Structure of the mitochondrial ATP synthase from Pichia angusta determined by electron cryo-microscopy

Abstract

The structure of the intact monomeric ATP synthase from the fungus, Pichia angusta, has been solved by electron cryo-microscopy. The structure provides insights into the mechanical coupling of the transmembrane proton motive force across mitochondrial membranes in the synthesis of ATP. This mechanism requires a strong and integral stator, consisting of the catalytic α3β3-domain, peripheral stalk, and, in the membrane domain, subunit a and associated supernumerary subunits, kept in contact with the rotor turning at speeds up to 350 Hz. The stator’s integrity is ensured by robust attachment of both the oligomycin sensitivity conferral protein (OSCP) to the catalytic domain and the membrane domain of subunit b to subunit a. The ATP8 subunit provides an additional brace between the peripheral stalk and subunit a. At the junction between the OSCP and the apparently stiff, elongated α-helical b-subunit and associated d- and h-subunits, an elbow or joint allows the stator to bend to accommodate lateral movements during the activity of the catalytic domain. The stator may also apply lateral force to help keep the static a-subunit and rotating c10-ring together. The interface between the c10-ring and the a-subunit contains the transmembrane pathway for protons, and their passage across the membrane generates the turning of the rotor. The pathway has two half-channels containing conserved polar residues provided by a bundle of four α-helices inclined at ∼30° to the plane of the membrane, similar to those described in other species. The structure provides more insights into the workings of this amazing machine.

Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase

“…4) and that therefore the multiple bands on the native gel are artifactual. It is likely that the removal of the c-ring and accompanying loss of subunits ATP6 and ATP8 destabilizes the dimerization interface, which probably relies on subunits e and g and may also depend on supernumerary subunits f, DAPIT, and 6.8PL (17,18). It is also possible that the vestigial complex remains dimeric in the membrane and that it is destabilized (monomerized) artifactually by the extraction process.…”

“…Recent cryo-electron microscopy analyses of the ATP synthase complexes suggest that at least subunits e and g are involved in the interface between monomers of the ATP synthase in the dimeric complexes (17,18). The gene disruption strategy followed for the c-subunit provides a possible approach toward resolving the issue of whether any or all of these subunits are involved in forming the PTP.…”

“…Because the sequences of the bovine and human c-subunits are identical, it is reasonable to assume that the human c-ring is identical. In the intact enzyme, the external surface of this ring is in contact with a single ATP6 (or a) subunit (15)(16)(17)(18). During ATP synthesis, the c 8 -ring turns with estimated speeds of up to 300 Hz.…”

“…In the absence of a proton-motive force, the F 1 -ATPase inhibitor protein, IF 1 , prevents the hydrolysis of ATP by binding to one of the catalytic interfaces (21). The main role of the peripheral stalk, a predominantly α-helical structure made from single copies of the oligomycin sensitivity conferral protein (OSCP), subunits b and d, and factor 6 (F 6 ) (22)(23)(24) is to link the α 3 β 3 domain to subunit ATP6, which interacts with the two predicted transmembrane α-helices of the b-subunit, so that together the α 3 β 3 domain, the peripheral stalk, and subunit a form the stator of the enzyme (11,18). The membrane domain of the mammalian ATP synthase also contains six other proteins-e, f, g, DAPIT (diabetesassociated protein in insulin sensitive tissues), 6.8PL (6.8-kDa proteolipid), and ATP8 (or A6L) (25)(26)(27)(28)(29).…”

Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry

“…S4). Furthermore, mapping of a homology model of ATP8 to cryo-EMderived structures of ATP synthase in multiple rotational states (55) revealed that links between ATP8 (K46 and K48) and the rotor subunit ATPD (K136) are possible only with ATP synthase rotational state 3. In contrast, the link between ATP8 (K48) and the rotor subunit ATP5E (K50) was possible only in rotational state 1 (Fig.…”