Ascospore formation in the yeast Saccharomyces cerevisiae.

Sporulation of the baker's yeast Saccharomyces cerevisiae is a response to nutrient depletion that allows a single diploid cell to give rise to four stress-resistant haploid spores. The formation of these spores requires a coordinated reorganization of cellular architecture. The construction of the spores can be broadly divided into two phases. The first is the generation of new membrane compartments within the cell cytoplasm that ultimately give rise to the spore plasma membranes. Proper assembly and growth of these membranes require modification of aspects of the constitutive secretory pathway and cytoskeleton by sporulation-specific functions. In the second phase, each immature spore becomes surrounded by a multilaminar spore wall that provides resistance to environmental stresses. This review focuses on our current understanding of the cellular rearrangements and the genes required in each of these phases to give rise to a wild-type spore.     

Recovery of Saccharomyces cerevisiae from ethanol-induced growth inhibition.

Ethanol caused altered mobility of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene in plasma membrane preparations of Saccharomyces cerevisiae. Because lipids had been shown to protect yeast cells against ethanol toxicity, sterols, fatty acids, proteins, and combinations of these were tested; however, protection from growth inhibition was not seen. Ethanol-induced, prolonged lag periods and diminished growth rates in S. cerevisiae were reduced by an autoconditioning of the medium by the inoculum.     

Flocculation of Saccharomyces cerevisiae: inhibition by sugars.

Flocculation is governed by the competition between electrostatic repulsion (nonspecific interactions) and polysaccharide-protein bonds (specific interactions). In our study, the inhibition of flocculation by sugars for 12 strains of Saccharomyces cerevisiae leads us to extend the classification described in the literature and to define three groups of yeasts: flocculation mannose sensitive (MS), flocculation glucose-mannose sensitive (GMS), and flocculation mannose insensitive (MI). Only the first two groups showed specific interactions between proteins and mannans. n the MI group, the sugars tested did not inhibit flocculation. To characterize the particularities of the stereochemistry of the cell-wall proteic receptors of strains belonging to the MS and GMS groups, 31 sugars were used as inhibitor probes on two representative strains. The results show that the lectin specificity of strains belonging to the GMS group is less restricted regarding C-1 and C-2 hydroxyl groups than the lectin from strains belonging to the MS group, which interacts with all of the hydroxyl groups of mannopyranose. The two groups also differ with respect to inhibition by sugars: strains belonging to the MS group are partially inhibited whereas strains of the GMS group are completely inhibited. We observed that the presence of ethanol increases sugar fixation by strains from the MS group, but not from the GMS group. Moreover, both receptors interact with disaccharides, provided the two monomers are linked by an alpha(1-4), alpha(1-3), or alpha(1-2) bond.     

Regulation of lysine transport by feedback inhibition in Saccharomyces cerevisiae.

A steady-state level of about 240 nmol/mg (dry wt) occurs during lysine transport in Saccharomyces cerevisiae. No subsequent efflux of the accumulated amino acid was detected. Two transport systems mediate lysine transport, a high-affinity, lysine-specific system and an arginine-lysine system for which lysine exhibits a lower affinity. Preloading with lysine, arginine, glutamic acid, or aspartic acid inhibited lysine transport activity; preloading with glutamine, glycine, methionine, phenylalanine, or valine had little effect; however, preloading with histidine stimulated lysine transport activity. These preloading effects correlated with fluctuations in the intracellular lysine and/or arginine pool: lysine transport activity was inhibited when increases in the lysine and/or arginine pool occurred and was stimulated when decreases in the lysine and/or arginine pool occurred. After addition of lysine to a growing culture, lysine transport activity was inhibited more than threefold in one-third of the doubling time of the culture. These results indicate that the lysine-specific and arginine-lysine transport systems are regulated by feedback inhibition that may be mediated by intracellular lysine and arginine.     

Induction and inhibition of the allantoin permease in Saccharomyces cerevisiae.

Allantoin uptake in Saccharomyces cerevisiae is mediated by an energy-dependent, low-Km, active transport system. However, there is at present little information concerning its regulation. In view of this, we investigated the control of alloantoin transport and found that it was regulated quite differently from the other pathway components. Preincubation of appropriate mutant cultures with purified allantoate (commercial preparations contain 17% allantoin), urea, or oxalurate did not significantly increase allantoin uptake. Preincubation with allantoin, however, resulted in a 10- to 15-fold increase in the rate of allantoin accumulation. Two allantoin analogs were also found to elicit dramatic increases in allantoin uptake. Hydantoin and hydantoin acetic acid were able to induce allantoin transport to 63 and 95% of the levels observed with allantoin. Neither of these compounds was able to serve as a sole nitrogen source for S. cerevisiae, and they may be non-metabolizable inducers of the allantoin permease. The rna1 gene product appeared to be required for allantoin permease induction, suggesting that control was exerted at the level of gene expression. In addition, we have shown that allantoin uptake is not unidirectional; efflux merely occurs at a very low rate. Allantoin uptake is also transinhibited by addition of certain amino acids to the culture medium, and several models concerning the operation of such inhibition were discussed.     

Autoconditioning factor relieves ethanol-induced growth inhibition of Saccharomyces cerevisiae.

Viable Saccharomyces cerevisiae suspended in medium containing growth-inhibiting concentrations of ethanol produce a metabolite that relieves growth inhibition. This autoconditioning of the medium by yeasts is due to the formation of small amounts (0.01%, vol/vol) of acetaldehyde. The effect is duplicated precisely in fresh medium by the addition of acetaldehyde. Acetaldehyde does not increase the yield of or accelerate ethanol production by the organism. Ethanol-induced modifications of membrane order in the plasma membranes, as measured by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, were not resolved by exogenously added acetaldehyde.     

Scanning electron microscope study of Saccharomyces cerevisiae spheroplast formation.

A suspension of Saccharomyces cerevisiae NCY366 in buffered 1.2 M sorbitol containing Zymolyase-5000 (a beta-glucanase-containing preparation/showed maximum osmotic sensitivity after 30 min of incubation at 30 degrees C. A scanning electron microscope study of spheroplast formation, using a very high resolution (4-nm) machine, revealed several new morphological features. The surface of the plug in bud scars on intact cells appeared warty. The wall, which assumed a beady appearance as digestion proceded, ultimately sloughed off to reveal the furrowed surface of the plasma membrane. Bud scars were resistant to digestion and. as incubation proceeded, they became surrounded by an outer annulus, which may be the seconary septum. Wall material was completely removed from the majority of cells only after 60 min of digestion. The surface of spheroplasts was studded with particles, about 25 to 30 nm in diameter. Many spheroplasts had a single large indentation, which may be in that part of the plasma membrane originally underlying the birth scar.     

The RSF1 gene regulates septum formation in Saccharomyces cerevisiae.

Septum formation in the mitotic cell cycle of the budding yeast Saccharomyces cerevisiae occurs by conversion of the chitin ring, laid down at bud formation, into the primary septum. We show here that under certain conditions this septation is dependent on the newly identified RSF1 gene. However, cells harboring the rsf1-1 mutation accumulated in a postcytokinesis state, with delayed conversion of the chitin-rich annulus into the primary septum. This rsf1-1-mediated inhibition of septum formation only occurred under conditions of biosynthetic stress and was correlated with biosynthetically mediated inhibition of the cell-cycle regulatory step START. The RSF1 gene is distinct from the CHS2 chitin synthase gene that is responsible for septation, and thus RSF1 most likely encodes a regulator of chitin synthesis. We hypothesize that RSF1 activity facilitates septum formation during times of biosynthetic stress, to allow efficient septation even under these conditions.     

Protein glycation in Saccharomyces cerevisiae. Argpyrimidine formation and methylglyoxal catabolism

Methylglyoxal is the most important intracellular glycation agent, formed nonenzymatically from triose phosphates during glycolysis in eukaryotic cells. Methylglyoxal-derived advanced glycation end-products are involved in neurodegenerative disorders (Alzheimer's, Parkinson's and familial amyloidotic polyneurophathy) and in the clinical complications of diabetes. Research models for investigating protein glycation and its relationship to methylglyoxal metabolism are required to understand this process, its implications in cell biochemistry and their role in human diseases. We investigated methylglyoxal metabolism and protein glycation in Saccharomyces cerevisiae. Using a specific antibody against argpyrimidine, a marker of protein glycation by methylglyoxal, we found that yeast cells growing on d-glucose (100 mM) present several glycated proteins at the stationary phase of growth. Intracellular methylglyoxal concentration, determined by a specific HPLC based assay, is directly related to argpyrimidine formation. Moreover, exposing nongrowing yeast cells to a higher d-glucose concentration (250 mM) increases methylglyoxal formation rate and argpyrimidine modified proteins appear within 1 h. A kinetic model of methylglyoxal metabolism in yeast, comprising its nonenzymatic formation and enzymatic catabolism by the glutathione dependent glyoxalase pathway and aldose reductase, was used to probe the role of each system parameter on methylglyoxal steady-state concentration. Sensitivity analysis of methylglyoxal metabolism and studies with gene deletion mutant yeast strains showed that the glyoxalase pathway and aldose reductase are equally important for preventing protein glycation in Saccharomyces cerevisiae.     

Selective inhibition of transition from sexual agglutination to zygote formation by ethyl N-phenylcarbamate in Saccharomyces cerevisiae

Effects of ethyl N-phenylcarbamate (EPC) on the mating reaction of Saccharomyces cerevisiae were studied, with special attention on the effect on the pheromone action. EPC inhibited zygote formation at a concentration which promoted induction of sexual agglutinability. EPC enhanced agglutinability induction by pheromone, but inhibited -pheromone-induced formation of large pearshaped cells in a mating type. The enhancement of agglutinability induction was accompanied with increased production of a agglutination substance and inhibition of pheromone inactivation. EPC arrested the cell cycle of a cells probably in the step controlled by CDC19, CDC35, cAMP etc., just before the step controlled by CDC28, pheromone etc.Abbreviations EPC Ethyl N-phenylcarbamate - PBS 0.01 M phosphate buffer solution, pH 5.5 - SPB spindle pole body     

Recovery of Saccharomyces cerevisiae from ethanol-induced growth inhibition

Ethanol caused altered mobility of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene in plasma membrane preparations of Saccharomyces cerevisiae. Because lipids had been shown to protect yeast cells against ethanol toxicity, sterols, fatty acids, proteins, and combinations of these were tested; however, protection from growth inhibition was not seen. Ethanol-induced, prolonged lag periods and diminished growth rates in S. cerevisiae were reduced by an autoconditioning of the medium by the inoculum.     

Regularities in formation of gamma-induced mutants of Saccharomyces cerevisiae

As shown by an example of the formation of reversions in the leu2 gene of Saccharomyces cerevisiae, the process of mutant formation after gamma-irradiation is associated with the post-irradiation replication phases. When no DNA replication takes place, the mutants are not formed. The periods of realizing premutation lesions into mutations depend on what cell cycle phase the cells were in when irradiated.     

Feed component inhibition in ethanolic fermentation by Saccharomyces cerevisiae

Inhibition by secondary feed components can limit productivity and restrict process options for the production of ethanol by fermentation. New fermentation processes (such as vacuum or extractive fermentation), while selectively removing ethanol, can concentrate nonmetabolized feed components in the remaining broth. Stillage recycle to reduce stillage waste treatment results in the buildup of nonmetabolized feed components. Continuous culture experiments are presented establishing an inhibition order: CaCl(2), (NH(4))(2)xSO(4) > NaCl, NH(4)Cl > KH(2)PO(4) > xylose, MgCl(2) > MgSO(4) > KCl. Reduction of the water activity alone is not an adequate predictor of the variation in inhibitory concentration among the different components tested. As a general trend, specific ethanol productivity increases and cell production decreases as inhibitors are added at higher concentration. We postulate that these results can be interpreted in terms of an increase in energy requirements for cell maintenance under hypertonic (stressed) conditions. Ion and carbohydrate transport and specific toxic effects are reviewed as they relate to the postulated inhibition mechanism. Glycerol production increases under hypertonic conditions and glycerol is postulated to function as a nontoxic osmoregulator. Calcium was the most inhibitory component tested, causing an 80%decline in cell mass production at 0.23 mol Ca(2+)/L and calcium is present at substantial concentration in many carbohydrate sources. For a typical final cane molasses feed, stillage recycle must be limited to less than onethird of the feed rate; otherwise inhibitory effects will be observed.