Evolution of the Inhibitory and Non-Inhibitory ε, ζ, and IF 1 Subunits of the F 1 F O -ATPase as Related to the Endosymbiotic Origin of Mitochondria.

The F 1 F O -ATP synthase nanomotor synthesizes >90% of the cellular ATP of almost all living beings by rotating in the "forward" direction, but it can also consume the same ATP pools by rotating in "reverse." To prevent futile F 1 F O -ATPase activity, several different inhibitory proteins or domains in bacteria (ε and ζ subunits), mitochondria (IF 1 ), and chloroplasts (ε and γ disulfide) emerged to block the F 1 F O -ATPase activity selectively. In this study, we analyze how these F 1 F O -ATPase inhibitory proteins have evolved. The phylogeny of the α-proteobacterial ε showed that it diverged in its C-terminal side, thus losing both the inhibitory function and the ATP-binding/sensor motif that controls this inhibition. The losses of inhibitory function and the ATP-binding site correlate with an evolutionary divergence of non-inhibitory α-proteobacterial ε and mitochondrial δ subunits from inhibitory bacterial and chloroplastidic ε subunits. Here, we confirm the lack of inhibitory function of wild-type and C-terminal truncated ε subunits of P. denitrificans . Taken together, the data show that ζ evolved to replace ε as the primary inhibitor of the F 1 F O -ATPase of free-living α-proteobacteria. However, the ζ inhibitory function was also partially lost in some symbiotic α-proteobacteria and totally lost in some strictly parasitic α-proteobacteria such as the Rickettsiales order. Finally, we found that ζ and IF 1 likely evolved independently via convergent evolution before and after the endosymbiotic origin mitochondria, respectively. This led us to propose the ε and ζ subunits as tracer genes of the pre-endosymbiont that evolved into the actual mitochondria.