Specific mutations in the α and ß subunits of the Kluyveromyces lactis F1-ATPase enhance ATP hydrolysis in the absence of the central γ-rotor

Abstract

In eukaryotic cells, the mitochondria are vital organelles which are required for cell viability. Mitochondrial stresses such as oxidative stress, loss of membrane potential or loss of mitochondrial DNA are considered extreme and are associated with many neurodegenerative diseases and aging. The mitochondrial FoF1-synthase, where the majority of cellular ATP is synthesized, is composed of one inner membrane bound Fo domain and a water soluble F1 domain in the mitochondrial matrix. F1 contains the hexameric α3β3core and the centrally located γ subunit. The γ subunit is believed to play a key role in inducing conformational changes while rotating within the α3β3 core during ATP hydrolysis/synthesis. Previous studies have shown that the α3β3 core alone from the Thermophilic bacterium PS3 has a detectable hydrolyzing activity. In recent years, evidence of the rotary catalysis of Thermophilic Bacillus sp. PS3 F1-ATPase without its rotor - subunit γ - was shown using high-speed atomic force microscopy[1]. Moreover, previous study undertaken in our lab had utilized a unique genetic screen that allowed the identification of two specific mutations in the α and β subunits in the aerobic yeast Kluyveromyces lactis that stimulate ATP hydrolysis by the mitochondrial F1-ATPase in the absence of γ. This allows cells to survive upon the loss of mitochondrial DNA. In current work, we confirmed that the αF446I and βG419D mutations on the DELSEED loop are sufficient to allow ρ0 cells to survive in the absence of γ. Biochemical experiments showed that the γ -less F1-ATPase can be assembled to actively hydrolyze iv ATP in vivo, but this activity becomes extremely labile in vitro. These studies give insights into the catalytic mechanism of the α3β3 subcomplex and help to better understand the evolutionary origin of the mitochondrial F1-ATPase.