Pedro S. de Araujo

Role of the trehalose carrier in dehydration resistance of Saccharomyces cerevisiae

Yeast cells are well known for their ability to survive complete dehydration, a phenomenon that is directly linked to the presence of the sugar trehalose in these cells. This sugar apparently endows the cells with the capacity to survive dehydration. Previous studies on in vitro models showed that trehalose must be present on both sides of the bilayer to stabilize dry membranes. The present report demonstrates that a specific trehalose carrier seems to enable the sugar to protect the yeast cell membrane by translocating trehalose from the cytosol to the extracellular environment. Saccharomyces cerevisiae mutant strains which lack the trehalose carrier did not survive after dehydration although they accumulated endogenous trehalose. Furthermore, when carrier mutants were dehydrated in the presence of exogenous trehalose the cells became more resistant showing increased survival.

Effect of lipid membranes on the apparent pK of the local anesthetic tetracaine spin label and titration studies

Electrometric titrations and spin label data demonstrate changes in the experimentally determined apparent pK of an ionizable drug in the presence of membranes. This effect is attributed to the difference in partition coefficients for the charged and uncharged forms of the drug. Investigation of the binding of a local anesthetic, tetracaine, to egg phosphatidylcholine membranes indicates that the drug apparent pK decreases in the presence of membranes, the decrease being a function of membrane concentration. The agreement between titration and spin label studies is very good and could be simulated by calculating membrane-bound and free populations of charged and uncharged tetracaine from the independently-measured partition coefficients for the two forms.

Effects of temperature and lipid composition on the serum albumin-induced aggregation and fusion of small unilamellar vesicles

Small unilamellar vesicles of egg phosphatidylcholine (PC) or dimyristoylphosphatidylcholine, mixed with small unilamellar vesicles labelled with 2-(10-(1-pyrene)decanoyl)phosphatidylcholine, exhibit a constant average size and excimer to monomer (E/M) ratio for several hours when incubated at pH 3.6 at a temperature higher than the phase transition temperature (Tc) of the lipids. Addition of bovine serum albumin to this system produces a transient turbidity increase, a fast decrease in the E/M ratio, a partial loss of vesicle-entrapped [14C]sucrose and a measurable leak-in of externally added sucrose. Sepharose 4B filtration of the system demonstrates that the E/M ratio decrease is strictly paralleled by the formation of liposomes which exhibit a low E/M ratio and a hydrodynamic radius larger than that of small unilamellar vesicles. These data demonstrate that the E/M ratio decrease can be unequivocally ascribed to a vesicle-vesicle fusion process induced by serum albumin. The rate of serum-albumin induced fusion of small unilamellar vesicles is: (a) maximal at a stoichiometric ratio of approx. 2 albumins per vesicle; (b) sensitive to the nature of the lipid and; (c) not altered when human serum albumin replaces bovine serum albumin. The rate of albumin-induced fusion of dimyristoylphosphatidylcholine small unilamellar vesicles is higher below the Tc of the lipid and increases with temperature above the Tc. The formation of protein-bound aggregates with defined stoichiometries and a high local vesicle concentration, as well as changes in the local degree of hydration, are proposed to be the driving forces for the protein-induced vesicle fusion in this system.

Subcellular distribution of low- and high-molecularweight acid phosphatases

Acid phosphatases (orthophosphoric-monoester phosphohydrolases (acid optimum), EC 3.1.3.2) of low and high molecular weight were separated by Sephadex G-75 filtration from extracts of rat brain, liver and kidney. The proportion of each phosphatase in the extract depends critically on the method employed for homogenate preparation, and no interconversion between high and low molecular weight forms was detected. In extracts obtained from subcellular organelles only high-molecular-weight acid phosphatase was detected, which is of lysosomal origin. Low-molecular-weight acid -phosphatase is restricted to the cell sap. Low- and high-molecular-weight acid phosphatases were characterized by their elution volumes, specific inhibition and activity with two substrates. It is suggested that the distribution pattern found om rat tissues could be common to all eukaryotic cells.

Molecular Analysis of Maltotriose Active Transport and Fermentation by Saccharomyces cerevisiae Reveals a Determinant Role for the AGT1 Permease

ABSTRACT Incomplete and/or sluggish maltotriose fermentation causes both quality and economic problems in the ale-brewing industry. Although it has been proposed previously that the sugar uptake must be responsible for these undesirable phenotypes, there have been conflicting reports on whether all the known α-glucoside transporters in Saccharomyces cerevisiae (MALx1, AGT1, and MPH2 and MPH3 transporters) allow efficient maltotriose utilization by yeast cells. We characterized the kinetics of yeast cell growth, sugar consumption, and ethanol production during maltose or maltotriose utilization by several S. cerevisiae yeast strains (both MAL constitutive and MAL inducible) and by their isogenic counterparts with specific deletions of the AGT1 gene. Our results clearly showed that yeast strains carrying functional permeases encoded by the MAL21, MAL31, and/or MAL41 gene in their plasma membranes were unable to utilize maltotriose. While both high- and low-affinity transport activities were responsible for maltose uptake from the medium, in the case of maltotriose, the only low-affinity (Km, 36 ± 2 mM) transport activity was mediated by the AGT1 permease. In conclusion, the AGT1 transporter is required for efficient maltotriose fermentation by S. cerevisiae yeasts, highlighting the importance of this permease for breeding and/or selection programs aimed at improving sluggish maltotriose fermentations.

Kinetics of active sucrose transport in Saccharomyces cerevisiae

The kinetic analysis of active sucrose-H+ uptake by Saccharomyces cerevisiae revealed the presence of two transport systems with high and low affinity for sucrose. The MAL2T permease has a low affinity (K(m) = 120 +/-20 mM) for sucrose, while the alpha-glucoside transporter encoded by the AGT1 gene is a high affinity sucrose-H+ symporter (K(m) = 7.9+/-0.8 mM) that increases the specific growth rate of cells growing on sucrose.

Switching the mode of sucrose utilization by Saccharomyces cerevisiae

BackgroundOverflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H+ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures.ResultsWe have deleted from the genome of a S. cerevisiae strain lacking invertase the high-affinity sucrose-H+ symporter encoded by the AGT1 gene. This strain could still grow efficiently on sucrose due to a low-affinity and low-capacity sucrose-H+ symport activity mediated by the MALx1 maltose permeases, and its further intracellular hydrolysis by cytoplasmic maltases. Although sucrose consumption by this engineered yeast strain was slower than with the parental yeast strain, the cells grew efficiently on sucrose due to an increased respiration of the carbon source. Consequently, this engineered yeast strain produced less ethanol and 1.5 to 2 times more biomass when cultivated in simple batch mode using 20 g/L sucrose as the carbon source.ConclusionHigher cell densities during batch cultures on 20 g/L sucrose were achieved by using a S. cerevisiae strain engineered in the sucrose uptake system. Such result was accomplished by effectively reducing sucrose uptake by the yeast cells, avoiding overflow metabolism, with the concomitant reduction in ethanol production. The use of this modified yeast strain in simpler batch culture mode can be a viable option to more complicated traditional sucrose-limited fed-batch cultures for biomass-directed processes of S. cerevisiae.

Phospholipase A2 activity in dehydrated systems: Effect of the physical state of the substrate

In the presence of excess water, enzymatic activity of phospholipase A2 (PLA2) depends on the physical state of the lipid substrate. In order to determine if this also holds true in dehydrated systems, the physical parameters of charge, hydration state, and head group spacing of liposome membranes and their effects on PLA2 lipid hydrolysis were studied. Liposomes of varying composition were freeze-dried in the presence of PLA2 and partially rehydrated at controlled relative humidities. Accumulation of free fatty acids in the liposomal membranes was used as a measure of PLA2 activity. We found that PLA2, which was not activated during lyophilization, was most active during partial rehydration of the liposomes. The hydration state, charge and headgroup spacing of the membrane were all important in determining PLA2 activity in the dehydrated system.

Ion exchange between n-alkyl carboxylates and bromide at the surface of cetyltrimethylammonium micelles

Abstract Selectivity coefficients for ion exchange between n -alkyl carboxylates and bromide at the surface of cetyltrimethylammonium micelles were measured by absorption and fluorescence spectroscopy techniques. Incorporation of the first methylene group (from formate to acetate) does not increase the binding capacity of the ion. After acetate, incorporation of each methylene group increases the binding constant by nearly a factor 2.3. These results can be interpreted in terms of the predominance of the alkyl chain hydrophobicity in determining the binding capacity of the organic counterion. A contribution of (500 ± 100) cal mole −1 per methylene group was obtained for the free energy of transfer from water to the micelle surface. This contribution is smaller than that corresponding to the transference of a methylene group from water to an hydrocarbon solvent. The possible factors determining this difference are discussed.

In silico and in vivo analysis reveal a novel gene in Saccharomyces cerevisiae trehalose metabolism

BackgroundThe ability to respond rapidly to fluctuations in environmental changes is decisive for cell survival. Under these conditions trehalose has an essential protective function and its concentration increases in response to enhanced expression of trehalose synthase genes, TPS1, TPS2, TPS3 and TSL1. Intriguingly, the NTH1 gene, which encodes neutral trehalase, is highly expressed at the same time. We have previously shown that trehalase remains in its inactive non-phosphorylated form by the action of an endogenous inhibitor. Recently, a comprehensive two-hybrid analysis revealed a 41-kDa protein encoded by the YLR270w ORF, which interacts with NTH1p.ResultsIn this work we investigate the correlation of this Trehalase Associated Protein, in trehalase activity regulation. The neutral trehalase activity in the ylr270w mutant strain was about 4-fold higher than in the control strain. After in vitro activation by PKA the ylr270w mutant total trehalase activity increased 3-fold when compared to a control strain. The expression of the NTH1 gene promoter fused to the heterologous reporter lacZ gene was evaluated. The mutant strain lacking YLR270w exhibited a 2-fold increase in the NTH1-lacZ basal expression when compared to the wild type strain.ConclusionsThese results strongly indicate a central role for Ylr270p in inhibiting trehalase activity, as well as in the regulation of its expression preventing a wasteful futile cycle of synthesis-degradation of trehalose.