Nicole H. Reifschneider

Architecture of Active Mammalian Respiratory Chain Supercomplexes

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 I1III2 and I1III2IV1 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 I1III2 and I1III2IV1 closely fits the x-ray structure of monocomplex IV and shows its location in the assembly. By comparing different views of supercomplex I1III2IV1, 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.

Supramolecular Organization of Cytochrome c Oxidase- and Alternative Oxidase-dependent Respiratory Chains in the Filamentous Fungus Podospora anserina

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 (Schägger, 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. I2 and I2III2), 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.

Proteome alterations in rat mitochondria caused by aging.

Abstract:  Analysis of the protein profile of mitochondria and its age‐dependent variation is a promising approach to unravel mechanisms involved in aging and age‐related diseases. Our studies focus on the mammalian mitochondrial membrane proteome, especially of the inner mitochondrial membrane with the respiratory chain complexes and other proteins possibly involved in life‐span control and aging. Variations of the mitochondrial proteome during aging, with the emphasis on the abundance, composition, structure, and activity of membrane proteins, are examined in various rat tissues by native polyacrylamide gel electrophoresis techniques in combination with MALDI‐TOF mass spectrometry. In rat brain, age‐modulated differences in the abundance of various mitochondrial and nonmitochondrial proteins, such as Na,K‐ATPase, HSP60, mitochondrial aconitase‐2, V‐type ATPase, MFoF1 ATP synthase, and the OXPHOS complexes I–IV are detected. During aging, a decrease in the amount of intact MFoF1 ATP synthase occurs in the cortex. As analytical technique, native PAGE separates not only individual proteins but also multi‐subunit (membrane) proteins, (membrane) protein supercomplexes as well as interacting proteins in their native state. It reveals the occurrence and architecture of supramolecular assemblies of proteins. The age‐related alterations in the oligomerization of the MFoF1 ATP synthase observed by us in rat cortex might be one clue for understanding the link between respiration and longevity. Also, the abundance of OXPHOS supercomplexes, that is, the natural assemblies of the respiratory complexes I, III, and IV into supramolecular stoichiometric entities, such as I1III2IV0‐4, can differ between young and aged cortex tissue. Age‐related changes in the supramolecular architecture of OXPHOS complexes might explain alterations in ROS production during aging.

OXPHOS Supercomplexes Respiration and Life-Span Control in the Aging Model Podospora anserina

Abstract:  Recent biochemical evidence has indicated the existence of respiratory supercomplexes as well as ATP synthase oligomers in the inner mitochondrial membrane of different eukaryotes. 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 aging model is able to respire by either the standard or the alternative pathway. In the latter, electrons are directly transferred from ubiquinol to the alternative oxidase (AOX) and thus bypass complexes III and IV. We showed that the two pathways are composed of distinct respiratory supercomplexes. These data are of significance for the understanding of both respiratory pathways as well as of life‐span control and aging.

Unraveling Age-Dependent Variation of the Mitochondrial Proteome

Abstract:  Blue‐native and colorless‐native gel electrophoresis combined with subsequent 2D‐SDS‐PAGE and MALDI mass spectrometry are successfully applied for understanding the role of mitochondria in cellular dysfunction, aging, and cellular death. The partial mitochondrial proteome maps of various tissues (liver, brain, kidney, heart, and skeletal muscle) obtained from rat serve now as a database for the elucidation of age‐dependent changes, including alterations in protein–protein interactions as well as in posttranslational modifications.