Author:
La Thuy,Clark-Walker George Desmond,Wang Xiaowen,Wilkens Stephan,Chen Xin Jie
Abstract
ABSTRACT
F
1
-ATPase is a rotary molecular machine with a subunit stoichiometry of α
3
β
3
γ
1
δ
1
ε
1
. It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central γ rotor dictates the sequential conformational changes to the catalytic sites in the α
3
β
3
core to achieve cooperativity. However, recent studies of the thermophilic
Bacillus
PS3 F
1
-ATPase have suggested that the α
3
β
3
core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this γ-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the α and β subunits that stimulate ATP hydrolysis by the mitochondrial F
1
-ATPase in the absence of γ. We found that the F446I mutation in the α subunit and G419D mutation in the β subunit suppress cell death by the loss of mitochondrial DNA (ρ
o
) in a
Kluyveromyces lactis
mutant lacking γ.
In organello
ATPase assays showed that the mutant but not the wild-type γ-less F
1
complexes retained 21.7 to 44.6% of the native F
1
-ATPase activity. The γ-less F
1
subcomplex was assembled but was structurally and functionally labile
in vitro
. Phe446 in the α subunit and Gly419 in the β 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 α or β subunit, thereby allowing robust ATP hydrolysis and cell survival under ρ
o
conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the α
3
β
3
subcomplex.
Publisher
American Society for Microbiology
Subject
Molecular Biology,General Medicine,Microbiology
Cited by
3 articles.
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