MeSH
• cinnamáty MeSH
• draslík MeSH
• hygromycin A MeSH Prohlížeč
• hygromycin B MeSH
• proteiny přenášející kationty MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• TRK1 protein, S cerevisiae MeSH Prohlížeč
• TRK2 protein, S cerevisiae MeSH Prohlížeč
• vápník MeSH

The existence of programmed cell death in Saccharomyces cerevisiae has been reported for many years. Glucose induces the death of S. cerevisiae in the absence of additional nutrients within a few hours, and the absence of active potassium uptake makes cells highly sensitive to this process. S. cerevisiae cells possess two transporters, Trk1 and Trk2, which ensure a high intracellular concentration of potassium, necessary for many physiological processes. Trk1 is the major system responsible for potassium acquisition in growing and dividing cells. The contribution of Trk2 to potassium uptake in growing cells is almost negligible, but Trk2 becomes crucial for stationary cells for their survival of some stresses, e.g. anhydrobiosis. As a new finding, we show that both Trk systems contribute to the relative thermotolerance of S. cerevisiae BY4741. Our results also demonstrate that Trk2 is much more important for the cell survival of glucose-induced cell death than Trk1, and that stationary cells deficient in active potassium uptake lose their ATP stocks more rapidly than cells with functional Trk systems. This is probably due to the upregulated activity of plasma-membrane Pma1 H+-ATPase, and consequently, it is the reason why these cells die earlier than cells with functional active potassium uptake.

• Klíčová slova
• ATP content, GICD, Saccharomyces cerevisiae, potassium uptake, stationary cells, thermotolerance,
• MeSH
• buněčná smrt MeSH
• draslík metabolismus   MeSH
• glukosa metabolismus   MeSH
• mikrobiální viabilita MeSH
• proteiny přenášející kationty genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae cytologie   růst a vývoj   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• draslík MeSH
• glukosa MeSH
• proteiny přenášející kationty MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• TRK1 protein, S cerevisiae MeSH Prohlížeč
• TRK2 protein, S cerevisiae MeSH Prohlížeč

In Saccharomyces cerevisiae, K+-uptake under K+-limiting conditions is largely mediated by the cation translocation systems Trk1 and Trk2 belonging to the family of SKT proteins. They are related to two-transmembrane-domain (inward rectifying K-) channels but unlike the symmetrical tetrameric structure of K-channels, a single Trk contains four pore-forming domains (A-D) encoded on one polypeptide chain. Between domains A and B Trks contain large cytosolic regions dubbed "long hydrophilic loop" (LHL). LHLs are not homologous/similar to any other identified protein (domain) and also show little similarity between Trk1 and Trk2. Here we demonstrate that Trk1 is functional without LHL. However, in growth experiments NaCl sensitivity of Trk1[ΔLHL] expressing cells is increased under K+-limiting conditions compared to full-length Trk1. Non-invasive ion flux measurements showed that K+-influx through Trk1 and Trk1[ΔLHL] is decreased in the presence of surplus Na+ due to permeability of the proteins for both cations and competition between them. Trk1[ΔLHL] is less affected than full-length Trk1 because it is more selective for K+ over Na+. Furthermore, K+ re-uptake after starvation is delayed and decreased in Trk1[ΔLHL]. Thus, a role of LHL is regulating cation fluxes through Trk1 by (i) allowing also Na+ to pass if monovalent cations (mainly K+) are limiting and (ii) by accelerating/enhancing a switch from low to high affinity ion translocation. We propose that LHL could modulate Trk1 transport properties via direct influence on a transmembrane helix (M2A) which can switch between bent and straight conformation, thereby directly modifying the radius of the pore and selectivity filter.

• Klíčová slova
• Bimolecular Fluorescence Complementation – BiFC, K(+)-transport, Long hydrophilic loop, LHL, Non-invasive ion-flux measurements – FLISE, Saccharomyces cerevisiae, Trk1–potassium translocation system,
• MeSH
• dimerizace MeSH
• draslík metabolismus   MeSH
• hydrofobní a hydrofilní interakce MeSH
• iontový transport MeSH
• kationty metabolismus   MeSH
• proteiny přenášející kationty chemie   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• draslík MeSH
• kationty MeSH
• proteiny přenášející kationty MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• TRK1 protein, S cerevisiae MeSH Prohlížeč

• MeSH
• aktivní transport MeSH
• draslík metabolismus   MeSH
• fungální proteiny metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• membránové proteiny metabolismus   MeSH
• proteiny přenášející kationty * MeSH
• protony * MeSH
• rubidium metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• thallium metabolismus   MeSH
• transportní proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• draslík MeSH
• fungální proteiny MeSH
• membránové proteiny MeSH
• proteiny přenášející kationty * MeSH
• protony * MeSH
• rubidium MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• thallium MeSH
• transportní proteiny MeSH
• TRK1 protein, S cerevisiae MeSH Prohlížeč
• TRK2 protein, S cerevisiae MeSH Prohlížeč

Yeasts usually have one or two high-affinity potassium transporters. Two complete and one interrupted gene encoding three types of putative potassium uptake system exist in Candida albicans SC5314. As high intracellular potassium is essential for many yeast cell functions, the existence of three transporters with differing transport mechanisms (Trk uniporter, Hak cation-proton symporter, Acu ATPase) may help pathogenic C. albicans cells to acquire the necessary potassium in various organs and tissues of the host. When expressed in Saccharomyces cerevisiae lacking their own potassium uptake systems, all three putative transporters were able to provide cells with the ability to grow with low amounts of potassium over a broad range of external pH. Only CaTrk1 was properly recognized and secreted to the plasma membrane. Nevertheless, even the small number of CaHak1 and mainly CaAcu1 molecules which reached the plasma membrane resulted in an improved growth of cells in low potassium concentrations, suggesting a high affinity and capacity of the transporters. A single-point mutation restored the complete CaACU1 gene, and the resulting protein not only provided cells with the necessary potassium but also improved their tolerance to toxic lithium. In contrast to its known homologues, CaAcu1 did not seem to transport sodium.

• Klíčová slova
• Acu1 ATPase, Candida, Hak1 transporter, Trk1 transporter, cation homeostasis, potassium uptake,
• MeSH
• Candida albicans genetika   metabolismus   MeSH
• draslík metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• kultivační média chemie   MeSH
• membránové transportní proteiny genetika   metabolismus   MeSH
• proteiny přenášející kationty nedostatek   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• Saccharomyces cerevisiae genetika   růst a vývoj   metabolismus   MeSH
• testy genetické komplementace MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• draslík MeSH
• kultivační média MeSH
• membránové transportní proteiny MeSH
• proteiny přenášející kationty MeSH
• rekombinantní proteiny MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• Trk1 protein, Candida albicans MeSH Prohlížeč
• TRK1 protein, S cerevisiae MeSH Prohlížeč
• TRK2 protein, S cerevisiae MeSH Prohlížeč

• MeSH
• draslík metabolismus   MeSH
• fungální proteiny genetika   fyziologie   MeSH
• iontový transport účinky léků   MeSH
• lithium farmakologie   MeSH
• membránové proteiny genetika   fyziologie   MeSH
• mutace MeSH
• proteiny přenášející kationty * MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• Saccharomyces cerevisiae účinky léků   metabolismus   MeSH
• sodík farmakologie   MeSH
• transportní proteiny genetika   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• draslík MeSH
• fungální proteiny MeSH
• lithium MeSH
• membránové proteiny MeSH
• proteiny přenášející kationty * MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• sodík MeSH
• transportní proteiny MeSH
• TRK1 protein, S cerevisiae MeSH Prohlížeč
• TRK2 protein, S cerevisiae MeSH Prohlížeč

Disruption of genes encoding endogenous transport proteins in Saccharomyces cerevisiae has facilitated the recent cloning, by functional expression, of cDNAs encoding K+ channels and amino acid transporters from the plant Arabidopsis thaliana [1-4]. In the present study, we demonstrate in whole-cell patch clamp experiments that the inability of trk1deltatrk2delta mutants of S. cerevisiae to grow on submillimolar K+ correlates with the lack of K+ inward currents, which are present in wild-type cells, and that transformation of the trk1deltatrk2delta double-deletion mutant with KAT1 from Arabidopsis thaliana restores this phenotype by encoding a plasma membrane protein that allows large K+ inward currents. Similar K+ inward currents are induced by transformation of a trk1 mutant with AKT1 from A. thaliana.

• MeSH
• Arabidopsis MeSH
• draslík metabolismus   MeSH
• draslíkové kanály dovnitř usměrňující * MeSH
• draslíkové kanály genetika   fyziologie   MeSH
• fungální proteiny genetika   MeSH
• homeodoménové proteiny genetika   fyziologie   MeSH
• iontový transport MeSH
• kineziny * MeSH
• membránové proteiny genetika   MeSH
• metoda terčíkového zámku MeSH
• proteiny huseníčku * MeSH
• proteiny přenášející kationty * MeSH
• rostlinné geny MeSH
• rostlinné proteiny genetika   fyziologie   MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• Saccharomyces cerevisiae genetika   fyziologie   MeSH
• sekvenční delece MeSH
• testy genetické komplementace MeSH
• trans-aktivátory genetika   fyziologie   MeSH
• transportní proteiny genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• ATK1 protein, Arabidopsis MeSH Prohlížeč
• draslík MeSH
• draslíkové kanály dovnitř usměrňující * MeSH
• draslíkové kanály MeSH
• fungální proteiny MeSH
• homeodoménové proteiny MeSH
• KAT1 protein, Arabidopsis MeSH Prohlížeč
• kineziny * MeSH
• membránové proteiny MeSH
• proteiny huseníčku * MeSH
• proteiny přenášející kationty * MeSH
• rostlinné proteiny MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• trans-aktivátory MeSH
• transportní proteiny MeSH
• TRK1 protein, S cerevisiae MeSH Prohlížeč
• TRK2 protein, S cerevisiae MeSH Prohlížeč

Maintenance of intracellular K+ homeostasis is one of the crucial requisites for the survival of yeast cells. In Saccharomyces cerevisiae, the high K+ content corresponds to a steady state between simultaneous influx and efflux across the plasma membrane. One of the transporters formerly believed to extrude K+ from the yeast cells (besides Ena1-4p and Nha1p) was named Kha1p and presumed as a putative plasma membrane K+/H+ antiporter. We prepared kha1 and tok1-kha1 deletion strains in the B31 and MAB 2d background. Both the strains contain the ena1-4 and nha1 deletions; that means they lack the main active sodium and potassium efflux systems. MAB 2d has additional trk1 and trk2 deletions, i.e. is impaired in active K+ uptake as well. We performed a large physiological study with these strains to specify the phenotype of kha1 deletion. In our experiments, no difference in K+ content or efflux was observed in strains lacking the KHA1 gene compared with control strains. Two main phenotype manifestations of the kha1 deletion were growth defect on high external pH and hygromycin sensitivity. The correlation between these phenotypes and the kha1 deletion was confirmed by plasmid complementation. Fluorescence microscopy of green fluorescent protein (GFP)-tagged Kha1p showed that this antiporter is localized preferentially intracellularly (in contrast to the plasma membrane Na+/H+ antiporter Nha1p). Based on these findings, Kha1p is probably not localized in plasma membrane and does not mediate efflux of alkali metal cations from cells, but is important for the regulation of intracellular cation homeostasis and optimal pH control, similarly as the Nhx1p.

• MeSH
• buněčná membrána MeSH
• delece genu * MeSH
• draslíko-vodíkové antiportéry genetika   metabolismus   MeSH
• homeostáza MeSH
• kationty metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• organely fyziologie   MeSH
• regulace genové exprese u hub MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   růst a vývoj   fyziologie   MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• draslíko-vodíkové antiportéry MeSH
• kationty MeSH
• KHA1 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• zelené fluorescenční proteiny MeSH

K+ is one of the cations (besides protons) whose transport across the plasma membrane is believed to contribute to the maintenance of membrane potential. To ensure K+ transport, Saccharomyces cerevisiae cells possess several types of active and passive transporters mediating the K+ influx and efflux, respectively. A diS-C3(3) assay was used to compare the contributions of various potassium transporters to the membrane potential changes of S. cerevisiae cells in the exponential growth phase. Altogether, the contributions of six K+ transporters to the maintenance of a stable membrane potential were tested. As confirmed by the observed hyperpolarization of trk1 trk2 deletion strains, the diS-C3(3) assay is a suitable method for comparative studies of the membrane potential of yeast strains differing in the presence/absence of one or more cation transporters. We have shown that the presence of the Tok1 channel strongly influences membrane potential: deletion of the TOK1 gene results in significant plasma membrane depolarization, whereas strains overexpressing the TOK1 gene are hyperpolarized. We have also proved that plasma membrane potential is not the only parameter determining the hygromycin B sensitivity of yeast cells, and that the role of intracellular transporters in protecting against its toxic effects must also be considered.

• MeSH
• buněčná membrána fyziologie   MeSH
• draslík metabolismus   MeSH
• draslíko-vodíkové antiportéry fyziologie   MeSH
• draslíkové kanály fyziologie   MeSH
• membránové potenciály MeSH
• Saccharomyces cerevisiae - proteiny fyziologie   MeSH
• Saccharomyces cerevisiae fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• draslík MeSH
• draslíko-vodíkové antiportéry MeSH
• draslíkové kanály MeSH
• KHA1 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• TOK1 protein, S cerevisiae MeSH Prohlížeč