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 most external double leaflet, so this membrane is also the inner mitochondrial membrane. However, gold particles that localize porin, a main component of the outer membrane, are also located in the periphery of wild-type mitochondria and are associated with the outermost membrane of onion-like structures, thus showing that the outer membrane of abnormal mitochondria constitutes a continuous envelope. The abnormal mitochondrial structure can be interpreted as the consequence of an uncontrolled biogenesis of the inner mitochondrial membrane. As a result, the folding of the inner membrane inside the organelle appears in cell sections as alternations of matricial and inter-membrane spaces. Digitations and onion-like structures are probably the same objects observed under different cross-sections. From immunolocalization studies it is clear that the inner mitochondrial membrane is contained inside a continuous envelope of outer membrane, a feature that is different from the mitochondrial Nebenkern occurring during Drosophila melanogaster spermatogenesis (Hales and Fuller, 1997).
Abnormal mitochondrial morphologies have already been described in yeast mutants (for reviews see Hermann and Shaw, 1998; Yaffe, 1999). The proteins of the outer mitochondrial membrane Mmm1p, Mdm10p and Mdm12p are required to maintain normal mitochondrial shape, mitochondrial segregation and mitochondrial DNA stability (Burgess et al., 1994; Sogo and Yaffe, 1994; Berger et al., 1997). In their absence, mitochondria appear as giant, spherical organelles under the fluorescence microscope. A striking loss of normal inner-membrane organization was also observed in the absence of Mmm1p. The tubular-shaped cristae were absent and the inner membrane was collapsed, forming stacks of membrane sheets inside the organelle (Aiken Hobbs et al., 2001). The maintenance of the mitochondrial reticulum in S.cerevisiae also requires opposing fission and fusion events that regulate organelle morphology and number. Fusion is regulated by the Fzo1p transmembrane GTPase (Hermann et al., 1998). Conversely, the dynamin-related GTPase, Dnm1p, associated with Mdv1p forms a complex associated with the outer mitochondrial membrane that controls mitochondrial morphology in yeast by regulating mitochondrial fission (Otsuga et al., 1998; Bleazard et al., 1999; Mozdy et al., 2000; Tieu and Nunnari, 2000). A second dynamin-related GTPase, Mgm1p, is also involved in genome maintenance and morphology (Shepard and Yaffe, 1999). The Mgm1 mutant is unable to grow with non-fermentable sources. Mgm1p plays a role in inner membrane remodelling events in yeast cells. In co-ordination with Dnm1p-dependent outer membrane fission, it regulates inner membrane division. It has recently been proposed that Mgm1p may help to form and/or stabilize inner membrane cristae or regulate inner membrane fission (Wong et al., 2000). The phenotypic alterations due to the loss of subunits e or g appear to be different from those described in the null mutants cited above. In the absence of subunits e or g, cells still grew on non-fermentable sources. rho− cells accumulated, but they still possessed mitochondrial DNA. Finally the large onion-like structures have not yet been observed in yeast mutants having a mitochondrial morphology deficiency. However, we cannot exclude a relationship between the C-terminal part of subunit e (amino acid residues 26–96) located in the inter-membrane space and other proteins involved in the maintenance of morphology, such as the inter-membrane protein Mgm1p, which is peripherically associated with the inner membrane. Cross-linking experiments will be performed to address this issue.
Earlier work concerning the yeast chondriome of wild-type yeast grown aerobically with glucose in the late S phase sometimes showed mitochondria to have small onion-like structures evoking the structures described in this paper (Yotsuyanagi, 1962a). It has also been reported that cross-sections of rho− cells display small, spherical mitochondria, the major alteration of which is the loss of cristae. Most of these mitochondria have no discernible internal organization, but sometimes display small onion-like structures (Yotsuyanagi, 1962b; Stevens, 1981). It should be remembered that rho− mitochondria do not have a functional F0 and are also devoid of subunits e and g (Arnold et al., 1998). Although some small onion-like structures have been observed in rho− cells, they cannot account for the extensive mitochondrial morphological alterations described in this paper. In fact, cross-sections of rho− cells isolated from the ΔATP20 strain also display small, spherical mitochondria without discernible internal organization (data not shown). It has also been reported that yeast mutants in apocytochrome b and cytochrome oxidase are characterized by mitochondria displaying normal-looking but less numerous cristae compared with normal respiring yeasts (Yotsuyanagi, 1988). Therefore, it is most likely the absence of either subunit e or g and the presence of a functional ATP synthase that are the cause of such large onion-like structures seen in the ΔATP20 and ΔTIM11 mutant strains. For these reasons we conclude that ATP synthase is also an essential element for normal mitochondrial morphology, and propose an alternative in the way mitochondrial cristae are generated.
Model of ATP synthase organization in mitochondrial cristae
The main purpose of this paper is to provide the link between the dimerization of the mitochondrial ATP synthase and the biogenesis of cristae. Allen (1995) proposed an exciting hypothesis that described the association of ATP synthase dimers as generating the tubular cristae. The association of identical ATP synthase complexes approximating truncated cones in overall shape might offer the potential to form a rigid arc, thus leading to a protrusion of the inner mitochondrial membrane, which in turn might amplify to form tubules upon association of additional complexes during mitochondrial biogenesis. The crucial point is the dimerization of the enzyme. Subunits e and g are involved in the latter process, since ATP synthase is not dimerized and cristae are not formed in their absence. The second point is the association of ATP synthase dimers to form higher complexes. In this paper we show such associations in digitonin extracts of wild-type mitochondria. Figure 7 shows a proposed model consisting of the association of F1F0 ATP synthase dimers. Such an association requires two different interfaces. The first is composed of subunits e and g (Arnold et al., 1998). The proximity between two subunits e and between subunits e and g has already been described in the bovine enzyme (Belogrudov et al., 1996). Indeed, the C-terminal part of subunit e (amino acids residues 26–60) has the potential for a coiled-coil configuration in the inter-membrane space, probably in association with another subunit e (Belogrudov et al., 1996). The model suggests intermolecular interactions between subunits e of two adjacent ATP synthase complexes. We now propose a second intermolecular interaction involving subunit b (subunit 4) (Velours et al., 2000), since spontaneous dimers of the subunit were found in mitochondrial membranes of cysteine mutants of subunit 4 in either the presence or absence of subunits e and g. The model also takes into account the intramolecular interaction of subunit g and the N-terminal part of subunit 4 (Soubannier et al., 1999). Subunits e, g and 4 are at least responsible for the association of ATP synthases through their F0 portions.
Figure 7. Schematic representation of associations of F1F0 ATP synthase dimers. The grey circles and blocks represent the membranous parts of yeast F0 components as observed from the intra-cristae space. Subunit 4 (subunit b) is represented by two dark grey circles (the two membrane-spanning segments) that are linked by a line corresponding to the inter-membrane hydrophilic loop (amino acid residues 46–56). The black dots represent the mutation 4D54C. This model displays two interfaces (black bars). One is mediated by subunit 4 and the other is mediated at least by subunits e and g.
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An apparent paradox was the dimerization of subunit 4 in ΔATP20 and ΔTIM11 membranes. The postulated existence of two different interfaces at the F0 level reconciles the observations of Arnold et al. (1998) with ours, because interactions could occur between two distinct faces in the membrane, one composed of subunits 4 and the other composed of subunits e and g. Thus, it is possible to obtain subunit 4 dimerization in the membrane in the absence of subunits e or g.
However, we have not yet been able to increase the oligomer amount in digitonin extracts by cross-linking experiments involving subunits 4 as targets. Although subunit 4 dimers were obtained, they were mainly present in the pellets after centrifugation of the digitonin extracts, thus precluding the observation of cross-linked ATP synthase complexes by BN–PAGE. In addition, an increase in the F1 concentration was observed in digitonin supernatant extracts, thus showing a destabilization of ATP synthase complexes upon cross-linking (not shown). We have already reported the lack of solubility of subunit 4 dimers in detergents other than SDS. The covalent linkage between subunits 4 of two different complexes resulted in a disconnection between the F1 and F0 sectors, leading to a decrease in oligomycin sensitivity (Spannagel et al., 1998). At present, a digitonin/protein ratio of 0.75 g/g is the best ratio to obtain a high amount of ATP synthase oligomer under native conditions. This probably reflects the lability of the interface involving the association of dimers in our experimental conditions.
The model in Figure 7 is still speculative and originates from the observation of the regular association of ATP synthases on the tubular cristae of P.multimicronucleatum mitochondria. Such an investigation has not yet been carried out on yeast cristae. Another point concerns the oligomerization of ATP synthase molecules. The question is whether digitonin extracts contain an association of ATP synthase dimers or ATP synthase aggregates, as with the detergent-solubilized chloroplast ATP synthase (Böttcher and Gräber, 2000). The observation by BN–PAGE of distinct bands having higher molecular weights than that of the dimeric form of the enzyme, and the absence of such bands in digitonin extracts of ΔATP20 and ΔTIM11 mitochondria while they are present in ΔATP18 extracts, are all characteristics not in favour of aggregates.
From the above data it appears that the F1F0 ATP synthase, the main role of which is to provide energy to the cell, is also involved in mitochondrial morphology. We suggest that in the absence of dimerization involving F0 interfaces, the role of ATP synthase in the organization of the tubular cristae is abolished and the inner membrane is produced without control, leading to onion-like structures, although small, similar objects are present in ΔATP4 mutant cells and sometimes in rho− cells. The small size of the onion-like structures of ΔATP4 and rho− mutants can result in a decrease in the biogenesis of mitochondrial membranes, due to a dramatic loss in energy. For instance, rho− cells are devoid of F0 and respiratory complexes and the energy source of rho− mitochondria is a transmembrane potential ΔΨ provided by ATP hydrolysis by F1, coupled with the adenine nucleotide carrier (Giraud and Velours, 1997). Similarly, ΔATP4 mutant is also devoid of F0 and a dramatic decrease in mitochondrial respiratory complexes has been reported previously (Paul et al., 1989). Experiments are now being performed to identify all the components involved in protein interfaces between ATP synthase complexes and allowing the oligomers of yeast ATP synthase to control tubular cristae biogenesis.
Subunits e and g have only been found as associated components of ATP synthase in organelles with cristae. We suggest an involvement of subunits e and g in the mitochondrial morphology of eukaryotic cells other than yeast, even if the cristae are not always tubular, since flattened lamellar cristae have been observed in brown adipose tissue and Neurospara crassa mitochondria (Perkins et al., 1998; Nicastro et al., 2000). This raises the question of the presence and stoichiometry of ATP synthase-associated proteins in various tissues and organisms, which may vary according to the metabolic state of the cell.