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ScienceDirect The role of mitochondria in fungal aging Dominik Bernhardt, Andrea Hamann and Heinz D Osiewacz Time-dependent impairments of mitochondrial function play a key role in biological aging. Work on fungal aging models has been instrumental in unraveling basic mechanisms leading to mitochondrial dysfunction and the identification of different pathways active in keeping mitochondria ‘healthy’ over time. Pathways including those involved in reactive oxygen scavenging, repair of damage, proteostasis, mitochondrial dynamics, and biogenesis, are interconnected and part of a complex quality control system. The individual components of this network are limited in capacity. However, if the capacity of one pathway is overwhelmed, another one may be activated. The mechanisms controlling the underlying cross-talk are poorly understood and subject of intensive investigation. Addresses Institute of Molecular Biosciences and Cluster of Excellence Frankfurt Macromolecular Complexes, Department of Biosciences, J.W. Goethe University, Frankfurt, Germany Corresponding author: Osiewacz, Heinz D ([email protected])
Saccharomyces cerevisiae can generate. This number is now defined as the replicative lifespan and the process is termed replicative aging [4,5]. Subsequently, strains of the genus Neurospora and Aspergillus with a limited replicative capacity were described and analyzed (reviewed in [6]). More recent surveys identified rapid senescence to occur frequently in species of coprophilous Sordariomycetes [7]. Apart from replicative aging, another type of aging was identified and studied in yeast. This process, termed chronological aging, describes the period of time a non-dividing yeast cell can survive. Both processes are controlled by common but also different traits and pathways. Here we specifically focus on replicative aging (also referred to as ‘proliferative’ or ‘mitotic’ aging). Our emphasis lies on genetic traits and molecular pathways that affect the pace of aging. More specifically, we concentrate on the role of mitochondria, which have been identified as key determinants of fungal aging and have stimulated aging research in other organisms.
Current Opinion in Microbiology 2014, 22:1–7 This review comes from a themed issue on Growth and development: eukaryotes Edited by Michael Bo¨lker
http://dx.doi.org/10.1016/j.mib.2014.09.007 1369-5274/# 2014 Elsevier Ltd. All rights reserved.
Introduction It is widely thought that fungi propagate indefinitely and may give rise to huge individual colonies, like it has been reported for a mycelium of Armillaria bulbosa, with an estimated weight of at least 10,000 kg and an age of approximately 1500 years [1]. This view is currently changing. More and more fungi with a limited vegetative growth have been described. Historically, the first description of limited fungal growth dates back to a study of George Rizet in the early 1950s [2]. He observed that, during vegetative growth, cultures of the filamentous ascomycete Podospora anserina show characteristic time-related changes which he collectively described as ‘senescence syndrome’. Finally, the peripheral hyphae die. Only a few years later, another type of fungal aging was described by Mortimer and Johnston [3], the finite number of daughter cells an individual mother cell of www.sciencedirect.com
Respiratory metabolism The best known function of mitochondria is oxygenic energy conservation: the generation of adenosine triphosphate from energy-rich compounds. Since it is known that part of this process relies on electron transport at the respiratory chain and that the majority of all cellular reactive oxygen species (ROS) are generated by this process, a link of mitochondria to aging has been conceptionalized in the ‘mitochondrial free radical theory of aging’ (MFRTA) [8]. This theory states that aging is caused by the accumulation of ROS-induced damage of biomolecules including nucleic acids, lipids, and proteins and the resulting adverse effects. In fungi, this link is well demonstrated. In particular in P. anserina which, in contrast to yeast, contains a standard respiratory chain organized in different supercomplexes (Figure 1) and additional, alternative respiratory components, an impact of respiration on aging has been demonstrated. In different mutants with impaired respiratory complex III or IV, an alternative terminal oxidase (PaAOX) is induced. Compared to standard respiration, the alternative respiration generates less ROS and the corresponding strains are characterized by an increased lifespan [9–12]. Also in yeast, which lacks complex I and alternative oxidase, a connection between ROS, lifespan, and respiration has been observed. Caloric restriction was found to lead to a decreased ROS release per consumed O2. This decrease in ROS levels is thought to be responsible for lifespan extension and can be mimicked by artificially increased respiration via uncouplers [13]. Current Opinion in Microbiology 2014, 22:1–7
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Mitochondrial protein quality control. A number of different proteolytic pathways are active to keep mitochondria functional. LON/PIM1 and CLPXP are matrix protease complexes involved in the degradation of damaged proteins, which cannot be repaired by the methionine sulfoxide reductases MSRA and MSRB. The peptides generated by these proteases are delivered to the cytosol via ABC transporters and PORIN. In the cytosol they induce the mitochondrial ‘unfolded protein response’ (UPRmt), signaling mitochondrial dysfunction to the nucleus. In the inner mitochondrial membrane proteases like i-AAA/YME1, m-AAA and others are involved in protein processing. In yeast, YME1 is also active in the degradation of access, non-assembled subunit 2 of the cytochrome c oxidase (COX2) of the respiratory chain complexes and supercomplexes. Also in yeast, m-AAA is active in the assembly of F1F0-ATP synthase (V). Damaged proteins from inside the mitochondrion can be retro-translocated to the outer membrane. These and other outer membrane proteins become ubiquitinated by E3 ligases. CDC48 and adaptor proteins like VMS1 deliver the ubiquitinated protein to the proteasome. Respiratory chain supercomplexes (I2II1III2IV, I1III2IV2, I1III2, III2) as they were described in mitochondria of P. anserina are indicated. IM: inner mitochondrial membrane, MM: mitochondrial matrix.
Mitochondrial protein quality control (PQC) Apart from reduced ROS production, scavenging of these molecules affects cellular ROS levels and can reduce oxidative stress and molecular damage. Mitochondrial Current Opinion in Microbiology 2014, 22:1–7
superoxide dismutase and peroxidases are part of a cellular scavenging network and specifically involved in mitochondrial ROS balancing. Protein damage can also be repaired by methionine sulfoxide reductases (MSRA/ www.sciencedirect.com
Fungal aging Bernhardt, Hamann and Osiewacz 3
B) via the reduction of oxidized methionine residues (Figure 1). Degradation of damaged proteins is yet another way of PQC. Several molecular chaperones and proteases are located in the different mitochondrial subcompartments [14], monitor the folding status of different target proteins and remove excess or damaged proteins. In the mitochondrial matrix, the proteases LON (PIM1 in yeast) and CLPXP (in hyphal fungi, yeasts lack the protease component CLPP) degrade damaged proteins. Other proteases, like i-AAA protease (YME1 in yeast, PaIAP in P. anserina), and m-AAA protease are located in the mitochondrial inner membrane. Both proteases are involved in processing and regulation of mitochondrial proteins like OPA1 or subunits 6, 8, and 9 of the F1F0-ATP synthase complex (reviewed in [15]). In yeast, YME1 is also known to degrade COX2, a subunit of respiratory complex IV. Recently, ‘mitochondria-associated degradation’ (MAD), a pathway which leads to the proteasomal degradation of mitochondrial proteins located at the outer mitochondrial membrane, has been described in yeast (Figure 1). The MAD target proteins are either genuine outer membrane proteins or proteins from other mitochondrial subcompartments which have been retro-translocated to the outer membrane. These proteins are delivered to the proteasome utilizing CDC48 and specific adaptor proteins in the cytoplasm [16]. Yet another PQC pathway is the degradation of excess and damaged mitochondria via mitophagy. The impact of this pathway on fungal aging is currently a matter of intensive investigations. The individual PQC pathways have a strong impact on cellular fitness and on fungal lifespan. For example, overexpression of Lon results in lifespan extension in P. anserina [17], while deletion of the gene shortens lifespan in P. anserina and yeast [18,19]. As another example, lack of the i-AAA protease PaIAP and of PaCLPP decreases lifespan of P. anserina at elevated growth temperature [20,21]. While these observations are in concordance with the role of these proteases in PQC pathways, other characteristics of deletion mutants are counter-intuitive. For instance, at 27 8C growth temperature the deletion of the PaIap and PaClpP lead to an extended lifespan [20,21]. Also in a yeast mutant lacking the inner membrane protease m-AAA lifespan extension has been described [22]. Interestingly, in this mutant, cytoplasmic mRNA translation is reduced, suggesting that mitochondrial proteases affect different cytoplasmic processes in a rather unexpected way. This idea is supported by several findings: the yeast i-AAA protease YME1 is necessary for processing of mitophagy receptor protein ATG32 and required for efficient mitophagy [23]. In the nematode Caenorhabditis elegans LON protease and the CLPXP complex have been implicated in the ‘mitochondrial unfolded protein response’ [24,25,26], a complex stress-induced pathway signaling from mitochondria to the nucleus. www.sciencedirect.com
Stability of mitochondrial DNA The degradation of damaged mitochondrial proteins is certainly an efficient strategy to counteract the adverse effects (e.g., ROS generation) resulting from damaged proteins. However, on the long-run it is also essential to replace the degraded proteins by new functional ones. For mitochondrial proteins this is a concerted action controlled by genes encoded in the nuclear DNA and the mitochondrial DNA (mtDNA). Thus, keeping the genetic information functional over the lifetime is of paramount importance. In fact, in particular in fungi, the mtDNA appears to be a molecular ‘achilles heel’. Early studies in P. anserina and some Neurospora species which display well-defined aging phenotypes identified extensive, age-related reorganizations of the mtDNA which are linked to activity of mobile genetic elements acting as mutators. In P. anserina they are circular in structure and derived from the standard mtDNA. In Neurospora both, circular as well as linear plasmids are active. In all cases, the autonomous elements integrate into the standard mtDNA causing deleterious mutations. During aging, functional mtDNA molecules encoding a number of essential proteins of the respiratory chain, as well as rRNAs and tRNAs crucial for mitochondrial protein biosynthesis, are replaced by defective molecules (Figure 2). Consequently, mitochondrial biogenesis is affected and mitochondrial propagation and remodeling of damaged components is impaired giving rise to degeneration and death of individual mycelia. At this point it should be noted that prior to the accumulation of gross mtDNA reorganization in aged cultures sexual reproduction via ascospores gives rise to a new generation of cultures. Those cultures which originate from ascospores to which non-reorganized mtDNA molecules have been distributed during spore formation give rise to new juvenile cultures, while those with grossly rearranged mtDNAs lead to cultures with a shortened life. A causal relationship between mtDNA instability and lifespan similar to what has been elaborated for some filamentous fungi is also implied in budding yeast. The demonstration and characterization of the corresponding processes have been repeatedly reviewed [6,27,28].
Mitochondrial dynamics Mitochondria are no static organelles but do represent a dynamic population of units which, via fission and fusion, are able to adapt to changing physiological situations (e.g. nutrients, stressors) [29]. Depending on the frequency of fission and fusion morphotypes differ from fragmented to network structure (Figure 3). Beside the adaptation to physiological conditions, mitochondrial fusion is important for stabilization and replication of mtDNA, a complex mechanism involving mitochondrial and cytosolic factors [30]. In yeast, fusion of mitochondria depends on three core components namely FZO1, MGM1 and UGO1. The two GTPases FZO1 and MGM1 orchestrate outer and inner membrane fusion, respectively, while UGO1 acts as Current Opinion in Microbiology 2014, 22:1–7
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Age-related reorganization of mtDNA. During aging of some filamentous fungi, mtDNA (green circles) becomes mutated (red circles with asterisks). These molecules accumulate, leading to a block in gene expression of mtDNA-encoded genes. Consequently, remodeling of affected components like the respiratory chain, which is depending on both, nuclear as well as mtDNA-encoded proteins is impaired. The scheme also indicates initial agerelated changes in mitochondrial ultra-structure from lamellar to vesicular architecture.
a linker between those two [31,32]. Mitochondrial fission relies on the mitochondrial outer membrane anchor protein FIS1, the WD40 domain containing linker protein MDV1, and the dynamin-related protein DNM1, which oligomerizes around mitochondria in spirals and separates them by GTP hydrolysis [33]. In mammalian cells, it has been shown that damaged mitochondrial parts can be separated from the network in a DRP1-(human DNM1 homolog)dependent manner, allowing for selective degradation of impaired mitochondria by mitophagy [34] and conserving a ‘healthy’ population of tubular mitochondria. This morphotype is strongly associated with respiratory active cells while fragmented mitochondria are predominant in resting, non-dividing cells [35]. During aging, mitochondria change their morphology from tubular to fragmented. The fragmentation process can be delayed by deletion of fission components. In some systems, mitochondrial morphology has been linked to programmed cell death (PCD) and aging. A first study reported that deletion of Dnm1 of yeast and P. anserina leads to an extended lifespan of the two fungi via an effective delay of PCD. This effect is linked to an extended period of time mitochondria are filamentous or of network-like morphology [36]. In concordance, in yeast, inhibition of fusion via deletion of Mgm1 or Fzo1deficiency leads to a shortened lifespan [37,38]. In addition, fragmentation of mitochondria can be induced by stressors like acetic acid [39], oxidative stress (H2O2) [40] or ethanol [41] as well as mutations in genes, regulating stress response (Whi2) [42], mRNA turnover (Lsm4) [43] or biosynthesis of glycoproteins (Wbp1-1) [44]. All these factors lead to the induction of PCD and lifespan shortening. A recent computational study, in which the effect of mitochondrial fission/fusion, mitophagy, ROS damaging and mitochondrial biogenesis was modelled revealed that mitochondrial dynamics is advantageous when mitochondria Current Opinion in Microbiology 2014, 22:1–7
are only marginally damaged. In contrast, a deceleration of fusion-fission cycles appears to be effective in maintaining mitochondrial quality when mitochondrial damage is increased [45,46].
Programmed cell death PCD has been demonstrated to occur in the unicellular yeast but also in multicellular, filamentous fungi like P. anserina [47]. This pathway is controlled by mitochondria and brings life of individuals to an end. Recent investigations in P. anserina have unraveled distinct molecular processes and changes in mitochondrial morphology and ultra-structure which occur in the last phase of the life cycle (Figure 3). While juvenile and middle-aged mitochondria are mainly filamentous in structure, they are punctate in senescent cultures [36]. In addition, the ultra-structure changes from mitochondria with few lamellar cristae to a vesicular structure in senescent mitochondria [48]. In parallel, PCD is induced. Cyclophilin D (CYPD) appears to be an important regulator of this process. The abundance of this peptidyl-cis,transprolyl-isomerase increases during aging [49]. Overexpression of the gene coding for CYPD accelerates PCD and aging. The application of the CYPD inhibitor cyclosporine A leads to a reversion of effects. CYPD is a welldefined regulator of the mitochondrial permeability transition pore (mPTP) consisting of a macromolecular protein complex of yet undefined composition. Recent studies suggest the F1F0-ATP synthase as a component of this complex [50]. The binding of CYPD to the ATP synthase may lead to mPTP opening, entrance of water and low molecular solutes and finally the rupture of the outer mitochondrial membrane and the release of apoptogens like cytochrome c. The F1F0-ATP synthase is also a key player in processes leading to changes in www.sciencedirect.com
Fungal aging Bernhardt, Hamann and Osiewacz 5
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Mitochondrial dynamics and aging. (A) During aging, the morphology of mitochondria changes from a tubular network in juvenile cultures to fragmented mitochondria (B). (a) In yeast, the fission factors DNM1, MDV1, and FIS1 act jointly together in separating impaired parts (red asterisks) of tubular mitochondria from respiratory-competent parts of the network. Subsequently, fragmented mitochondria can undergo mitophagy initiated via the interaction of outer membrane-bound ATG32 to the linker protein ATG11 and ATG8 at the pre-autophagosomal membrane (phagophore). Consecutively, growth of the separation membrane of the phagophore leads to the engulfment of the mitochondrion in an autophagosome that fuses with the vacuole. The mitochondrion is subsequently degraded. (b) In yeast, mitochondrial fusion depends on FZO1, MGM1 and UGO1 which lead to the tethering of the membranes of two adjacent mitochondria. Fusion of the membranes results in the dissociation of mitochondrial respiratory supercomplex III2IV2 (1), and the YME1-dependent degradation of complex IV (2). This is followed by generation of a ROS signal which activates mtDNA replication (3). (B) Aging of mitochondria leads to changes of membrane architecture. In P. anserina, mitochondria with lamellar cristae are functional (blue), while those with a vesicular structure are defective (pink). (c) Finally, rupture of the outer membrane leads to the release of apoptogens like cytochrome c (green) and the induction of programmed cell death.
mitochondrial membrane architecture. Dimers of F1F0ATP synthase are normally located at the ridges of the inner mitochondrial membrane [51]. During aging, the lamellar cristae recede and finally the F1F0-ATP synthase dimers dissociate leading to the formation of the vesicular architecture of senescent mitochondria. According to a model, the process is initiated by binding of CYPD to F1F0-ATP synthase dimers [52]. The inner mitochondrial membrane comes occasionally into close contact with the outer membrane. At these contact sites electron dense material appears. In electron micrographs, breakage of the outer membrane in the vicinity of contact sites and the release of vesicles from mitochondria has been observed [52]. It is now of particular interest to unravel the detailed molecular mechanisms controlling the observed series of events. www.sciencedirect.com
Conclusions and perspectives Various pathways have been identified acting at different levels to constitute a complex network of pathways controlling mitochondrial quality. These pathways act at the molecular (e.g., ROS scavenging, repair or degradation of impaired molecules) and the organellar (i.e., mitochondrial dynamics, mitophagy) level. Each individual pathway is limited in its capacity, but failure of one pathway may be compensated by the induction of a pathway of higher hierarchy. Currently neither the individual pathways and their regulation nor the cross-talk between pathways are unraveled in sufficient detail. This knowledge holds a key to understand and intervene into processes leading to the induction of PCD in fungi which brings life of these organisms to an end. In contrast, in mammals PCD constitutes another cellular surveillance Current Opinion in Microbiology 2014, 22:1–7
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level protecting against the development of cancer. The detailed understanding of the involved mechanisms by which mitochondria control the induction of PCD thus is an important issue in biomedicine. Fungi are of important value to experimentally unravel the involved mechanism.
Acknowledgements
15. Luce K, Weil AC, Osiewacz HD: Mitochondrial protein quality control systems in aging and disease. Adv Exp Med Biol 2010, 694:108-125. 16. Taylor EB, Rutter J: Mitochondrial quality control by the ubiquitin–proteasome system. Biochem Soc Trans 2011, 39:1509-1513. This review summarizes ‘mitochondria-associated degradation’ (MAD) of mitochondrial proteins by the ubiquitin–proteasome system. 17. Luce K, Osiewacz HD: Increasing organismal healthspan by enhancing mitochondrial protein quality control. Nat Cell Biol 2009, 11:852-858.
HDO is supported by the German Ministry of Education and Research (GerontoMitoSys, FKZ0315584A), the Deutsche Forschungsgemeinschaft (Os75/15-1), the Frankfurt Autophagy Network (FAN), the state of Hesse via the LOEWE instrument (projects: Integrated Fungal Research and Ubiquitin Networks), the Center of Membrane Proteomics (CMP), Frankfurt, and the Cluster of Excellence Frankfurt ‘Macromolecular Complexes’ (CEF).
18. Adam C, Picard M, Dequard-Chablat M, Sellem CH, HermannLe DS, Contamine V: Biological roles of the Podospora anserina mitochondrial Lon protease and the importance of its Ndomain. PLoS ONE 2012, 7:e38138.
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30. Hori A, Yoshida M, Ling F: Mitochondrial fusion increases the mitochondrial DNA copy number in budding yeast. Genes to Cells 2011, 16:527-544. The authors monitored mitochondrial fusion using Bimolecular Fluorescence Complementation (BiFC) and demonstrated that mitochondrial fusion induces mtDNA synthesis in a YME1-dependent manner, which prevents the generation of mtDNA-lacking mitochondria. 31. Anton F, Fres JM, Schauss A, Pinson B, Praefcke GJ, Langer T, Escobar-Henriques M: Ugo1 and Mdm30 act sequentially during Fzo1-mediated mitochondrial outer membrane fusion. J Cell Sci 2011, 124:1126-1135. 32. Abutbul-Ionita I, Rujiviphat J, Nir I, McQuibban GA, Danino D: Membrane tethering and nucleotide-dependent conformational changes drive mitochondrial genome maintenance (Mgm1) protein-mediated membrane fusion. J Biol Chem 2012, 287:36634-36638. Using cryo-electron microscopy and in vitro liposome fusion assays it is demonstrated that the dynamin-related protein MGM1 is able to tether opposing membranes. This study provides first insights into the mechanistic details of membrane fusion and cristae maintenance. 33. Westermann B: Mitochondrial fusion and fission in cell life and death. Nat Rev Mol Cell Biol 2010, 11:872-884. This review provides detailed information on the mechanisms and components involved in mitochondrial dynamics. It stresses the importance of mitochondrial dynamics for other cellular pathways like mitophagy and apoptosis. 34. Twig G, Elorza A, Molina AJ, Mohamed H, Wikstrom JD, Walzer G, Stiles L, Haigh SE, Katz S, Las G, Alroy J, Wu M, Py BF, Yuan J, Deeney JT, Corkey BE, Shirihai OS: Fission and selective fusion govern mitochondrial segregation and elimination by autophagy. EMBO J 2008, 27:433-446. 35. Merz S, Hammermeister M, Altmann K, Du¨rr M, Westermann B: Molecular machinery of mitochondrial dynamics in yeast. Biol Chem 2007, 388:917-926. 36. Scheckhuber CQ, Erjavec N, Tinazli A, Hamann A, Nystro¨m T, Osiewacz HD: Reducing mitochondrial fission results in increased life span and fitness of two fungal ageing models. Nat Cell Biol 2007, 9:99-105. The study describes for the first time a link between mitochondrial dynamics and lifespan control in two fungi. Mutants impaired in different mitochondrial fission factors (DNM1, FIS1) were shown to have an increased lifespan without deleterious side effects. 37. Scheckhuber CQ, Wanger RA, Mignat CA, Osiewacz HD: Unopposed mitochondrial fission leads to severe lifespan shortening. Cell Cycle 2011, 10:3105-3110. 38. Kurashima K, Chae M, Inoue H, Hatakeyama S, Tanaka S: A uvs-5 strain is deficient for a mitofusin gene homologue, fzo1, involved in maintenance of long life span in Neurospora crassa. Eukaryot Cell 2013, 12:233-243. 39. Pereira C, Chaves S, Alves S, Salin B, Camougrand N, Manon S, Sousa MJ, Corte-Real M: Mitochondrial degradation in acetic acid-induced yeast apoptosis: the role of Pep4 and the ADP/ ATP carrier. Mol Microbiol 2010, 76:1398-1410. 40. Palermo V, Falcone C, Mazzoni C: Apoptosis and aging in mitochondrial morphology mutants of S. cerevisiae. Folia Microbiol (Praha) 2007, 52:479-483. 41. Kitagaki H, Araki Y, Funato K, Shimoi H: Ethanol-induced death in yeast exhibits features of apoptosis mediated by mitochondrial fission pathway. FEBS Lett 2007, 581:2935-2942. 42. Leadsham JE, Miller K, Ayscough KR, Colombo S, Martegani E, Sudbery P, Gourlay CW: Whi2p links nutritional sensing to actin-dependent Ras-cAMP-PKA regulation and apoptosis in yeast. J Cell Sci 2009, 122:706-715. 43. Palermo V, Falcone C, Calvani M, Mazzoni C: Acetyl-L-carnitine protects yeast cells from apoptosis and aging and inhibits mitochondrial fission. Aging Cell 2010, 9:570-579.
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44. Sesaki H, Jensen RE: Ugo1p links the Fzo1p and Mgm1p GTPases for mitochondrial fusion. J Biol Chem 2004, 279:28298-28303. 45. Figge MT, Reichert AS, Meyer-Hermann M, Osiewacz HD: Deceleration of fusion–fission cycles improves mitochondrial quality control during aging. PLoS Comput Biol 2012, 8:e1002576. 46. Figge MT, Osiewacz HD, Reichert AS: Quality control of mitochondria during aging: is there a good and a bad side of mitochondrial dynamics? Bioessays 2013, 35:314-322. 47. Hamann A, Brust D, Osiewacz HD: Apoptosis pathways in fungal growth, development and ageing. Trends Microbiol 2008, 16:276-283. 48. Brust D, Daum B, Breunig C, Hamann A, Ku¨hlbrandt W, Osiewacz HD: Cyclophilin D links programmed cell death and organismal aging in Podospora anserina. Aging Cell 2010, 9:761-775. In this work, changes in the mitochondrial membrane architecture are demonstrated to occur during aging of Podospora anserina. These changes are linked to the peptidyl-cis,trans-prolyl-isomerase cyclophilin D, a known regulator of the mitochondrial transition pore (mPTP). During aging, the abundance of this protein increases. Overexpression of the gene coding for cyclophilin D accelerates the remodeling of the inner mitochondrial membrane and programmed cell death and decreases lifespan. These effects can be reversed by the cyclophilin D inhibitor cyclosporine A. The established system bears a great potential to experimentally address the composition and function of the yet insufficiently characterized mPTP. 49. Groebe K, Krause F, Kunstmann B, Unterluggauer H, Reifschneider NH, Scheckhuber CQ, Sastri C, Stegmann W, Wozny W, Schwall GP, Poznanovic S, Dencher NA, Jansen-Du¨rr P, Osiewacz HD, Schrattenholz A: Differential proteomic profiling of mitochondria from Podospora anserina, rat and human reveals distinct patterns of age-related oxidative changes. Exp Gerontol 2007, 42:887-898. 50. Giorgio V, von Stockum S, Antoniel M, Fabbro A, Fogolari F, Forte M, Glick GD, Petronilli V, Zoratti M, Szabo I, Lippe G, Bernardi P: Dimers of mitochondrial ATP synthase form the permeability transition pore. Proc Natl Acad Sci USA 2013, 110:5887-5892. In this study, the binding site of cyclophilin D, the regulator of mitochondrial permeability transition pore (mPTP), to the F1F0-ATP synthase is reported. The data suggest this mitochondrial protein complex as a component of the mitochondrial permeability transition pore, which, although intensively investigated, is currently elusive in its protein composition. 51. Davies KM, Strauss M, Daum B, Kief JH, Osiewacz HD, Rycovska A, Zickermann V, Ku¨hlbrandt W: Macromolecular organization of ATP synthase and complex I in whole mitochondria. Proc Natl Acad Sci USA 2011, 108:14121-14126. The study describes the molecular arrangement of respiratory chain complexes in mitochondria of different fungi, bovine heart and potato by the use of electron cryotomography. In all species the arrangement of proton pumping complexes on flat cristae membranes and ATP synthase dimer rows along cristae edges was conserved, suggesting that this positioning ensures optimal conditions for ATP synthesis. 52. Daum B, Walter A, Horst A, Osiewacz HD, Ku¨hlbrandt W: Age dependent dissociation of ATP synthase dimers and loss of inner-membrane cristae in mitochondria. Proc Natl Acad Sci USA 2013, 110:15301-15306. The authors unravel a series of events by electron cryotomography leading to age-related changes in mitochondrial ultra-structure and suggest a model leading to the rupture of the outer membrane and the induction of programmed cell death during aging of Podospora anserina. In mitochondria from juvenile cultures cristae are predominantly lamellar and contain rows of F1F0 ATP synthase dimers at the sites of the greatest convex curvature. During aging the cristae recede, F1F0 ATP synthase dimers dissociate leading to a vesicular architecture of mitochondria. At this time the inner membrane shows a concave structure and occasionally comes into contact with the outer member. These sides appear to be prone to rupture of the outer membrane and release of apoptogens leading to the induction of programmed cell death.
Current Opinion in Microbiology 2014, 22:1–7
ScienceDirect The role of mitochondria in fungal aging Dominik Bernhardt, Andrea Hamann and Heinz D Osiewacz Time-dependent impairments of mitochondrial function play a key role in biological aging. Work on fungal aging models has been instrumental in unraveling basic mechanisms leading to mitochondrial dysfunction and the identification of different pathways active in keeping mitochondria ‘healthy’ over time. Pathways including those involved in reactive oxygen scavenging, repair of damage, proteostasis, mitochondrial dynamics, and biogenesis, are interconnected and part of a complex quality control system. The individual components of this network are limited in capacity. However, if the capacity of one pathway is overwhelmed, another one may be activated. The mechanisms controlling the underlying cross-talk are poorly understood and subject of intensive investigation. Addresses Institute of Molecular Biosciences and Cluster of Excellence Frankfurt Macromolecular Complexes, Department of Biosciences, J.W. Goethe University, Frankfurt, Germany Corresponding author: Osiewacz, Heinz D ([email protected])
Saccharomyces cerevisiae can generate. This number is now defined as the replicative lifespan and the process is termed replicative aging [4,5]. Subsequently, strains of the genus Neurospora and Aspergillus with a limited replicative capacity were described and analyzed (reviewed in [6]). More recent surveys identified rapid senescence to occur frequently in species of coprophilous Sordariomycetes [7]. Apart from replicative aging, another type of aging was identified and studied in yeast. This process, termed chronological aging, describes the period of time a non-dividing yeast cell can survive. Both processes are controlled by common but also different traits and pathways. Here we specifically focus on replicative aging (also referred to as ‘proliferative’ or ‘mitotic’ aging). Our emphasis lies on genetic traits and molecular pathways that affect the pace of aging. More specifically, we concentrate on the role of mitochondria, which have been identified as key determinants of fungal aging and have stimulated aging research in other organisms.
Current Opinion in Microbiology 2014, 22:1–7 This review comes from a themed issue on Growth and development: eukaryotes Edited by Michael Bo¨lker
http://dx.doi.org/10.1016/j.mib.2014.09.007 1369-5274/# 2014 Elsevier Ltd. All rights reserved.
Introduction It is widely thought that fungi propagate indefinitely and may give rise to huge individual colonies, like it has been reported for a mycelium of Armillaria bulbosa, with an estimated weight of at least 10,000 kg and an age of approximately 1500 years [1]. This view is currently changing. More and more fungi with a limited vegetative growth have been described. Historically, the first description of limited fungal growth dates back to a study of George Rizet in the early 1950s [2]. He observed that, during vegetative growth, cultures of the filamentous ascomycete Podospora anserina show characteristic time-related changes which he collectively described as ‘senescence syndrome’. Finally, the peripheral hyphae die. Only a few years later, another type of fungal aging was described by Mortimer and Johnston [3], the finite number of daughter cells an individual mother cell of www.sciencedirect.com
Respiratory metabolism The best known function of mitochondria is oxygenic energy conservation: the generation of adenosine triphosphate from energy-rich compounds. Since it is known that part of this process relies on electron transport at the respiratory chain and that the majority of all cellular reactive oxygen species (ROS) are generated by this process, a link of mitochondria to aging has been conceptionalized in the ‘mitochondrial free radical theory of aging’ (MFRTA) [8]. This theory states that aging is caused by the accumulation of ROS-induced damage of biomolecules including nucleic acids, lipids, and proteins and the resulting adverse effects. In fungi, this link is well demonstrated. In particular in P. anserina which, in contrast to yeast, contains a standard respiratory chain organized in different supercomplexes (Figure 1) and additional, alternative respiratory components, an impact of respiration on aging has been demonstrated. In different mutants with impaired respiratory complex III or IV, an alternative terminal oxidase (PaAOX) is induced. Compared to standard respiration, the alternative respiration generates less ROS and the corresponding strains are characterized by an increased lifespan [9–12]. Also in yeast, which lacks complex I and alternative oxidase, a connection between ROS, lifespan, and respiration has been observed. Caloric restriction was found to lead to a decreased ROS release per consumed O2. This decrease in ROS levels is thought to be responsible for lifespan extension and can be mimicked by artificially increased respiration via uncouplers [13]. Current Opinion in Microbiology 2014, 22:1–7
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Mitochondrial protein quality control. A number of different proteolytic pathways are active to keep mitochondria functional. LON/PIM1 and CLPXP are matrix protease complexes involved in the degradation of damaged proteins, which cannot be repaired by the methionine sulfoxide reductases MSRA and MSRB. The peptides generated by these proteases are delivered to the cytosol via ABC transporters and PORIN. In the cytosol they induce the mitochondrial ‘unfolded protein response’ (UPRmt), signaling mitochondrial dysfunction to the nucleus. In the inner mitochondrial membrane proteases like i-AAA/YME1, m-AAA and others are involved in protein processing. In yeast, YME1 is also active in the degradation of access, non-assembled subunit 2 of the cytochrome c oxidase (COX2) of the respiratory chain complexes and supercomplexes. Also in yeast, m-AAA is active in the assembly of F1F0-ATP synthase (V). Damaged proteins from inside the mitochondrion can be retro-translocated to the outer membrane. These and other outer membrane proteins become ubiquitinated by E3 ligases. CDC48 and adaptor proteins like VMS1 deliver the ubiquitinated protein to the proteasome. Respiratory chain supercomplexes (I2II1III2IV, I1III2IV2, I1III2, III2) as they were described in mitochondria of P. anserina are indicated. IM: inner mitochondrial membrane, MM: mitochondrial matrix.
Mitochondrial protein quality control (PQC) Apart from reduced ROS production, scavenging of these molecules affects cellular ROS levels and can reduce oxidative stress and molecular damage. Mitochondrial Current Opinion in Microbiology 2014, 22:1–7
superoxide dismutase and peroxidases are part of a cellular scavenging network and specifically involved in mitochondrial ROS balancing. Protein damage can also be repaired by methionine sulfoxide reductases (MSRA/ www.sciencedirect.com
Fungal aging Bernhardt, Hamann and Osiewacz 3
B) via the reduction of oxidized methionine residues (Figure 1). Degradation of damaged proteins is yet another way of PQC. Several molecular chaperones and proteases are located in the different mitochondrial subcompartments [14], monitor the folding status of different target proteins and remove excess or damaged proteins. In the mitochondrial matrix, the proteases LON (PIM1 in yeast) and CLPXP (in hyphal fungi, yeasts lack the protease component CLPP) degrade damaged proteins. Other proteases, like i-AAA protease (YME1 in yeast, PaIAP in P. anserina), and m-AAA protease are located in the mitochondrial inner membrane. Both proteases are involved in processing and regulation of mitochondrial proteins like OPA1 or subunits 6, 8, and 9 of the F1F0-ATP synthase complex (reviewed in [15]). In yeast, YME1 is also known to degrade COX2, a subunit of respiratory complex IV. Recently, ‘mitochondria-associated degradation’ (MAD), a pathway which leads to the proteasomal degradation of mitochondrial proteins located at the outer mitochondrial membrane, has been described in yeast (Figure 1). The MAD target proteins are either genuine outer membrane proteins or proteins from other mitochondrial subcompartments which have been retro-translocated to the outer membrane. These proteins are delivered to the proteasome utilizing CDC48 and specific adaptor proteins in the cytoplasm [16]. Yet another PQC pathway is the degradation of excess and damaged mitochondria via mitophagy. The impact of this pathway on fungal aging is currently a matter of intensive investigations. The individual PQC pathways have a strong impact on cellular fitness and on fungal lifespan. For example, overexpression of Lon results in lifespan extension in P. anserina [17], while deletion of the gene shortens lifespan in P. anserina and yeast [18,19]. As another example, lack of the i-AAA protease PaIAP and of PaCLPP decreases lifespan of P. anserina at elevated growth temperature [20,21]. While these observations are in concordance with the role of these proteases in PQC pathways, other characteristics of deletion mutants are counter-intuitive. For instance, at 27 8C growth temperature the deletion of the PaIap and PaClpP lead to an extended lifespan [20,21]. Also in a yeast mutant lacking the inner membrane protease m-AAA lifespan extension has been described [22]. Interestingly, in this mutant, cytoplasmic mRNA translation is reduced, suggesting that mitochondrial proteases affect different cytoplasmic processes in a rather unexpected way. This idea is supported by several findings: the yeast i-AAA protease YME1 is necessary for processing of mitophagy receptor protein ATG32 and required for efficient mitophagy [23]. In the nematode Caenorhabditis elegans LON protease and the CLPXP complex have been implicated in the ‘mitochondrial unfolded protein response’ [24,25,26], a complex stress-induced pathway signaling from mitochondria to the nucleus. www.sciencedirect.com
Stability of mitochondrial DNA The degradation of damaged mitochondrial proteins is certainly an efficient strategy to counteract the adverse effects (e.g., ROS generation) resulting from damaged proteins. However, on the long-run it is also essential to replace the degraded proteins by new functional ones. For mitochondrial proteins this is a concerted action controlled by genes encoded in the nuclear DNA and the mitochondrial DNA (mtDNA). Thus, keeping the genetic information functional over the lifetime is of paramount importance. In fact, in particular in fungi, the mtDNA appears to be a molecular ‘achilles heel’. Early studies in P. anserina and some Neurospora species which display well-defined aging phenotypes identified extensive, age-related reorganizations of the mtDNA which are linked to activity of mobile genetic elements acting as mutators. In P. anserina they are circular in structure and derived from the standard mtDNA. In Neurospora both, circular as well as linear plasmids are active. In all cases, the autonomous elements integrate into the standard mtDNA causing deleterious mutations. During aging, functional mtDNA molecules encoding a number of essential proteins of the respiratory chain, as well as rRNAs and tRNAs crucial for mitochondrial protein biosynthesis, are replaced by defective molecules (Figure 2). Consequently, mitochondrial biogenesis is affected and mitochondrial propagation and remodeling of damaged components is impaired giving rise to degeneration and death of individual mycelia. At this point it should be noted that prior to the accumulation of gross mtDNA reorganization in aged cultures sexual reproduction via ascospores gives rise to a new generation of cultures. Those cultures which originate from ascospores to which non-reorganized mtDNA molecules have been distributed during spore formation give rise to new juvenile cultures, while those with grossly rearranged mtDNAs lead to cultures with a shortened life. A causal relationship between mtDNA instability and lifespan similar to what has been elaborated for some filamentous fungi is also implied in budding yeast. The demonstration and characterization of the corresponding processes have been repeatedly reviewed [6,27,28].
Mitochondrial dynamics Mitochondria are no static organelles but do represent a dynamic population of units which, via fission and fusion, are able to adapt to changing physiological situations (e.g. nutrients, stressors) [29]. Depending on the frequency of fission and fusion morphotypes differ from fragmented to network structure (Figure 3). Beside the adaptation to physiological conditions, mitochondrial fusion is important for stabilization and replication of mtDNA, a complex mechanism involving mitochondrial and cytosolic factors [30]. In yeast, fusion of mitochondria depends on three core components namely FZO1, MGM1 and UGO1. The two GTPases FZO1 and MGM1 orchestrate outer and inner membrane fusion, respectively, while UGO1 acts as Current Opinion in Microbiology 2014, 22:1–7
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Age-related reorganization of mtDNA. During aging of some filamentous fungi, mtDNA (green circles) becomes mutated (red circles with asterisks). These molecules accumulate, leading to a block in gene expression of mtDNA-encoded genes. Consequently, remodeling of affected components like the respiratory chain, which is depending on both, nuclear as well as mtDNA-encoded proteins is impaired. The scheme also indicates initial agerelated changes in mitochondrial ultra-structure from lamellar to vesicular architecture.
a linker between those two [31,32]. Mitochondrial fission relies on the mitochondrial outer membrane anchor protein FIS1, the WD40 domain containing linker protein MDV1, and the dynamin-related protein DNM1, which oligomerizes around mitochondria in spirals and separates them by GTP hydrolysis [33]. In mammalian cells, it has been shown that damaged mitochondrial parts can be separated from the network in a DRP1-(human DNM1 homolog)dependent manner, allowing for selective degradation of impaired mitochondria by mitophagy [34] and conserving a ‘healthy’ population of tubular mitochondria. This morphotype is strongly associated with respiratory active cells while fragmented mitochondria are predominant in resting, non-dividing cells [35]. During aging, mitochondria change their morphology from tubular to fragmented. The fragmentation process can be delayed by deletion of fission components. In some systems, mitochondrial morphology has been linked to programmed cell death (PCD) and aging. A first study reported that deletion of Dnm1 of yeast and P. anserina leads to an extended lifespan of the two fungi via an effective delay of PCD. This effect is linked to an extended period of time mitochondria are filamentous or of network-like morphology [36]. In concordance, in yeast, inhibition of fusion via deletion of Mgm1 or Fzo1deficiency leads to a shortened lifespan [37,38]. In addition, fragmentation of mitochondria can be induced by stressors like acetic acid [39], oxidative stress (H2O2) [40] or ethanol [41] as well as mutations in genes, regulating stress response (Whi2) [42], mRNA turnover (Lsm4) [43] or biosynthesis of glycoproteins (Wbp1-1) [44]. All these factors lead to the induction of PCD and lifespan shortening. A recent computational study, in which the effect of mitochondrial fission/fusion, mitophagy, ROS damaging and mitochondrial biogenesis was modelled revealed that mitochondrial dynamics is advantageous when mitochondria Current Opinion in Microbiology 2014, 22:1–7
are only marginally damaged. In contrast, a deceleration of fusion-fission cycles appears to be effective in maintaining mitochondrial quality when mitochondrial damage is increased [45,46].
Programmed cell death PCD has been demonstrated to occur in the unicellular yeast but also in multicellular, filamentous fungi like P. anserina [47]. This pathway is controlled by mitochondria and brings life of individuals to an end. Recent investigations in P. anserina have unraveled distinct molecular processes and changes in mitochondrial morphology and ultra-structure which occur in the last phase of the life cycle (Figure 3). While juvenile and middle-aged mitochondria are mainly filamentous in structure, they are punctate in senescent cultures [36]. In addition, the ultra-structure changes from mitochondria with few lamellar cristae to a vesicular structure in senescent mitochondria [48]. In parallel, PCD is induced. Cyclophilin D (CYPD) appears to be an important regulator of this process. The abundance of this peptidyl-cis,transprolyl-isomerase increases during aging [49]. Overexpression of the gene coding for CYPD accelerates PCD and aging. The application of the CYPD inhibitor cyclosporine A leads to a reversion of effects. CYPD is a welldefined regulator of the mitochondrial permeability transition pore (mPTP) consisting of a macromolecular protein complex of yet undefined composition. Recent studies suggest the F1F0-ATP synthase as a component of this complex [50]. The binding of CYPD to the ATP synthase may lead to mPTP opening, entrance of water and low molecular solutes and finally the rupture of the outer mitochondrial membrane and the release of apoptogens like cytochrome c. The F1F0-ATP synthase is also a key player in processes leading to changes in www.sciencedirect.com
Fungal aging Bernhardt, Hamann and Osiewacz 5
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Mitochondrial dynamics and aging. (A) During aging, the morphology of mitochondria changes from a tubular network in juvenile cultures to fragmented mitochondria (B). (a) In yeast, the fission factors DNM1, MDV1, and FIS1 act jointly together in separating impaired parts (red asterisks) of tubular mitochondria from respiratory-competent parts of the network. Subsequently, fragmented mitochondria can undergo mitophagy initiated via the interaction of outer membrane-bound ATG32 to the linker protein ATG11 and ATG8 at the pre-autophagosomal membrane (phagophore). Consecutively, growth of the separation membrane of the phagophore leads to the engulfment of the mitochondrion in an autophagosome that fuses with the vacuole. The mitochondrion is subsequently degraded. (b) In yeast, mitochondrial fusion depends on FZO1, MGM1 and UGO1 which lead to the tethering of the membranes of two adjacent mitochondria. Fusion of the membranes results in the dissociation of mitochondrial respiratory supercomplex III2IV2 (1), and the YME1-dependent degradation of complex IV (2). This is followed by generation of a ROS signal which activates mtDNA replication (3). (B) Aging of mitochondria leads to changes of membrane architecture. In P. anserina, mitochondria with lamellar cristae are functional (blue), while those with a vesicular structure are defective (pink). (c) Finally, rupture of the outer membrane leads to the release of apoptogens like cytochrome c (green) and the induction of programmed cell death.
mitochondrial membrane architecture. Dimers of F1F0ATP synthase are normally located at the ridges of the inner mitochondrial membrane [51]. During aging, the lamellar cristae recede and finally the F1F0-ATP synthase dimers dissociate leading to the formation of the vesicular architecture of senescent mitochondria. According to a model, the process is initiated by binding of CYPD to F1F0-ATP synthase dimers [52]. The inner mitochondrial membrane comes occasionally into close contact with the outer membrane. At these contact sites electron dense material appears. In electron micrographs, breakage of the outer membrane in the vicinity of contact sites and the release of vesicles from mitochondria has been observed [52]. It is now of particular interest to unravel the detailed molecular mechanisms controlling the observed series of events. www.sciencedirect.com
Conclusions and perspectives Various pathways have been identified acting at different levels to constitute a complex network of pathways controlling mitochondrial quality. These pathways act at the molecular (e.g., ROS scavenging, repair or degradation of impaired molecules) and the organellar (i.e., mitochondrial dynamics, mitophagy) level. Each individual pathway is limited in its capacity, but failure of one pathway may be compensated by the induction of a pathway of higher hierarchy. Currently neither the individual pathways and their regulation nor the cross-talk between pathways are unraveled in sufficient detail. This knowledge holds a key to understand and intervene into processes leading to the induction of PCD in fungi which brings life of these organisms to an end. In contrast, in mammals PCD constitutes another cellular surveillance Current Opinion in Microbiology 2014, 22:1–7
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level protecting against the development of cancer. The detailed understanding of the involved mechanisms by which mitochondria control the induction of PCD thus is an important issue in biomedicine. Fungi are of important value to experimentally unravel the involved mechanism.
Acknowledgements
15. Luce K, Weil AC, Osiewacz HD: Mitochondrial protein quality control systems in aging and disease. Adv Exp Med Biol 2010, 694:108-125. 16. Taylor EB, Rutter J: Mitochondrial quality control by the ubiquitin–proteasome system. Biochem Soc Trans 2011, 39:1509-1513. This review summarizes ‘mitochondria-associated degradation’ (MAD) of mitochondrial proteins by the ubiquitin–proteasome system. 17. Luce K, Osiewacz HD: Increasing organismal healthspan by enhancing mitochondrial protein quality control. Nat Cell Biol 2009, 11:852-858.
HDO is supported by the German Ministry of Education and Research (GerontoMitoSys, FKZ0315584A), the Deutsche Forschungsgemeinschaft (Os75/15-1), the Frankfurt Autophagy Network (FAN), the state of Hesse via the LOEWE instrument (projects: Integrated Fungal Research and Ubiquitin Networks), the Center of Membrane Proteomics (CMP), Frankfurt, and the Cluster of Excellence Frankfurt ‘Macromolecular Complexes’ (CEF).
18. Adam C, Picard M, Dequard-Chablat M, Sellem CH, HermannLe DS, Contamine V: Biological roles of the Podospora anserina mitochondrial Lon protease and the importance of its Ndomain. PLoS ONE 2012, 7:e38138.
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10. Borghouts C, Werner A, Elthon T, Osiewacz HD: Coppermodulated gene expression and senescence in the filamentous fungus Podospora anserina. Mol Cell Biol 2001, 21:390-399. 11. Stumpferl SW, Stephan O, Osiewacz HD: Impact of a disruption of a pathway delivering copper to mitochondria on Podospora anserina metabolism and life span. Eukaryotic Cell 2004, 3:200-211. 12. Gredilla R, Grief J, Osiewacz HD: Mitochondrial free radical generation and lifespan control in the fungal aging model Podospora anserina. Exp Gerontol 2006, 41:439-447. 13. Barros MH, Bandy B, Tahara EB, Kowaltowski AJ: Higher respiratory activity decreases mitochondrial reactive oxygen release and increases life span in Saccharomyces cerevisiae. J Biol Chem 2004, 279:49883-49888. 14. Koppen M, Langer T: Protein degradation within mitochondria: versatile activities of AAA proteases and other peptidases. Crit Rev Biochem Mol Biol 2007, 42:221-242. This review gives a comprehensive overview on the different pathways of mitochondrial protein degradation. Current Opinion in Microbiology 2014, 22:1–7
20. Weil A, Luce K, Dro¨se S, Wittig I, Brandt U, Osiewacz HD: Unmasking a temperature-dependent effect of the P. anserina i-AAA protease on aging and development. Cell Cycle 2011, 10:4280-4290. 21. Fischer F, Weil A, Hamann A, Osiewacz HD: Human CLPP reverts the longevity phenotype of a fungal ClpP deletion strain. Nat Commun 2013, 4:1397. 22. Delaney JR, Ahmed U, Chou A, Sim S, Carr D, Murakami CJ, Schleit J, Sutphin GL, An EH, Castanza A, Fletcher M, Higgins S, Jelic M, Klum S, Muller B, Peng ZJ, Rai D, Ros V, Singh M, Wende HV, Kennedy BK, Kaeberlein M: Stress profiling of longevity mutants identifies Afg3 as a mitochondrial determinant of cytoplasmic mRNA translation and aging. Aging Cell 2013, 12:156-166. 23. Wang K, Jin M, Liu X, Klionsky DJ: Proteolytic processing of Atg32 by the mitochondrial i-AAA protease Yme1 regulates mitophagy. Autophagy 2013, 9:1828-1836. The authors demonstrate that YME1-mediated processing of ATG32 is required for mitophagy providing new insights into the regulation of mitophagy activity. 24. Haynes CM, Petrova K, Benedetti C, Yang Y, Ron D: ClpP mediates activation of a mitochondrial unfolded protein response in C. elegans. Dev Cell 2007, 13:467-480. 25. Haynes CM, Yang Y, Blais SP, Neubert TA, Ron D: The matrix peptide exporter HAF-1 signals a mitochondrial UPR by activating the transcription factor ZC376.7 in C. elegans. Mol Cell 2010, 37:529-540. 26. Nargund AM, Pellegrino MW, Fiorese CJ, Baker BM, Haynes CM: Mitochondrial import efficiency of ATFS-1 regulates mitochondrial UPR activation. Science 2012, 337:587-590. The authors elaborate the mechanism by which mitochondrial dysfunction can be communicated to the nucleus. The transcription factor ATFS1 plays a key role in this process. Under normal conditions it becomes imported into mitochondria and is degraded. Mitochondrial stress however, leads to reduced import efficiency allowing ATFS-1 to translocate to the nucleus. 27. Scheckhuber CQ, Osiewacz HD: Podospora anserina: a model organism to study mechanisms of healthy ageing. Mol Genet Genomics 2008, 280:365-374. 28. Dunn CD: Running on empty: does mitochondrial DNA mutation limit replicative lifespan in yeast?: Mutations that increase the division rate of cells lacking mitochondrial DNA also extend replicative lifespan in Saccharomyces cerevisiae. Bioessays 2011, 33:742-748. The review summarizes the current state of knowledge about the impact of mtDNA mutations on yeast lifespan. It highlights the potential link between mtDNA loss and replicative lifespan. 29. Knorre DA, Popadin KY, Sokolov SS, Severin FF: Roles of mitochondrial dynamics under stressful and normal conditions in yeast cells. Oxid Med Cell Longev 2013, 2013:139491. www.sciencedirect.com
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30. Hori A, Yoshida M, Ling F: Mitochondrial fusion increases the mitochondrial DNA copy number in budding yeast. Genes to Cells 2011, 16:527-544. The authors monitored mitochondrial fusion using Bimolecular Fluorescence Complementation (BiFC) and demonstrated that mitochondrial fusion induces mtDNA synthesis in a YME1-dependent manner, which prevents the generation of mtDNA-lacking mitochondria. 31. Anton F, Fres JM, Schauss A, Pinson B, Praefcke GJ, Langer T, Escobar-Henriques M: Ugo1 and Mdm30 act sequentially during Fzo1-mediated mitochondrial outer membrane fusion. J Cell Sci 2011, 124:1126-1135. 32. Abutbul-Ionita I, Rujiviphat J, Nir I, McQuibban GA, Danino D: Membrane tethering and nucleotide-dependent conformational changes drive mitochondrial genome maintenance (Mgm1) protein-mediated membrane fusion. J Biol Chem 2012, 287:36634-36638. Using cryo-electron microscopy and in vitro liposome fusion assays it is demonstrated that the dynamin-related protein MGM1 is able to tether opposing membranes. This study provides first insights into the mechanistic details of membrane fusion and cristae maintenance. 33. Westermann B: Mitochondrial fusion and fission in cell life and death. Nat Rev Mol Cell Biol 2010, 11:872-884. This review provides detailed information on the mechanisms and components involved in mitochondrial dynamics. It stresses the importance of mitochondrial dynamics for other cellular pathways like mitophagy and apoptosis. 34. Twig G, Elorza A, Molina AJ, Mohamed H, Wikstrom JD, Walzer G, Stiles L, Haigh SE, Katz S, Las G, Alroy J, Wu M, Py BF, Yuan J, Deeney JT, Corkey BE, Shirihai OS: Fission and selective fusion govern mitochondrial segregation and elimination by autophagy. EMBO J 2008, 27:433-446. 35. Merz S, Hammermeister M, Altmann K, Du¨rr M, Westermann B: Molecular machinery of mitochondrial dynamics in yeast. Biol Chem 2007, 388:917-926. 36. Scheckhuber CQ, Erjavec N, Tinazli A, Hamann A, Nystro¨m T, Osiewacz HD: Reducing mitochondrial fission results in increased life span and fitness of two fungal ageing models. Nat Cell Biol 2007, 9:99-105. The study describes for the first time a link between mitochondrial dynamics and lifespan control in two fungi. Mutants impaired in different mitochondrial fission factors (DNM1, FIS1) were shown to have an increased lifespan without deleterious side effects. 37. Scheckhuber CQ, Wanger RA, Mignat CA, Osiewacz HD: Unopposed mitochondrial fission leads to severe lifespan shortening. Cell Cycle 2011, 10:3105-3110. 38. Kurashima K, Chae M, Inoue H, Hatakeyama S, Tanaka S: A uvs-5 strain is deficient for a mitofusin gene homologue, fzo1, involved in maintenance of long life span in Neurospora crassa. Eukaryot Cell 2013, 12:233-243. 39. Pereira C, Chaves S, Alves S, Salin B, Camougrand N, Manon S, Sousa MJ, Corte-Real M: Mitochondrial degradation in acetic acid-induced yeast apoptosis: the role of Pep4 and the ADP/ ATP carrier. Mol Microbiol 2010, 76:1398-1410. 40. Palermo V, Falcone C, Mazzoni C: Apoptosis and aging in mitochondrial morphology mutants of S. cerevisiae. Folia Microbiol (Praha) 2007, 52:479-483. 41. Kitagaki H, Araki Y, Funato K, Shimoi H: Ethanol-induced death in yeast exhibits features of apoptosis mediated by mitochondrial fission pathway. FEBS Lett 2007, 581:2935-2942. 42. Leadsham JE, Miller K, Ayscough KR, Colombo S, Martegani E, Sudbery P, Gourlay CW: Whi2p links nutritional sensing to actin-dependent Ras-cAMP-PKA regulation and apoptosis in yeast. J Cell Sci 2009, 122:706-715. 43. Palermo V, Falcone C, Calvani M, Mazzoni C: Acetyl-L-carnitine protects yeast cells from apoptosis and aging and inhibits mitochondrial fission. Aging Cell 2010, 9:570-579.
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44. Sesaki H, Jensen RE: Ugo1p links the Fzo1p and Mgm1p GTPases for mitochondrial fusion. J Biol Chem 2004, 279:28298-28303. 45. Figge MT, Reichert AS, Meyer-Hermann M, Osiewacz HD: Deceleration of fusion–fission cycles improves mitochondrial quality control during aging. PLoS Comput Biol 2012, 8:e1002576. 46. Figge MT, Osiewacz HD, Reichert AS: Quality control of mitochondria during aging: is there a good and a bad side of mitochondrial dynamics? Bioessays 2013, 35:314-322. 47. Hamann A, Brust D, Osiewacz HD: Apoptosis pathways in fungal growth, development and ageing. Trends Microbiol 2008, 16:276-283. 48. Brust D, Daum B, Breunig C, Hamann A, Ku¨hlbrandt W, Osiewacz HD: Cyclophilin D links programmed cell death and organismal aging in Podospora anserina. Aging Cell 2010, 9:761-775. In this work, changes in the mitochondrial membrane architecture are demonstrated to occur during aging of Podospora anserina. These changes are linked to the peptidyl-cis,trans-prolyl-isomerase cyclophilin D, a known regulator of the mitochondrial transition pore (mPTP). During aging, the abundance of this protein increases. Overexpression of the gene coding for cyclophilin D accelerates the remodeling of the inner mitochondrial membrane and programmed cell death and decreases lifespan. These effects can be reversed by the cyclophilin D inhibitor cyclosporine A. The established system bears a great potential to experimentally address the composition and function of the yet insufficiently characterized mPTP. 49. Groebe K, Krause F, Kunstmann B, Unterluggauer H, Reifschneider NH, Scheckhuber CQ, Sastri C, Stegmann W, Wozny W, Schwall GP, Poznanovic S, Dencher NA, Jansen-Du¨rr P, Osiewacz HD, Schrattenholz A: Differential proteomic profiling of mitochondria from Podospora anserina, rat and human reveals distinct patterns of age-related oxidative changes. Exp Gerontol 2007, 42:887-898. 50. Giorgio V, von Stockum S, Antoniel M, Fabbro A, Fogolari F, Forte M, Glick GD, Petronilli V, Zoratti M, Szabo I, Lippe G, Bernardi P: Dimers of mitochondrial ATP synthase form the permeability transition pore. Proc Natl Acad Sci USA 2013, 110:5887-5892. In this study, the binding site of cyclophilin D, the regulator of mitochondrial permeability transition pore (mPTP), to the F1F0-ATP synthase is reported. The data suggest this mitochondrial protein complex as a component of the mitochondrial permeability transition pore, which, although intensively investigated, is currently elusive in its protein composition. 51. Davies KM, Strauss M, Daum B, Kief JH, Osiewacz HD, Rycovska A, Zickermann V, Ku¨hlbrandt W: Macromolecular organization of ATP synthase and complex I in whole mitochondria. Proc Natl Acad Sci USA 2011, 108:14121-14126. The study describes the molecular arrangement of respiratory chain complexes in mitochondria of different fungi, bovine heart and potato by the use of electron cryotomography. In all species the arrangement of proton pumping complexes on flat cristae membranes and ATP synthase dimer rows along cristae edges was conserved, suggesting that this positioning ensures optimal conditions for ATP synthesis. 52. Daum B, Walter A, Horst A, Osiewacz HD, Ku¨hlbrandt W: Age dependent dissociation of ATP synthase dimers and loss of inner-membrane cristae in mitochondria. Proc Natl Acad Sci USA 2013, 110:15301-15306. The authors unravel a series of events by electron cryotomography leading to age-related changes in mitochondrial ultra-structure and suggest a model leading to the rupture of the outer membrane and the induction of programmed cell death during aging of Podospora anserina. In mitochondria from juvenile cultures cristae are predominantly lamellar and contain rows of F1F0 ATP synthase dimers at the sites of the greatest convex curvature. During aging the cristae recede, F1F0 ATP synthase dimers dissociate leading to a vesicular architecture of mitochondria. At this time the inner membrane shows a concave structure and occasionally comes into contact with the outer member. These sides appear to be prone to rupture of the outer membrane and release of apoptogens leading to the induction of programmed cell death.
Current Opinion in Microbiology 2014, 22:1–7
