1. The fatty acid composition of the ole-1 and ole-1 petite mutants of Saccharomyces cerevisiae was manipulated by growing the organism in the presence of defined supplements of Tween 80 or by allowing cells that had first been grown in the presence of Tween 80 to deplete their unsaturated fatty acids by sequent growth in the absence of Tween 80. 2. The transition temperature of Arrhenius plots of mitochondrial ATPase (adenosine triphosphatase) increases as the unsaturated fatty acid content is lowered. 3. Cells require larger amounts of unsaturated fatty acids to grow on ethanol at lower temperatures. 4. Cells that stop growing owing to unsaturated fatty acid depletion at low temperatures are induced to grow further by raising the temperature and this results in a further depletion of unsaturated acids. This is due to a higher rate, but not a greater efficiency, of mitochondrial ATP synthesis. 5. Arrhenius plots of the passive permeability of mitochondria to protons between 4 and 37°C are linear. The rate and the Arrhenius activation energy of proton entry increase greatly as the unsaturated fatty acid content is lowered. 6. Unsaturated fatty acid depletion has the same effects on the proton permeability of ole-1 petite mitochondria, indicating that the mitochondrially synthesized subunits of the ATPase are not involved in the enhanced rates of proton entry. 7. The adenylate energy charge of depleted ole-1 cells is greatly decreased by growth on ethanol medium. 8. The adenylate energy charge of isolated mitochondria is also lowered by unsaturated fatty acid depletion. 9. The results confirm that unsaturated fatty acid depletion uncouples oxidative phosphorylation in yeast both in vivo and in vitro, and is a consequence of changes in the lipid part of the membrane.

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