Role of the delta subunit in enhancing proton conduction through the F0 of the Escherichia coli F1F0 ATPase

Abstract

We studied the effect of the 8 subunit of the Escherichia coli F1 ATPase on the proton permeability of the Fo proton channel synthesized and assembled in vivo. Membranes isolated from an unc deletion strain carrying a plasmid containing the genes for the Fo subunits and the 8 subunit were significantly more permeable to protons than membranes isolated from the same strain carrying a plasmid containing the genes for the Fo subunits alone. This increased proton permeability could be blocked by treatment with either dicyclohexylcarbodiimide or purified Fl, both of which block proton conduction through the Fo. After reconstitution with purified F1 in vitro, both membrane preparations could couple proton pumping to ATP hydrolysis. These results demonstrate that an interaction between the 8 subunit and the Fo during synthesis and assembly produces a significant change in the proton permeability of the Fo proton channel. We have proposed a model for Fo assembly in which the Fo is synthesized and assembled in a relatively proton-impermeable form (3,15). Instead of the F1 subunits catalyzing membrane insertion of certain Fo subunits, the Fo subunits are inserted spontaneously and assemble in an immature form. It is through interactions with F1 subunits that this immature "closed" channel is opened, and the flow of protons through the complex is then regulated by the activity of the F1 sector. In this study, we examined the role of the 8 subunit of the F1 sector during assembly of the Fo sector. The 8 subunit is coded for by the uncH gene, which is the first F1 gene in the operon, located immediately following the Fo genes. Previous genetic studies implicated the 8 subunit as causing the F1-dependent proton permeability of the Fo (1 MATERIALS AND METHODSStrains and plasmids. These studies used E. coli JM103 A(uncB-uncD), which is strain JM103 (11) with a deletion of seven of the nine unc genes, including all the Fo genes (10).Plasmids pEA5, pWSB30.0, and pWSB33 were described previously (13, 22). pRM1, which carries the Fo genes plus uncH (S subunit) cloned behind the lac promoter, was constructed by digesting pWSB30.0 with HindIll and EcoRI and ligating the resulting fragments with the pUC9 (23) vector, which had been digested with HindIIl and EcoRI. pRM1 was digested with AflII, which cuts at the stop codon for uncH, treated with mung bean nuclease to form blunt ends, and then ligated with a Sall adapter (5'-TGGTGTCGACACCA-3') to produce plasmid pRM6. pRM7, which carries the Fo genes and all of uncH fused in frame to a biotinylation sequence, was constructed by ligating the SalI-EcoRI fragment from the biotinylation vector YEp352-Bio7 into pRM6, which had been digested with the same enzymes. YEp352-Bio7 was a generous gift from A. Tzagoloff. Treatment with mung bean nuclease and insertion of the Sall adapter removed the stop codon for uncH and allowed the biotin attachment sequence to be cloned in frame. This region of the resultant plasmid was sequenced to verify the construction. pRM8, which encodes the uncH-biotin a...

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

“…Furthermore, purification of F O subunits and reconstitution into liposomes revealed the same characteristics (51,52). In contrast, after synthesis of F O subunits in the absence of F 1 , only low levels of proton permeability were observed in membranes as well as after reconstitution into liposomes, implying a dependence on F 1 for the proton translocation by F O (53,54), although the additional presence of subunit ␦ enhances the proton conduction through F O (55,56).…”

“…However, whether subunit ␦ simply increases the affinity of ab 2 for the rest of the complex or whether it changes the conformation of F O to an active one, as suggested previously (55,56), remains to be solved in further investigations.…”

The b and δ Subunits of the Escherichia coli ATP Synthase Interact via Residues in their C-terminal Regions

“…In subsequent work, Brusilow and co-workers (53,54) demonstrated that the ␦ subunit was required for the formation of the proton pore from the cloned F 0 subunits. Here we have shown that the same region of b implicated in the F 0 assembly process is essential for binding to ␦, strengthening the argument that the b-␦ interaction is critical for the assembly of functional F 0 .…”

Assembly of the Escherichia coli FoF1 ATP synthase involves distinct subcomplex formation

“…Furthermore, purification of F o subunits from assembled F o F 1 and their reconstitution into liposomes revealed the same characteristics [5,56]. In contrast, after synthesis of F o subunits in the absence of F 1 , only low levels of proton permeability were observed in membranes as well as after reconstitution of purified subunits into liposomes, implying a dependence on F 1 for the proton translocation by F o [15,16], although the additional presence of subunit δ slightly enhances the proton conduction through F o [57,58]. The interdependence between F o and F 1 during assembly has been proposed to ensure that an open proton channel does not exist as an intermediate [59,16].…”