Heterologous expression reveals unique properties of Trk K+ importers from nonconventional biotechnologically relevant yeast species together with their potential to support Saccharomyces cerevisiae growth

In the model yeast Saccharomyces cerevisiae, Trk1 is the main K⁺ importer. It is involved in many important physiological processes, such as the maintenance of ion homeostasis, cell volume, intracellular pH, and plasma-membrane potential. The ScTrk1 protein can be of great interest to industry, as it was shown that changes in its activity influence ethanol production and tolerance in S. cerevisiae and also cell performance in the presence of organic acids or high ammonium under low K⁺ conditions. Nonconventional yeast species are attracting attention due to their unique properties and as a potential source of genes that encode proteins with unusual characteristics. In this work, we aimed to study and compare Trk proteins from Debaryomyces hansenii, Hortaea werneckii, Kluyveromyces marxianus, and Yarrowia lipolytica, four biotechnologically relevant yeasts that tolerate various extreme environments. Heterologous expression in S. cerevisiae cells lacking the endogenous Trk importers revealed differences in the studied Trk proteins' abilities to support the growth of cells under various cultivation conditions such as low K⁺ or the presence of toxic cations, to reduce plasma-membrane potential or to take up Rb⁺. Examination of the potential of Trks to support the stress resistance of S. cerevisiae wild-type strains showed that Y. lipolytica Trk1 is a promising tool for improving cell tolerance to both low K⁺ and high salt and that the overproduction of S. cerevisiae's own Trk1 was the most efficient at improving the growth of cells in the presence of highly toxic Li⁺ ions.     

Characterization of the respiration‐induced yeast mitochondrial permeability transition pore

When isolated mitochondria from the yeast Saccharomyces cerevisiae oxidize respiratory substrates in the absence of phosphate and ADP, the yeast mitochondrial unselective channel, also called the yeast permeability transition pore (yPTP), opens in the inner membrane, dissipating the electrochemical gradient. ATP also induces yPTP opening. yPTP opening allows mannitol transport into isolated mitochondria of laboratory yeast strains, but mannitol is not readily permeable through the yPTP in an industrial yeast strain, Yeast Foam. The presence of oligomycin, an inhibitor of ATP synthase, allowed for respiration‐induced mannitol permeability in mitochondria from this strain. Potassium (K⁺) had varied effects on the respiration‐induced yPTP, depending on the concentration of the respiratory substrate added. At low respiratory substrate concentrations K⁺ inhibited respiration‐induced yPTP opening, while at high substrate concentrations this effect diminished. However, at the high respiratory substrate concentrations, the presence of K⁺ partially prevented phosphate inhibition of yPTP opening. Phosphate was found to inhibit respiration‐induced yPTP opening by binding a site on the matrix space side of the inner membrane in addition to its known inhibitory effect of donating protons to the matrix space to prevent the pH change necessary for yPTP opening. The respiration‐induced yPTP was also inhibited by NAD, Mg²⁺, NH₄⁺ or the oxyanion vanadate polymerized to decavanadate. The results demonstrate similar effectors of the respiration‐induced yPTP as those previously described for the ATP‐induced yPTP and reconcile previous strain‐dependent differences in yPTP solute selectivity. Copyright © 2013 John Wiley & Sons, Ltd.     

Monovalent cation transporters at the plasma membrane in yeasts

Maintenance of proper intracellular concentrations of monovalent cations, mainly sodium and potassium, is a requirement for survival of any cell. In the budding yeast Saccharomyces cerevisiae, monovalent cation homeostasis is determined by the active extrusion of protons through the Pma1 H⁺-ATPase (reviewed in another chapter of this issue), the influx and efflux of these cations through the plasma membrane transporters (reviewed in this chapter), and the sequestration of toxic cations into the vacuoles. Here, we will describe the structure, function, and regulation of the plasma membrane transporters Trk1, Trk2, Tok1, Nha1, and Ena1, which play a key role in maintaining physiological intracellular concentrations of Na⁺, K⁺, and H⁺, both under normal growth conditions and in response to stress.     

Deletion of the N-terminal domain of the yeast vacuolar (Na+,K+)/H+ antiporter Vnx1p improves salt tolerance in yeast and transgenic Arabidopsis

Cation/proton antiporters play a major role in the control of cytosolic ion concentrations in prokaryotes and eukaryotes organisms. In yeast, we previously demonstrated that Vnx1p is a vacuolar monovalent cation/H⁺ exchanger showing Na⁺/H⁺ and K⁺/H⁺ antiporter activity. We have also shown that disruption of VNX1 results in an almost complete abolishment of vacuolar Na⁺/H⁺ exchange, but yeast cells overexpressing the complete protein do not show improved salinity tolerance. In this study, we have identified an autoinhibitory N-terminal domain and have engineered a constitutively activated version of Vnx1p, by removing this domain. Contrary to the wild type protein, the activated protein has a pronounced effect on yeast salt tolerance and vacuolar pH. Expression of this truncated VNX1 gene also improves Arabidopsis salt tolerance and increases Na⁺ and K⁺ accumulation of salt grown plants thus suggesting a biotechnological potential of activated Vnx1p to improve salt tolerance of crop plants.     

Evidence for a selective and electroneutral K+/H+-exchange in Saccharomyces cerevisiae using plasma membrane vesicles

The existence of a K⁺/H⁺ transport system in plasma membrane vesicles from Saccharomyces cerevisiae is demonstrated using fluorimetric monitoring of proton fluxes across vesicles (ACMA fluorescence quenching). Plasma membrane vesicles used for this study were obtained by a purification/reconstitution protocol based on differential and discontinuous sucrose gradient centrifugations followed by an octylglucoside dilution/gel filtration procedure. This method produces a high percentage of tightly-sealed inside-out plasma membrane vesicles. In these vesicles, the K⁺/H⁺ transport system, which is able to catalyse both K⁺ influx and efflux, is mainly driven by the K⁺ transmembrane gradient and can function even if the plasma membrane H⁺-ATPase is not active. Using the anionic oxonol VI and the cationic DISC₂(5) probes, it was shown that a membrane potential is not created during K⁺ fluxes. Such a dye response argues for the presence of a K⁺/H⁺ exchange system in S. cerevisiae plasma membrane and established the non-electrogenic character of the transport. The maximal rate of exchange is obtained at pH 6·8. This reversible transport system presents a high selectivity for K⁺ among other monovalent cations and a higher affinity for the K⁺ influx into the vesicles (exit from cells). The possible role of this K⁺/H⁺ exchange system in regulation of internal potassium concentration in S. cerevisiae is discussed.     

Genome‐wide analysis of caesium and strontium accumulation in Saccharomyces cerevisiae

¹³⁷Cs and ⁹⁰Sr contribute to significant and long‐lasting contamination of the environment with radionuclides. Due to their relatively high biological availability, they are transferred rapidly into biotic systems and may enter the food chain. In this study, we analysed 4862 haploid yeast knockout strains of Saccharomyces cerevisiae to identify genes involved in caesium (Cs⁺) and/or strontium (Sr²⁺) accumulation. According to this analysis, 212 mutant strains were associated with reproducible altered Cs⁺ and/or Sr²⁺ accumulation. These mutants were deficient for a wide range of cellular processes. Among those, the vacuolar function and biogenesis turned out to be crucial for both Cs⁺ and Sr²⁺ accumulation. Disruption of the vacuole diminished Cs⁺ accumulation, whereas Sr²⁺ enrichment was enhanced. Further analysis with a subset of the identified candidates were undertaken comparing the accumulation of Cs⁺ and Sr²⁺ with their essential counterparts potassium (K⁺) and calcium (Ca²⁺). Sr²⁺ and Ca²⁺ accumulation was highly correlated in yeast excluding the possibility of a differential regulation or uptake mechanisms. In direct contrast, the respective results suggest that Cs⁺ uptake is at least partially dependent on mechanisms distinct from K⁺ uptake. Single candidates (e.g. KHA1) are presented which might be specifically responsible for Cs⁺ homeostasis. Copyright © 2010 John Wiley & Sons, Ltd.     

Evidence for the presence of a FAD pyrophosphatase and a FMN phosphohydrolase in yeast mitochondria: a possible role in flavin homeostasis

Despite the crucial roles of flavin cofactors in metabolism, we know little about the enzymes responsible for the turnover of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) and their subcellular localization. The mechanism by which mitochondria obtain their own flavin cofactors is an interesting point of investigation, because FMN and FAD are mainly located in mitochondria, where they act as redox cofactors of a number of dehydrogenases and oxidases that play a crucial function in both bioenergetics and cellular regulation. In this context, the capability of yeast mitochondria to metabolize externally added and endogenous FAD and FMN was investigated and use was made of purified and bioenergetically active mitochondria prepared starting from the Saccharomyces cerevisiae cell. To determine whether flavin metabolism can occur, the amounts of flavins in aliquots of neutralized perchloric extracts of both spheroplasts and mitochondria were measured by HPLC, and the competence of S. cerevisiae mitochondria to metabolize FAD and FMN was investigated both spectroscopically and via HPLC. FAD deadenylation and FMN dephosphorylation were studied with respect to dependence on substrate concentration, pH profile and inhibitor sensitivity. The existence of two novel mitochondrial FAD pyrophosphatase (diphosphatase) (EC 3.6.1.18) and FMN phosphohydrolase (EC 3.1.3.2) activities, which catalyse the reactions FAD + H₂O → FMN + AMP and FMN + H₂O → riboflavin + Pi respectively, is here shown by fractionation studies. Considering cytosolic riboflavin, FMN and FAD concentrations, as calculated by measuring both spheroplast and mitochondrial contents via HPLC, probably mitochondria play a major role in regulating the flavin pool in yeast and in relation to flavin homeostasis. Copyright © 2011 John Wiley & Sons, Ltd.     

Identification and Preliminary Characterization of p31, a New PSTAIRE-related Protein in Fission Yeast

One of the defining characteristics of the catalytic subunit of the cyclin-dependent protein kinases (cdks) is the so-called PSTAIRE motif. Western blots of fission yeast cytosolic extracts using a monoclonal antibody against the PSTAIRE peptide revealed two bands at 34 kDa (p34^cdc2) and 31 kDa (p31). Polyclonal antibodies to the C-terminus of p34^cdc2 or to the full-length protein recognized the 34 kDa band but not p31. Overexpression of the cdc2⁺ gene resulted in the increase of the 34 kDa band but not p31. Like p34 the level of p31 revealed no obvious cell cycle regulation but the protein was present in spores where p34^cdc2 was barely detectable. p31 expression was unaffected by removal of either phosphate or ammonium from the growth medium, although the level of p34^cdc2 was reduced in the absence of phosphate. p31 was not associated with cyclin B, nor was it adsorbed to p13^suc1 Sepharose beads, two characteristics of p34^cdc2. p31 did, however, interact with p15, the starfish homologue of p13^suc1. p31 was present in cells in which cdc2⁺ was replaced by its budding yeast homologue CDC28. When fission yeast cytosolic extracts were subjected to gel filtration chromatography, p31 eluted in two peaks, one at approximately 100 kDa, the other at approximately 30 kDa. We conclude that p31 is a novel fission yeast PSTAIRE protein and therefore, potentially, a new cdk. © 1997 John Wiley & Sons, Ltd.     

The ALD6 gene of Saccharomyces cerevisiae encodes a cytosolic,Mg2+-activated acetaldehyde dehydrogenase

The deduced translation product of an open reading frame on the left arm of chromosome XVI of Saccharomyces cerevisiae, with the systematic name of YPL061w, is 500 amino acids in length and shares significant homology with aldehyde dehydrogenases. Amino acids 2 to 16 of the protein encoded by YPL061w were found to be identical to the N-terminal 15 amino acids of the purified cytosolic, Mg²⁺-activated acetaldehyde dehydrogenase (ACDH) of S. cerevisiae. This enzyme is thought to be involved in the production of acetate from which cytosolic acetyl-CoA is then synthesized. Deletion of YPL061w was detrimental to the growth of haploid strains of yeast; an analysis of one deletion mutant revealed a maximum specific growth rate (in complex medium containing glucose) of one-third of that displayed by the wild-type strain. Mutants deleted in YPL061w were also unable to use ethanol as a carbon source. As expected, the cytosolic, Mg²⁺-activated ACDH activity had been lost from the mutants, although the mitochondrial, K⁺-activated ACDH was readily detected. YPL061w has been registered with the name of ALD6 in the Saccharomyces Genome Database and the nucleotide sequence submitted to GenBank as part of accession number U39205. © 1997 John Wiley & Sons, Ltd.     

Mechanisms of Salt Tolerance Conferred by Overexpression of the HAL1 Gene in Saccharomyces cerevisiae

Overexpression of the HAL1 gene improves the tolerance of Saccharomyces cerevisiae to NaCl by increasing intracellular K⁺ and decreasing intracellular Na⁺. The effect of HAL1 on intracellular Na⁺ was mediated by the PMR2/ENA1 gene, corresponding to a major Na⁺ efflux system. The expression level of ENA1 was dependent on the gene dosage of HAL1 and overexpression of HAL1 suppressed the salt sensitivity of null mutants in calcineurin and Hal3p, other known regulators of ENA1 expression. The effect of HAL1 on intracellular K⁺ was independent of the TRK1 and TOK1 genes, corresponding to a major K⁺ uptake system and to a K⁺ efflux system activated by depolarization, respectively. Overexpression of HAL1 reduces K⁺ loss from the cells upon salt stress, a phenomenon mediated by an unidentified K⁺ efflux system. Overexpression of HAL1 did not increase NaCl tolerance in galactose medium. NaCl poses two types of stress, osmotic and ionic, counteracted by glycerol synthesis and sodium extrusion, respectively. As compared to glucose, with galactose as carbon source glycerol synthesis is reduced and the expression of ENA1 is increased. As a consequence, osmotic adjustment through glycerolsynthesis, a process not affected by HAL1, is the limiting factor for growth on galactose under NaCl stress. © 1997 John Wiley & Sons, Ltd.     

An overview of membrane transport proteins in Saccharomyces cerevisiae

All eukaryotic cells contain a wide variety of proteins embedded in the plasma and internal membranes, which ensure transmembrane solute transport. It is now established that a large proportion of these transport proteins can be grouped into families apparently conserved throughout organisms. This article presents the data of an in silicio analysis aimed at establishing a preliminary classification of membrane transport proteins in Saccharomyces cerevisiae. This analysis was conducted at a time when about 65% of all yeast genes were available in public databases. In addition to ～60 transport proteins whose function was at least partially known, ～100 deduced protein sequences of unknown function display significant sequence similarity to membrane transport proteins characterized in yeast and/or other organisms. While some protein families have been well characterized by classical genetic experimental approaches, others have largely if not totally escaped characterization. The proteins revealed by this in silicio analysis also include a putative K⁺ channel, proteins similar to aquaporins of plant and animal origin, proteins similar to Na⁺-solute symporters, a protein very similar to electroneural cation-chloride co-transporters, and a putative Na⁺-H⁺ antiporter. A new research area is anticipated: the functional analysis of many transport proteins whose existence was revealed by genome sequencing.     

The role of K+ and H+ transport systems during glucose‐ and H2O2‐induced cell death in Saccharomyces cerevisiae

Glucose, in the absence of additional nutrients, induces programmed cell death in yeast. This phenomenon is independent of yeast metacaspase (Mca1/Yca1) and of calcineurin, requires ROS production and it is concomitant with loss of cellular K⁺ and vacuolar collapse. K⁺ is a key nutrient protecting the cells and this effect depends on the Trk1 uptake system and is associated with reduced ROS production. Mutants with decreased activity of plasma membrane H⁺‐ATPase are more tolerant to glucose‐induced cell death and exhibit less ROS production. A triple mutant ena1‐4 tok1 nha1, devoid of K⁺ efflux systems, is more tolerant to both glucose‐ and H₂O₂‐induced cell death. We hypothesize that ROS production, activated by glucose and H⁺‐ATPase and inhibited by K⁺ uptake, triggers leakage of K⁺, a process favoured by K⁺ efflux systems. Loss of cytosolic K⁺ probably causes osmotic lysis of vacuoles. The nature of the ROS‐producing system sensitive to K⁺ and H⁺ transport is unknown. Copyright © 2010 John Wiley & Sons, Ltd.     

SHM1: A multicopy suppressor of a temperature-sensitive null mutation in the HMG1-like abf2 gene

HM, an HMG1-like mitochondrial DNA-binding protein, is required for maintenance of the yeast mitochondrial genome when cells are grown in glucose. To better understand the role of HM in mitochondria, we have isolated several multicopy suppressors of the temperature-sensitive defect associated with an abf2 null mutation (lacking HM protein). One of these suppressors, SHM1, has been characterized at the molecular level and is described herein. SHM1 encodes a protein (SHM1p) that shares sequence similarity to a family of mitochondrial carrier proteins. On glycerol medium, where mitochondrial function is required for growth, shm1 deletion mutants are able to grow, whereas shm1 abf2 double mutants are severely inhibited. These results suggest that SHM1p plays an accessory role to HM in the mitochondrion. The GenBank Accession Number for the SHM1 sequence is U08352.     

Cloning and Characterization of the KlDIM1 Gene from Kluyveromyces lactis Encoding the m26A Dimethylase of the 18S rRNA

The KlDIM1 gene encoding the m₂⁶A rRNA dimethylase was cloned from a Kluyveromyces lactis genomic library using a PCR amplicon from the Saccharomyces cerevisiae ScDIM1 gene as probe. The KlDIM1 gene encodes a 320-amino acid protein which shows 81% identity to ScDim1p from S. cerevisiae and 25% identity to ksgAp from Escherichia coli. Complementation of the kasugamycin-resistant ksgA-mutant of E. coli lacking dimethylase activity demonstrates that KlDim1p is the functional homologue of the bacterial enzyme. Multiple alignment of dimethylases from prokaryotes and yeasts shows that the two yeast enzymes display distinctive structural motives including a putative nuclear localization signal. © 1997 John Wiley & Sons, Ltd.     

Removing gluconic acid by using different treatments with a Schizosaccharomyces pombe mutant: Effect on fermentation byproducts

Three different treatments involving inoculation with Schizosaccharomyces pombe YGS-5 and Saccharomyces cerevisiae G1 strains were tested with a view to reducing the amount of gluconic acid in synthetic medium. The treatments involved (a) simultaneous inoculation with S. cerevisiae and S. pombe (SpSc); (b) depletion of gluconic acid with S. pombe and subsequent inoculation with S. cerevisiae following removal of S. pombe from the medium (Sp − Sp + Sc); and (c) as (b) but without removing S. pombe from the medium (Sp + Sc). The results thus obtained were compared with those for a control treatment involving fermentation with S. cerevisiae alone (Sc). The amounts of volatile compounds quantified in the fermented media were similar with the treatments where gluconic acid was previously depleted (viz.Sp − Sp + Sc and Sp + Sc). Amino acids were used in large amounts by S. pombe during removal of gluconic acid; this affected subsequent fermentation by S. cerevisiae and the formation of byproducts. Based on the gluconic acid uptake, fermentation kinetics, volatile composition and absence of off-flavours in the fermented media, both treatments (Sp − Sp + Sc and Sp + Sc) can be effectively used in winemaking processes to remove gluconic acid from must prior to fermentation.     

BTN1, a Yeast Gene Corresponding to the Human Gene Responsible for Batten's Disease,is Not Essential for Viability,Mitochondrial Function,or Degradation of Mitochondrial ATP Synthase

The Saccharomyces cerevisiae gene BTN1, encodes a 408 amino acid putative integral membrane protein, which is 39% identical and 59% similar to the human Cln3p, whose mutant forms are responsible for Batten's disease and for a diminished degradation of mitochondrial ATPase synthase subunit c. Disruption experiments established that Btn1p is not essential for viability, mitochondrial function, or degradation of mitochondrial ATP synthase in yeast. © 1997 John Wiley & Sons, Ltd.     

Mutations in the N‐terminal region of the Schizosaccharomyces pombe glutathione transporter pgt1+ allows functional expression in Saccharomyces cerevisiae

Pgt1p encodes a glutathione transporter in Schizosaccharomyces pombe, orthologous to the Saccharomyces cerevisiae glutathione transporter, Hgt1p. Despite high similarity to Hgt1p, Pgt1p failed to display functionality during heterologous expression in S. cerevisiae. In the present study we employed a genetic strategy to investigate the reason behind the non‐functionality of pgt1⁺ in S. cerevisiae. Functional mutants were isolated after in vitro mutagenesis. Several mutants were obtained and four mutants analysed. Among these, three yielded different point mutations in the N‐terminal region (301–350 bp) of the transporter before the first transmembrane domain, while one mutant contained a deletion of 42 nucleotides within the same region. The mutant pgt1⁺ proteins not only expressed and localized correctly, but displayed high‐affinity glutathione transport capabilities in S. cerevisae. Comparison of wild‐type pgt1⁺ with the functional mutants revealed that a loss in protein expression was responsible for lack of functionality of wild‐type pgt1⁺ in S. cerevisiae. The mRNA levels in wild‐type and mutants were comparable, suggesting that the block was in translation. The formation of a strong stem–loop structure appeared to be responsible for inefficient translation in pgt1⁺ and disruption of these structures in the mutants was probably permitting translation. This was confirmed by making silent mutations in this region of wild‐type pgt1⁺, which led to their functionality in S. cerevisiae. This genetic strategy to relieve functional blocks in expression should greatly facilitate the study of these and other transporters from more intractable genetic organisms in a heterologous expression system. Copyright © 2012 John Wiley & Sons, Ltd.