Assembly of the ATP synthase (FOF1) of Escherichia coli

• Applicant: Dr. Gabriele Deckers-Hebestreit
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2009 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 160974601

The ATP synthase (FOF1) of Escherichia coli couples the translocation of H+ across the cytoplasmic membrane by FO to ATP synthesis/hydrolysis in F1. In the first funding period, FOF1 consisting of 22 membrane-integral as well as peripheral, hydrophilic polypeptides has been shown to be assembled in a modular way from subcomplexes. Surprisingly, during biogenesis of FOF1, F1 subunit delta is the key player in generating stable FO. Subunit delta serves as clamp between subcomplexes a-b2 and c10-alpha3-beta3-gamma-epsilon and guarantees that an open proton channel is concomitantly assembled within coupled FOF1 to maintain the low membrane proton permeability essential for viability.In the second funding period, the project focusses on three main objectives:I. Molecular interactions essential to generate a functional proton translocation unit during assembly. Particularly, the interaction between assembly intermediates ab2 and c10 triggered by subunit delta, the conformation of the two half channels in subunit a in the ab2 assembly intermediate as well as the appearance of subunit delta in the assembly process will be studied on the molecular level. To address this issue, our recently developed time-delayed in vivo assembly system, nearest neighbour analyses by intermolecular disulfide bond formation, cysteine accessibility analysis or affinity purification of partially assembled FOF1 will be combined with the use of mutants synthesizing different sets of FOF1 subunits. II. Stepwise assembly of the ATP-hydrolyzing sub-complex alpha3-beta3-gamma-(delta)-epsilon formed in the cytoplasm, whose formation is the last part missing in the assembly pathway of FOF1. Especially the formation of the alternate alpha3-beta3 hexamer from single subunits demands special attention. The experimental tools described will be adapted and amended by developing a cysteine-based in vivo cross-linking assay.III. Participation of chaperon(s) in the assembly process of FOF1. AtpI is known to be involved at least in rotor ring oligomerization of Na+-dependent ATP synthases. Therefore, the interaction of AtpI with subunit c will be studied in detail for E. coli as well as Acetobacterium woodii FOF1. Moreover, a monovalent fluorescent labeling of the A. woodii subunit c rotor ring is possible and enables the dynamic visualization of subunit c-AtpI interactions in vivo by super-resolution microscopy (FPALM). Further chaperons and, simultaneously, proteases involved in FOF1 turnover, will be identified by in vivo crosslinking as well as the BioID technique (proximity-dependent biotin identification), affinity purification and subsequent mass spectrometry, followed by a specifically tailored characterization of the interacting partners. As a result, a detailed picture for the entire assembly pathway of E. coli ATP synthase is expected.

• DFG Programme: Research Grants