Membrane potential in yeast cells measured by direct and indirect methods

Vaçata, V.; Kotyk, Arnošt; Sigler, Karel

doi:10.1016/0005-2736(81)90241-8

Cited by 67 publications

(29 citation statements)

References 4 publications

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“…Furthermore, while not affecting the initial binding event of Hst-5, C. albicans cells treated with uncouplers of the electrochemical gradient, such as carbonylcyanide-m-chlorophenylhydrazone, dinitrophenol, and sodium azide, as well as petite respiration-deficient mutants have increased resistance to Hst-5 [12],[18]. The membrane potential of S. cerevisiae has been measured at −71 mV, a value closer to that of mammalian cells (−90 to −110 mV), whereas C. albicans membrane potential is −120 mV, a value more in line with bacterial pathogens (−130 to −150 mV) [34],[54],[55]. The trans-negative electrochemical gradient may potentially act as an underlying mechanism providing Hst-5 resistance to S. cerevisiae and Hst-5 susceptibility to C. albicans .…”

Section: Discussionmentioning

confidence: 99%

The Antimicrobial Peptide Histatin-5 Causes a Spatially Restricted Disruption on the Candida albicans Surface, Allowing Rapid Entry of the Peptide into the Cytoplasm

2008

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Antimicrobial peptides play an important role in host defense against microbial pathogens. Their high cationic charge and strong amphipathic structure allow them to bind to the anionic microbial cell membrane and disrupt the membrane bilayer by forming pores or channels. In contrast to the classical pore-forming peptides, studies on histatin-5 (Hst-5) have suggested that the peptide is transported into the cytoplasm of Candida albicans in a non-lytic manner, and cytoplasmic Hst-5 exerts its candicidal activities on various intracellular targets, consistent with its weak amphipathic structure. To understand how Hst-5 is internalized, we investigated the localization of FITC-conjugated Hst-5. We find that Hst-5 is internalized into the vacuole through receptor-mediated endocytosis at low extracellular Hst-5 concentrations, whereas under higher physiological concentrations, Hst-5 is translocated into the cytoplasm through a mechanism that requires a high cationic charge on Hst-5. At intermediate concentrations, two cell populations with distinct Hst-5 localizations were observed. By cell sorting, we show that cells with vacuolar localization of Hst-5 survived, while none of the cells with cytoplasmic Hst-5 formed colonies. Surprisingly, extracellular Hst-5, upon cell surface binding, induces a perturbation on the cell surface, as visualized by an immediate and rapid internalization of Hst-5 and propidium iodide or rhodamine B into the cytoplasm from the site using time-lapse microscopy, and a concurrent rapid expansion of the vacuole. Thus, the formation of a spatially restricted site in the plasma membrane causes the initial injury to C. albicans and offers a mechanism for its internalization into the cytoplasm. Our study suggests that, unlike classical channel-forming antimicrobial peptides, action of Hst-5 requires an energized membrane and causes localized disruptions on the plasma membrane of the yeast. This mechanism of cell membrane disruption may provide species-specific killing with minimal damage to microflora and the host and may be used by many other antimicrobial peptides.

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Section: Discussionmentioning

confidence: 99%

The Antimicrobial Peptide Histatin-5 Causes a Spatially Restricted Disruption on the Candida albicans Surface, Allowing Rapid Entry of the Peptide into the Cytoplasm

2008

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“…Because of the large degree of homology that exists between bacterial and fungal FEX genes, the eukaryotic FEX proteins are likely to also function as selective F − channels. Furthermore, the resting membrane voltage of fungi is normally highly negative (about −200 mV) (44,45), which would provide a sufficient thermodynamic gradient to expel F − by the same mechanism. The results from both eubacteria and eukaryotes indicate that FEX proteins have a conserved function in maintaining low levels of intracellular fluoride across multiple domains of life and that these proteins promote the survival of cells under fluoride stress.…”

Section: Discussionmentioning

confidence: 99%

Eukaryotic resistance to fluoride toxicity mediated by a widespread family of fluoride export proteins

Smith²,

Davis

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

110

132

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Fluorine is an abundant element and is toxic to organisms from bacteria to humans, but the mechanisms by which eukaryotes resist fluoride toxicity are unknown. The Escherichia coli gene crcB was recently shown to be regulated by a fluoride-responsive riboswitch, implicating it in fluoride response. There are >8,000 crcB homologs across all domains of life, indicating that it has an important role in biology. Here we demonstrate that eukaryotic homologs [renamed FEX (fluoride exporter)] function in fluoride export. FEX KOs in three eukaryotic model organisms, Neurospora crassa, Saccharomyces cerevisiae, and Candida albicans, are highly sensitized to fluoride (>200-fold) but not to other halides. Some of these KO strains are unable to grow in fluoride concentrations found in tap water. Using the radioactive isotope of fluoride, 18 F, we developed an assay to measure the intracellular fluoride concentration and show that the FEX deletion strains accumulate fluoride in excess of the external concentration, providing direct evidence of FEX function in fluoride efflux. In addition, they are more sensitive to lower pH in the presence of fluoride. These results demonstrate that eukaryotic FEX genes encode a previously unrecognized class of fluoride exporter necessary for survival in standard environmental conditions. toxicity resistance | environmental toxin | ion transport | dual membrane topology

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“…The lipophilic cation triphenylmethylphosphonium (Ph3MeP+) and the related compound tetraphenylphosphonium are widely used as membrane potential sensors for whole cells (1,2), subcellular fractions such as synaptosomes (3), and membrane vesicles (4). These cations accumulate into the membrane-enclosed compartment, when a potential (interior negative) is generated across the membrane.…”

mentioning

confidence: 99%

Triphenylmethylphosphonium is an ion channel ligand of the nicotinic acetylcholine receptor.

Lauffer¹,

Hucho²

1982

Proc. Natl. Acad. Sci. U.S.A.

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The lipophilic cation triphenylmethylphosphonium (Ph3MeP+), which is widely used as a sensor for membrane potential with cells, organelles, and membrane vesicles, is shown also to accumulate in membranes rich in nicotinic acetylcholine receptor in a voltage-independent way. Evidence is presented that Ph3MeP+ in this sytem is bound to a cation-binding site of the ion channel that is part ofthe acetylcholine receptor complex. Binding is stimulated by cholinergic effectors (Kd = 13 ,gM in the absence of carbamoylcholine; Kd = 1.5 ,uM in the presence of 10 jLM carbamoylcholine), and this stimulation is blocked by a-bungarotoxin.Ph3MeP+ blocks efflux of 'Na from receptor-rich microsacs and appears to compete with the channel ligand phencyclidine for a common binding site. In contrast to the binding of other proven channel ligands, Ph3MeP+-binding is not affected by desensitization.The lipophilic cation triphenylmethylphosphonium (Ph3MeP+) and the related compound tetraphenylphosphonium are widely used as membrane potential sensors for whole cells (1, 2), subcellular fractions such as synaptosomes (3), and membrane vesicles (4). These cations accumulate into the membrane-enclosed compartment, when a potential (interior negative) is generated across the membrane. Using radioactively labeled ions, it is possible to determine the amount ofions accumulated by a simple filtration assay (5). The concentration difference of cations between inside and outside can be used to calculate the membrane potential with the Nernst equation.We intended to apply this method for the investigation ofthe potentials across membranes ofvesicles prepared from the electric organ of Torpedo marmorata. Surprisingly, we found that with these vesicles accumulation ofPh3MeP+ not only is voltage dependent but also is stimulated in a voltage-independent manner by a variety of cholinergic effectors. t MATERIALS AND METHODS

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Membrane potential in yeast cells measured by direct and indirect methods

Cited by 67 publications

References 4 publications

The Antimicrobial Peptide Histatin-5 Causes a Spatially Restricted Disruption on the Candida albicans Surface, Allowing Rapid Entry of the Peptide into the Cytoplasm

The Antimicrobial Peptide Histatin-5 Causes a Spatially Restricted Disruption on the Candida albicans Surface, Allowing Rapid Entry of the Peptide into the Cytoplasm

Eukaryotic resistance to fluoride toxicity mediated by a widespread family of fluoride export proteins

Triphenylmethylphosphonium is an ion channel ligand of the nicotinic acetylcholine receptor.

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