Growth of sod1Δ and wild yeast in glucose or raffinose. WT (EG103) (filled symbols) and sod1Δ (EG118) (empty symbols) were grown in SC medium with 2% glucose (circles and solid lines) or raffinose (triangles and broken lines) as the carbon source. At the indicated times the turbidity of the culture was measured as OD₆₀₀ as an indication of growth. Data represent an average of 3 independent colonies, errors were 5% or less, and, for clarity, are not shown. This experiment was repeated at least five times, with similar results.

sod1Δ cells do not accumulate glycogen. WT and sod1Δ cells (EG103 and EG118) were grown in SDC medium. At the indicated times, cells were harvested, digested, and their glycogen content was determined as described in Materials and Methods. Data represent an average of 3 independent colonies and the experiment was repeated at least 3 times.

Oxygen consumption is elevated in sod1Δ as compared to WT cultures grown in glucose or raffinose as the carbon source. A. WT (EG103, closed triangles) and isogenic sod1Δ yeast (EG118, open squares) were grown in SDC medium until they reached an OD₆₀₀ of 1.0 and diluted 10-fold into fresh SDC medium (time zero). At the indicated times, oxygen consumption was measured as described in Materials and Methods. Values represent the average of 3 separate colonies with error bars indicating the standard deviation. B. WT (EG103, black bars) and isogenic sod1Δ yeast (EG118, gray bars) were seeded at OD₆₀₀ of 0.1 in SC medium containing 2% raffinose as the carbon source and grown under standard (high aeration) conditions. When cultures reached an OD₆₀₀ of 0.5, oxygen consumption was measured. Values represent the average of 3 separate colonies with error bars indicating the standard deviation. Experiments were repeated at least five different times with similar results.

sod1Δ yeast show decreased glucose repression. A. sod1Δ yeast show increased expression from the CYC1 promoter when grown on glucose and this expression is Hap2,3,4,5 driven. WT (EG103) and sod1Δ (EG314) strains containing the reporter construct pLG669Z, which has the full CYC1 promoter fused to the lacZ gene (CYC1-whole), or the construct pLG669Z-ΔAX, containing the CYC1 promoter with a mutated Hap2,3,4,5 binding site (CYC1-ΔUAS2) were grown to OD₆₀₀ of 0.5 to 1.0 (exponential phase), and the mean beta-galactosidase activities were determined. It is apparent that there is higher CYC1 induction in sod1Δ yeast compared to WT, and that this effect is greatly reduced if the Hap2,3,4,5 binding site is absent. Graphs represent the results of 3 colonies per strain, done in duplicate assays.

Glucose consumption and ethanol production by WT and sod1Δ yeast. WT (EG103) and sod1Δ (EG118) cells were grown in SC media at pH 6.0 containing 2% glucose. Every 2 to 4 hours, a small aliquot of the culture was removed and the A. glucose content or B. ethanol content of the medium was measured as described in Materials and Methods. Because the sod1Δ cells grow more slowly than WT, the data are plotted versus culture density rather than time. Experiments were repeated at least twice with similar results. A representative one is shown. Closed squares, WT; open circles, sod1Δ.

sod1Δ cells are defective in executing the switch from glucose-based to ethanol-based growth. A. sod1Δ cells grown in glucose are unable to switch to growth in ethanol after 24 hours, although they can switch if grown in raffinose. WT and sod1Δ cells were grown in either SDC (bars labeled “from glucose”) or SRC (bars labeled “from raffinose”) for 24 h and were then inoculated into either SDC, SRC, or SEC medium and grown for an additional 72 h before their optical density was measured. B. sod1Δ cultures or WT cultures treated with paraquat are able to switch from growth in glucose to growth in ethanol at earlier stages of growth but not at later stages. WT with or without 1 mM paraquat and sod1Δ cells were grown in SDC medium for the indicated amount of time, and then inoculated into SC medium containing either 2 % glucose or 2% ethanol. After 72 h the OD₆₀₀ was measured. Data represent the average of 3 independent colonies and each experiment was repeated at least twice.

Relative levels of mitochondrial proteins and DNA indicate a higher mitochondrial mass in sod1Δ yeast. A. Expression of mitochondrial proteins relative to cytosolic ones is increased in sod1Δ. WT (EG103) and sod1Δ (EG118) cells were grown to an OD₆₀₀ of 0.5 in SDC medium and lysates were prepared as described in “Materials and Methods”. Immunoblotting was performed on equal amounts of lysate protein using antibodies that recognize porin, actin, and cytochrome oxidase (COX1). This experiment was repeated at least three different times with similar results. A representative experiment is shown in which it can be seen that there are increased amounts of mitochondrial proteins porin and COX1 in the sod1Δ yeast relative to WT, while the amount of the cytosolic protein, actin, is slightly decreased. The break indicates different exposures of the same gel. B. The ratio of mitochondrial DNA to nuclear DNA is increased in sod1Δ compared to WT yeast. WT EG103 and isogenic sod1Δ yeast were seeded at OD₆₀₀ of 0.1 in SDC medium and grown to an OD₆₀₀ of 0.5 (early exponential phase). Cells were harvested and whole cell DNA was purified as described in Materials and Methods. Real time - PCR on segments of the mitochondrial genes COX3 (M1) and ATP9 (M2) and nuclear genes FRE1 (C1) and VMA1 (C2) was used to quantitate each gene by fluorescence measurements using SYBR Green as the fluorescent reporter. The data are reported as the ratio of each mitochondrial DNA segment to each nuclear DNA segment. This experiment was repeated three times and a representative trial is shown. The inset represents the sod1Δ:WT ratio, showing that sod1Δ cells have a 2 to 4 fold higher ratio of mitochondrial to nuclear DNA.