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.

• Klíčová slova
• NPR/Hal family, Nha1, Pma1 activity, Saccharomyces cerevisiae, acetic acid tolerance, plasma membrane H+-ATPase, yeast kinases,
• Publikační typ
• časopisecké články 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

Carbocyanine dye diS-C3(3) was repeatedly employed in monitoring the plasma membrane potential of yeast and other living cells. Four methods of measuring and evaluating probe fluorescence signal were used in different studies, based on following fluorescence parameters: fluorescence intensity emitted within a certain spectral interval, F(580)/F(560) fluorescence emission ratio, wavelength of emission spectrum maximum, and the ratio of respective fluorescence intensities corresponding to the diS-C3(3) bound to cytosolic macromolecules and remaining dissolved in the aqueous cell medium (i.e., unbound, or free). Here we show that data corresponding to the three latter spectral assessments of diS-C3(3) accumulation in cells is mutually convertible, which means that their alternative use cannot lead to ambiguities in the interpretation of the results of biological experiments. On the other hand, experiments based on the effortless measurements of fluorescence intensities should be interpreted cautiously because controversial results can be obtained, depending on the particular choice of cell-to-dye concentration ratio and emission wavelength.

• Klíčová slova
• Fluorescent probe, Plasma membrane potential, Saccharomyces cerevisiae, Spectral analysis, Yeast,
• MeSH
• fluorescenční barviva chemie   MeSH
• fluorescenční spektrometrie metody   MeSH
• karbocyaniny chemie   MeSH
• membránové potenciály * MeSH
• Saccharomyces cerevisiae chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3,3'-dipropylthiacarbocyanine MeSH Prohlížeč
• fluorescenční barviva MeSH
• karbocyaniny MeSH

Recently we introduced a fluorescent probe technique that makes possible to convert changes of equilibrium fluorescence spectra of 3,3'-dipropylthiadicarbocyanine, diS-C3(3), measured in yeast cell suspensions under defined conditions into underlying membrane potential differences, scaled in millivolts (Plasek et al. in J Bioenerg Biomembr 44: 559-569, 2012). The results presented in this paper disclose measurements of real early changes of plasma membrane potential induced by the increase of extracellular K(+), Na(+) and H(+) concentration in S. cerevisiae with and without added glucose as energy source. Whereas the wild type and the ∆tok1 mutant cells exhibited similar depolarization curves, mutant cells lacking the two Trk1,2 potassium transporters revealed a significantly decreased membrane depolarization by K(+), particularly at lower extracellular potassium concentration [K(+)]out. In the absence of external energy source plasma membrane depolarization by K(+) was almost linear. In the presence of glucose the depolarization curves exhibited an exponential character with increasing [K(+)]out. The plasma membrane depolarization by Na(+) was independent from the presence of Trk1,2 transporters. Contrary to K(+), Na(+) depolarized the plasma membrane stronger in the presence of glucose than in its absence. The pH induced depolarization exhibited a fairly linear relationship between the membrane potential and the pHo of cell suspensions, both in the wild type and the Δtrk1,2 mutant strains, when cells were energized by glucose. In the absence of glucose the depolarization curves showed a biphasic character with enhanced depolarization at lower pHo values.

• MeSH
• buněčná membrána metabolismus   MeSH
• draslík metabolismus   MeSH
• fluorescenční barviva chemie   MeSH
• fluorometrie MeSH
• kationty jednomocné metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• membránové potenciály účinky léků   MeSH
• Saccharomyces cerevisiae účinky léků   metabolismus   MeSH
• sodík metabolismus   MeSH
• vodík metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• draslík MeSH
• fluorescenční barviva MeSH
• kationty jednomocné MeSH
• sodík MeSH
• vodík MeSH

Three different transport systems exist to accumulate a sufficient amount of potassium cations in yeasts. The most common of these are Trk-type transporters, which are used by all yeast species. Though most yeast species employ two different types of transporters, we only identified one gene encoding a potassium uptake system (Trk-type) in the genome of the highly osmotolerant yeast Zygosaccharomyces rouxii, and our results showed that ZrTrk1 is its major (and probably only) specific potassium uptake system. When expressed in Saccharomyces cerevisiae, the product of the ZrTRK1 gene is localized to the plasma membrane and its presence efficiently complements the phenotypes of S. cerevisiae trk1∆ trk2∆ cells. Deletion of the ZrTRK1 gene resulted in Z. rouxii cells being almost incapable of growth at low K(+) concentrations and it changed some cell physiological parameters in a way that differs from S. cerevisiae. In contrast to S. cerevisiae, Z. rouxii cells without the TRK1 gene contained less potassium than the control cells and their plasma membrane was significantly hyperpolarized compared with those of the parental strain when grown in the presence of 100 mM KCl. On the other hand, subsequent potassium starvation led to a substantial depolarization which is again different from S. cerevisiae. Plasma-membrane hyperpolarization did not prevent the efflux of potassium from Z. rouxii trk1Δ cells during potassium starvation, and the activity of ZrPma1 is less affected by the absence of ZrTRK1 than in S. cerevisiae. The use of a newly constructed Z. rouxii-specific plasmid for the expression of pHluorin showed that the intracellular pH of the Z. rouxii wild type and the trk1∆ mutant is not significantly different. Together with the fact that Z. rouxii cells contain a significantly lower amount of intracellular potassium than identically grown S. cerevisiae cells, our results suggest that this highly osmotolerant yeast species maintain its intracellular pH and potassium homeostasis in way(s) partially distinct from S. cerevisiae.

• MeSH
• biologická adaptace MeSH
• biologický transport MeSH
• buněčná membrána fyziologie   MeSH
• delece genu MeSH
• DNA fungální genetika   metabolismus   MeSH
• draslík metabolismus   MeSH
• geny hub * MeSH
• homeostáza MeSH
• homologní rekombinace MeSH
• koncentrace vodíkových iontů MeSH
• membránové potenciály MeSH
• proteiny přenášející kationty genetika   metabolismus   MeSH
• regulace genové exprese u hub * MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   fyziologie   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie MeSH
• sekvenční seřazení MeSH
• Zygosaccharomyces genetika   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA fungální MeSH
• draslík MeSH
• proteiny přenášející kationty MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• TRK1 protein, S cerevisiae MeSH Prohlížeč

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