Aluminum does not play any role in essential biological processes, but in acidic conditions it becomes biologically reactive and highly toxic even at low concentrations. Eukaryotes have developed numerous strategies for detoxification, one of which relies on membrane proteins. Fungi possess so-called aluminum resistance proteins (Alr1/2p), which are distant homologs of CorA Mg2+-channels but are claimed to confer resistance to aluminum. However, the mechanism behind this resistance remains poorly understood. In this study we aimed to conduct a thorough functional and structural characterization of Alr1p from Pichia pastoris (PpAlr1p). Our results confirm that PpAlr1p similar to other members of the CorA family functions as a pentamer and can mediate Zn2+ and Cd2+ transport across the membrane independently from a pH gradient. Studies on the truncated version of PpAlr1p define the core functional unit of CorA proteins but pinpoint that the intracellular domain is important for the correct localization. Competitive uptake assays with Al3+ showed aluminum to activate the transport of divalent cations in PpAlr1p. In addition, the growth assay of the ALR2 knock-out Saccharomyces cerevisiae strain indicates some role of Alr2p in promoting tolerance to Ni2+. Together, these results provide the functional and structural characterization of a eukaryotic CorA-like aluminum resistance protein, highlight the role of its N-terminal domain in proper cellular localization and define the functional core of the CorA family.

Erv14 is a cargo receptor of COPII vesicles, which is necessary for the efficient trafficking of various membrane proteins. In this work, we demonstrate that the deletion of the ERV14 gene impacts various physiological functions of the vacuole. Compared to the wild-type cells, cells lacking the ERV14 gene exhibited higher vacuolar pH and altered vacuolar morphology with increased fragmentation. In addition, erv14Δ cells exhibit a thinner cell wall and an impaired process of endocytosis. We also found the importance of ERV14 for cells to overcome environmental stresses, such as neutral external pH, increased zinc and calcium concentrations, and high temperature. Furthermore, comparing gene expression, proteome analysis, and structural modeling revealed new interactions between Erv14 and several vacuolar proteins, including subunits of V-ATPase and other proteins involved in carbon metabolism.

Sterols perform essential structural and signalling functions in living organisms. Ergosterol contributes to the fluidity, permeability, microdomain formation and functionality of proteins in the yeast membrane. In our study, desmosterol was the most successful at compensating for the lack of ergosterol in Saccharomyces cerevisiae, besides stigmasterol and sitosterol. These three sterols supported cell growth without causing severe morphological defects, unlike cholesterol, 7-dehydrocholesterol, lathosterol, cholestanol or lanosterol. Together with ergosterol, they were also able to bring the plasma membrane potential of hem1Δ cells closer to the level of the wild type. In addition, desmosterol conferred even higher thermotolerance to yeast than ergosterol. Some sterols counteracted the antifungal toxicity of polyenes, azoles and terbinafine to hem1Δ cells. Plant sterols (stigmasterol, sitosterol) and desmosterol ensured the glucose-induced activation of H+-ATPase in hem1Δ cells analogously to ergosterol, whereas cholesterol and 7-dehydrocholesterol were less effective. Exogenous ergosterol, stigmasterol, sitosterol, desmosterol and cholesterol also improved the growth of Candida glabrata and Candida albicans in the presence of inhibitory concentration of fluconazole. The proper incorporation of exogenous sterols into the membrane with minimal adverse side effects on membrane functions was mainly influenced by the structure of the sterol acyl chain, and less by their ring structures.

Yeast plasma -membrane Na+/H+ antiporters (Nha/Sod) ensure the optimal intracellular level of alkalimetal 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. (c) 2024 Elsevier Ltd. All rights reserved.

Trk1 is the main K+ importer of Saccharomyces cerevisiae. Its proper functioning enables yeast cells to grow in environments with micromolar amounts of K+. Although the structure of Trk1 has not been experimentally determined, the transporter is predicted to be composed of four MPM (transmembrane segment - pore loop - transmembrane segment) motifs which are connected by intracellular loops. Of those, in particular the first loop (IL1) is unique in its length; it forms more than half of the entire protein. The deletion of the majority of IL1 does not abolish the transport activity of Trk1. However IL1 is thought to be involved in the modulation of the transporter's functioning. In this work, we prepared a series of internally shortened versions of Trk1 that lacked various parts of IL1, and we studied their properties in S. cerevisiae cells without chromosomal copies of TRK genes. Using this approach, we were able to determine that both N- and C-border regions of IL1 are necessary for the proper localization of Trk1. Moreover, the N-border part of IL1 is also important for the functioning of Trk1, as its absence resulted in a decrease in the transporter's substrate affinity. In addition, in the internal part of IL1, we newly identified a stretch of amino-acid residues that are indispensable for retaining the transporter's maximum velocity, and another region whose deletion affected the ability of Trk1 to adjust its affinity in response to external levels of K+.

The non-conventional yeast Kluyveromyces marxianus has recently emerged as a promising candidate for many food, environment, and biotechnology applications. This yeast is thermotolerant and has robust growth under many adverse conditions. Here, we show that its ability to grow under potassium-limiting conditions is much better than that of Saccharomyces cerevisiae, suggesting a very efficient and high-affinity potassium uptake system(s) in this species. The K. marxianus genome contains two genes for putative potassium transporters: KmHAK1 and KmTRK1. To characterize the products of the two genes, we constructed single and double knock-out mutants in K. marxianus and also expressed both genes in an S. cerevisiae mutant, that lacks potassium importers. Our results in K. marxianus and S. cerevisiae revealed that both genes encode efficient high-affinity potassium transporters, contributing to potassium homeostasis and maintaining plasma-membrane potential and cytosolic pH. In K. marxianus, the presence of HAK1 supports growth at low K+ much better than that of TRK1, probably because the substrate affinity of KmHak1 is about 10-fold higher than that of KmTrk1, and its expression is induced similar to 80-fold upon potassium starvation. KmHak1 is crucial for salt stress survival in both K. marxianus and S. cerevisiae. In co-expression experiments with ScTrk1 and ScTrk2, its robustness contributes to an increased tolerance of S. cerevisiae cells to sodium and lithium salt stress.

The effectivity of utilization of exogenous sterols in the yeast Saccharomyces cerevisiae exposed to hypoxic stress is dependent on the sterol structure. The highly imported sterols include animal cholesterol or plant sitosterol, while ergosterol, typical of yeasts, is imported to a lesser extent. An elevated utilization of non-yeast sterols is associated with their high esterification and relocalization to lipid droplets (LDs). Here we present data showing that LDs and sterol esterification play a critical role in the regulation of the accumulation of non-yeast sterols in membranes. Failure to form LDs during anaerobic growth in media supplemented with cholesterol or sitosterol resulted in an extremely long lag phase, in contrast to normal growth in media with ergosterol or plant stigmasterol. Moreover, in hem1∆, which mimics anaerobiosis, neither cholesterol nor sitosterol supported the growth in an LD-less background. The incorporation of non-ergosterol sterols into the membranes affected fundamental membrane characteristics such as relative membrane potential, permeability, tolerance to osmotic stress and the formation of membrane domains. Our findings reveal that LDs assume an important role in scenarios wherein cells are dependent on the utilization of exogenous lipids, particularly under anoxia. Given the diverse lipid structures present in yeast niches, LDs fulfil a protective role, mitigating the risk of excessive accumulation of potentially toxic steroids and fatty acids in the membranes. Finally, we present a novel function for sterols in a model eukaryotic cell - alleviation of the lipotoxicity of unsaturated fatty acids.

Acetic acid-induced stress is a common challenge in natural environments and industrial bioprocesses, significantly affecting the growth and metabolic per-formance 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, particu-larly 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 implicat-ed 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 tol-erance of the hrk1∆ 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.

Microbiology Society journals contain high-quality research papers and topical review articles. We are a not-for-profit publisher and we support and invest in the microbiology community, to the benefit of everyone. This supports our principal goal to develop, expand and strengthen the networks available to our members so that they can generate new knowledge about microbes and ensure that it is shared with other communities.

Potassium is an essential intracellular ion, and a sufficient intracellular concentration of it is crucial for many processes; therefore it is fundamental for cells to precisely regulate K+ uptake and efflux through the plasma membrane. The uniporter Trk1 is a key player in K+ acquisition in yeasts. The TRK1 gene is expressed at a low and stable level; thus the activity of the transporter needs to be regulated at a posttranslational level. S. cerevisiae Trk1 changes its activity and affinity for potassium ion quickly and according to both internal and external concentrations of K+, as well as the membrane potential. The molecular basis of these changes has not been elucidated, though phosphorylation is thought to play an important role. In this study, we examined the role of the second, short, and highly conserved intracellular hydrophilic loop of Trk1 (IL2), and identified two phosphorylable residues (Ser882 and Thr900) as very important for 1) the structure of the loop and consequently for the targeting of Trk1 to the plasma membrane, and 2) the upregulation of the transporter's activity reaching maximal affinity under low external K+ conditions. Moreover, we identified three residues (Thr155, Ser414, and Thr900) within the Trk1 protein as strong candidates for interaction with 14-3-3 regulatory proteins, and showed, in an in vitro experiment, that phosphorylated Thr900 of the IL2 indeed binds to both isoforms of yeast 14-3-3 proteins, Bmh1 and Bmh2.

The strong antifungal effect of sulfonated polystyrene nanoparticles (NPs) with an encapsulated tetraphenylporphyrin (TPP) photosensitizer is reported here. TPP is activated by visible light, resulting in the generation of singlet oxygen. Its antifungal action is potentiated in the presence of potassium iodide, yielding I2/I3⁻, another antifungal species. The NPs exhibit no dark toxicity, but a broad spectrum of antifungal photodynamic effects. The efficiency of this rapid killing (on the order of minutes) depends on the concentration of TPP NPs, potassium iodide, yeast species and temperature. A strong antifungal activity of TPP NPs is demonstrated on eleven pathogenic and opportunistic pathogenic yeast species (six Candida species and other yeast species, including melanized Hortaea werneckii). The composition and architecture of yeast cell envelope structures clearly influence the efficacy of photodynamic therapy. Candida krusei is the most sensitive to photodynamic therapy. Despite expectations, melanin does not provide Hortaea cells with marked resistance compared to white yeast species. The kinetics of the interaction of NPs with yeast cells is also described. This study may inspire and promote the fabrication of a new type of antiseptic for various skin injuries in clinical medicine.

Microbiology Society journals contain high-quality research papers and topical review articles. We are a not-for-profit publisher and we support and invest in the microbiology community, to the benefit of everyone. This supports our principal goal to develop, expand and strengthen the networks available to our members so that they can generate new knowledge about microbes and ensure that it is shared with other communities.

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.

The human Na + /H + antiporter NHA2 ( SLC9B2 ) transports Na + or Li + across the plasma membrane in exchange for protons, and is implicated in various pathologies. It is a 537 amino acids protein with an 82 residues long hydrophilic cytoplasmic N-terminus followed by a transmembrane part comprising 14 transmembrane helices. We optimized the functional expression of Hs NHA2 in the plasma membrane of a salt-sensitive Saccharomyces cerevisiae strain and characterized a set of mutated or truncated versions of Hs NHA2 in terms of their substrate specificity, transport activity, localization and protein stability. We identified a highly conserved proline 246, located in the core of the protein, as being crucial for ion selectivity. The replacement of P246 with serine or threonine resulted in antiporters with altered substrate specificity and increased resistance to the Hs NHA2-specific inhibitor phloretin that were not only highly active at an acidic pH of 4.0 (like the native antiporter), but also at neutral pH. We also experimentally confirmed the importance of a putative salt bridge between E215 and R432 for antiporter function and structural integrity. Truncations of the first 50 - 70 residues of the N-terminus doubled the transport activity of Hs NHA2, whilst changes in the charge at positions E47, E56, K57, or K58 decreased the antiporter’s transport activity. Thus, the hydrophilic N-terminal part of the protein appears to allosterically autoinhibit its cation transport. Our data also show this in vivo approach to be useful for a rapid screening of SNP’s effect on Hs NHA2 activity.

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.

The existence of programmed cell death in Saccharomyces cerevisiae has been reported for many years. Glucose induces the death of S. cerevisiae in the absence of additional nutrients within a few hours, and the absence of active potassium uptake makes cells highly sensitive to this process. S. cerevisiae cells possess two transporters, Trk1 and Trk2, which ensure a high intracellular concentration of potassium, necessary for many physiological processes. Trk1 is the major system responsible for potassium acquisition in growing and dividing cells. The contribution of Trk2 to potassium uptake in growing cells is almost negligible, but Trk2 becomes crucial for stationary cells for their survival of some stresses, e.g. anhydrobiosis. As a new finding, we show that both Trk systems contribute to the relative thermotolerance of S. cerevisiae BY4741. Our results also demonstrate that Trk2 is much more important for the cell survival of glucose-induced cell death than Trk1, and that stationary cells deficient in active potassium uptake lose their ATP stocks more rapidly than cells with functional Trk systems. This is probably due to the upregulated activity of plasma-membrane Pma1 H+-ATPase, and consequently, it is the reason why these cells die earlier than cells with functional active potassium uptake.

Erv14, a conserved cargo receptor of COPII vesicles, helps the proper trafficking of many but not all transporters to the yeast plasma membrane, for example, three out of five alkali-metal-cation transporters in Saccharomyces cerevisiae. Among them, the Nha1 cation/proton antiporter, which participates in cell cation and pH homeostasis, is a large membrane protein (985 aa) possessing a long hydrophilic C-terminus (552 aa) containing six conserved regions (C1-C6) with unknown function. A short Nha1 version, lacking almost the entire C-terminus, still binds to Erv14 but does not need it to be targeted to the plasma membrane. Comparing the localization and function of ScNha1 variants shortened at its C-terminus in cells with or without Erv14 reveals that only ScNha1 versions possessing the complete C5 region are dependent on Erv14. In addition, our broad evolutionary conservation analysis of fungal Na+ /H+ antiporters identified new conserved regions in their C-termini, and our experiments newly show C5 and other, so far unknown, regions of the C-terminus, to be involved in the functionality and substrate specificity of ScNha1. Taken together, our results reveal that also relatively small hydrophilic parts of some yeast membrane proteins underlie their need to interact with the Erv14 cargo receptor.

Two Saccharomyces cerevisiae strains, BY4741 and BY4741-derived strain lacking the IST2 gene (ist2Δ), were used to characterise the possible role of cortical endoplasmic reticulum (ER) protein Ist2 upon cell dehydration and subsequent rehydration. For the first time, we show that not only protein components of the plasma membrane (PM), but also at least one ER membrane protein (Ist2) play an important role in the maintenance of the viability of yeast cells during dehydration and subsequent rehydration. The low viability of the mutant strain ist2∆ upon dehydration-rehydration stress was related to the lack of Ist2 protein in the ER. We revealed that the PM of ist2∆ strain is not able to completely restore its molecular organisation during reactivation from the state of anhydrobiosis. As the result, the permeability of the PM remains high regardless of the type of reactivation (rapid or gradual rehydration). We conclude that ER protein Ist2 plays an important role in ensuring the stability of molecular organisation and functionality of the PM during dehydration-rehydration stress. These results indicate an important role of ER-PM interactions during cells transition into the state of anhydrobiosis and the subsequent restoration of their physiological activities.

Wild-type cells of Candida albicans, the most common human fungal pathogen, are able to grow at very low micromolar concentrations of potassium in the external milieu. One of the reasons behind that behaviour is the existence of three different types of K+ transporters in their plasma membrane: Trk1, Acu1 and Hak1. This work shows that the transporters are very differently regulated at the transcriptional level upon exposure to saline stress, pH alterations or K+ starvation. We propose that different transporters take the lead in the diverse environmental conditions, Trk1 being the "house-keeping" one, and Acu1/Hak1 dominating upon K+ limiting conditions. Heterologous expression of the genes coding for the three transporters in a Saccharomyces cerevisiae strain lacking its endogenous potassium transporters showed that all of them mediated cation transport but with very different efficiencies. Moreover, expression of the transporters in S. cerevisiae also affected other physiological characteristics such as sodium and lithium tolerance, membrane potential or intracellular pH, being, in general, CaTrk1 the most effective in keeping these parameters close to the usual wild-type physiological levels.

The maintenance of K+ and Ca2+ homeostasis is crucial for many cellular functions. Potassium is accumulated in cells at high concentrations, while the cytosolic level of calcium, to ensure its signalling function, is kept at low levels and transiently increases in response to stresses. We examined Ca2+ homeostasis and Ca2+ signalling in Saccharomyces cerevisiae strains lacking plasma-membrane K+ influx (Trk1 and Trk2) or efflux (Tok1, Nha1 and Ena1-5) systems. The lack of K+ exporters slightly increased the cytosolic Ca2+, but did not alter the Ca2+ tolerance or Ca2+-stress response. In contrast, the K+-importers Trk1 and Trk2 play important and distinct roles in the maintenance of Ca2+ homeostasis. The presence of Trk1 was vital mainly for the growth of cells in the presence of high extracellular Ca2+, whilst the lack of Trk2 doubled steady-state intracellular Ca2+ levels. The absence of both K+ importers highly increased the Ca2+ response to osmotic or CaCl2 stresses and altered the balance between Ca2+ flux from external media and intracellular compartments. In addition, we found Trk2 to be important for the tolerance to high KCl and hygromycin B in cells growing on minimal media. All the data describe new interconnections between potassium and calcium homeostasis in S. cerevisiae.