Phylogenetic analyses of the homologous transmembrane channel-forming proteins of the F0F1-ATPases of bacteria, chloroplasts and mitochondria

101

F 1 F 0 ATP synthases catalyze the formation of ATP utilizing the energy of a transmembrane H ϩ electrochemical gradient, generated by electron transport complexes and other ion pumping systems. Closely related ATP synthases are found in the plasma membrane of eubacteria, the inner membrane of mitochondria, and the thylakoid membrane of chloroplasts. The enzyme is a multisubunit complex with distinct extramembranous and transmembrane domains, termed F 1 and F 0 , respectively. Ion movement through F 0 is coupled to ATP synthesis/ hydrolysis at sites in F 1 (1, 2). The simplest F 1 sectors, as found in Escherichia coli, consist of five subunits in an ␣ 3 ␤ 3 ␥␦⑀ stoichiometry. Homologous subunits are found in mitochondria and chloroplasts. A high resolution structure of a substantial part of bovine F 1 shows the three ␣-and three ␤-subunits to alternate around a central core through which subunit ␥ extends and protrudes (3). The structure fits well with the binding change mechanism proposed by Boyer and co-workers (4), where each of the three ␤-subunits alternates between the loose binding of ADP plus P i , tight ADP plus P i binding and ATP synthesis, and ATP release during catalytic turnover.

Section: Discussionsupporting

confidence: 65%

Section: Effect Of Single Cys Substitutions On Function-singlementioning

confidence: 87%

See 1 more Smart Citation

Arrangement of the Multicopy H+-translocating Subunit c in the Membrane Sector of the Escherichia coliF1F0 ATP Synthase

Jones

Jiang

Fillingame

1998

101

“…Hydrophobic residues are conserved at positions corresponding to E. coli residues 153 and 156 in the b subunit of many organisms (51). It is tempting to suggest that the importance of these hydrophobic residues in the b-␦ interaction may provide the explanation for the observation that low ionic strength disrupts the binding of F 1 to F 0 .…”

Section: Discussionmentioning

confidence: 99%

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

McLachlin¹,

Bestard

Dunn

1998

An affinity resin for the F 1 sector of the Escherichia coli ATP synthase was prepared by coupling the b subunit to a solid support through a unique cysteine residue in the N-terminal leader. b 24 -156 , a form of b lacking the N-terminal transmembrane domain, was able to compete with the affinity resin for binding of F 1 . Truncated forms of b 24 -156 , in which one or four residues from the C terminus were removed, competed poorly for F 1 binding, suggesting that these residues play an important role in b-F 1 interactions. Sedimentation velocity analytical ultracentrifugation revealed that removal of these C-terminal residues from b 24 -156 resulted in a disruption of its association with the purified ␦ subunit of the enzyme. To determine whether these residues interact directly with ␦, cysteine residues were introduced at various C-terminal positions of b and modified with the heterobifunctional cross-linker benzophenone-4-maleimide. Cross-links between b and ␦ were obtained when the reagent was incorporated at positions 155 and 158 (two residues beyond the normal C terminus) in both the reconstituted b 24 -156 -F 1 complex and the membranebound F 1 F 0 complex. CNBr digestion followed by peptide sequencing showed the site of cross-linking within the 177-residue ␦ subunit to be C-terminal to residue 148, possibly at Met-158. These results indicate that the b and ␦ subunits interact via their C-terminal regions and that this interaction is instrumental in the binding of the F 1 sector to the b subunit of F 0 .In the process of oxidative phosphorylation or photophosphorylation, the electron transport chain generates a transmembrane proton gradient. The ATP synthase, or F 1 F 0 -ATPase, allows protons to flow down this electrochemical gradient and uses the energy obtained to synthesize ATP (for reviews, see Refs. 1-4). Under appropriate conditions ATP synthase can hydrolyze ATP to pump protons. The enzyme is composed of two sectors; the F 0 sector is membrane-integral and is responsible for proton translocation, and the F 1 sector is attached to the membrane via F 0 and houses the catalytic sites for ATP synthesis. F 1 is easily detached from the membrane and can be purified as a soluble protein with ATPase activity.ATP synthases contain at least eight types of subunits. In the relatively simple enzyme from Escherichia coli, the F 1 sector has the stoichiometry ␣ 3 ␤ 3 ␥ 1 ␦ 1 ⑀ 1 , whereas F 0 is composed of three subunits of stoichiometry a 1 b 2 c 9 -12 . The a and c subunits, but not b, contain residues essential for the translocation of protons across the membrane (3). The 156-residue b subunit is believed to span the membrane once at its hydrophobic N terminus, whereas the remainder of the protein is very hydrophilic. b is thought to exist as a dimer in the complex (5-7), and proteolysis studies have shown that the hydrophilic region of b is required for the association of F 1 with the membrane (7-9). Removal of two residues from the C terminus of b disrupts normal assembly of the complex (10), as does mut...

“…Cross-linking of R36C in F 1 F 0 -Arg-36 is conserved in b subunits of ATP synthase from many species (43,44), and certain mutations at this position in E. coli ATP synthase cause functional defects in the enzyme (44). Because a transmembrane domain exists at the N-terminal end of b, the region of the subunit around residue 36 may be close to the surface of the membrane and may make important contacts with subunits of F 0 .…”

Section: Figmentioning

confidence: 99%

Site-directed Cross-linking of b to the α, β, anda Subunits of the Escherichia coli ATP Synthase

McLachlin¹,

Coveny

Clark

et al. 2000

The b subunit dimer of the Escherichia coli ATP synthase, along with the ␦ subunit, is thought to act as a stator to hold the ␣ 3 ␤ 3 hexamer stationary relative to the a subunit as the ␥⑀c 9 -12 complex rotates. Despite their essential nature, the contacts between b and the ␣, ␤, and a subunits remain largely undefined. We have introduced cysteine residues individually at various positions within the wild type membrane-bound b subunit, or within b 24 -156 , a truncated, soluble version consisting only of the hydrophilic C-terminal domain. The introduced cysteine residues were modified with a photoactivatable cross-linking agent, and cross-linking to subunits of the F 1 sector or to complete ATP synthase, or F 1 F 0 -ATPase, utilizes a transmembrane proton gradient to synthesize ATP and is responsible for the final step in oxidative phosphorylation and photophosphorylation. The enzyme (reviewed in Refs. 1-3) is composed of two sectors. The membrane-integral F 0 sector is a proton pore, and in Escherichia coli has a subunit composition of ab 2 c 9 -12 . The membrane-peripheral F 1 sector has a subunit stoichiometry of ␣ 3 ␤ 3 ␥␦⑀. A key feature of the F 1 sector, as seen in the bovine heart mitochondrial crystal structure (4), is that the ␣ and ␤ subunits alternate in a ring around a lengthy pair of ␣-helices of ␥. Each ␤ subunit bears one catalytic nucleotide-binding site, while non-catalytic nucleotide-binding sites are found on the ␣ subunits. These nucleotide-binding sites are located close to the interfaces between ␣ and ␤ subunits, with one site near each of the six interfaces.Subunits from each sector contribute to the formation of two stalks that join F 1 and F 0 . The ␥ and ⑀ subunits form the central stalk, rotation of which is believed to be caused by translocation of protons across the membrane by the a and c subunits. This rotation is thought to cause conformational changes in the catalytic sites, driving synthesis of ATP (1). It is believed that the ␣ and ␤ subunits are prevented from rotating by a peripheral stalk consisting of ␦ and the two b subunits (5, 6) that joins the ␣ 3 ␤ 3 complex to the a subunit.In recent years the interaction of the b dimer and ␦ has been well established by a variety of evidence (7-10). The ␦ subunit appears to be located near the crown of the F 1 complex, the part of F 1 furthest from the membrane (11-15). Because b has a single membrane-spanning region at its N terminus, the remainder of the subunit must span a distance of over 100 Å to come in contact with ␦. Consistent with this proposed arrangement, the region of interaction between b and ␦ has been localized to the C terminus of b (10, 16). The hydrophilic domain of b by itself is mostly ␣-helical as measured by circular dichroism (17), and an isolated complex composed of ␦ with the hydrophilic domain of b was demonstrated by sedimentation velocity ultracentrifugation to be highly extended (9). The dimerization domain of b, encompassing residues 53-122, was also shown to be highly extended (18). Therefore the b 2...