Rotor architecture in the yeast and bovine F1-c-ring complexes of F-ATP synthase.

The F(1)F(O)-ATP synthase is a rotary molecular nanomotor. F(1) is a chemical motor driven by ATP hydrolysis while F(O) is an electrical motor driven by the proton flow. The two stepping motors are mechanically coupled through a common rotary shaft. Up to now, the three available crystal structures of the F(1)c(10) sub-complex of the yeast F(1)F(O)-ATP synthase were isomorphous and then named yF(1)c(10)(I). In this crystal form, significant interactions of the c(10)-ring with the F(1)-head of neighboring molecules affected the overall conformation of the F(1)-c-ring complex. The symmetry axis of the F(1)-head and the inertia axis of the c-ring were tilted near the interface between the F(1)-central stalk and the c-ring rotor, resulting in an unbalanced machine. We have solved a new crystal form of the F(1)c(10) complex, named yF(1)c(10)(II), inhibited by adenylyl-imidodiphosphate (AMP-PNP) and dicyclohexylcarbodiimide (DCCD), at 6.5Å resolution in which the crystal packing has a weaker influence over the conformation of the F(1)-c-ring complex. yF(1)c(10)(II) provides a model of a more efficient generator. yF(1)c(10)(II) and bovine bF(1)c(8) structures share a common rotor architecture with the inertia center of the F(1)-stator close to the rotor axis.

Supramolecular organization of the yeast F1Fo-ATP synthase.

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.

Incorporating clickers into an enzymology course improves student performance.

Here, we describe how poor exam results of undergraduate students enrolled in an enzymology course at the University of Bordeaux were improved through the introduction of 'clickers' as an audience response system. By using clickers only in a small-group tutorial element of a large theoretical course, we observed an improvement in exam scores that resulted in a lower failure rate for the course. Furthermore, students of all abilities were found to benefit from their use. Students reported better retention of both lecture and tutorial content. An analysis of how clickers were employed within the tutorials indicated that the use of clickers to promote discussion and impart knowledge likely resulted in a moderate improvement of exam scores. We hypothesize that students were more prepared for exams through greater reflection of exam questions, resulting in an enhanced ability to retrieve memorized information and apply it within a time-limited exam setting.

Is there a relationship between the supramolecular organization of the mitochondrial ATP synthase and the formation of cristae?

Blue native polyacrylamide gel electrophoresis (BN-PAGE) analyses of detergent mitochondrial extracts have provided evidence that the yeast ATP synthase could form dimers. Cross-linking experiments performed on a modified version of the i-subunit of this enzyme indicate the existence of such ATP synthase dimers in the yeast inner mitochondrial membrane. We also show that the first transmembrane segment of the eukaryotic b-subunit (bTM1), like the two supernumerary subunits e and g, is required for dimerization/oligomerization of ATP synthases. Unlike mitochondria of wild-type cells that display a well-developed cristae network, mitochondria of yeast cells devoid of subunits e, g, or bTM1 present morphological alterations with an abnormal proliferation of the inner mitochondrial membrane. From these observations, we postulate that an anomalous organization of the inner mitochondrial membrane occurs due to the absence of ATP synthase dimers/oligomers. We provide a model in which the mitochondrial ATP synthase is a key element in cristae morphogenesis.

Human F1F0 ATP synthase, mitochondrial ultrastructure and OXPHOS impairment: a (super-)complex matter?

Mitochondrial morphogenesis is a key process of cell physiology. It is essential for the proper function of this double membrane-delimited organelle, as it ensures the packing of the inner membrane in a very ordered pattern called cristae. In yeast, the mitochondrial ATP synthase is able to form dimers that can assemble into oligomers. Two subunits (e and g) are involved in this supramolecular organization. Deletion of the genes encoding these subunits has no effect on the ATP synthase monomer assembly or activity and only affects its dimerization and oligomerization. Concomitantly, the absence of subunits e and g and thus, of ATP synthase supercomplexes, promotes the modification of mitochondrial ultrastructure suggesting that ATP synthase oligomerization is involved in cristae morphogenesis. We report here that in mammalian cells in culture, the shRNA-mediated down-regulation of subunits e and g affects the stability of ATP synthase and results in a 50% decrease of the available functional enzyme. Comparable to what was shown in yeast, when subunits e and g expression are repressed, ATP synthase dimers and oligomers are less abundant when assayed by native electrophoresis. Unexpectedly, mammalian ATP synthase dimerization/oligomerization impairment has functional consequences on the respiratory chain leading to a decrease in OXPHOS activity. Finally these structural and functional alterations of the ATP synthase have a strong impact on the organelle itself leading to the fission of the mitochondrial network and the disorganization of mitochondrial ultrastructure. Unlike what was shown in yeast, the impairment of the ATP synthase oligomerization process drastically affects mitochondrial ATP production. Thus we propose that mutations or deletions of genes encoding subunits e and g may have physiopathological implications.

ATP synthase oligomerization: from the enzyme models to the mitochondrial morphology.

Mitochondrial F(1)F(o) ATP synthase is an enzymatic complex involved in the aerobic synthesis of ATP. It is well known that several enzymes are organized in supramolecular complexes in the inner mitochondrial membrane. The ATP synthase supramolecular assembly is mediated through two interfaces. One leads to dimer formation and the other to oligomer formation. In yeast, the presence of ATP synthase oligomers has been described as essential to the maintenance of the mitochondrial cristae ultrastructure. Indeed, the destabilization of the interactions between monomers was shown to alter the organization of the inner mitochondrial membrane, leading to the formation of onion-like structures similar to those observed in some mitochondrial pathologies. By using information obtained this decade (structure modeling, electron microscopy and cross-linking), this paper (i) reviews the actual state of the art and (ii) proposes a topological model of the transmembrane domains and interfaces of the ATP synthase's tetramer. This review also discusses the physiological role of this oligomerization process and its potential implications in mammal pathology. This article is part of a Directed Issue entitled: Bioenergetic Dysfunction, adaptation and therapy.

A rivet model for channel formation by aerolysin-like pore-forming toxins.

The bacterial toxin aerolysin kills cells by forming heptameric channels, of unknown structure, in the plasma membrane. Using disulfide trapping and cysteine scanning mutagenesis coupled to thiol-specific labeling on lipid bilayers, we identify a loop that lines the channel. This loop has an alternating pattern of charged and uncharged residues, suggesting that the transmembrane region has a beta-barrel configuration, as observed for Staphylococcal alpha-toxin. Surprisingly, we found that the turn of the beta-hairpin is composed of a stretch of five hydrophobic residues. We show that this hydrophobic turn drives membrane insertion of the developing channel and propose that, once the lipid bilayer has been crossed, it folds back parallel to the plane of the membrane in a rivet-like fashion. This rivet-like conformation was modeled and sequence alignments suggest that such channel riveting may operate for many other pore-forming toxins.

In the absence of the first membrane-spanning segment of subunit 4(b), the yeast ATP synthase is functional but does not dimerize or oligomerize.

The N-terminal portion of the mitochondrial b-subunit is anchored in the inner mitochondrial membrane by two hydrophobic segments. We investigated the role of the first membrane-spanning segment, which is absent in prokaryotic and chloroplastic enzymes. In the absence of the first membrane-spanning segment of the yeast subunit (subunit 4), a strong decrease in the amount of subunit g was found. The mutant ATP synthase did not dimerize or oligomerize, and mutant cells displayed anomalous mitochondrial morphologies with onion-like structures. This phenotype is similar to that of the null mutant in the ATP20 gene that encodes subunit g, a component involved in the dimerization/oligomerization of ATP synthase. Our data indicate that the first membrane-spanning segment of the mitochondrial b-subunit is not essential for the function of the enzyme since its removal did not directly alter the oxidative phosphorylation. It is proposed that the unique membrane-spanning segment of subunit g and the first membrane-spanning segment of subunit 4 interact, as shown by cross-linking experiments. We hypothesize that in eukaryotic cells the b-subunit has evolved to accommodate the interaction with the g-subunit, an associated ATP synthase component only present in the mitochondrial enzyme.

Flucytosine-fluconazole cross-resistance in purine-cytosine permease-deficient Candida lusitaniae clinical isolates: indirect evidence of a fluconazole uptake transporter.

An unusual interaction between flucytosine and fluconazole was observed when a collection of 60 Candida lusitaniae clinical isolates was screened for cross-resistance. Among eight isolates resistant to flucytosine (MIC >/= 128 micro g/ml) and susceptible to fluconazole (0.5 < MIC < 2 micro g/ml), four became flucytosine-fluconazole cross resistant when both antifungals were used simultaneously. Fluconazole resistance occurred only in the presence of high flucytosine concentrations, and the higher the fluconazole concentration used, the greater the flucytosine concentration necessary to trigger the cross-resistance. When the flucytosine- and fluconazole-resistant cells were grown in the presence of fluconazole alone, the cells reversed to fluconazole susceptibility. Genetic analyses of the progeny from crosses between resistant and sensitive isolates showed that resistance to flucytosine was derived from a recessive mutation in a single gene, whereas cross-resistance to fluconazole seemed to vary like a quantitative trait. We further demonstrated that the four clinical isolates were susceptible to 5-fluorouracil and that cytosine deaminase activity was unaffected. Kinetic transport studies with [(14)C]flucytosine showed that flucytosine resistance was due to a defect in the purine-cytosine permease. Our hypothesis was that extracellular flucytosine would subsequently behave as a competitive inhibitor of fluconazole uptake transport. Finally, in vitro selection of spontaneous and induced mutants indicated that such a cross-resistance mechanism could also affect other Candida species, including C. albicans, C. tropicalis, and C. glabrata. This is the first report of a putative fluconazole uptake transporter in Candida species and of a possible resistance mechanism associated with a deficiency in the uptake of this drug.

Two ATP synthases can be linked through subunits i in the inner mitochondrial membrane of Saccharomyces cerevisiae.

Cross-linking experiments showed that the supernumerary subunit i is close to the interface between two ATP synthases. These data were used to demonstrate the presence of ATP synthase dimers in the inner mitochondrial membrane of Saccharomyces cerevisiae. A cysteine residue was introduced into the inter-membrane space located C-terminal part of subunit i. Cross-linking experiments revealed a dimerization of subunit i. This cross-linking occurred only with the dimeric form of the enzyme after incubating intact mitochondria with a bis-maleimide reagent, thus indicating an inter-ATP synthase cross-linking, whereas the monomeric form of the enzyme exhibited only an intra-ATP synthase cross-linking with subunit 6, another component of the membranous domain of the ATP synthase.

The ATP synthase is involved in generating mitochondrial cristae morphology.

The inner membrane of the mitochondrion folds inwards, forming the cristae. This folding allows a greater amount of membrane to be packed into the mitochondrion. The data in this study demonstrate that subunits e and g of the mitochondrial ATP synthase are involved in generating mitochondrial cristae morphology. These two subunits are non-essential components of ATP synthase and are required for the dimerization and oligomerization of ATP synthase. Mitochondria of yeast cells deficient in either subunits e or g were found to have numerous digitations and onion-like structures that correspond to an uncontrolled biogenesis and/or folding of the inner mitochondrial membrane. The present data show that there is a link between dimerization of the mitochondrial ATP synthase and cristae morphology. A model is proposed of the assembly of ATP synthase dimers, taking into account the oligomerization of the yeast enzyme and earlier data on the ultrastructure of mitochondrial cristae, which suggests that the association of ATP synthase dimers is involved in the control of the biogenesis of the inner mitochondrial membrane.

Conversion of a transmembrane to a water-soluble protein complex by a single point mutation.

Proteins exist in one of two generally incompatible states: either membrane associated or soluble. Pore-forming proteins are exceptional because they are synthesized as a water-soluble molecule but end up being located in the membrane -- that is, they are nonconstitutive membrane proteins. Here we report the pronounced effect of the single point mutation Y221G of the pore-forming toxin aerolysin. This mutation blocks the hemolytic activity of the toxin but does not affect its initial structure, its ability to bind to cell-surface receptors or its capacity to form heptamers, which constitute the channel-forming unit. The overall structure of the Y221G protein as analyzed by cryo-negative staining EM and three-dimensional reconstruction is remarkably similar to that of the wild type heptamer. The mutant protein forms a mushroom-shaped complex whose stem domain is thought to be within the membrane in the wild type toxin. In contrast to the wild type heptamer, which is a hydrophobic complex, the Y221G heptamer is fully hydrophilic. This point mutation has, therefore, converted a normally membrane-embedded toxin into a soluble complex.