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Escherichia coli F O F 1 ATP synthase, a rotary nanomachine, is composed of eight different subunits in a α 3 β 3 γδe ab 2 c 10 stoichiometry. Whereas F O F 1 has been studied in detail with regard to its structure and function, much less is known about how this multisubunit enzyme complex is assembled. Single-subunit atp deletion mutants are known to be arrested in assembly, thus leading to formation of partially assembled subcomplexes. To determine whether those subcomplexes are preserved in a stable standby mode, a time-delayed in vivo assembly system was developed. To establish this approach, we targeted the time-delayed assembly of membrane-integrated subunit a into preformed F O F 1 lacking subunit a (F O F 1 - a ) which is known to form stable subcomplexes in vitro . Two expression systems ( araBADp and T7 p-laco ) were adjusted to provide compatible, mutually independent, and sufficiently stringent induction and repression regimens. In detail, all structural atp genes except atpB (encoding subunit a ) were expressed under the control of araBADp and induced by arabinose. Following synthesis of F O F 1 - a during growth, expression was repressed by glucose/d-fucose, and degradation of atp mRNA controlled by real-time reverse transcription-PCR. A time-delayed expression of atpB under T7 p-laco control was subsequently induced in trans by addition of isopropyl-β-d-thiogalactopyranoside. Formation of fully assembled, and functional, F O F 1 complexes was verified. This demonstrates that all subunits of F O F 1 - a remain in a stable preformed state capable to integrate subunit a as the last subunit. The results reveal that the approach presented here can be applied as a general method to study the assembly of heteromultimeric protein complexes in vivo .

The subunit c stoichiometry of Escherichia coli ATP synthase was studied by intermolecular cross-linking via oxidation of bi-cysteine-substituted subunit c ( c A21C/ c M65C). Independent of the carbon source used for growth and independent of the presence of other F o F 1 subunits, an equal pattern of cross-link formation stopping at the formation of decamers was obtained.