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

Abstract

Escherichia coli F O F 1 ATP synthase, a rotary nanomachine, is composed of eight different subunits in a ␣ 3 ␤ 3 ␥␦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 T7p-laco) were adjusted to provide compatible, mutually independent, and sufficiently stringent induction and repression regimens. In detail, all structural atp genes except atpB (

Assembly of F1F0-ATP synthases

“…Both complexes appear to be true assembly intermediates as demonstrated by the use of a delayed expression system in which subunit δ synthesis was induced in the Δδ mutant background. In a similar approach, a-subunits synthesized after a delay could be functionally integrated into a preformed α 3 β 3 γεδb 2 c 10 complex in Δa mutants [182]. A comparable result was obtained with the thermophilic bacterium Bacillus PS3, which is able to assemble an ATP synthase complex lacking only the a-subunit.…”

“…A combination of nearest-neighbor analyses based on substituted cysteine pairs for disulfide cross-linking and His-tag affinity purification of membrane-bound subcomplexes in different mutant strain backgrounds has identified potential intermediates in the assembly process ( [12,182,183] summarized in [118]). F 0 intermediates can act as uncouplers of the pmf in bacteria [184,185], thus formation of the membranous ATP synthase subcomplex requires also tight regulation and a coordinated assembly process.…”

Subunit δ Is the Key Player for Assembly of the H+-translocating Unit of Escherichia coli FOF1 ATP Synthase

“…Although subunit a is missing and the proton pathway cannot be established (30), the ATPase activity of ⌬a is in a range comparable with wild type activities, providing evidence for a stable assembly of the remaining F O F 1 subunits. In membranes of ⌬␦ and ⌬b, the ATPase activities were reduced by a factor of 5-7, corresponding well to the amount of F O F 1 proteins present in the membrane.…”

“…The primer annealing sites are indicated in parentheses according to the numbering of plasmid pBWU13 (21), starting with 1 at the second HindIII restriction site in atpI. The calculated threshold cycle values were normalized against those of housekeeping gene rpsL (5Ј-GGTACGCAAACCACGTGCTCG-3Ј and 5Ј-CAGGTTGTGACCTTCACCACC-3Ј) (30).…”

“…After degradation of atp mRNA within 20 min, the expression of atpH and T7 gene1 (28) 9 viable cells at OD ϭ 1.0) were mixed with 2 volumes of RNAprotect bacteria reagent (Qiagen), incubated for 5 min at room temperature, harvested at 5,000 ϫ g, and stored at Ϫ20°C. RNA extraction, synthesis of cDNA, and rt-RT-PCR were performed as described (30,35) using the following primer pairs: atpEЈF (5Ј-CAGGCGCAG-GCGGAAATTG-3Ј (bp 1626 -1645) and 5Ј-CCGTAATA-AATTCAGACATCAGCCCC-3Ј (bp 1821-1796)) and atpA (5Ј-GCGAACTGATCAAGCAGCGC-3Ј (bp 2374 -2393) and 5Ј-ACCCATAACAACCGCACCTAC-3Ј (bp 2579 -2559)). The primer annealing sites are indicated in parentheses according to the numbering of plasmid pBWU13 (21), starting with 1 at the second HindIII restriction site in atpI.…”