Effect of nutrient starvation on the cellular composition and metabolic capacity of Saccharomyces cerevisiae

Abstract

This investigation addresses the following question: what are the important factors for maintenance of a high catabolic capacity under various starvation conditions? Saccharomyces cerevisiae was cultured in aerobic batch cultures, and during the diauxic shift cells were transferred and subjected to 24 h of starvation. The following conditions were used: carbon starvation, nitrogen starvation in the presence of glucose or ethanol, and both carbon starvation and nitrogen starvation. During the starvation period changes in biomass composition (including protein, carbohydrate, lipid, and nucleic acid contents), metabolic activity, sugar transport kinetics, and the levels of selected enzymes were recorded. Subsequent to the starvation period the remaining catabolic capacity was measured by addition of 50 mM glucose. The results showed that the glucose transport capacity is a key factor for maintenance of high metabolic capacity in many, but not all, cases. The results for cells starved of carbon, carbon and nitrogen, or nitrogen in the presence of glucose all indicated that the metabolic capacity was indeed controlled by the glucose transport ability, perhaps with some influence of hexokinase, phosphofructokinase, aldolase, and enolase levels. However, it was also demonstrated that there was no such correlation when nitrogen starvation occurred in the presence of ethanol instead of glucose.

FIG. 1.

Activities of selected enzymes. The influence of starvation on the activities is expressed as ratios of activity after 24 h of starvation to activity in nonstarved cells. Light gray columns, carbon starvation; black columns, dual carbon and nitrogen starvation; open columns, nitrogen starvation in the presence of glucose; dark gray columns, nitrogen starvation in the presence of ethanol. The data are averages for two to four independently grown cultures, and the mean standard deviations, estimated using data for all cell types, are indicated by error bars. Abbreviations: G6Pdh, glucose-6-phosphate dehydrogenase; Glt, glucose transport; Hk, hexokinase; Pgi, phosphoglucose isomerase; Pfk, phosphofructokinase; Aldo, aldolase; Tpi, triose phosphate isomerase; G3Pdh, glycerol-3-phosphate dehydrogenase; Gpp, glycerol-3-phosphate phosphatase; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; Pgk, phosphoglycerate kinase; Pgm, phosphoglycerate mutase; Eno, enolase; Pyk, pyruvate kinase; Pdc, pyruvate decarboxylase; Adh, alcohol dehydrogenase; NAD-Ald, NAD-acetaldehyde dehydrogenase; NADP-Ald, NADP-acetaldehyde dehydrogenase; PEPck, phosphoenolpyruvate carboxykinase; NM, not measured. The effects of the various starvation conditions were statistically evaluated by using a two-tailed Student t test and comparison with nonstarved cells. Significant differences at P values of <0.01 (three asterisks), <0.02 (two asterisks), and <0.05 (one asterisk) are indicated. The degrees of freedom ranged from 4 to 22.

FIG. 2.

Comparison between zero trans-influx rates (open columns) and consumption rates (black columns) at an extracellular sugar concentration of 50 mM in cells from the diauxic shift (No) and after subsequent starvation (C, carbon starvation; NC, dual nitrogen and carbon starvation; Ng, nitrogen starvation with glucose; Ne, nitrogen starvation with ethanol). The sugars used were glucose (G), fructose (F), and mannose (M). Glucose consumption rates were obtained from Table 3, the zero trans-influx rates were measured by determining [¹⁴C]glucose uptake, and the protein content was measured by the method of Lowry et al. (24). The standard deviations are indicated by error bars. The differences between sugar consumption rates and uptake capacities were statistically evaluated by using a two-tailed Student t test. Significantly lower consumption rates compared to the uptake capacity are indicated by three asterisks (P < 0.01) and two asterisks (P < 0.02). The degrees of freedom were always more than 30.

FIG. 3.

Plot of influx of glucose (J_glc) versus the maximal initial uptake rate (V_max) for the diauxic shift (No) and for starved cells (C, carbon starvation; NC, dual nitrogen and carbon starvation; Ng, nitrogen starvation with glucose; Ne, nitrogen starvation with ethanol). The linear relationship for carbon-starved cells, nitrogen- and carbon-starved cells, and nitrogen-starved cells in the presence of glucose is indicated, with a slope of 0.83 (R² = 0.999).