The effects of beta-pinene on yeast cells were studied. This terpene inhibited respiration with glucose or ethanol as the substrate. The inhibition depended on the ratio of the terpene to the amount of yeast cells; for a fixed concentration of pinene, inhibition decreased as the amount of yeast cells increased. Pinene also inhibited the pumping of protons and K+ transport, but this inhibition was more marked with with ethanol than with glucose as the substrate, indicating the mitochondrial localization of the inhibition. The studies on isolated mitochondria showed a series of effects, starting with the disappearance of the respiratory control and deenergization of the organelles and followed by an inhibition of respiration at higher concentrations of the terpene. The effect on respiration could be localized to the cytochrome b region of the electron transport chain. No effect could be detected on the activity of ATPase. The effects can be ascribed to a localization of pinene on membranes which was also accompanied by a decrease in the fluorescence polarization of diphenyl hexatriene, probably meaning an increase in the fluidity of the membrane, localized preferentially to the mitochondria.

The effects of low molecular weight (96.5 KDa) chitosan on the pathogenic yeast Candida albicans were studied. Low concentrations of chitosan, around 2.5 to 10 μg·mL−1 produced (a) an efflux of K+ and stimulation of extracellular acidification, (b) an inhibition of Rb+ uptake, (c) an increased transmembrane potential difference of the cells, and (d) an increased uptake of Ca2+. It is proposed that these effects are due to a decrease of the negative surface charge of the cells resulting from a strong binding of the polymer to the cells. At higher concentrations, besides the efflux of K+, it produced (a) a large efflux of phosphates and material absorbing at 260 nm, (b) a decreased uptake of Ca2+, (c) an inhibition of fermentation and respiration, and (d) the inhibition of growth. The effects depend on the medium used and the amount of cells, but in YPD high concentrations close to 1 mg·mL−1 are required to produce the disruption of the cell membrane, the efflux of protein, and the growth inhibition. Besides the findings at low chitosan concentrations, this work provides an insight of the conditions required for chitosan to act as a fungistatic or antifungal and proposes a method for the permeabilization of yeast cells.

Debaryomyces hansenii showed an increased growth in the presence of either 1 M, KCl or 1 M NaCl and a low acidification of the medium, higher for the cells grown in the presence of NaCl. These cells accumulated high concentrations of the cations, and showed a very fast capacity to exchange either Na+ or K+ for the opposite cation. They showed a rapid uptake of 86Rb+ and 22Na+. 86Rb+ transport was saturable, with K(m) and Vmax values higher for cells grown in 1 M NaCl. 22Na+ uptake showed a diffusion component, also higher for the cells grown with NaCl. Changes depended on growth conditions, and not on further incubation, which changed the internal ion concentration. K+ stimulated proton pumping produced a rapid extrusion of protons, and also a decrease of the membrane potential. Cells grown in 1 M KCl showed a higher fermentation rate, but significantly lower respiratory capacity. ATP levels were higher in cells grown in the presence of NaCl; upon incubation with glucose, those grown in the presence of KCl reached values similar to the ones grown in the presence of NaCl. In both, the addition of KCl produced a transient decrease of the ATP levels. As to ion transport mechanisms, D. hansenii appears to have (a) an ATPase functioning as a proton pump, generating a membrane potential difference which drives K+ through a uniporter; (b) a K+/H+ exchange system; and (c) a rapid cation/cation exchange system. Most interesting is that cells grown in different ionic environments change their studied capacities, which are not dependent on the cation content, but on differences in their genetic expression during growth.

In the Saccharomyces cerevisiae glycolytic pathway, 11 enzymes catalyze the stepwise conversion of glucose to two molecules of ethanol plus two CO 2 molecules. In the highly crowded cytoplasm, this pathway would be very inefficient if it were dependent on substrate/enzyme diffusion. Therefore, the existence of a multi-enzymatic glycolytic complex has been suggested. This complex probably uses the cytoskeleton to stabilize the interaction of the various enzymes. Here, the role of filamentous actin (F-actin) in stabilization of a putative glycolytic metabolon is reported. Experiments were performed in isolated enzyme/actin mixtures, cytoplasmic extracts and permeabilized yeast cells. Polymerization of actin was promoted using phalloidin or inhibited using cytochalasin D or latrunculin. The polymeric filamentous F-actin, but not the monomeric globular G-actin, stabilized both the interaction of isolated glycolytic pathway enzyme mixtures and the whole fermentation pathway, leading to higher fermentation activity. The associated complexes were resistant against inhibition as a result of viscosity (promoted by the disaccharide trehalose) or inactivation (using specific enzyme antibodies). In S. cerevisiae, a glycolytic metabolon appear to assemble in association with F-actin. In this complex, fermentation activity is enhanced and enzymes are partially protected against inhibition by trehalose or by antibodies. Structured digital abstract• ALD physically interacts with PGK and GAPDH by anti bait coimmunoprecipitation (View interaction)• ALD physically interacts with GAPDH and PGK by affinity chromatography technology (View interaction) IntroductionThe cytoplasm is a highly concentrated suspension of proteins, polysaccharides, nucleic acids and small solutes [1,2]. It has been proposed that saturation promotes specific protein-protein interactions [1,3], and, once associated, enzymes in a given pathway team up to catalyze several consecutive reactions; these enzyme complexes are called metabolons [4,5]. In metabolons, intermediaries are channeled, i.e. enzymes that catalyze consecutive reactions transfer intermediaries directly to each other [2,6,7]. Substrate channeling confers a number of benefits, including altered reaction kinetics, preservation of cellular solvation capacity [8] or sequestration of toxic intermediaries [9]. The highly dynamic nature of enzyme-enzyme interactions Abbreviations ADH, alcohol dehydrogenase; ALD, aldolase; ENO, enolase; F-actin, filamentous actin; G-actin, globular (monomeric) actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPI, glucose-6-phosphate isomerase; HXK, hexokinase; PFK, phosphofructokinase; PGAM, phosphoglyceromutase; PGK, phosphoglycerate kinase; PK, pyruvate kinase; TPI, triosephosphate isomerase. 3887[10] probably regulates their reaction rate, and channels substrates through specific pathway(s) [2,6,11]. Various groups have described metabolons in the cysteine synthase complex, the Calvin cycle, cyanogenic glucoside synthesis and the phenylpropanoid pathway of ...

A gene, SIT4, was identified as corresponding to a serine/threonine protein phosphatase and when overexpressed confers lithium tolerance in galactose medium to the budding yeast Saccharomyces cerevisiae. This gene has been previously identified as a regulator of the cell cycle and involved in nitrogen sensing. It is shown that the transcription levels of SIT4 are induced by low concentrations of Li ؉ in a time-dependent manner. Na ؉ and K ؉ at high concentrations, but not sorbitol, also induce transcription. As a response to Na ؉ or Li ؉ stress, yeast cells lower the intracellular K ؉ content. This effect is enhanced in cells overexpressing SIT4, which also increase 86 Rb efflux after the addition of Na ؉ or Li ؉ to the extracellular medium. Another feature of SIT4-overexpressing cells is that they maintain a more alkaline pH of 6.64 compared with 6.17 in the wild type cells. It has been proposed that the main pathway of salt tolerance in yeast is mediated by a P-type ATPase, encoded by PMR2A/ENA1. However, our results show that in a sit4 strain, expression of ENA1 is still induced by monovalent cations, and overexpression of SIT4 does not alter the amount of ENA1 transcript. These results show that SIT4 acts in a parallel pathway not involving induction of transcription of ENA1 and suggest a novel function for SIT4 in response to salt stress.

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Debaryomyces hansenii, a halophile yeast found in shallow sea waters and salty food products grows optimally in 0.

The measurement of internal pH in microorganisms, in yeast cells and in cells in general, has been studied for many years. Several mechanisms are involved in the regulation of the internal pH of the cell, many cellular processes are regulated by the internal pH, and many transport processes depend on the H ϩ cycle. In yeast cells, very crude procedures were initially used, with disruption of the cells by boiling or freezing and thawing, after which the pH of the resulting sap was measured. In 1950, the group of Conway used the distribution of weak acids, such as carbonic or propionic acid, to measure the internal pH of yeast cells (5,6). By these methods, the pH was probably obtained as an average of that of the entire cell interior, including all internal compartments. More recently, other methods have been used; among them, the shift of the P i peak in nuclear magnetic resonance spectra has been useful but complicated and expensive (1, 3).In yeast cells, the use of ionizable fluorescent probes capable of crossing the membrane and distributing between the cells, organelles, or vesicles depending on the internal and external pH (16) has proven useless. Slavík (17) first introduced indicators into yeast cells whose fluorescence depends on the surrounding pH; some of these, which are available commercially, can be introduced into the cells as acetoxymethyl esters (the permeant form), which are cleaved inside by esterases, transforming them into an impermeant form and preventing their efflux. A recent report on the use of one of these dyes to measure the internal pH of yeast cells has appeared (9). However, Slayman et al. (18) have pointed out some of the drawbacks of these dyes; because of the concentration of esterases in some intracellular compartments, they are preferentially hydrolyzed and accumulated in vacuoles or other internal compartments which accumulate hydrolytic enzymes.Kano and Fendler (10) introduced the use of pyranine (8-hydroxy-1,3,6-pyrene-trisulfonic acid), a fluorescent dye with a ionizable -OH group, which shows remarkable pH dependence in fluorescence. The dye could be trapped in liposomes and used to estimate their internal pH (see also reference 4), the most important advantage being the relatively low interaction with the bilayer or proteins, because of its hydrophilic nature.Unfortunately, this property makes its use with whole cells difficult, because of problems with entry. However, a controlled electric shock of high intensity and short duration appears to produce transient openings of the cell membrane that are closed in a short time after the treatment (11). This phenomenon has been used to introduce even substances with high molecular weights into cells by a procedure known as electroporation. This method has been successfully used in yeast cells, and great improvements have been made recently (2,8).The present communication deals with the use of pyranine to measure the internal pH of yeast cells. MATERIALS AND METHODSCells from an isolated colony of commercial yeast cells (La Azt...