We have shown in previous research that the loss of phosphatidylglycerol and cardiolipin caused by disruption of the PGS1 gene is lethal for the petite-negative yeast Kluyveromyces lactis . This present study demonstrates the role and mechanism of atp2.1 in the suppression of pgs1 lethality in K. lactis cells. Phenotypic characterization has shown that a strain lacking the phosphatidylglycerolphosphate synthase (atp2.1pgs1Δ) possessed a markedly impaired respiratory chain, very low endogenous respiration, and uncoupled mitochondria. As a result the mutant strain was unable to generate a sufficient mitochondrial membrane potential via respiration. The atp2.1 suppressor mutation enabled an increase in the affinity of F(1)-ATPase for ATP in the hydrolytic reaction, resulting in the maintenance of sufficient membrane potential for the biogenesis of mitochondria and survival of cells lacking anionic phospholipid biosynthesis.