#SUPEROXIDE D SPLITT FREE#

Extracts were prepared under argon or nitrogen to minimize enzyme inactivation, as Fe-S clusters are very sensitive to oxidation. Activities of Leu1p, Lys4p, and aconitase were measured on extracts from EG103 ( WT), EG103 + 1 m m paraquat ( PQ), EG118 ( sod1Δ), and EG110 ( sod2Δ) yeast strains. The above cultures were allowed to grow at 30 ☌, 220 rpm until mid-log phase (assays) or with timed absorbance readings as indicated.įig. Yeast for aconitase experiments were inoculated in SDC medium at an A 600 of 0.05. For Leu1p experiments, cultures were inoculated in SD-Leu at an A 600 of 0.05 for EG103L and EG118L. These cultures were then incubated at 30 ☌, 220 rpm and collected at the indicated times. This medium was used so that the effect of the absence of lysine could be measured under conditions where neither strain could grow, because removing only lysine would have allowed the wild type but not the sod1Δ mutant to grow. These cultures were allowed to grow at 30 ☌, 220 rpm until mid-log phase ( A 600 of 1–2), spun down, washed, and resuspended in SD-Leu-Lys-Trp. For the Lys4p experiments, cultures were inoculated in SDC at an A 600 of 0.025 for EG103/EG110 and an A 600 of 0.05 for EG118. For paraquat studies, yeast pre-cultures were grown in standard YPD media and inoculated at an initial A 600 of 0.05, although all other experiments used pre-cultures grown in SDC. Growth was followed by monitoring the turbidity at 600 nm ( A 600 A 600 of 1 = 1 × 10 7 cells/ml). Specific amino acids were dropped out as indicated ( e.g.

In addition, a 4-fold excess of the supplements tryptophan, leucine, uracil, adenine, methionine, and histidine was added (SDC). In the case of Lys4p, the protection by Sod1p is exerted from one compartment (the IMS) across a membrane barrier to the matrix and is required despite the presence of Mn-SOD in the matrix.

Overall, we conclude that both auxotrophies are caused by direct inactivation of the respective protein by superoxide and that Sod1p protects them by lowering superoxide levels.

#SUPEROXIDE D SPLITT FREE#

In addition, we localize Lys4p in the mitochondrial matrix and show that levels of mitochondrial total and free iron are similar in wild type and sod1Δ strains. In this study we demonstrate inactivation of the iron-sulfur cluster enzymes Lys4p and Leu1p in the sod1Δ mutant under aerobic conditions. A leucine auxotrophy has not been reported for sod1Δ yeast, but based on the above information we decided to look for and found a previously undescribed leaky leucine auxotrophy in sod1Δ yeast. We hypothesized that Lys4p is susceptible to superoxide inactivation and that this sensitivity leads to the lysine auxotrophy. Thus, we conclude that when Sod1p is absent a lysine auxotrophy is induced because Lys4p is inactivated in the matrix by superoxide that originates in the intermembrane space and diffuses across the inner membrane.Ī tight lysine auxotrophy is a classic phenotype of sod1Δ yeast.

We measured only slight changes in total mitochondrial iron and found no detectable difference in mitochondrial “free” (EPR-detectable) iron making it unlikely that a gross defect in mitochondrial iron metabolism is the cause of the decreased enzyme activities. The activity of a second matrix protein, the tricarboxylic acid cycle enzyme aconitase, was similarly lowered in sod1Δ mutants. Surprisingly, Lys4p does not share a compartment with Sod1p but is located in the mitochondrial matrix. Located in the cytosol and intermembrane space of the mitochondria, Sod1p likely provides direct protection of the cytosolic enzyme Leu1p. We present evidence that direct aerobic inactivation of these enzymes in sod1Δ yeast results in the auxotrophies. The lysine and leucine biosynthetic pathways each contain a 4Fe-4S cluster enzyme homologous to aconitase and likely to be superoxide-sensitive, homoaconitase (Lys4p) and isopropylmalate dehydratase (Leu1p), respectively.

Glycobiology and Extracellular MatricesĪmong the phenotypes of Saccharomyces cerevisiae mutants lacking CuZn-superoxide dismutase (Sod1p) is an aerobic lysine auxotrophy in the current work we show an additional leaky auxotrophy for leucine.

Superoxide d splitt