title: Mutations on the N-terminal edge of the DELSEED loop in either the α or β subunit of the mitochondrial F1-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 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York, USA.
authors.forename: Thuy
authors.initials: T
authors.lastname: La
authors.forename: George Desmond
authors.initials: GD
authors.lastname: Clark-Walker
authors.forename: Xiaowen
authors.initials: X
authors.lastname: Wang
authors.forename: Stephan
authors.initials: S
authors.lastname: Wilkens
authors.forename: Xin Jie
authors.initials: XJ
authors.lastname: Chen
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007716:Kluyveromyces / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D025261:Mitochondrial Proton-Translocating ATPases