Mitochondria are dynamic organelles, the morphology of which results from an equilibrium between two opposing processes, fusion and fission. Mitochondrial fusion relies on dynamin-related GTPases, the mitofusins (MFN1 and 2) in the outer mitochondrial membrane and OPA1 (optic atrophy 1) in the inner mitochondrial membrane. Apart from a role in the maintenance of mitochondrial DNA, little is known about the physiological role of mitochondrial fusion. Here we report that mitochondria hyperfuse and form a highly interconnected network in cells exposed to selective stresses. This process precedes mitochondrial fission when it is triggered by apoptotic stimuli such as UV irradiation or actinomycin D. Stress-induced mitochondrial hyperfusion (SIMH) is independent of MFN2, BAX/ BAK, and prohibitins, but requires L-OPA1, MFN1, and the mitochondrial inner membrane protein SLP-2. In the absence of SLP-2, L-OPA1 is lost and SIMH is prevented. SIMH is accompanied by increased mitochondrial ATP production and represents a novel adaptive pro-survival response against stress.

In the inner mitochondrial membrane, the respiratory chain complexes generate an electrochemical proton gradient, which is utilized to synthesize most of the cellular ATP. According to an increasing number of biochemical studies, these complexes are assembled into supercomplexes. However, little is known about the architecture of the proposed multicomplex assemblies. Here, we report the electron microscopic characterization of the two respiratory chain supercomplexes I 1 III 2 and I 1 III 2 IV 1 in bovine heart mitochondria, which are also two major supercomplexes in human mitochondria. After purification and demonstration of enzymatic activity, their structures in projection were determined by single particle image analysis. A difference map between the supercomplexes I 1 III 2 and I 1 III 2 IV 1 closely fits the x-ray structure of monomeric complex IV and shows its location in the assembly. By comparing different views of supercomplex I 1 III 2 IV 1 , the location and mutual arrangement of complex I and the complex III dimer are discussed. Detailed knowledge of the architecture of the active supercomplexes is a prerequisite for a deeper understanding of energy conversion by mitochondria in mammals.All living organisms use a series of integral membrane protein complexes for energy conversion and ATP synthesis. In eukaryotes, electrons are transported by the respiratory chain, starting from NADH via complex I (NADH:ubiquinone oxidoreductase) or from succinate via complex II (succinate:ubiquinone oxidoreductase), the membrane integral electron carrier ubiquinol, complex III (ubiquinol:cytochrome c oxidoreductase), the peripheral electron carrier cytochrome c, and complex IV (cytochrome c oxidase) to the terminal acceptor molecular oxygen (1). The electron transport chain generates a proton gradient across the inner mitochondrial membrane, which is used by complex V (F O F 1 -ATP synthase) to synthesize ATP. In the last decade, structures of the individual respiratory chain complexes from various organisms have been determined. Atomic models exist for bovine heart mitochondrial complex III (2) and IV (3). A high resolution structure of complex I is not yet available, but electron microscopy indicates that it is L-shaped in all organisms investigated, and a 2.2-nm resolution map from cryoelectron microscopy exists for the bovine heart complex I (4).Two alternative models for the arrangement of the respiratory chain complexes in the membrane have been proposed. According to the currently favored random collision model (5), all components of the respiratory chain diffuse individually in the membrane, and electron transfer depends on the random, transient encounter of the individual protein complexes and the smaller electron carriers. In the solid state model (6) proposed 50 years ago, the substrate is channeled directly from one enzyme to the next. Recently isolated stoichiometric assemblies, so-called supercomplexes, support this model. Respiratory supercomplexes of different compositions have been described in bact...

It is an essential and challenging task to unravel protein-protein interactions in their actual in vivo context. Native gel systems provide a separation platform allowing the analysis of protein complexes on a rather proteome-wide scale in a single experiment. This review focus on blue-native (BN)-PAGE as the most versatile and successful gel-based approach to separate soluble and membrane protein complexes of intricate protein mixtures derived from all biological sources. BN-PAGE is a charge-shift method with a running pH of 7.5 relying on the gentle binding of anionic CBB dye to all membrane and many soluble protein complexes, leading to separation of protein species essentially according to their size and superior resolution than other fractionation techniques can offer. The closely related colorless-native (CN)-PAGE, whose applicability is restricted to protein species with intrinsic negative net charge, proved to provide an especially mild separation capable of preserving weak protein-protein interactions better than BN-PAGE. The essential conditions determining the success of detecting protein-protein interactions are the sample preparations, e.g. the efficiency/mildness of the detergent solubilization of membrane protein complexes. A broad overview about the achievements of BN- and CN-PAGE studies to elucidate protein-protein interactions in organelles and prokaryotes is presented, e.g. the mitochondrial protein import machinery and oxidative phosphorylation supercomplexes. In many cases, solubilization with digitonin was demonstrated to facilitate an efficient and particularly gentle extraction of membrane protein complexes prone to dissociation by treatment with other detergents. In general, analyses of protein interactomes should be carried out by both BN- and CN-PAGE.

To elucidate the molecular basis of the link between respiration and longevity, we have studied the organization of the respiratory chain of a wild-type strain and of two long-lived mutants of the filamentous fungus Podospora anserina. This established aging model is able to respire by either the standard or the alternative pathway. In the latter pathway, electrons are directly transferred from ubiquinol to the alternative oxidase and thus bypass complexes III and IV. We show that the cytochrome c oxidase pathway is organized according to the mammalian "respirasome" model (Schä gger, H., and Pfeiffer, K. (2000) EMBO J. 19, 1777-1783). In contrast, the alternative pathway is composed of distinct supercomplexes of complexes I and III (i.e. I 2 and I 2 III 2 ), which have not been described so far. Enzymatic analysis reveals distinct functional properties of complexes I and III belonging to either cytochrome c oxidase-or alternative oxidase-dependent pathways. By a gentle colorless-native PAGE, almost all of the ATP synthases from mitochondria respiring by either pathway were preserved in the dimeric state. Our data are of significance for the understanding of both respiratory pathways as well as lifespan control and aging.

No abstract