Abstract 1397: ATP Synthase Activity can be Modulated by Phosphorylation of Selected Residues of the β Subunit: Implications to Preconditioning.
Introduction: Recently, we discovered that the β subunit of the mitochondrial ATP synthase undergoes modification upon treatment of myocytes with the preconditioning agent, adenosine and identified 5 novel phosphorylation sites on the protein. Two sites are buried within the complex and the others are located on the external face. The functional consequences of phosphorylation via ATPase rate modulation and assembly effects are assessed.
Methods: A model system, S. cerevisiae, was chosen for high sequence homology between yeast and mammalian β subunits and for ease of cloning and mutation protocols. Strains with non-phosphorylatable (S/T to A) and pseudophosphorylated (T to E or S to D) analogs of 4 sites were created, T91, S246, T295 and T351. Strains were compared to WT and a deletion strain for growth, ATPase activity of mitochondria (measuring release of Pi from ATP), and complex assembly (Blue Native (BN)-PAGE) of mitochondria).
Results: Growth assays under conditions of obligate mitochondria function, showed that the internal residue strain T295E, grew slowly compared to WT at 30C had no growth at 16C, where other mutants had WT growth. ATPase assays on T295E strain showed a significant reduction in activity compared to WT (0.48 ± 0.10 and 1.69 ± 0.25 respectively, p<0.0001) and was equivalent to a deletion strain (0.43 ± 0.04). The other internal strain mutation T351E, had significantly increased function (2.53 ± 0.31, p<0.0001). One external site strain T91A had decreased function (1.39 ± 0.25, p<0.005). BN-PAGE gels revealed a complex assembly defect in the T295E mutants that lack the free F1 component, normally found in abundance. Other strains had little change in assembly. Activity changes of phosphorylation mutants T91 and T351 are likely functionally regulatory, as they have little effect on assembly.
Conclusions: Mutations of ATP synthase β subunit showed pseudophosphorylation of T351 increases ATPase activity, a non-phosphorylatable T91A decreases activity and T295 pseudophosphorylation impedes assembly resulting in a “dead” protein complex. In conclusion, this data suggests the novel proposal that ATP synthase can be modulated by phosphorylation and has implications to preconditioning where the phosphorylations were first identified.