Mutations on the N-Terminal Edge of the DELSEED Loop in either the α or β Subunit of the Mitochondrial F ₁ -ATPase Enhance ATP Hydrolysis in the Absence of the Central γ Rotor

Abstract

F 1 -ATPase is a rotary molecular machine with a subunit stoichiometry of ␣ 3 ␤ 3 ␥ 1 ␦ 1 1 . It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central ␥ rotor dictates the sequential conformational changes to the catalytic sites in the ␣ 3 ␤ 3 core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F 1 -ATPase have suggested that the ␣ 3 ␤ 3 core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this ␥-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the ␣ and ␤ subunits that stimulate ATP hydrolysis by the mitochondrial F 1 -ATPase in the absence of ␥. We found that the F446I mutation in the ␣ subunit and G419D mutation in the ␤ subunit suppress cell death by the loss of mitochondrial DNA ( o ) in a Kluyveromyces lactis mutant lacking ␥. In organello ATPase assays showed that the mutant but not the wild-type ␥-less F 1 complexes retained 21.7 to 44.6% of the native F 1 -ATPase activity. The ␥-less F 1 subcomplex was assembled but was structurally and functionally labile in vitro. Phe446 in the ␣ subunit and Gly419 in the ␤ subunit are located on the N-terminal edge of the DELSEED loops in both subunits. Mutations in these two sites likely enhance the transmission of catalytically required conformational changes to an adjacent ␣ or ␤ subunit, thereby allowing robust ATP hydrolysis and cell survival under o conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the ␣ 3 ␤ 3 subcomplex.

Possible Involvement of F1F0-ATP synthase and Intracellular ATP in Keratinocyte Differentiation in normal skin and skin lesions

“…F1F0-ATP synthase is composed of 16–18 subunits, including α, β, γ, δ, ε and a, b, c, that respectively constitute two major sectors, the F1 and F0 11 . The β subunit (ATP5B) contains the ATP and ADP binding site (the catalytic site), which makes it the active center of the enzyme 12 13 .…”