Yeast plasma-membrane Na+/H+ antiporters (Nha/Sod) ensure the optimal intracellular level of alkali-metal cations and protons in cells. They are predicted to consist of 13 transmembrane segments (TMSs) and a large hydrophilic C-terminal cytoplasmic part with seven conserved domains. The substrate specificity, specifically the ability to recognize and transport K+ cations in addition to Na+ and Li+, differs among homologs. In this work, we reveal that the composition of the C-terminus impacts the ability of antiporters to transport particular cations. In the osmotolerant yeast Zygosaccharomyces rouxii, the Sod2-22 antiporter only efficiently exports Na+ and Li+, but not K+. The introduction of a negative charge or removal of a positive charge in one of the C-terminal conserved regions (C3) enabled ZrSod2-22 to transport K+. The same mutations rescued the low level of activity and purely Li+ specificity of ZrSod2-22 with the A179T mutation in TMS6, suggesting a possible interaction between this TMS and the C-terminus. The truncation or replacement of the C-terminal part of ZrSod2-22 with the C-terminus of a K+-transporting Nha/Sod antiporter (Saccharomyces cerevisiae Nha1 or Z. rouxii Nha1) also resulted in an antiporter with the capacity to export K+. In addition, in ScNha1, the replacement of three positively charged arginine residues 539-541 in the C3 region with alanine caused its inability to provide cells with tolerance to Li+. All our results demonstrate that the physiological functions of yeast Nha/Sod antiporters, either in salt tolerance or in K+ homeostasis, depend on the composition of their C-terminal parts.

• Klíčová slova
• C-terminus, cation/H(+) antiport, fungi, potassium homeostasis, salt tolerance,
• MeSH
• draslík * metabolismus   MeSH
• fungální proteiny * chemie   genetika   MeSH
• lithium metabolismus   MeSH
• Na(+)-H(+) antiport * genetika   chemie   MeSH
• protony MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   MeSH
• sodík metabolismus   MeSH
• Zygosaccharomyces * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• draslík * MeSH
• fungální proteiny * MeSH
• lithium MeSH
• Na(+)-H(+) antiport * MeSH
• NHA1 protein, S cerevisiae MeSH Prohlížeč
• protony MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• SOD2 protein, Zygosaccharomyces rouxii MeSH Prohlížeč
• sodík MeSH

The so-called nonconventional yeasts are becoming increasingly attractive in food and industrial biotechnology. Among them, Zygosaccharomyces rouxii is known to be halotolerant, osmotolerant, petite negative, and poorly Crabtree positive. These traits and the high fermentative vigour make this species very appealing for industrial and food applications. Nevertheless, the biotechnological exploitation of Z. rouxii has been biased by the low availability of genetic engineering tools and the recalcitrance of this yeast towards the most conventional transformation procedures. Centromeric and episomal Z. rouxii plasmids have been successfully constructed with prototrophic markers, which limited their usage to auxotrophic strains, mainly derived from the Z. rouxii haploid type strain Centraalbureau voor Schimmelcultures (CBS) 732T . By contrast, the majority of industrially promising Z. rouxii yeasts are prototrophic and allodiploid/aneuploid strains. In order to expand the genetic tools for manipulating these strains, we developed two centromeric and two episomal vectors harbouring KanMXR and ClonNATR as dominant drug resistance markers, respectively. We also constructed the plasmid pGRCRE that allows the Cre recombinase-mediated marker recycling during multiple gene deletions. As proof of concept, pGRCRE was successfully used to rescue the kanMX-loxP module in Z. rouxii ATCC 42981 G418-resistant mutants previously constructed by replacing the MATαP expression locus with the loxP-kanMX-loxP cassette.

• Klíčová slova
• Cre-loxP system, Zygosaccharomyces rouxii, dominant drug resistance marker, nonconventional yeast, plasmid,
• MeSH
• antibakteriální látky farmakologie   MeSH
• centromera genetika   MeSH
• fungální léková rezistence účinky léků   genetika   MeSH
• genetické inženýrství MeSH
• genetické markery MeSH
• integrasy genetika   MeSH
• plazmidy genetika   MeSH
• Zygosaccharomyces účinky léků   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• Cre recombinase MeSH Prohlížeč
• genetické markery MeSH
• integrasy MeSH

A specific technique of nuclear magnetic resonance (NMR) spectroscopy, filter-exchange spectroscopy (FEXSY), was employed to investigate water transport through the plasma membrane in intact yeast cells. This technique allows water transport to be monitored directly, thus avoiding the necessity to subject the cells to any rapid change in the external conditions, e.g. osmotic shock. We established a sample preparation protocol, a data analysis procedure and verified the applicability of FEXSY experiments. We recorded the exchange rates in the temperature range 10-40°C for Saccharomyces cerevisiae. The resulting activation energy of 29 kJ mol-1 supports the hypothesis that water exchange is facilitated by water channels-aquaporins. Furthermore, we measured for the first time water exchange rates in three other phylogenetically unrelated yeast species (Schizosaccharomyces pombe, Candida albicans and Zygosaccharomyces rouxii) and observed remarkably different water exchange rates between these species. Findings of our work contribute to a better understanding of as fundamental a cell process as the control of water transport through the plasma membrane.

• Klíčová slova
• Saccharomyces cerevisiae, activation energy, membrane permeability, nuclear magnetic resonance spectroscopy, water transport, yeast,
• MeSH
• akvaporiny metabolismus   MeSH
• biologický transport MeSH
• buněčná membrána metabolismus   MeSH
• Candida albicans metabolismus   MeSH
• kinetika MeSH
• magnetická rezonanční spektroskopie MeSH
• Schizosaccharomyces metabolismus   MeSH
• teplota MeSH
• termodynamika MeSH
• voda metabolismus   MeSH
• Zygosaccharomyces metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• akvaporiny MeSH
• voda MeSH

The accumulation of glycerol is essential for yeast viability upon hyperosmotic stress. Here we show that the osmotolerant yeast Zygosaccharomyces rouxii has two genes, ZrSTL1 and ZrSTL2, encoding transporters mediating the active uptake of glycerol in symport with protons, contributing to cell osmotolerance and intracellular pH homeostasis. The growth of mutants lacking one or both transporters is affected depending on the growth medium, carbon source, strain auxotrophies, osmotic conditions and the presence of external glycerol. These transporters are localised in the plasma membrane, they transport glycerol with similar kinetic parameters and besides their expected involvement in the cell survival of hyperosmotic stress, they surprisingly both contribute to an efficient survival of hypoosmotic shock and to the maintenance of intracellular pH homeostasis under non-stressed conditions. Unlike STL1 in Sa. cerevisiae, the two Z. rouxii STL genes are not repressed by glucose, but their expression and activity are downregulated by fructose and upregulated by non-fermentable carbon sources, with ZrSTL1 being more influenced than ZrSTL2. In summary, both transporters are highly important, though Z. rouxii CBS 732(T) cells do not use external glycerol as a source of carbon.

• MeSH
• biologický transport MeSH
• delece genu MeSH
• fyziologický stres MeSH
• glycerol metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• kultivační média chemie   MeSH
• mikrobiální viabilita MeSH
• organické látky metabolismus   MeSH
• osmoregulace * MeSH
• osmotický tlak MeSH
• regulace genové exprese u hub účinky léků   MeSH
• symportéry genetika   metabolismus   MeSH
• Zygosaccharomyces genetika   růst a vývoj   metabolismus   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glycerol MeSH
• kultivační média MeSH
• organické látky MeSH
• symportéry MeSH

Zygosaccharomyces rouxii is an osmotolerant yeast growing in the presence of high concentrations of salts and/or sugars. The maintenance of intracellular potassium homeostasis is essential for osmostress adaptation. Zygosaccharomyces rouxii is endowed with only one typical potassium transporter (ZrTrk1). We characterized ZrTrk1 activity and its contribution to various physiological parameters in detail. Our results show that ZrTrk1 is a high-affinity K(+) transporting system efficiently discriminating between K(+) and Li(+) and indicate the presence of another, currently unknown K(+) importing system with a low affinity in Z. rouxii cells. Upon ZrTrk1 heterologous expression in Saccharomyces cerevisiae, it confers cells with a remarkably high lithium tolerance (even to wild-type strains) due to preventing Li(+) influx into cells, and is able to complement a plasma-membrane hyperpolarization and cell sensitivity to cationic compounds caused by the lack of endogenous K(+) transporters. Intracellular pH measurements with pHluorin, whose coding sequence was integrated into the genome, showed that the expression of ZrTrk1 also complements a decrease in intracellular pH in S. cerevisiae trk1Δ trk2Δ cells. Our data corroborate a tight connection between potassium and proton transporters in yeasts and provide new insights into Z. rouxii cation homeostasis and the basis of its high osmotolerance.

• Klíčová slova
• Zygosaccharomyces rouxii, intracellular pH, lithium tolerance, membrane potential, pHluorin integration, potassium transport,
• MeSH
• cytosol chemie   MeSH
• draslík metabolismus   MeSH
• exprese genu MeSH
• koncentrace vodíkových iontů MeSH
• lithium metabolismus   toxicita   MeSH
• proteiny přenášející kationty genetika   metabolismus   MeSH
• Saccharomyces cerevisiae účinky léků   genetika   metabolismus   MeSH
• tolerance léku * MeSH
• Zygosaccharomyces účinky léků   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• draslík MeSH
• lithium MeSH
• proteiny přenášející kationty MeSH

Na(+)/H(+) antiporters may recognize all alkali-metal cations as substrates but may transport them selectively. Plasma-membrane Zygosaccharomyces rouxii Sod2-22 antiporter exports Na(+) and Li(+), but not K(+). The molecular basis of this selectivity is unknown. We combined protein structure modeling, site-directed mutagenesis, phenotype analysis and cation efflux measurements to localize and characterize the cation selectivity region. A three-dimensional model of the ZrSod2-22 transmembrane domain was generated based on the X-ray structure of the Escherichia coli NhaA antiporter and primary sequence alignments with homologous yeast antiporters. The model suggested a close proximity of Thr141, Ala179 and Val375 from transmembrane segments 4, 5 and 11, respectively, forming a hydrophobic hole in the putative cation pathway's core. A series of mutagenesis experiments verified the model and showed that structural modifications of the hole resulted in altered cation selectivity and transport activity. The triple ZrSod2-22 mutant T141S-A179T-V375I gained K(+) transport capacity. The point mutation A179T restricted the antiporter substrate specificity to Li(+) and reduced its transport activity, while serine at this position preserved the native cation selectivity. The negative effect of the A179T mutation can be eliminated by introducing a second mutation, T141S or T141A, in the preceding transmembrane domain. Our experimental results confirm that the three residues found through modeling play a central role in the determination of cation selectivity and transport activity in Z. rouxii Na(+)/H(+) antiporter and that the cation selectivity can be modulated by repositioning a single local methyl group.

• Klíčová slova
• plasma membrane, potassium transport, sodium proton exchanger, substrate specificity, yeast,
• MeSH
• bodová mutace MeSH
• draslík metabolismus   MeSH
• fungální proteiny chemie   genetika   metabolismus   MeSH
• hydrofobní a hydrofilní interakce MeSH
• kationty metabolismus   MeSH
• konformace proteinů MeSH
• lithium metabolismus   MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• Na(+)-H(+) antiport chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sodík metabolismus   MeSH
• substrátová specifita MeSH
• Zygosaccharomyces chemie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• draslík MeSH
• fungální proteiny MeSH
• kationty MeSH
• lithium MeSH
• Na(+)-H(+) antiport MeSH
• sodík MeSH

Zygosaccharomyces rouxii is a fructophilic yeast that consumes fructose preferably to glucose. This behavior seems to be related to sugar uptake. In this study, we constructed Z. rouxii single-, double-, and triple-deletion mutants in the UL4 strain background (a ura3 strain derived from CBS 732(T)) by deleting the genes encoding the specific fructose facilitator Z. rouxii Ffz1 (ZrFfz1), the fructose/glucose facilitator ZrFfz2, and/or the fructose symporter ZrFsy1. We analyzed the effects on the growth phenotype, on kinetic parameters of fructose and glucose uptake, and on sugar consumption profiles. No growth phenotype was observed on fructose or glucose upon deletion of FFZ genes. Deletion of ZrFFZ1 drastically reduced fructose transport capacity, increased glucose transport capacity, and eliminated the fructophilic character, while deletion of ZrFFZ2 had almost no effect. The strain in which both FFZ genes were deleted presented even higher consumption of glucose than strain Zrffz1Δ, probably due to a reduced repressing effect of fructose. This study confirms the molecular basis of the Z. rouxii fructophilic character, demonstrating that ZrFfz1 is essential for Z. rouxii fructophilic behavior. The gene is a good candidate to improve the fructose fermentation performance of industrial Saccharomyces cerevisiae strains.

• MeSH
• biologický transport genetika   MeSH
• fermentace genetika   MeSH
• fruktosa metabolismus   MeSH
• fungální proteiny genetika   metabolismus   MeSH
• genový knockdown MeSH
• glukosa metabolismus   MeSH
• proliferace buněk genetika   MeSH
• regulace genové exprese u hub MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• Zygosaccharomyces genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fruktosa MeSH
• fungální proteiny MeSH
• glukosa MeSH

Owing to its high resistance to weak-acid preservatives and extreme osmotolerance, Zygosaccharomyces rouxii is one of the main spoilage yeasts of sweet foods and beverages. In contrast with Saccharomyces cerevisiae, Z. rouxii is a fructophilic yeast; it consumes fructose faster than glucose. So far, to our knowledge, no specific Z. rouxii proteins responsible for this fructophilic behaviour have been characterized. We have identified two genes encoding putative fructose transporters in the Z. rouxii CBS 732 genome. Heterologous expression of these two Z. rouxii ORFs in a S. cerevisiae strain lacking its own hexose transporters (hxt-null) and subsequent kinetic analysis of sugar transport showed that both proteins are functionally expressed at the plasma membrane: ZrFfz1 is a high-capacity fructose-specific facilitator (K(m)∼400 mM and V(max)∼13 mmol h(-1) g(-1)) and ZrFfz2 is a facilitator transporting glucose and fructose with similar capacity and affinity (K(m)∼200 mM and V(max)∼4 mmol h(-1) g(-1)). These two proteins together with the Zygosaccharomyces bailii Ffz1 fructose-specific transporter belong to a new family of sugar transport systems mediating the uptake of hexoses via the facilitated diffusion mechanism, and are more homologous to drug/H(+) antiporters (regarding their primary protein structure) than to other yeast sugar transporters of the Sugar Porter family.

• MeSH
• biologický transport MeSH
• DNA fungální genetika   MeSH
• fruktosa metabolismus   MeSH
• fungální proteiny genetika   metabolismus   MeSH
• glukosa metabolismus   MeSH
• klonování DNA MeSH
• proteiny přenášející monosacharidy genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• substrátová specifita MeSH
• Zygosaccharomyces genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA fungální MeSH
• fruktosa MeSH
• fungální proteiny MeSH
• glukosa MeSH
• proteiny přenášející monosacharidy MeSH

The osmotolerant yeast Zygosaccharomyces rouxii is sensitive to the toxic L-proline analogue, L-azetidine-2-carboxylate (AZC). The possibility of use of the Saccharomyces cerevisiae MPR1 gene (ScMPR1) encoding the AZC-detoxifying enzyme as a dominant selection marker in Z. rouxii was examined. The heterologous expression of ScMPR1 in two Z. rouxii strains resulted in AZC-resistant colonies, but that of ScMPR1 as a dominant marker gene in vectors was affected by a high frequency of spontaneously resistant colonies. The same was found for an AZC-sensitive S. cerevisiae strain in which the ScMPR1 was expressed. In both yeasts, ScMPR1 can be used only as an auxiliary marker gene.

• MeSH
• acetyltransferasy genetika   metabolismus   MeSH
• antibiotická rezistence genetika   MeSH
• bakteriální transformace MeSH
• genetické inženýrství MeSH
• genetické markery MeSH
• kultivační média chemie   MeSH
• kyselina azetidinkarboxylová antagonisté a inhibitory   farmakologie   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   fyziologie   MeSH
• selekce (genetika) MeSH
• technika přenosu genů MeSH
• Zygosaccharomyces genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetyltransferasy MeSH
• genetické markery MeSH
• kultivační média MeSH
• kyselina azetidinkarboxylová MeSH
• Mpr1 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH

Yeast plasma membrane Na+/H+ antiporters are divided according to their substrate specificity in two distinct subfamilies. To identify amino acid residues responsible for substrate specificity determination (recognition of K+), the Zygosaccharomyces rouxii Sod2-22 antiporter (non-transporting K+) was mutagenized and a collection of ZrSod2-22 mutants that improved the KCl tolerance of a salt-sensitive Saccharomyces cerevisiae strain was isolated. Several independent ZrSod2-22 mutated alleles contained the replacement of a highly conserved proline 145 with a residue containing a hydroxyl group (Ser, Thr). Site-directed mutagenesis of Pro145 proved that an amino acid with a hydroxyl group at this position is enough to enable ZrSod2-22p to transport K+. Simultaneously, the P145(S/T) mutation decreased the antiporter transport activity for both Na+ and Li+. Replacement of Pro145 with glycine resulted in a ZrSod2-22p with extremely low activity only for Na+, and the exchange of a charged residue (Asp, Lys) for Pro145 completely stopped the activity. Mutagenesis of the corresponding proline in the S. cerevisiae Nha1 antiporter (Pro146) confirmed that this proline of the fifth transmembrane domain is a critical residue for antiporter function. This is the first evidence that a non-polar amino acid residue is important for the substrate specificity and activity of yeast Nha antiporters.

• MeSH
• alely MeSH
• biologický transport MeSH
• buněčná membrána metabolismus   MeSH
• časové faktory MeSH
• draslík chemie   metabolismus   MeSH
• druhová specificita MeSH
• fungální proteiny genetika   fyziologie   MeSH
• glycin chemie   MeSH
• kationty MeSH
• konformace proteinů MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• mutageneze cílená MeSH
• Na(+)-H(+) antiport MeSH
• plazmidy metabolismus   MeSH
• polymerázová řetězová reakce MeSH
• prolin chemie   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• soli farmakologie   MeSH
• substrátová specifita MeSH
• transportní proteiny genetika   fyziologie   MeSH
• ultrafialové záření MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Zygosaccharomyces metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• draslík MeSH
• fungální proteiny MeSH
• glycin MeSH
• kationty MeSH
• Na(+)-H(+) antiport MeSH
• prolin MeSH
• SOD22 protein, Zygosaccharomyces MeSH Prohlížeč
• soli MeSH
• transportní proteiny MeSH