In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits.

We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa: Trypanosoma brucei, a lethal human parasite, and Euglena gracilis, a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F1 head, in which the catalytic (αß)3 assembly forms a triangular pyramid rather than the pseudo-sixfold ring arrangement typical of all other ATP synthases investigated so far. Fitting of known X-ray structures reveals that this unusual geometry results from a phylum-specific cleavage of the α subunit, in which the C-terminal αC fragments are displaced by ∼20 Å and rotated by ∼30° from their expected positions. In this location, the αC fragment is unable to form the conserved catalytic interface that was thought to be essential for ATP synthesis, and cannot convert γ-subunit rotation into the conformational changes implicit in rotary catalysis. The new arrangement of catalytic subunits suggests that the mechanism of ATP generation by rotary ATPases is less strictly conserved than has been generally assumed. The ATP synthases of these organisms present a unique model system for discerning the individual contributions of the α and ß subunits to the fundamental process of ATP synthesis.

Helical arrays of U-shaped ATP synthase dimers form tubular cristae in ciliate mitochondria.

F1Fo-ATP synthases are universal energy-converting membrane protein complexes that synthesize ATP from ADP and inorganic phosphate. In mitochondria of yeast and mammals, the ATP synthase forms V-shaped dimers, which assemble into rows along the highly curved ridges of lamellar cristae. Using electron cryotomography and subtomogram averaging, we have determined the in situ structure and organization of the mitochondrial ATP synthase dimer of the ciliate Paramecium tetraurelia. The ATP synthase forms U-shaped dimers with parallel monomers. Each complex has a prominent intracrista domain, which links the c-ring of one monomer to the peripheral stalk of the other. Close interaction of intracrista domains in adjacent dimers results in the formation of helical ATP synthase dimer arrays, which differ from the loose dimer rows in all other organisms observed so far. The parameters of the helical arrays match those of the cristae tubes, suggesting the unique features of the P. tetraurelia ATP synthase are directly responsible for generating the helical tubular cristae. We conclude that despite major structural differences between ATP synthase dimers of ciliates and other eukaryotes, the formation of ATP synthase dimer rows is a universal feature of mitochondria and a fundamental determinant of cristae morphology.

Central role of Mic10 in the mitochondrial contact site and cristae organizing system.

The mitochondrial contact site and cristae organizing system (MICOS) is a conserved multi-subunit complex crucial for maintaining the characteristic architecture of mitochondria. Studies with deletion mutants identified Mic10 and Mic60 as core subunits of MICOS. Mic60 has been studied in detail; however, topogenesis and function of Mic10 are unknown. We report that targeting of Mic10 to the mitochondrial inner membrane requires a positively charged internal loop, but no cleavable presequence. Both transmembrane segments of Mic10 carry a characteristic four-glycine motif, which has been found in the ring-forming rotor subunit of F1Fo-ATP synthases. Overexpression of Mic10 profoundly alters the architecture of the inner membrane independently of other MICOS components. The four-glycine motifs are dispensable for interaction of Mic10 with other MICOS subunits but are crucial for the formation of large Mic10 oligomers. Our studies identify a unique role of Mic10 oligomers in promoting the formation of inner membrane crista junctions.

Visualization of ATP synthase dimers in mitochondria by electron cryo-tomography.

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.