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The ATP synthase is involved in generating mitochondrial cristae morphology

2002; Springer Nature; Volume: 21; Issue: 3 Linguagem: Inglês

10.1093/emboj/21.3.221

1460-2075

Patrick Paumard, Jacques Vaillier, Bénédicte Coulary, Jacques Schaëffer, Vincent Soubannier, David M. Mueller, Daniel Brèthes, Jean‐Paul di Rago, Jean Velours,

Photosynthetic Processes and Mechanisms

Article1 February 2002free access The ATP synthase is involved in generating mitochondrial cristae morphology Patrick Paumard Patrick Paumard Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Ségalen, Bordeaux 2, 1 rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author Jacques Vaillier Jacques Vaillier Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Ségalen, Bordeaux 2, 1 rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author Bénédicte Coulary Bénédicte Coulary Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Ségalen, Bordeaux 2, 1 rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author Jacques Schaeffer Jacques Schaeffer Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Ségalen, Bordeaux 2, 1 rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author Vincent Soubannier Vincent Soubannier Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Ségalen, Bordeaux 2, 1 rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author David M. Mueller David M. Mueller Department of Biochemistry and Molecular Biology, The Chicago Medical School, 3333 Greenbay Road, North Chicago, IL, 60064 USA Search for more papers by this author Daniel Brèthes Daniel Brèthes Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Ségalen, Bordeaux 2, 1 rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author Jean-Paul di Rago Jean-Paul di Rago Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Ségalen, Bordeaux 2, 1 rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author Jean Velours Corresponding Author Jean Velours Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Ségalen, Bordeaux 2, 1 rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author Patrick Paumard Patrick Paumard Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Ségalen, Bordeaux 2, 1 rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author Jacques Vaillier Jacques Vaillier Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Ségalen, Bordeaux 2, 1 rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author Bénédicte Coulary Bénédicte Coulary Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Ségalen, Bordeaux 2, 1 rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author Jacques Schaeffer Jacques Schaeffer Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Ségalen, Bordeaux 2, 1 rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author Vincent Soubannier Vincent Soubannier Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Ségalen, Bordeaux 2, 1 rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author David M. Mueller David M. Mueller Department of Biochemistry and Molecular Biology, The Chicago Medical School, 3333 Greenbay Road, North Chicago, IL, 60064 USA Search for more papers by this author Daniel Brèthes Daniel Brèthes Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Ségalen, Bordeaux 2, 1 rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author Jean-Paul di Rago Jean-Paul di Rago Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Ségalen, Bordeaux 2, 1 rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author Jean Velours Corresponding Author Jean Velours Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Ségalen, Bordeaux 2, 1 rue Camille Saint Saëns, 33077 Bordeaux, cedex, France Search for more papers by this author Author Information Patrick Paumard1, Jacques Vaillier1, Bénédicte Coulary1, Jacques Schaeffer1, Vincent Soubannier1, David M. Mueller2, Daniel Brèthes1, Jean-Paul di Rago1 and Jean Velours 1 1Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Ségalen, Bordeaux 2, 1 rue Camille Saint Saëns, 33077 Bordeaux, cedex, France 2Department of Biochemistry and Molecular Biology, The Chicago Medical School, 3333 Greenbay Road, North Chicago, IL, 60064 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:221-230https://doi.org/10.1093/emboj/21.3.221 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction The mitochondrion is referred to as the ‘power house’ of the cell, because it is responsible for the synthesis of the majority of ATP under aerobic conditions. The inner membrane of the mitochondrion contains the components of the electron transport chain. Oxidation/reduction reactions along the components of the electron transport chain generate a proton gradient that is used by ATP synthase to phosphorylate ADP, thereby producing ATP. To increase the capacity of the mitochondrion to synthesize ATP, the inner membrane is folded to form cristae. These folds allow a much greater amount of electron transport chain enzymes and ATP synthase to be packed into the mitochondrion. However, until now, little was known about how the inner membrane is able to form cristae. This study provides evidence that subunits of ATP synthase are involved in cristae formation. ATP synthase, or F1F0 ATP synthase, is composed of a hydrophilic catalytic unit (F1), which is located in the mitochondrial matrix, and a membranous domain (F0), which anchors the enzyme in the inner mitochondrial membrane and mediates the conduction of protons that participate indirectly in ATP synthesis (Fillingame, 1999; Pedersen et al., 2000). Electron microscopy of negatively stained mitochondria revealed 9 nm diameter projections in the mitochondrial matrix (Fernández-Morán, 1962), which were identified as the hydrophilic catalytic units (F1) of the F1F0 ATP synthase (Racker et al., 1965). These projections were observed by electron microscopy to be arranged in a non-random, tightly ordered pattern on tubular cristae in Paramecium multimicronucleatum mitochondria using rapid techniques of freezing followed by fracturing, etching and replication (Allen et al., 1989). In this organism, the F1 complexes are arranged as a double row of particles along the full length of the helically shaped tubular cristae. Allen (1995) proposed a model where the association of ATP synthase dimers promotes a distortion of the plane of the inner mitochondrial membrane, leading to the formation of 50 nm diameter tubular cristae. Arnold et al. (1998) recently demonstrated the presence of ATP synthase dimers in both Triton X-100 and digitonin extracts of wild-type yeast using blue native polyacrylamide gel electrophoresis (BN–PAGE) (Schägger and von Jagow, 1991). The proximity between yeast ATP synthases has also been shown by the formation in the inner mitochondrial membrane of a disulfide bridge between two subunits 4 (subunit b) belonging to two ATP synthases (Spannagel et al., 1998). Such a dimerization also involves the two ATP synthase-associated subunits e and g (Arnold et al., 1998). These two components are supernumerary subunits that are not essential for cellular growth and are found only in mitochondria (Walker et al., 1991; Higuti et al., 1992; Collinson et al., 1994; Boyle et al., 1999). In Saccharomyces cerevisiae, of the 20 different subunits that compose ATP synthase, four are supernumerary subunits (subunits e, g, i/j and k) that are associated with F0. Inactivation of the corresponding genes does not significantly alter growth on non-fermentable sources (Tokatlidis et al., 1996; Arnold et al., 1997, 1998, 1999; Boyle et al., 1999; Vaillier et al., 1999). The e, g and i subunits are small proteins with a unique transmembrane-spanning segment, and are probably located in the periphery of the yeast enzyme (Soubannier et al., 1999; Paumard et al., 2000). A regulatory role of subunit e in cellular ATP production has been proposed (Levy and Kelly, 1997). The involvement of at least subunits e and g in the dimerization of the ATP synthase led us to investigate the structure of null mutant ATP synthases and to examine yeast cells devoid of these two subunits by electron microscopy. Surprisingly, there were extensive changes in the morphology of the mitochondrion in mutants lacking subunits e or g. The results of this study indicate that the ATP synthase could be involved, through subunits e and g, in the formation of the cristae by an oligomerization process. Results Phenotypic analyses of mutants devoid of ATP synthase-associated subunits e, g and i The strains used in this study contain null mutations in ATP18, ATP20 or TIM11 genes, encoding subunits i, g and e, respectively. The null mutant in the ATP18 gene was used as a control as it is partially defective for ATP synthesis (Vaillier et al., 1999), yet still contains a stable ATP synthase and thus more closely mimics the mutations in ATP20 and TIM11 genes. A number of biochemical and genetic studies were performed to provide an initial characterization of the mutant strains (Table I). ΔATP20 and ΔTIM11 mutant cells grew using lactate as carbon source either at 28 or 37°C, thus indicating that they were able to generate ATP via oxidative phosphorylation. However, they had generation times longer than that of the wild-type strain. Indeed, 40% of ΔATP20 and ΔTIM11 mutant cells spontaneously converted to rho− cells, which explains both the 44–48% inhibition of ATPase activity by oligomycin and the 34% decrease in the CCCP-stimulated respiration rate. Our interpretation of these data is different from that of Boyle et al. (1999), who showed that the absence of subunit g only reduced cytochrome oxidase activity. The different parental strains used could explain this discrepancy. Our data indicate that although growing on non-fermentable medium, a part of the cellular population, while converting to rho− cells, did not grow but accumulated. As a result, the mitochondrial preparation is a mixture of rho− and rho+ mitochondria. The ATP/O ratio, which is independent of the protein concentration, indicates that the efficiency of the oxidative phosphorylation machinery of ΔATP20 and ΔTIM11 mitochondria was not altered, whereas that of ΔATP18 mitochondria was slightly lowered as a consequence of a proton leak occurring during phosphorylation (Vaillier et al., 1999). As a consequence, ATP synthases of ΔATP20 and ΔTIM11 rho+ mitochondria are fully functional, and the high percentage of rho− cells appearing in cultures is probably the cause of the increase in the generation time of ΔATP20 and ΔTIM11 cell populations. 4,6-diamidino-2-phenylindole staining revealed that ΔATP20 rho− cells still contained mitochondrial DNA (P.Paumard, unpublished observation). Table 1. Phenotypic analyses of yeast strains useda Strains Doubling time (min) % of rho− cells in cultures Uncoupled respiration rate (nmol of O/min/mg of protein)b ATP/Oc ATPase activityd − oligomycine + oligomycine (%) inhibition Wild type 150 0.9 ± 0.2 1199 ± 42 1.09 ± 0.13 4.98 ± 0.10 0.67 ± 0.02 87 ΔATP20 221 40.0 ± 2.4 795 ± 50 1.22 ± 0.04 6.61 ± 0.04 3.68 ± 0.09 44 ΔTIM11 229 40.9 ± 1.1 868 ± 24 0.89 ± 0.03 5.79 ± 0.29 2.99 ± 0.03 48 ΔATP18 456 11.1 ± 0.8 1380 ± 130 0.79 ± 0.06 2.83 ± 0.02 2.15 ± 0.06 24 a Yeast cells were grown at 28°C on complete medium containing lactate as carbon source. rho− production in cultures was measured on glycerol plates supplemented with 0.1% glucose. Mitochondria were prepared from protoplasts. b Uncoupled respiration rates were determined in the presence of 3 μM CCCP (carbonyl cyanide m-chlorophenylhydrazone). c ATP/O ratios were determined with NADH as substrate. d ATPase activities and the sensitivity to the F0 inhibitor oligomycin were measured at pH 8.4 in the presence of 0.375% Triton X-100 to remove the natural inhibitor IF1. e Units: μmol of Pi/min/mg of protein. Dimerization of subunit 4 in mitochondria devoid of subunits g or e In a previous study, we demonstrated the dimerization of the ATP synthase by monitoring the formation of the disulfide bond between two subunit 4 molecules containing the mutation D54C, a position located in the inter-membrane space (Spannagel et al., 1998). Subunit 4 is the product of the ATP4 gene and is homologous to the bovine subunit b. It is present as only one per ATP synthase molecule, and constitutes an essential component of the second stalk (Collinson et al., 1994, 1996; Spannagel et al., 1998; Bateson et al., 1999). As a result, the disulfide bond between subunit 4 molecules is probably due to the dimerization of ATP synthases. Since subunits e and g are also involved in the dimerization of the yeast ATP synthases (Arnold et al., 1998), we analysed the dimerization state of subunit 4 in either the ΔATP20 or the ΔTIM11 context. Figure 1A shows the presence of a subunit 4 dimer in ΔATP20-4D54C and ΔTIM11-4D54C mitochondria, thus showing that ATP synthase dimers are probably present in the inner mitochondrial membrane lacking subunits g or e. The efficiency of the spontaneous dimerization was quantified. Subunit 4–subunit 4 dimer ratios were found to be 1.6, 1.3 and 1.9 in 4D54C, ΔATP20-4D54C and ΔTIM11-4D54C mitochondria, respectively. The presence of such dimers is apparently inconsistent with the results of Arnold et al. (1998), who did not find any ATP synthase dimers in Triton X-100 extracts of mitochondria isolated from either ΔATP20 or ΔTIM11 mutant strains. However, our experimental conditions were different from those of Arnold et al. (1998), as we analysed whole mitochondrial membranes by SDS–PAGE and not native solubilized complexes. Figure 1.Dimerization of subunit 4. Mutant and wild-type mitochondria (50 μg of protein) were incubated with 40 mM NEM, dissolved in sample buffer containing SDS and submitted to western blot analysis using polyclonal antibodies against subunit 4 of the ATP synthase. (A) Mitochondria were isolated from wild-type yeast (lane 1), and from 4D54C (lane 2), ΔATP20-4D54C (lane 3) and ΔTIM11-4D54C (lane 4) mutant strains. (B and C) Mitochondria isolated from cysteine mutants of subunit 4 were analysed as above. Download figure Download PowerPoint Other sites in subunit 4 are involved in the dimerization of subunit 4 Cysteine mutants replacing lysine residues have been built to determine the environment of subunit 4 (Soubannier et al., 1999). Spontaneous subunit 4 dimer formation was found in mitochondrial membranes of mutants modified in the matricial C-terminal domain of the subunit. In addition to position 54, three other positions gave strong subunit 4 dimers, i.e. positions 86, 98 and 104, while positions 14, 71, 151, 188 and 192 gave low intensity adducts (Figure 1B and C). Surprisingly, the apparent molecular weight of subunit 4 dimers varied, probably reflecting the shape of the SDS-solubilized covalently linked molecules. With a stoichiometry of one subunit 4 per ATP synthase, the C-terminal domain of subunit 4 probably participates in an interface involved in the dimerization of ATP synthases. The identification of subunit 4 dimers was not performed by two-dimensional analysis of oxidized ATP synthases, unlike in a previous study with mutant 4D54C (Spannagel et al., 1998). However, as for mutant 4D54C, we observed that Triton X-100 extraction destabilized the subunit 4 dimers that were separated from the solubilized enzyme during centrifugation of extracts and were recovered in the pellet (not shown). Digitonin extracts of yeast mitochondria contain oligomers of ATP synthases The association of ATP synthases was studied by BN–PAGE. Digitonin was used as detergent, because it solubilizes the enzyme without significantly altering interactions between the mitochondrial complexes (Schägger and Pfeiffer, 2000). After digitonin extraction and centrifugation of the extract, the proteins contained in the supernatant were separated under native conditions on a 3–13% linear gradient acrylamide gel. The slab gel was either stained with Coomassie Blue (Figure 2A) or analysed for ATPase activity. Four bands showing an ATPase activity migrated at acrylamide concentrations of 9, 7.5, 6 and 4.8% (Figure 2B). The band running at an acrylamide concentration of 9% with a relative mol. wt of ∼450 kDa corresponds to free F1, while the band at 7.5% (650 kDa) corresponds to the monomeric form of the enzyme devoid of subunits e and g (Arnold et al., 1998). With detergent/protein ratios 1 g/g destabilized the oligomeric forms. The major oligomeric form could correspond at least to a tetrameric form of the enzyme. Usually, the estimation of the molecular weight of proteins migrating on pore gradient electrophoresis is determined from the relationship between the log (molecular weight) and log (%T) (acrylamide concentration) (Poduslo and Rodbard, 1980). However, in our experiments, the absence of protein standards with molecular weights of several million Daltons led to a weight estimation of the oligomer that was probably erroneous. Whatever the molecular weight of the oligomer, this result shows that oligomeric forms of the yeast ATP synthase with a molecular weight higher than that of the dimeric form are present in digitonin extracts. In agreement with Arnold et al. (1998), detergent/protein ratios of 0.75 or 1 g/g led to digitonin extracts of ΔATP20 mitochondria containing the monomeric form of the ATP synthase (Figure 2C). However, a very faint band of the dimeric form was observed only by using the ATPase activity assay, whereas the oligomeric forms of the enzyme were absent. Similar observations were made with digitonin extracts of ΔTIM11 mitochondria (not shown). On the other hand, digitonin extracts of ΔATP18 mitochondria contained the dimeric and oligomeric forms of ATP synthase. The high amount of F1 present in ΔATP18 extracts may be correlated with the low oligomycin-sensitive ATPase activity due to the lack of subunit i (Table I). The mitochondrial morphology in cells devoid of subunits e or g is altered Transmission electron micrographs of yeast cell sections were performed to determine the effect of the loss of subunits e and g on the ultrastructure of the mitochondrial membrane. Mitochondria from either wild-type or ΔATP18 mutant cells appeared normal, with numerous cristae (Figure 3A and B). Transmission electron micrographs of null mutant cells devoid of either subunits e or g showed mitochondria with much different morphologies, i.e. onion-like structures (Figure 3D and F), and large digitations surrounding other organelles (Figure 3C and E). These abnormal structures were observed with cells harvested during both exponential and stationary growth phases. This kind of data has not previously been reported with mutant strains deficient in α- or β-subunits or in F1 assembly. Indeed, mitochondria of atp1, atp2 and atp12 mutants appear to be small and entirely devoid of cristae (J.P.diRago, unpublished result). The interpretation of this is difficult, owing to the defect in energy production of strains devoid of components of the ATP synthase catalytic domain. Nevertheless, in the absence of subunit 4 (subunit b), a structural component of the stator, ΔATP4 cells displayed mitochondria showing very small onion-like structures (Figure 4A). The ATP synthase of ΔATP4 mitochondria is not functional, although it contains an active catalytic sector F1, but it is devoid of the F0 sector (Paul et al., 1989). Subunits e and g were detected in ΔATP4 mitochondrial membranes, albeit in low concentrations (Figure 4B), showing that subunit 4 and therefore a functional ATP synthase are involved in generating mitochondrial cristae morphology. Figure 3.Yeast cells devoid of either subunit e or g have abnormal mitochondria. Samples were prepared as described in Materials and methods. They were observed by transmission electron microscopy. The arrows indicate abnormal mitochondria. (A) Wild-type, (B) ΔATP18, (C and D) ΔATP20 and (E and F) ΔTIM11. Bars indicate 0.5 μm. Download figure Download PowerPoint Figure 4.ΔATP4 yeast cells have abnormal mitochondria. (A) Cells were grown with 2% galactose as carbon source, and observed by transmission electron microscopy. The arrow indicates abnormal mitochondria. Bar indicates 0.5 μm. (B) Wild-type (lane 1) and ΔATP4 mitochondria (lane 2) were dissociated and samples (50 μg of protein) were submitted to western blot analysis. Blots were probed with polyclonal antibodies raised against subunits γ, 4, g and e. Download figure Download PowerPoint To better characterize the unusual mitochondrial morphology of mutant strains, immunolocalization studies were performed with a polyclonal antibody raised against the β-subunit of the ATP synthase. The gold particles were mainly found in cristae of wild-type mitochondria (Figure 5A), but also in the digitations and onion-like structures of null mutant cells (Figure 5B–D). A statistical analysis of the location of gold particles revealed that 82% of them were associated with digitations and onion-like structures (Table II). Thus, these membranes clearly contain ATP synthase and the organelles are in fact mitochondria with an altered morphology. Similarly, an immunolocalization study was performed with a monoclonal antibody raised against the yeast mitochondrial porin (Figure 6). Eighty percent of the gold particles were found in the periphery of wild-type mitochondria and were restricted to the outermost membranes of onion-like structures (Table II). These observations point to a role of subunits e and g in cristae formation. The ΔATP18 mutant, which is devoid of the ATP synthase-associated subunit i, did not display such morphological modifications (Figure 3B). Consistent with the role of subunits e and g in cristae formation, western blot analysis of mitochondrial membranes devoid of subunit i indicated the presence of both subunits e and g (not shown). The morphological modifications described above were also investigated by fluorescence microscopy (not shown). ΔATP20 yeast cells were 1.5-fold larger than wild-type cells. Mitochondria were labelled with a matrix-targeted green fluorescent protein (Okamoto et al., 1998). A peripheral distribution of mitochondria was observed in wild-type and ΔATP20 yeast cells, but the latter also displayed diffuse large mitochondria that invaded the cells. Figure 5.Immunological detection in yeast cells of β-subunit of yeast ATP synthase Immunogold electron microscopy was carried as described in Materials and methods. The pictures are representative of experiments performed with wild-type (A) and ΔATP20 (B–D) cells. Bars indicate 0.5 μm. Download figure Download PowerPoint Figure 6.Immunological detection in yeast cells of yeast mitochondrial porin. Immunogold electron microscopy was carried out as described in Materials and methods. The images are representative of experiments performed with wild-type (A) and ΔATP20 (B) cells. Bars indicate 0.5 μm. Download figure Download PowerPoint Table 2. Location of immunogold particles in yeast cellsa Strains Cytoplasm and plasma membrane Membrane-associated Inside organellesd anti-βATPase anti-porin anti-βATPaseb anti-porinc anti-βATPase anti-porin Wild type 187 (21%) 32 (12%) 629 (71%) 223 (84%) 69 (8%) 11 (4%) ΔATP20 564 (15%) 63 (14%) 3056 (83%) 354 (80%) 80 (2%) 27 (6%) a Gold particles corresponding to β and porin immunocomplexes were counted from electron micrographs. b Mitochondria or onion-like structures. c Outermost membranes of mitochondria or onion-like structures. d Mitochondria or onion-like structures, but not clearly associated with membranes. Discussion As discussed by Frey and Mannela (2000), the formation of tubular cristae and cristae junctions at the inter-membrane compartment level probably has important functional implications in terms of efficiency of oxidative phosphorylation by providing high surface-to-volume ratios. This would limit the diffusion of ions and substrates involved in ATP synthesis. The ultrastructural data shown above indicate that the ATP synthase-associated subunits e and g are indispensable for the biogenesis of the mitochondrial cristae. These observations lead to the conclusion that in the absence of either subunits e or g, the cristae are also absent, even though the inner membrane is conspicuously present. Thus, in mutant strains devoid of either subunit e or g, the inner membrane is organized differently than it is in wild-type mitochondria. Immunogold electron microscopy showed onion-like structures composed of two to three concentric layers of double leaflets of membranes containing a material dense to electrons. Figure 3D clearly displays three of these dense layers that are separated by two white spaces. The dense layers might correspond to the matrix space, as gold particles that localize F1 are mainly associated with membranes, which are therefore the inner mitochondrial membrane (Figure 5D). The white spaces could correspond to the inter-membrane space. Many gold particles that localize F1 are also associated with the