A conserved putative dimerization GxxxG motif located in the unique membrane-spanning segment of the ATP synthase subunit e was altered in yeast both by insertion of an alanine residue and by replacement of glycine by leucine residues. These alterations led to the loss of subunit g and the loss of dimeric and oligomeric forms of the yeast ATP synthase. Furthermore, as in null mutants devoid of either subunit e or subunit g, mitochondria displayed anomalous morphologies with onion-like structures. By taking advantage of the presence of the endogenous cysteine 28 residue in the wild-type subunit e, disulfide bond formation between subunits e in intact mitochondria was found to increase the stability of an oligomeric structure of the ATP synthase in digitonin extracts. The data show the involvement of the dimerization motif of subunit e in the formation of supramolecular structures of mitochondrial ATP synthases and are in favour of the existence in the inner mitochondrial membrane of associations of ATP synthases whose masses are higher than those of ATP synthase dimers.

Subunits e and g of Saccharomyces cerevisiae ATP synthase are required to maintain ATP synthase dimeric forms. Mutants devoid of these subunits display anomalous mitochondrial morphologies. An expression system regulated by doxycycline was used to modulate the expression of the genes encoding the subunits e and g. A decrease in the amount of subunit e induces a decrease in the amount of subunit g, but a decrease in the amount of subunit g does not affect subunit e. The loss of subunit e or g leads to the loss of supramolecular structures of ATP synthase, which is fully reversible upon removal of doxycycline. In the absence of doxycycline, mitochondria present poorly defined cristae. In the presence of doxycycline, onion-like structures are formed after five generations. When doxycycline is removed after five generations, cristae are mainly observed. The data demonstrate that the inner structure of mitochondria depends upon the ability of ATP synthase to make supramolecular structures.F 0 F 1 -ATP synthase is a molecular rotary motor that is responsible for aerobic synthesis of ATP. It exhibits a head piece (catalytic sector), a base piece (membrane sector), and two connecting stalks. The sector F 1 containing the head piece is a water-soluble unit that retains the ability to hydrolyze ATP when in soluble form. F 0 is embedded in the membrane and is mainly composed of hydrophobic subunits forming a specific proton-conducting pathway. When the F 1 and F 0 sectors are coupled, the enzyme functions as a reversible H ϩ -transporting ATPase or ATP synthase (1-4). The two connecting stalks are made of components from F 1 and F 0 . The central stalk is a part of the rotor of the enzyme. The second stalk, which is part of the stator, connects F 1 and hydrophobic membranous components of the enzyme probably via a flexible region (5). High resolution x-ray crystallographic data have led to solving the structure of F 1 (6 -9) from different sources. Stock et al. (10) reported the 3.9-Å resolution x-ray diffraction structure of the Saccharomyces cerevisiae F 1 associated with a c 10 -ring oligomer.In Escherichia coli, F 0 is composed of only 3 subunits, whereas the mitochondrial F 0 of mammals is composed of 10 different subunits (11). The same 10 components have been identified in the S. cerevisiae enzyme (12-14). Among these additional subunits not present in bacterial and chloroplast ATP synthases, subunits e and g are not involved in ATP synthesis function but are involved in the dimerization/oligomerization of the mitochondrial ATP synthase (13, 15) because the absence of subunits e and g in the respective null mutants abolishes the ability of ATP synthase to make supramolecular structures. Subunits e and g are small hydrophobic proteins with an N in -C out orientation in the inner mitochondrial membrane (12, 16) with a unique transmembrane span probably located at the interface between two ATP synthase monomers. Subunit e can form homodimers upon oxidation via its unique cysteine residue (17), and it has been reported...

Mitochondrial diseases are severe and largely untreatable. Owing to the many essential processes carried out by mitochondria and the complex cellular systems that support these processes, these diseases are diverse, pleiotropic, and challenging to study. Much of our current understanding of mitochondrial function and dysfunction comes from studies in the baker's yeast Saccharomyces cerevisiae. Because of its good fermenting capacity, S. cerevisiae can survive mutations that inactivate oxidative phosphorylation, has the ability to tolerate the complete loss of mitochondrial DNA (a property referred to as ‘petite-positivity’), and is amenable to mitochondrial and nuclear genome manipulation. These attributes make it an excellent model system for studying and resolving the molecular basis of numerous mitochondrial diseases. Here, we review the invaluable insights this model organism has yielded about diseases caused by mitochondrial dysfunction, which ranges from primary defects in oxidative phosphorylation to metabolic disorders, as well as dysfunctions in maintaining the genome or in the dynamics of mitochondria. Owing to the high level of functional conservation between yeast and human mitochondrial genes, several yeast species have been instrumental in revealing the molecular mechanisms of pathogenic human mitochondrial gene mutations. Importantly, such insights have pointed to potential therapeutic targets, as have genetic and chemical screens using yeast.

Devastating human neuromuscular disorders have been associated to defects in the ATP synthase. This enzyme is found in the inner mitochondrial membrane and catalyzes the last step in oxidative phosphorylation, which provides aerobic eukaryotes with ATP. With the advent of structures of complete ATP synthases, and the availability of genetically approachable systems such as the yeast Saccharomyces cerevisiae, we can begin to understand these molecular machines and their associated defects at the molecular level. In this review, we describe what is known about the clinical syndromes induced by 58 different mutations found in the mitochondrial genes encoding membrane subunits 8 and a of ATP synthase, and evaluate their functional consequences with respect to recently described cryo-EM structures.

I-III)). An ADP molecule was bound in both ␤ DP and ␤ TP catalytic sites. The ␣ DP -␤ DP pair is slightly open and resembles the novel conformation identified in yF 1 , whereas the ␣ TP -␤ TP pair is very closed and resembles more a DP pair. yF 1 c 10 ⅐ADP provides a model of a new Mg⅐ADP-inhibited state of the yeast F 1 . As for the original yF 1 and yF 1 c 10 structures, the foot of the central stalk is rotated by ϳ40°with respect to bovine structures. The assembly of the F 1 central stalk with the F 0 c-ring rotor is mainly provided by electrostatic interactions. On the rotor ring, the essential cGlu 59 carboxylate group is surrounded by hydrophobic residues and is not involved in hydrogen bonding.The F 1 F 0 -ATP synthase is an essential membrane rotary motor that uses transmembrane electrochemical ion gradients to synthesize ATP. To date, only cryo-electron microscopy has provided a complete view of the yeast ATP synthase (1). The structure of the Saccharomyces cerevisiae F 1 c 10 -ATP synthase subcomplex provided the first model at 3.9-Å resolution of the molecular assembly between the membrane rotor ring and the central stalk of a F 1 F 0 -ATP synthase (2). Both adenylyl imidodiphosphate (AMP-PNP) 3 and ADP were added to the crystallization medium, a structure that will be referred to as yF 1 c 10 . It was solved by molecular replacement using the C␣ coordinates of the crystal structures of the AMP-PNP-inhibited bovine F 1 -ATPase containing (␣␤) 3 ␥-subunits, which is considered as the bovine reference structure bF 1 ⅐AMP-PNP (3), and of the Escherichia coli ⑀-subunit, the homologous of the mitochondrial ␦-subunit (4), as models. A decameric ring of c-subunits (also named subunit 9 in yeast) was afterward found in the residual electron densities and the NMR solution structure of the E. coli c-monomer (5), which shares only 18% of identity with the yeast subunit, was used to build the c 10 -ring in the maps. The smallest subunit (⑀-subunit) was indistinguishable in electron densities but was present as shown by a SDS-PAGE analysis of the crystal (2). Unfortunately and unexpectedly, the peripheral stalk of the enzyme dissociated during the crystallization process. Owing to the lack of crystals that diffracted at high enough resolution and lack of crystal structures of yeast subunits or subcomplexes, refinement of the yF 1 c 10 was impossible a few years ago. Although the model deposited in the Protein Data Bank is a mix of x-ray and NMR unrefined C␣ atom coordinates of bovine and E. coli homologous proteins, the structure was fitted successfully in the envelope of the electron microscopy map of F 1 F 0 -ATP synthase to obtain the envelope of the peripheral stalk (1, 6).The first F 1 -ATPase x-ray structure (3) supports Boyer's binding change mechanism for catalysis (7). It was proposed that (i) the empty and open catalytic site ␤ E was the open site with low affinity for nucleotides, (ii) the ␤ TP site filled by ATP (or AMP-PNP) was the loose conformation, and (iii) the ␤ DP site filled by ADP was the tig...

Horse-spleen apoferritin is known to crystallize in three different space groups, cubic F432, tetragonal P4212 and orthorhombic P2~212. A structure comparison of the cubic and tetragonal forms is presented here. Both crystal forms were obtained by the vapor-diffusion technique and data were collected at 2.26/~ (cubic crystal) and 2.60/~ (tetragonal crystal) resolution. Two main differences were observed between these crystal structures: (i) whereas intermolecular contacts only involve saltbridge type interactions via cadmium ions in the cubic structure, two types of interactions are observed in the tetragonal crystal (cadmium-ion-mediated salt bridges and hydrogen-bonding interactions) and (ii) cadmium ions bound in the threefold axes of ferritin molecules exhibit lower site-occupation factors in the tetragonal structure than in the cubic one.

Background information. The yeast mitochondrial F 1 F o -ATP synthase is a large complex of 600 kDa that uses the proton electrochemical gradient generated by the respiratory chain to catalyse ATP synthesis from ADP and P i . For a large range of organisms, it has been shown that mitochondrial ATP synthase adopts oligomeric structures. Moreover, several studies have suggested that a link exists between ATP synthase and mitochondrial morphology.Results and discussion. In order to understand the link between ATP synthase oligomerization and mitochondrial morphology, more information is needed on the supramolecular organization of this enzyme within the inner mitochondrial membrane. We have conducted an electron microscopy study on wild-type yeast mitochondria at different levels of organization from spheroplast to isolated ATP synthase complex. Using electron tomography, freeze-fracture, negative staining and image processing, we show that cristae form a network of lamellae, on which ATP synthase dimers assemble in linear and regular arrays of oligomers. Conclusions.Our results shed new light on the supramolecular organization of the F 1 F o -ATP synthase and its potential role in mitochondrial morphology.

Nucleic acid triplexes are formed by sequence-specific interactions between single-stranded polynucleotides and the double helix. These triplexes are implicated in genetic recombination in vivo and have application to areas that include genome analysis and antigene therapy. Despite the importance of the triple helix, only limited high-resolution structural information is available. The x-ray crystal structure of the oligonucleotide d(GGCCAATTGG) is described; it was designed to contain the d(G middle dotGC)2 fragment and thus provide the basic repeat unit of a DNA triple helix. Parameters derived from this crystal structure have made it possible to construct models of both parallel and antiparallel triple helices.