Arrangement of subunits in intact mammalian mitochondrial ATP synthase determined by cryo-EM

Abstract

Mitochondrial ATP synthase is responsible for the synthesis of ATP, a universal energy currency in cells. Whereas X-ray crystallography has revealed the structure of the soluble region of the complex and the membrane-intrinsic c-subunits, little is known about the structure of the six other proteins (a, b, f, A6L, e, and g) that comprise the membrane-bound region of the complex in animal mitochondria. Here, we present the structure of intact bovine mitochondrial ATP synthase at ∼18 Å resolution by electron cryomicroscopy of single particles in amorphous ice. The map reveals that the a-subunit and c 8 -ring of the complex interact with a small contact area and that the b-subunit spans the membrane without contacting the c 8 -ring. The e-and g-subunits extend from the a-subunit density distal to the c 8 -ring. The map was calculated from images of a preparation of the enzyme solubilized with the detergent dodecyl maltoside, which is visible in electron cryomicroscopy maps. The structure shows that the micelle surrounding the complex is curved. The observed bend in the micelle of the detergent-solubilized complex is consistent with previous electron tomography experiments and suggests that monomers of ATP synthase are sufficient to produce curvature in lipid bilayers.A TP synthases are responsible for the synthesis of ATP from ADP and inorganic phosphate. In mammalian mitochondria, the enzyme is an ∼600-kDa membrane protein complex comprised of a catalytic F 1 region and a membrane-bound F O region. The F 1 region consists of subunits α 3 β 3 γδε (1, 2) and the F O region consists of subunits abc 8 defg(A6L)F 6 (3). The F 1 and F O regions are connected by a central stalk, comprised of the γ-, δ-, and ε-subunits from the F 1 region, and a peripheral stalk, comprised of the oligomycin sensitivity conferral protein (OSCP) and subunits b, d, and F 6 from the F O region (4, 5). Previously, electron cryomicroscopy (cryo-EM) of intact detergent-solubilized bovine ATP synthase particles embedded in a thin layer of amorphous ice revealed the overall shape of the complex at 32 Å resolution (6), and subsequent analysis of the Saccharomyces cerevisiae enzyme produced a similar map at 24 Å resolution (7). X-ray crystallography of subcomplexes of the bovine enzyme has defined the arrangement of subunits in the F 1 region (8) and the peripheral stalk (9, 10) and showed the presence of a ring of eight c-subunits in the F O region (11). The structure and arrangement of the remaining subunits in the membrane-bound region of the enzyme are not known.The ATP synthase functions by a rotary catalytic mechanism. Proton translocation through the F O region requires the a-, b-, and c-subunits (12-14) and induces rotation of the membranebound c 8 -ring (15). The structure of the c 8 -ring is thought to be stabilized by binding of cardiolipin to a lysine residue conserved throughout animalia that has been shown to be trimethylated at the ε-amino group in all animal ATP synthases tested (11,16,17). The c 8 -ring is attached to the central st...

Age-dependent dissociation of ATP synthase dimers and loss of inner-membrane cristae in mitochondria

“…It is most likely a matrix protein that interacts with the ATP synthase dimer before the dimers finally dissociate. This protein may be a factor acting on the ATP synthase subunits that are required for the formation of ATP synthase dimers (35), dimer rows (1), and regular cristae (6,7).…”

Load-dependent destabilization of the γ-rotor shaft in F O F 1 ATP synthase revealed by hydrogen/deuterium-exchange mass spectrometry

“…X-ray crystallography (5,8,10,11) and cryoelectron microscopy (12,13) have provided valuable snapshots of subcomplexes and solubilized F o F 1 . Molecular dynamics (MD) simulations (14)(15)(16) can help interpret such data in a dynamic context.…”

Escherichia coli F1Fo-ATP Synthase with a b/δ Fusion Protein Allows Analysis of the Function of the Individual b Subunits

“…The assignment of b (and not bЈ) as the helix that is more distant from the c ring (Fig. 3) is tentative; it is based on the observation that in mitochondrial ATP synthase the single b subunit, which is of the full-length b type with a C-terminal helix, appears to be on the side of the stator stalk that points away from the c ring (44). This question could be addressed in a b/c cross-linking experiment as in Ref.…”