title: FRET reveals changes in the F1-stator stalk interaction during activity of F1F0-ATP synthase.
abstract: A stator is proposed as necessary to prevent futile rotation of the F(1) catalytic sector of mitochondrial ATP synthase (mtATPase) during periods of ATP synthesis or ATP hydrolysis. Although the second stalk of mtATPase is generally believed to fulfil the role of a stator capable of withstanding the stress produced by rotation of the central rotor, there is little evidence to directly support this view. We show that interaction between two candidate proteins of the second stalk, OSCP and subunit b, fused at their C-termini to GFP variants and assembled into functional mtATPase can be monitored in mitochondria using fluorescence resonance energy transfer (FRET). Substitution of native OSCP with a variant containing a glycine 166 to asparagine (G166N) substitution yielded a metastable complex. In contrast to the enzyme containing native OSCP, FRET could be irreversibly lowered for the enzyme containing G166N at a rate that correlated closely with the rate of enzyme activity (ATP hydrolysis). The non-hydrolysable ATP analogue, AMP-PCP did not have this effect. We conclude that two candidate proteins of the stator stalk, OSCP and b, are subject to stresses during enzyme catalytic activity commensurate with their role as a part of a stator stalk.
authors.affiliation: Department of Biochemistry and Molecular Biology, ARC Center for Structural and Functional Genomics, Monash University, Clayton Campus, Victoria 3800, Australia.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Gavin
authors.forename: Rodney J
authors.initials: RJ
authors.lastname: Devenish
authors.forename: Mark
authors.initials: M
authors.lastname: Prescott
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D017930:Genes, Reporter; D025261:Mitochondrial Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010710:Phosphates / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence
chemical_list: D010710:Phosphates; C474451:F1F0-ATP synthase; D025261:Mitochondrial Proton-Translocating ATPases