Assembly of the ATP synthase (FOF1) of Escherichia coli
Final Report Abstract
FOF1 ATP synthase, a rotary nanomachine, catalyzes the formation of ATP from ADP and inorganic phosphate by utilizing the energy stored in the ion motive force of the cytoplasmic membrane. To study its assembly, which comprises in Escherichia coli 22 polypeptides, is of particular interest, (i) due to the presence of membrane-integral as well as peripherally associated, soluble subunits, (ii) due to the different binding affinities between subunits necessary to enable rotation of the rotor subunits c10γε towards the stator subunits ab2α3β3δ, and (iii) due to the necessity to avoid assembly intermediates that may be lethal for the cell. Based on the pathway defined for the assembly of FOF1 from its subcomplexes, we now elucidate in detail the assembly of the preassembled F1 subcomplex (pre-F1) in the cytosol. Interactions between subunits α, β, and γ in pre-F1 are comparable to those in FOF1. For assembly of this minimal ATPase activity-exhibiting unit, we demonstrate that a stable α3β3 hexamer can be formed in the absence of subunit γ. Using our time-delayed in vivo assembly system, we revealed that central stalk subunit(s) γ or γε is (are) subsequently integrated into a preformed α3β3 hexamer. Subunit ε is present in pre-F1 in its elongated conformation leading to autoinhibition of ATP hydrolysis, thereby avoiding the exhaustion of cytosolic ATP during pre-F1 formation from α3β3 hexamer and γε subcomplex. Subunit δ, working as a clamp between ab2 and c10α3β3γε, is the key player in generating the H+-translocating FO. Due to this arrangement an open H+ channel is only assembled within coupled FOF1, thereby preventing membrane H+ permeability. To fulfill this task, subunit δ, a two-domain protein interacting with b2 and α, changes its interactions with the α subunits in pre-F1 when making contact to b2 of the ab2 subcomplex to generate functional FOF1. The C- terminal region of δ essential for b2 binding is rapidly degraded in the absence of ab2, however, can be stabilized by a C-terminal His-tag fusion. Several recent Cryo-EM structures of FOF1 showed a quite unexpected localization of subunits a and b in the membrane. Therefore, the work program on the molecular interactions essential to generate a functional H+ translocation unit had to be reorganized. After biochemical verification of interaction points between a and the transmembrane helices (TMHs) of b2, we observed conformational changes within the part of the α-helical regions of hairpin TMH-4 and TMH-5 of subunit a located close to the border of the periplasmic side of the membrane. In the ab2 subcomplex, that is in the absence of c10, this region shows stronger interactions with residues located in TMH-2 and TMH-3, while it is in close contact to the c ring in FOF1. The results demonstrate that this region undergoes a conformational shift, when the contact between preassembled ab2 and c10α3β3γε is formed via δ to generate a functional FOF1 complex and thereby the H+ translocation pathway. The topology of AtpI, a possible chaperone in subunit c oligomerization, was determined to contain four TMHs with a hydrophobic core of 14-18 amino acid residues. However, verification of a four-helix bundle within AtpI or of its interaction with subunit c monomer/oligomer failed. Whereas studies with the bacterial holotranslocon (in cooperation with I. Collinson and C. Schaffitzel, Bristol University, UK) revealed that the holotranslocon or the SecYEG translocon are sufficient to obtain a fully assembled E. coli c10 ring. Nevertheless, for the Na+-FOF1 ATP synthase of Acetobacterium woodii, AtpI or at least the first gene of the atp operon is essential for the assembly of functional FOF1. However, all results obtained so far for A. woodii or E. coli AtpI/atpI, raised more questions than answers. Even the possibility that the protein itself is probably not essential for the assembly process, but that the DNA region comprising atpI within the atp operon might be essential, is now under discussion.
Publications
- (2015) 3D-localization microscopy and tracking of FOF1-ATP synthases in living bacteria. Proceedings of SPIE 9331, 93310D-1
Renz, A., Renz, M., Klütsch, D., Deckers-Hebestreit, G., Börsch, M.
(See online at https://doi.org/10.1117/12.2080981) - (2016) Membrane protein insertion and assembly by the bacterial holo-translocon SecYEG- SecDF-YajC-YidC. Biochemical Journal 473, 3341-3354
Komar, J., Alvira, S., Schulze, R.J., Martin, R., Lycklama a Nijeholt, J.A., Lee, S.C., Dafforn, T.R., Deckers-Hebestreit, G., Berger, I., Schaffitzel, C., and Collinson I.
(See online at https://doi.org/10.1042/BCJ20160545) - (2016) Observing single FOF1-ATP synthase at work using an improved fluorescent protein mNeonGreen as FRET donor. Proceedings of SPIE 9714, 97140B-1
Heitkamp, T., Deckers-Hebestreit, G., Börsch, M.
(See online at https://doi.org/10.1117/12.2209123) - (2016) Refined method to study the posttranslational regulation of alternative oxidases from Arabidopsis thaliana in vitro. Physiologia Plantarum 157, 264-279
Selinski, J., Hartmann, A., Höfler, S., Deckers-Hebestreit, G., and Scheibe, R.
(See online at https://doi.org/10.1111/ppl.12418)
