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

In baker's yeast (Saccharomyces cerevisiae), Trk1, a member of the superfamily of K-transporters (SKT), is the main K+ uptake system under conditions when its concentration in the environment is low. Structurally, Trk1 is made up of four domains, each similar and homologous to a K-channel α subunit. Because most K-channels are proteins containing four channel-building α subunits, Trk1 could be functional as a monomer. However, related SKT proteins TrkH and KtrB were crystallised as dimers, and for Trk1, a tetrameric arrangement has been proposed based on molecular modelling. Here, based on Bimolecular Fluorescence Complementation experiments and single-molecule fluorescence microscopy combined with molecular modelling; we provide evidence that Trk1 can exist in the yeast plasma membrane as a monomer as well as a dimer. The association of monomers to dimers is regulated by the K+ concentration.

• Klíčová slova
• K+ translocation, MD simulation, Saccharomyces cerevisiae, bimolecular fluorescence complementation, dimerisation, molecular modelling,
• MeSH
• biologický transport MeSH
• buněčná membrána metabolismus   MeSH
• draslík metabolismus   MeSH
• fungální proteiny metabolismus   MeSH
• proteiny přenášející kationty * genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny * genetika   metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• translokace genetická MeSH
• transportní proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• draslík MeSH
• fungální proteiny MeSH
• proteiny přenášející kationty * MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• transportní proteiny MeSH
• TRK1 protein, S cerevisiae MeSH Prohlížeč

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