Functional comparison of plasma-membrane Na+/H+ antiporters from two pathogenic Candida species

<p>Abstract</p> <p>Background</p> <p>The virulence of <it>Candida </it>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 <it>Candida </it>species has not been studied so far.</p> <p>Results</p> <p>The tolerance/sensitivity of four pathogenic <it>Candida </it>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, <it>C. dubliniensis </it>and <it>C. parapsilosis </it>respectively, were identified, cloned and functionally expressed in the plasma membranes of <it>Saccharomyces cerevisiae </it>cells lacking their own cation exporters. Both <it>Cp</it>Cnh1 and <it>Cd</it>Cnh1 antiporters had broad substrate specificity and transported Na⁺, K⁺, Li⁺, and Rb⁺. Their activity in <it>S. cerevisiae </it>cells differed; <it>Cp</it>Cnh1p provided cells with a much higher salt tolerance than the <it>Cd</it>Cnh1 antiporter. The observed difference in activity was confirmed by direct measurements of sodium and potassium efflux mediated by these antiporters.</p> <p>Conclusion</p> <p>We have cloned two genes encoding putative Na⁺/H⁺antiporters in <it>C. parapsilosis </it>and <it>C. dubliniensis</it>, 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 <it>Candida </it>species to high external concentrations of alkali metal cations.</p

Alternative Glycerol Balance Strategies among Saccharomyces Species in Response to Winemaking Stress

Production and balance of glycerol is essential for the survival of yeast cells in certain stressful conditions as hyperosmotic or cold shock that occur during industrial processes as winemaking. These stress responses are well-known in S. cerevisiae, however, little is known in other phylogenetically close related Saccharomyces species associated with natural or fermentation environments such as S. uvarum, S. paradoxus or S. kudriavzevii. In this work we have investigated the expression of four genes (GPD1, GPD2, STL1, and FPS1) crucial in the glycerol pool balance in the four species with a biotechnological potential (S. cerevisiae; S. paradoxus; S. uvarum; and S. kudriavzevii), and the ability of strains to grow under osmotic and cold stresses. The results show different pattern and level of expression among the different species, especially for STL1. We also studied the function of Stl1 glycerol symporter in the survival to osmotic changes and cell growth capacity in winemaking environments. These experiments also revealed a different functionality of the glycerol transporters among the different species studied. All these data point to different strategies to handle glycerol accumulation in response to winemaking stresses as hyperosmotic or cold-hyperosmotic stress in the different species, with variable emphasis in the production, influx, or efflux of glycerol.BO was supported by CAPES the Brazilian Federal Agency for the Support and Evaluation of Graduate Education (Brazilian Ministry of Education). This work has been supported by grants AGL2012-39937-C02-01 and AGL2015-67504-C3-1-R from the Spanish Government, FEDER, and Generalitat Valenciana PROMETEOII/2014/042 to AQ, GA CR 15-03708S from the Czech National Foundation to HS, and by the European Commission FP7: Marie Curie Initial Training Network CORNUCOPIA no. 264717 to AQ and HS.Peer reviewe

Analysis of the mKir2.1 channel activity in potassium influx defective Saccharomyces cerevisiae strains determined as changes in growth characteristics

AbstractPotassium uptake defective Saccharomyces cerevisiae strains (Δtrk1,2 and Δtrk1,2 Δtok1) were used for the phenotypic analysis of the mouse inward rectifying Kir2.1 channel by growth analysis. Functional expression of both, multi-copy plasmid and chromosomally expressed GFP-mKir2.1 fusion constructs complemented the potassium uptake deficient phenotype in a pHout dependent manner. Upon application of Hygromycin B to chromosomally mKir2.1 expressing cells, significantly lower toxin sensitivity (EC50 15.4μM) compared to Δtrk1,2 Δtok1 cells (EC50 2.6μM) was observed. Growth determination of mKir2.1 expressing strains upon application of Ag+, Cs+ and Ba2+ as known blockers of mKir2.1 channels revealed significantly decreased channel function. Cells with mKir2.1 were about double sensitive to AgNO3, 350-fold more sensitive to CsCl and 1500-fold more sensitive to BaCl2 in comparison to the respective controls indicating functional expression and correct pharmacology

Yarrowia lipolytica possesses two plasma membrane alkali metal cation/H+ antiporters with different functions in cell physiology

AbstractThe family of Nha antiporters mediating the efflux of alkali metal cations in exchange for protons across the plasma membrane is conserved in all yeast species. Yarrowia lipolytica is a dimorphic yeast, phylogenetically very distant from the model yeast Saccharomyces cerevisiae. A search in its sequenced genome revealed two genes (designated as YlNHA1 and YlNHA2) with homology to the S. cerevisiae NHA1 gene, which encodes a plasma membrane alkali metal cation/H+ antiporter. Upon heterologous expression of both YlNHA genes in S. cerevisiae, we showed that Y. lipolytica antiporters differ not only in length and sequence, but also in their affinity for individual substrates. While the YlNha1 protein mainly increased cell tolerance to potassium, YlNha2p displayed a remarkable transport capacity for sodium. Thus, Y. lipolytica is the first example of a yeast species with two plasma membrane alkali metal cation/H+ antiporters differing in their putative functions in cell physiology; cell detoxification vs. the maintenance of stable intracellular pH, potassium content and cell volume

Heterologous expression of mammalian Na/H antiporters in Saccharomyces cerevisiae

Na+/H+ antiporters, integral membrane proteins that exchange protons for alkali metal cations, play multiple roles in probably all living organisms (preventing cells from excessive amounts of alkali metal cations, regulating intracellular pH and cell volume). In this work, we studied the functionality of rat plasma membrane NHE1-3 exchangers upon their heterologous expression in alkali-metal-cation sensitive Saccharomyces cerevisiae, and searched for conditions that would increase their level in the plasma membrane and improve their functionality. Though three tested exchangers were partially localized to the plasma membrane (and two of them (NHE2 and NHE3) in an active form), the bulk of the synthesized proteins were arrested along the secretory pathway, mainly in the ER. To increase the level of exchangers in the yeast plasma membrane several approaches (truncation of C-terminal regulatory sequences, expression in mutant yeast strains, construction of rat/yeast protein chimeras, various growth conditions and chemical chaperones) were tested. The only increase in the amount of NHE exchangers in the plasma membrane was obtained upon expression in a strain with the npi1 mutation, which significantly lowers the level of Rsp5 ubiquitin ligase in cells. This mutation helped to stabilize proteins in the plasma membrane

Charakterizace Na+/H+-antiportnich systému plasmatické membrany kvasinek

Le maintien d'ions à des concentrations intracellulaires constantes est essentiel à la survie des cellules. Les principaux cations concernés sont le potassium (K+), qui est indispensable au bon fonctionnement des levures, ainsi que le sodium (Na+) et le lithium (Li+) qui sont toxiques lorsqu'ils sont présents en concentration trop importante dans la cellule. En effet, le potassium est indispensable à de nombreuses fonctions physiologiques et intervient dans le maintien de la pression osmotique. Ce cation ne présente pas de toxicité pour la cellule mais sa concentration doit être limitée afin que la pression osmotique intracellulaire soit compatible avec le bon fonctionnement de la cellule. Par contre, les ions Na+ et Li+ sont toxiques pour la plupart des levures. Les seuils de tolérance aux ions toxiques et à la pression osmotique sont très variables d'une souche de levure à une autre. Le maintien de la concentration intracellulaire de ces ions compatible avec le fonctionnement cellulaire est assurée chez les levures par des mécanismes actifs ayant pour fonction de transporter les cations monovalents. Chez Saccharomyces cerevisiae, K+, Na+ et Li+ sont exportés par les mêmes systèmes de transport (Bañuelos et al., 1998). Le système le plus efficace, et en particulier à pH voisin de la neutralité, est constitué d'ATPases codées par les gènes ENA1-4. La dégradation de l'ATP fournit dans ce cas l'énergie nécessaire au transport. Par ailleurs, il existe un antiporteur codé par le gène NHA1 qui fonctionne à pH acide parce qu'il utilise le flux entrant de protons (H+) pour assurer l'efflux de cations. L'expression du gène NHA1 (YLR138w) est constitutive et faible. Il code pour une protéine de 985 aminoacides localisée dans la membrane plasmique. Enfin il faut noter que des travaux antérieurs effectués au laboratoire suggèrent que le transporteur Nha1p aurait des fonctions plus générales dans la cellule et il participerait en particulier à la régulation du pH intracellulaire (Sychrová et al., 1999). Sur le plan de la structure, Nha1p possède une extrémité N-terminale courte, un domaine intermédiaire hydrophobe formé de 12 segments transmembranaires et un domaine C-terminal hydrophile probablement à localisation intracellulaire. La structure a été comparée aux produits de gènes orthologues des levures Schizosaccharomyces pombe (Jia et al., 1992) et Zygosaccharomyces rouxii (Iwaki et al., 1998; Watanabe et al., 1995) les 2 seuls connus au début de ce travail, ainsi qu'à celui trouvé plus récemment de Candida albicans (Soong et al., 2000). Le degré de similarité entre les protéines est important dans les parties N-terminales et intermédiaires tandis que le segment C-terminal présente des différences notables: chez Saccharomyces cerevisiae, il est exceptionnellement long (554 aa, 56,2 % de la protéine total) en comparaison des autres levures. Par ailleurs, les antiporteurs des autres levures avaient été décrits comme étant uniquement des transporteurs à Na+ et Li+. Ceci semblait donc suggérer un rôle particulier pour ce long domaine hydrophile de Nha1p: rôle dans le transport de plusieurs cations, dans la réponse au stress osmotique ou encore dans la régulation du pH intracellulaire. C'est ce que nous nous sommes attaché à tester au cours de ce travail.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceCzech RepublicFRC

Yeast Membrane Transport. Preface.

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