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

Abstract

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. 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. The inner mitochondrial membrane harbors the four major respiratory complexes (I–IV) of the standard respiratory chain and the F0F1-ATP synthase (complex V) designated as OXPHOS 1The abbreviations used are: OXPHOS, oxidative phosphorylation; AOX, alternative oxidase; MALDI, matrix-assisted laser desorption ionization; BN-PAGE, blue-native PAGE; CN-PAGE, colorless-native PAGE; COX, cytochrome c oxidase; cyt, cytochrome; DBQ, decylubiquinone; MOPS, 4-morpholinepropanesulfonic acid.-complexes (oxidative phosphorylation). The OXPHOS-complexes I (NADH:ubiquinone oxidoreductase), III (ubiquinol:cytochrome c oxidoreductase), and IV (cytochrome c oxidase, also referred to as COX) transduce the energy of nutritional compounds, ultimately leading to the generation of ATP by the F0F1-ATP synthase. According to the generally accepted “random collision model”, the OXPHOS-complexes are distributed independently from each other in the membrane (1Hackenbrock C.R. Chazotte B. Gupte S.S. J. Bioenerg. Biomembr. 1986; 18: 331-368Crossref PubMed Scopus (289) Google Scholar). In contrast to this textbook view, it was proposed recently that the OXPHOS-complexes of mammals were organized as very large supramolecular structures termed the “respirasome” consisting of two different supercomplexes (I1III2IV4 and III2IV4) as building blocks in a 2:1 ratio (2Schägger H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1039) Google Scholar, 3Schägger H. Pfeiffer K. J. Biol. Chem. 2001; 276: 37861-37867Abstract Full Text Full Text PDF PubMed Google Scholar, 4Schägger H. IUBMB Life. 2001; 2: 119-128Google Scholar). This model was based on results by blue-native PAGE (BN-PAGE) after mild solubilization of bovine and human mitochondria with digitonin by which most of complexes I and III, as well as a smaller but significant proportion of complex IV were resolved as specific supercomplexes. As an important functional consequence of the respirasome architecture, a kinetic advantage through “substrate channeling” between the OXPHOS-complexes was suggested (2Schägger H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1039) Google Scholar). Apart from the standard respiratory chain, plants, some fungi, and protozoa contain alternative respiratory chains utilizing an alternative oxidase (AOX). Recently, in higher plant mitochondria, a stable supercomplex formed by complexes I and III was described. Importantly, the interactions of the plant AOX with other respiratory complexes could not be found (5Eubel H. Jänsch L. Braun H.P. Plant Physiol. (Bethesda). 2003; 133: 274-286Crossref PubMed Scopus (280) Google Scholar). Until now, nothing was known about the occurrence of supercomplexes in the branched respiratory chain of fungi possessing complex I. In obligate aerobes like mammals and most fungi, mitochondria produce >90% of cellular ATP and therefore are indispensable to satisfy the cellular energy demands. Impairments of this machinery lead to severe diseases and are thought to be causatively involved in aging. The latter is conceptualized in the “mitochondrial theory of aging” (reviewed in Ref. 6Finkel T. Holbrook N.J. Nature. 2000; 408: 239-247Crossref PubMed Scopus (7349) Google Scholar). A paradigm of an organism in which mitochondria play an etiological role in aging is the filamentous fungus Podospora anserina. In contrast to almost all of the other fungi, which are capable of infinite growth, all of the examined wild-type strains of P. anserina exhibit a characteristic “senescence syndrome” (7Rizet G. C. R. Acad. Sci. (Paris). 1953; 237: 838-855Google Scholar). After prolonged vegetative propagation, the growth rate of the culture decreases and the pigmentation of the mycelium changes. Finally, the filamentous “cells” of the mycelium, the hyphae, die at their tips. These features and the fact that a number of mutants were selected in which lifespan is significantly increased led to extensive investigations aimed to understand the molecular basis of aging in this model system (reviewed in Refs. 8Osiewacz H.D. Ageing Res. Rev. 2002; 1: 425-442Crossref PubMed Scopus (69) Google Scholar and 9Osiewacz H.D. Gene (Amst.). 2002; 286: 65-71Crossref PubMed Scopus (58) Google Scholar). The lifespan of P. anserina is determined by environmental factors (e.g. growth temperature, nutrition) and by both nuclear as well as extranuclear genetic traits. The most significant insight into the molecular mechanisms involved in lifespan control of P. anserina is derived from the characterization of different long-lived mutants. Interestingly, some mutants are characterized by impairments of the standard respiratory chain at complex IV. They respire via an AOX (see above), which circumvents the electron transport to complexes III and IV and directly transfers electrons from ubiquinol to (reviewed in Refs. 8Osiewacz H.D. Ageing Res. Rev. 2002; 1: 425-442Crossref PubMed Scopus (69) Google Scholar and 9Osiewacz H.D. Gene (Amst.). 2002; 286: 65-71Crossref PubMed Scopus (58) Google Scholar). was that respiration via this pathway to a in generation J. Acad. Sci. 2000; PubMed Scopus Google Scholar). In in the was found to be J. Acad. Sci. 2000; PubMed Scopus Google Scholar, C. H.D. Acad. Sci. PubMed Scopus Google Scholar, C. H.D. 2000; PubMed Scopus Google Scholar, K. Scopus Google Scholar, H.D. PubMed Scopus Google Scholar). the and features of the AOX pathway are in T. 2001; PubMed Scopus Google Scholar, C. 2002; PubMed Scopus Google Scholar). In an to elucidate the molecular basis of the link between respiration and lifespan control in P. we an extensive of mitochondrial from wild-type strain as well as from the mutants and with on the characterization of the We mitochondria by as described (2Schägger H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1039) Google but analysis the gentle colorless-native that the of We on the properties of and we show that the respiratory machinery in P. anserina is organized according to the respirasome model of the mammalian respiratory we the dimeric complex I and the large supercomplex as distinct of the respiratory The of supercomplexes via complex and complex interactions is by Enzymatic analysis reveals distinct functional properties of complexes I and III belonging to either or respiratory pathways. as in mammalian and mitochondria (2Schägger H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1039) Google Scholar, I. Pfeiffer K. H. EMBO J. PubMed Scopus Google ATP synthases to as by analysis almost all of the ATP synthases were in the dimeric in all of the P. anserina We the of the results for the respirasome model of the standard respiratory chain as well as significance as a molecular basis for lifespan control in this aging and wild-type strain and the two and of P. anserina were in this K. Scopus Google Scholar, K. of Scholar, H. K. PubMed Scopus Google Scholar, H.D. B. K. PubMed Scopus Google Scholar). were derived from a on of in of K. of for at in the of the mycelium were transferred to an After of at in the of the mycelium from the growth were transferred to for C. H.D. 2000; PubMed Scopus Google Scholar). were at and used to a of was by the of to the at a of of of the were at mycelium was in and in of MOPS, bovine of and of were The was for and at for The was through two of and for at the was in of MOPS, bovine and a of were and of the were of in a were at for The was in of MOPS, of mitochondria were at and were of from and from P. anserina strains were as described (2Schägger H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1039) Google Scholar, I. Pfeiffer K. H. EMBO J. PubMed Scopus Google a ratio of for the and with as well as a ratio of for the with was with the from mitochondria, each of BN-PAGE, with a were was according to H. G. PubMed Scopus Google with of with a which were both with either or from the or CN-PAGE, were and used for a as described H. G. PubMed Scopus Google Scholar). The were and were the characteristic of the (e.g. and V) were from to and by as described H. 2003; PubMed Scopus Google Scholar). The molecular of the OXPHOS-complexes and their supercomplexes were by bovine mitochondria (2Schägger H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1039) Google to the The supercomplexes as well as dimeric ATP synthase and dimeric complex I were according to their and molecular The of the supercomplex was by of of with of supercomplex a from the with was not to the of supercomplexes resolved by that were designated as and of was a Enzymatic of respiratory chain complexes were as described (2Schägger H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1039) Google with some P. anserina mitochondrial of in and were with digitonin to After of on all of the were at and as the and of were to the (complex was by the of and as electron c (complex was by the of cytochrome c cytochrome was used as the for complex c (complex was by the and of cytochrome c as electron The was determined with or The of complex I was by a described by 1986; PubMed Scopus Google and and PubMed Scopus Google and the cytochrome c oxidase of complex IV by of and a of L. 18: PubMed Scopus Google Scholar). after BN-PAGE, were for at either in and or in cytochrome c and the were in for in of P. a respirasome organization was found in mitochondria of P. we of mitochondria from of wild-type strain of P. anserina. resolved the characteristic of the OXPHOS-complexes by the of OXPHOS-complexes and their supercomplexes of wild-type mitochondria was to that of mammalian mitochondria (2Schägger H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1039) Google Scholar, 3Schägger H. Pfeiffer K. J. Biol. Chem. 2001; 276: 37861-37867Abstract Full Text Full Text PDF PubMed Google Scholar, 4Schägger H. IUBMB Life. 2001; 2: 119-128Google Scholar). of the complex I and complex III were found in each with a of complex I and dimeric complex III as well as of complex IV and The most supercomplex with an molecular of was As in the of mammalian mitochondria, the of supercomplexes was by the ratio at Importantly, the supercomplexes were as by of and cytochrome oxidase according to their of complex I and complex IV A specific of complex III is not L. 18: PubMed Scopus Google Scholar). complex III known to as molecular an and ATP synthase (complex V) at the in the with an molecular of This was to which play a role for and respiratory of complex III H. T. B. G. J. PubMed Scopus Google Scholar, C. C. EMBO J. 2001; PubMed Scopus Google Scholar). or mitochondrial from all of the strains used in this we could not of complex C. H. and H. This that in complex III most into to a of A complex between supercomplex a and complex I with the as ATP synthase was molecular of that this complex dimeric ATP synthase. ATP synthase to of the ATP synthase. The ATP synthase. This is to the in mitochondria (2Schägger H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1039) Google Scholar, I. Pfeiffer K. H. EMBO J. PubMed Scopus Google and is a of dimeric ATP synthases was from bovine mitochondria the (2Schägger H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1039) Google Scholar, 3Schägger H. Pfeiffer K. J. Biol. Chem. 2001; 276: 37861-37867Abstract Full Text Full Text PDF PubMed Google Scholar, 4Schägger H. IUBMB Life. 2001; 2: 119-128Google of after The complex I on as in A and and on of the were wild-type strain in a I in the of P. the AOX respiratory pathway was in strains but not in C. T. H.D. Biol. 2001; PubMed Scopus Google Scholar, J. 2001; PubMed Scopus (58) Google or wild-type C. T. H.D. Biol. 2001; PubMed Scopus Google standard To the and supramolecular organization of the respiratory system in P. anserina by we examined mitochondria of the which via AOX of the of a for K. Scopus Google Scholar, Ageing PubMed Scopus Google Scholar). As (complex was in either or in supercomplexes. in this complex III was not as an electron to of the inner mitochondrial it was found to be a major In contrast to the cytochrome respiratory system of the in which most of complexes I and III were in supercomplexes most of complex III was as a and a to complex I after mitochondria by of which solubilization most of the complexes I and III were found as resolved supercomplexes the supercomplex a were two supercomplexes not in wild-type mitochondria, and The smaller with a molecular of the as complex I and a of complex I. The was composed of the of complexes I and of the in and the of in that it of each a complex I and a complex III their after the ratio to at all supercomplexes were found in the in the ATP synthase was found in the ratio of dimeric to complexes as in the A and The supercomplexes and a complex I and dimeric complex This that most or all of the complex I and dimeric complex III as well as the smaller supercomplexes I2 and are into the large supercomplex or supercomplexes. These are from complex I and supercomplexes of the respiratory pathway. We a stable of the AOX with supramolecular to electron from complex I to analysis of a the AOX of L. Plant Physiol. (Bethesda). PubMed Google interactions of complex I and two at the of were to and dimeric AOX of and in digitonin solubilization of mitochondrial and two distinct of both complexes I and III to the and respiration of the wild-type and the A and To the supramolecular organization of both respiratory pathways are not to specific we the nuclear which via both pathways. The of this strain is the of a in the nuclear for the C. H.D. PubMed Scopus Google Scholar). the of a for the C. H.D. J. Biol. 2002; PubMed Scopus Google the is characterized by a significant of the the be to wild-type by of the growth with of C. T. H.D. Biol. 2001; PubMed Scopus Google Scholar, K. J. PubMed Scopus Google Scholar). via a system and is to the respiration and all other wild-type specific of two independently of the as well as a culture of this in with were The culture to the cytochrome pathway as wild-type mitochondria and both of a different the of OXPHOS-complexes as with of and supercomplex and an with a of in which is by a higher of supercomplex and a proportion of complex I and In this the of both of OXPHOS-complexes to the in wild-type and mitochondria was These results that the respiratory at in the P. anserina are each organized in a characteristic not to in the AOX described results are with the that and AOX respiratory pathways are organized as supramolecular A by analysis was that the specific supercomplexes of the AOX pathway were significantly of the pathway. This be the of a of the supercomplexes in the AOX pathway to the the properties of are which or interactions between complexes Res. PubMed Scopus Google Scholar, K. C. H. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). this we of P. anserina mitochondria by the gentle The is based on the system as In contrast to BN-PAGE, the of is and mild are in a the to of We digitonin or to the CN-PAGE, the complexes from to their the is the molecular of the as in the other the of is to that of but was found for mitochondrial OXPHOS-complexes H. G. PubMed Scopus Google Scholar). of wild-type mitochondria by with digitonin as a to and interactions in wild-type mitochondria, an of in the was and almost complex III was all of complex III was found with complex I complex IV. In dimeric complex IV was complex IV as well as ATP synthase found in the dimeric the mitochondria also almost dimeric ATP synthase as well as a of supercomplexes of complexes I and III supercomplexes were resolved and in a by the of some ATP synthase To the of supercomplexes and to the we increased the in to and digitonin and The of the digitonin in the a of supercomplexes into complex I and dimeric complex III the of supercomplexes were dimeric ATP synthase also into ATP synthase. a of digitonin in CN-PAGE, a of OXPHOS-complexes and as in was the of in features with This that a of complexes I and III of the AOX pathway are into supercomplexes that are to of complex I to complex I in supercomplexes after I was by of the in the of complex I and the supercomplexes or on data not are in and wild-type strain in a in the AOX the of analysis by with between mitochondria of and complex III was found in the as a in both complex interactions were as well as complex interactions in wild-type mitochondria A and In contrast to wild-type mitochondria in which most of complex I was found in the complex I from mitochondria a of molecular as in These which at to be to by A and and an of the distinct supramolecular of both of complex I. in mitochondria, complex which were by of dimeric complex I by BN-PAGE, are for the of the to a of are also with the results of that complex interactions of the AOX pathway were we that dimeric complex I with complex III a major of the respiratory pathway in P. anserina. in the respiratory pathway of wild-type mitochondria, analysis dimeric complex I. In with this the that in the respiratory pathway of wild-type mitochondria, complex interactions to complex I after of complex interactions and complex interactions by analysis of from mitochondria that AOX was not to complex I of in P. functional of complexes I and III in the and we of mitochondria from the wild-type and the according to the from and Pfeiffer (2Schägger H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1039) Google Scholar). The were on mitochondria, each with the and to The electron transport rate of complex I (NADH:ubiquinone of wild-type mitochondria with the of was found to be increased after digitonin solubilization In contrast, rate of mitochondria was that from wild-type mitochondria and after solubilization by digitonin The of complex I from wild-type mitochondria after digitonin solubilization to of the rate of mitochondria, which to the of complex I from the mitochondrial A with this distinct of solubilization be by different in both the and the AOX respiratory the of complex I from are in the membrane the of complex I from wild-type A of c rate (complex but with a different of both wild-type and mitochondria was solubilization the c rate of mitochondria, in the a significantly specific with the the higher of complex III in mitochondria A with c complex of wild-type mitochondria were higher of mitochondria, in the state. the of increased wild-type c c of were rate of wild-type mitochondria was a of that In rate of mitochondria was a of that it in c are determined by the c and This to the fact in the of AOX complex III is not as electron electron from complex I to complex III is not for the for the supramolecular organization of the mitochondrial standard respiratory chain in with the recently proposed respirasome model (2Schägger H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1039) Google Scholar, 3Schägger H. Pfeiffer K. J. Biol. Chem. 2001; 276: 37861-37867Abstract Full Text Full Text PDF PubMed Google Scholar, 4Schägger H. IUBMB Life. 2001; 2: 119-128Google Scholar). The occurrence of distinct of complexes I and III in the and of P. anserina and to important After the to mitochondrial of Podospora the of complexes I and III was as complexes by BN-PAGE, to supercomplexes in wild-type This was the ratio of ATP synthase and is the A and The of in a of supercomplexes to and respiration as well as almost dimeric ATP synthase in both the supercomplexes of the pathway after digitonin were was used as of of complex and interactions the and of the respiratory chain are not a consequence of by solubilization as but the of the standard respiratory machinery in the inner mitochondrial which to be between filamentous fungi and mammals (2Schägger H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1039) Google Scholar, 3Schägger H. Pfeiffer K. J. Biol. Chem. 2001; 276: 37861-37867Abstract Full Text Full Text PDF PubMed Google Scholar, 4Schägger H. IUBMB Life. 2001; 2: 119-128Google Scholar). and AOX from by that complex I and as specific supercomplexes of the respiration not found in the respiratory pathway and A higher of supercomplexes via complex and complex interactions could be by and The specific supercomplex of the AOX pathway is not an of respirasome supercomplexes composed of complexes III, and IV by the of complex in which the supercomplex a it is that dimeric complex I is an in the In the other four OXPHOS-complexes are known to stable or and that dimeric complex I a for and electron to The rate as well as the cytochrome c rate (complex and mitochondria in contrast to wild-type mitochondria a are two of complexes I and III that are by and as well as This that both of complexes I and III in the two pathways different which be or membrane is of AOX with the distinct supercomplexes of the AOX pathway for electron which a of and AOX pathways. As by the supercomplexes of the AOX pathway are significantly to the of the of the pathway. In the of the pathway were almost resolved as supercomplexes by of A and and a stable complex as of the supercomplexes from the AOX pathway but to by of mitochondrial In with the respirasome model (see for Refs. H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1039) Google Scholar, 3Schägger H. Pfeiffer K. J. Biol. Chem. 2001; 276: 37861-37867Abstract Full Text Full Text PDF PubMed Google Scholar, 4Schägger H. IUBMB Life. 2001; 2: 119-128Google we the distinct supramolecular organization of both respiratory chains and respiratory III for of III is not involved in electron transport in the AOX respiratory the at all is complex III in large and at to complex I of as well as supercomplexes with complex I is an and this respiratory complex important and most The respiratory chain complexes of mitochondria of different of which a directly in electron The of most that not contain are III and both of complex III are to the mitochondrial which from of H.P. Sci. Full Text PDF PubMed Scopus Google Scholar). In contrast to and the plant mitochondrial is an of complex In a of P. is an I is to the of mitochondrial H. H. Nature. PubMed Scopus Google Scholar). as in the mitochondria from the in fungi is found in the H. B. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The respiration on complex I as the of the the inner mitochondrial membrane to ATP and other This the that complex III is other for of complex I. The to the proposed role of complex III for of complex I is the specific of a for an of complex III (e.g. which the of complex the is the be the of complexes III and I. These are in with data from the analysis of a respirasome Pfeiffer K. T. B. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google in of human with mitochondrial diseases H. IUBMB Life. 2001; 2: 119-128Google Scholar). A of complex III of specific in the with a of complex leading this to the that complex III is for the the of complex I H. IUBMB Life. 2001; 2: 119-128Google Scholar). and in the P. of a distinct supramolecular organization of and AOX respiratory pathways a basis to a of respiration on the of in the filamentous fungus P. anserina J. Acad. Sci. 2000; PubMed Scopus Google Scholar, C. H.D. Acad. Sci. PubMed Scopus Google Scholar, C. H.D. 2000; PubMed Scopus Google Scholar, K. Scopus Google Scholar, H.D. PubMed Scopus Google Scholar, C. T. H.D. Biol. 2001; PubMed Scopus Google Scholar). strains with different genetic as the mutants and in this were found to respire or via AOX of a or severe of COX, In two a significantly of mitochondrial in the wild-type strain was J. Acad. Sci. 2000; PubMed Scopus Google Scholar). Interestingly, the plant AOX was also to mitochondrial L. Acad. Sci. PubMed Scopus Google Scholar). The results of the that all of the which the of complex IV lead to the of the AOX respiratory of the to and the AOX respiratory which as a system to are The of respiratory chain complexes as well as their supramolecular the and number of supercomplexes and the of the of the respiratory the of the AOX is to be on the specific of the AOX respiratory the occurrence of dimeric complex I. an of the AOX is to lead to features of the strains from of it was that of AOX in wild-type strains of P. anserina not and with the J. 2001; PubMed Scopus (58) Google Scholar). a significant of AOX in the led to in ATP and in and a significantly J. 2001; PubMed Scopus (58) Google Scholar). In it that the of the AOX respiratory as a respiratory machinery in with the of the is for the lifespan in a number of long-lived P. anserina We of and for the of the AOX and