Time-Delayed In Vivo Assembly of Subunit a into Preformed Escherichia coli FoF1 ATP Synthase

Escherichia coli FOF1 ATP synthase, a rotary nanomachine, is composed of eight different subunits in alpha(3)beta(3)gamma delta epsilon ab(2)c(10) stoichiometry. Whereas FOF1 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 FOF1 lacking subunit a (FOF1-a) which is known to form stable subcomplexes in vitro. Two expression systems (araBADp and T7p-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 FOF1-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 T7p-laco control was subsequently induced in trans by addition of isopropyl-beta-D-thiogalactopyranoside. Formation of fully assembled, and functional, FOF1 complexes was verified. This demonstrates that all subunits of FOF1-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.