Energy requirement for amino acid uptake inSaccharomyces cerevisiae

The uptake of glycine and α-aminoisobutyric acid by baker’s yeast was substantially increased by preincubation withd-glucose,d-fructose, sucrose, and maltose, but much less with ethanol or acetate. The increments in uptake are in rough agreement with the intracellular amount of acid-non-extractable high-energy phosphate (probably polyphosphate). The energy for amino acid transport is thus provided predominantly by the nonmitochondrial catabolic processes.     

Mating reaction inSaccharomyces cerevisiae

The effect of proteolytic enzymes on sexual agglutinability of haploid cells of the yeastSaccharomyces cerevisiae was examined. Sexual agglutinability of cells of botha and α types was lost on treatment with alkaline protease and two kinds of neutral proteases ofBacillus subtilis, pronase and α-chymotrypsin. Agglutinability of α type cells was lost after treatment with acid protease ofRhizopus chinensis and trypsin, but that ofa type cells was not. These results indicate that the sex-specific substance responsible for the sexual agglutination (agglutination factor) ina type cells differs from that in α type cells. Agglutination factors were solubilized from cell-wall fractions of both mating types by Glusulase treatment. These crude factors specifically inhibited the agglutinability of cells of the opposite mating type with little effect on the agglutinability of cells of the same mating type.     

Transport ofl-tryptophan inSaccharomyces cerevisiae

In addition to the general amino acid transport system (GAP) ofS. cerevisiae l-tryptophan is transported by another system with approximately 25% capacity of GAP, with aK _T of 0.41±0.08 mmol/L and with a similar specificity as GAP (lower inhibition by Met, Pro, Ser, Thr and 2-aminoisobutyric acid; greater inhibition by Glu and His). The pH optimum of this system is at 5.0–5.5, activation energy above the transition point (20°C) was 20 kJ/mol, below the transition point 55 kJ/mol. The transport by this system was virtually unidirectional, efflux amounting to at most 10% into a tryptophan-free medium. The transport itself was blocked by 2,4-dinitrophenol, antimycin A and uranyl nitrate. The system was synthesized de novo during preincubation with glucose=fructose>trehalose >ethanol within 30 min, and was degraded with a half-time of 15 min in the absence of further synthesis. The accumulation ratios ofl-tryptophan ingap1 mutants were concentration-dependent (200∶1 at 1 μmoll-Trp/L, 4∶1 at 2.5 mmoll-Trp/L) and decreased with increasing suspension density from 200∶1 to 5∶1 (for 10 μmoll-Trp/L). The involvement of hydrogen ions in the uptake was clearly demonstrated by the effect of D₂O even if it could not be established by either shifts of pH_out or membrane depolarization.     

Computer-assisted nonlinear regression analysis of the multicomponent glucose uptake kinetics of Saccharomyces cerevisiae.

The kinetics of glucose uptake in Saccharomyces cerevisiae are complex. An Eadie-Hofstee (rate of uptake versus rate of uptake over substrate concentration) plot of glucose uptake shows a nonlinear form typical of a multicomponent system. The nature of the constituent components is a subject of debate. It has recently been suggested that this nonlinearity is due to either a single saturable component together with free diffusion of glucose or a single constitutive component with a variable Km, rather than the action of multiple hexose transporters. Genetic data support the existence of a family of differentially regulated glucose transporters, encoded by the HXT genes. In this work, kinetic expressions and nonlinear regression analysis, based on an improved zero trans-influx assay, were used to address the nature of the components of the transport system. The results indicate that neither one component with free diffusion nor a single permease with a variable Km can explain the observed uptake rates. Results of uptake experiments, including the use of putative alternative substrates as inhibitory compounds, support the model derived from genetic analyses of a multicomponent system with at least two components, one a high-affinity carrier and the other a low-affinity carrier. This approach was extended to characterize the activity of the SNF3 protein and identify its role in the depression of high-affinity uptake. The kinetic data support a role of SNF3 as a regulatory protein that may not itself be a transporter.     

The chitin-glucan complex inSaccharomyces cerevisiae

The content of glucosamine in the walls of daughter (without bud scars) and mother (multiscar) cells ofSaccharomyces cerevisiae was examined in a control and after treatment with dilute alkali, acid and buffer. The occurrence of chitin in the bud and birth scars is discussed. The results of IR and X-ray analysis of cell-wall fractions indicate the presence of α-chitin which is a part of the chitin-glucan complex. The size of the crystallite of α-chitin in this complex is about 60 Å.     

Transport of acyclic polyols inSaccharomyces cerevisiae

Acyclic polyols (erythritol, xylitol, ribitol, D-arabinitol, mannitol, sorbitol and galactitol) are not metabolized by Saccharomyces cerevisiae. They are taken up by a fast non-active process, reaching 40-70% distribution referred to total cell water. The uptake is insensitive to temperature, pH (between 4 and 8), 2,4-dinitrophenol and uranyl ions. Its initial rate rises linearly with concentration from 10(-5)M to 1M. The process resembles simple diffusion through large pores or the trapping of the whole solution on the surface. Protoplasts behave like whole cells in this respect. Only erythritol shows a second type of uptake which is inhibitor-insensitive but temperature-dependent.     

Turnover of canavanine-containing proteins inSaccharomyces cerevisiae

Saccharomyces cerevisiae grown for 2 h in the presence of 0.5 mmol/L canavanine in a synthetic medium with ethanol as the sole carbon source (OEC) exhibited a slowing down of protein synthesis for 3–4 h after a shift to fresh ethanolbased medium containing 1.0 mmol/L arginine (OEA) in comparison with untreated cells grown on OEA. The change of carbon source from ethanol to glucose (OGA) after growth in the OEC medium resulted in an even deeper decline of protein synthesis. The degradation of canavanine-containing proteins in cells pregrown and labelled in an OEC medium after transfer to OEA was more rapid than in the OGA medium. The initial rate of protein degradation during the first hour in the OGA medium was less than 1%/h whereas in the OEA medium it reached almost 10%/h. The fraction of proteins with high turnover (half-life 0.46 h) constituted 8.3% on OEA, while during subsequent growth on OGA it was only 0.75% with a half-life of 0.12 h.     

The relationship between transport kinetics and glucose uptake by Saccharomyces cerevisiae in aerobic chemostat cultures

The steady-state residual glucose concentrations in aerobic chemostat cultures of Saccharomyces cerevisiae ATCC 4126, grown in a complex medium, increased sharply in the respiro-fermentative region, suggesting a large increase in the apparent k_s value. By contrast, strain CBS 8066 exhibited much lower steady-state residual glucose concentrations in this region. Glucose transport assays were conducted with these strains to determine the relationship between transport kinetics and sugar assimilation. With strain CBS 8066, a high-affinity glucose uptake system was evident up to a dilution rate of 0.41 h^–1, with a low-affinity uptake system and high residual glucose levels only evident at the higher dilution rates. With strain ATCC 4126, the high-affinity uptake system was present up to a dilution rate of about 0.38 h^–1, but a low-affinity uptake system was discerned already from a dilution rate of 0.27 h^–1, which coincided with the sharp increase in the residual glucose concentration. Neither of the above yeast strains had an absolute vitamin requirement for aerobic growth. Nevertheless, in the same medium supplemented with vitamins, no low-affinity uptake system was evident in cells of strain ATCC 4126 even at high dilution rates and the steady-state residual glucose concentration was much lower. The shift in the relative proportions of the high and low-affinity uptake systems of strain ATCC 4126, which might have been mediated by an inositol deficiency through its effect on the cell membrane, may offer an explanation for the unusually high steady-state residual glucose concentrations observed at dilution rates above 52% of the wash-out dilution rate.     

Regulation of sterol biosynthesis inSaccharomyces cerevisiae

Sterol synthesis inSaccharomyces cerevisiae was primarily controlled by the growth rate. At low specific growth rates the intermediates of ergosterol biosynthesis prevailed in cells. At the same time, the total sterol content reached about 6% of dry matter whereas the content of ergosterol was only 2–2.5%, which seems to be the maximum value forS. cerevisiae. After esterification with fatty acids these sterol intermediates are stored in lipid globules together with reserve triacylglycerols. The sporulatingS. cerevisiae cells contained 3.5% sterols and 1.5% ergosterol of dry matter.     

Patterns of wall synthesis inSaccharomyces cerevisiae

Wall formation inSaccharomyces cerevisiae seems to be the result of two main patterns of wall material deposition: (i) around the whole periphery of the cell in nonbudding ones, and (ii) mainly at the tip of the daughter cell or at the cross wall that separates dividing cells. This interpretation has been obtained following experiments in which RNA or protein synthesis has been inhibited. Under these conditions, glucan formation takes place, and wall thickening is probably due to the accumulation of this polysaccharide. Furthermore, once a pattern of wall deposition has been established, it is not modified by inhibition of RNA or protein synthesis.     

Aspects of glucose uptake in Saccharomyces cerevisiae.

A wild-type Saccharomyces cerevisiae strain showed simple saturation kinetics for glucose uptake, with a Km of 4 mM when cells were obtained from exponential growth on glucose, and a similar, single Km of 2 to 8 mM was found under a variety of other growth conditions. Later in growth on glucose, and during ethanol utilization, a second kinetic component was observed, which might reflect either artifacts of membrane alteration or a Km in the molar range.