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

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.

• Klíčová slova
• K+ transport, Nha1, Ppz1 phosphatase, Saccharomyces cerevisiae, cation homeostasis, intracellular pH,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: The virulence of Candida species depends on many environmental conditions. Extracellular pH and concentration of alkali metal cations belong among important factors. Nevertheless, the contribution of transporters mediating the exchange of alkali metal cations for protons across the plasma membrane to the cell salt tolerance and other physiological properties of various Candida species has not been studied so far. RESULTS: The tolerance/sensitivity of four pathogenic Candida species to alkali metal cations was tested and the role of one of the cation transporters in that tolerance (presumed to be the plasma-membrane Na+/H+ antiporter) was studied. The genes encoding these antiporters in the most and least salt sensitive species, C. dubliniensis and C. parapsilosis respectively, were identified, cloned and functionally expressed in the plasma membranes of Saccharomyces cerevisiae cells lacking their own cation exporters. Both CpCnh1 and CdCnh1 antiporters had broad substrate specificity and transported Na+, K+, Li+, and Rb+. Their activity in S. cerevisiae cells differed; CpCnh1p provided cells with a much higher salt tolerance than the CdCnh1 antiporter. The observed difference in activity was confirmed by direct measurements of sodium and potassium efflux mediated by these antiporters. CONCLUSION: We have cloned two genes encoding putative Na+/H+ antiporters in C. parapsilosis and C. dubliniensis, and characterized the transport properties of encoded proteins. Our results show that the activity of plasma-membrane Na+/H+ antiporters is one of the factors determining the tolerance of pathogenic Candida species to high external concentrations of alkali metal cations.

• MeSH
• alkalické kovy metabolismus   MeSH
• Candida genetika   růst a vývoj   metabolismus   patogenita   MeSH
• draslík metabolismus   MeSH
• fluorescenční mikroskopie MeSH
• fungální proteiny genetika   metabolismus   MeSH
• kationty metabolismus   MeSH
• lithium metabolismus   MeSH
• membránové proteiny genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• Na(+)-H(+) antiport genetika   metabolismus   MeSH
• proteiny přenášející kationty genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   růst a vývoj   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• sekvence nukleotidů MeSH
• soli metabolismus   MeSH
• substrátová specifita MeSH
• superoxiddismutasa 1 MeSH
• superoxiddismutasa metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• alkalické kovy MeSH
• CNH1 protein, Candida albicans MeSH Prohlížeč
• draslík MeSH
• fungální proteiny MeSH
• kationty MeSH
• lithium MeSH
• membránové proteiny MeSH
• Na(+)-H(+) antiport MeSH
• NHA1 protein, S cerevisiae MeSH Prohlížeč
• proteiny přenášející kationty MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• soli MeSH
• superoxiddismutasa 1 MeSH
• superoxiddismutasa MeSH

The Saccharomyces cerevisiae genome contains three genes encoding alkali metal cation/H+ antiporters (Nha1p, Nhx1p, Kha1p) that differ in cell localization, substrate specificity and physiological function. Systematic genome sequencing of other yeast species revealed highly conserved homologous ORFs in all of them. We compared the yeast sequences both at DNA and protein levels. The subfamily of yeast endosomal/prevacuolar Nhx1 antiporters is closely related to mammalian plasma membrane NHE proteins and to both plasma membrane and vacuolar plant antiporters. The high sequence conservation within this subfamily of yeast antiporters suggests that Nhx1p is of great importance in cell physiology. Yeast Kha1 proteins probably belong to the same subfamily as bacterial antiporters, whereas Nhal proteins form a distinct subfamily.

• MeSH
• DNA fungální analýza   MeSH
• draslíko-vodíkové antiportéry chemie   klasifikace   genetika   MeSH
• fylogeneze MeSH
• membránové proteiny chemie   klasifikace   genetika   MeSH
• molekulární sekvence - údaje MeSH
• Na(+)-H(+) antiport chemie   klasifikace   genetika   MeSH
• proteiny přenášející kationty chemie   klasifikace   genetika   MeSH
• Saccharomyces cerevisiae - proteiny chemie   klasifikace   genetika   MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza DNA MeSH
• sekvenční homologie aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA fungální MeSH
• draslíko-vodíkové antiportéry MeSH
• KHA1 protein, S cerevisiae MeSH Prohlížeč
• membránové proteiny MeSH
• Na(+)-H(+) antiport MeSH
• NHA1 protein, S cerevisiae MeSH Prohlížeč
• NHX1 protein, S cerevisiae MeSH Prohlížeč
• proteiny přenášející kationty MeSH
• Saccharomyces cerevisiae - proteiny MeSH

Saccharomyces cerevisiae uses different mechanisms to adapt to changes in environmental osmolarity. Upon hyperosmotic shock, cells first mobilize a rapid rescue system that prevents excessive loss of ions and water; then in the adaptation period they accumulate a compatible solute (glycerol). When subjected to hypoosmotic shock, they rapidly release intracellular stocks of glycerol to reduce intracellular osmolarity and prevent bursting. The plasma membrane Nha1 alkali metal cation/H+ antiporter is not important in helping the cells to survive a sudden drop in external osmolarity, but is involved in the cell response to hyperosmotic shock. For this role, its long hydrophilic C-terminus is indispensable. The capacity of the Nha1 antiporter to transport potassium is regulated by Hog1 kinase. Upon sorbitol-mediated stress, the Nha1p potassium export activity decreases in order to maintain a higher intracellular concentration of solutes. The C-terminal-less Nha1 version is not inactivated and its potassium efflux activity renders cells very sensitive to hyperosmotic shock. Taken together, our results suggest an important role of Nha1p and its C-terminus in the immediate response to hyperosmotic shock as part of the rapid rescue mechanism.

• MeSH
• fyziologická adaptace fyziologie   MeSH
• iontový transport fyziologie   MeSH
• membránové proteiny genetika   metabolismus   MeSH
• mitogenem aktivované proteinkinasy genetika   metabolismus   MeSH
• Na(+)-H(+) antiport genetika   metabolismus   MeSH
• osmotický tlak MeSH
• proteiny přenášející kationty genetika   metabolismus   MeSH
• regulace genové exprese u hub fyziologie   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• terciární struktura proteinů genetika   MeSH
• upregulace fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HOG1 protein, S cerevisiae MeSH Prohlížeč
• membránové proteiny MeSH
• mitogenem aktivované proteinkinasy MeSH
• Na(+)-H(+) antiport MeSH
• NHA1 protein, S cerevisiae MeSH Prohlížeč
• proteiny přenášející kationty MeSH
• Saccharomyces cerevisiae - proteiny MeSH

A triple mutant strain of Saccharomyces cerevisiae lacking its own Na+-ATPases and Na+/H+ antiporters (enal-4delta nha1delta nhx1delta) was used for the expression of the Oryza sativa NHX1 gene encoding a putative vacuolar Na+/H+ exchanger. Upon expression in yeast cells, the OsNhx 1p is not a transport system specific only for sodium cations but it has a broad substrate specificity for at least four alkali metal cations (Na+, Li+, K+ and Rb+) and is able to substitute for the endogenous yeast ScNhx1 antiporter. Its activity contributes to sequestration of alkali metal cations in intracellular vesicles.

• MeSH
• DNA rostlinná genetika   MeSH
• geny hub MeSH
• kovy metabolismus   farmakologie   MeSH
• mutace MeSH
• Na(+)-H(+) antiport genetika   metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• rostlinné geny MeSH
• rýže (rod) genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae účinky léků   genetika   metabolismus   MeSH
• sekvence nukleotidů MeSH
• testy genetické komplementace MeSH
• vakuoly metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH
• kovy MeSH
• Na(+)-H(+) antiport MeSH
• rekombinantní proteiny MeSH
• Saccharomyces cerevisiae - proteiny MeSH