Mitochondrial DNA from Podospora anserina. I. Isolation and characterization.

Mitochondrial (Mt) DNA from Podospora anserina was isolated and characterized with respect to density in CsCl, contour length and endonuclease restriction enzymes. The density of Mt DNA for four races examined was 1.694 g/cm3, compared with 1.712 g/cm3 for nuclear DNA. Extraction in the presence of a nuclease inhibitor, aurintricarboxylic acid and isolation in DAPI CsCl gradients allowed us to isolate high molecular weight DNA. Mt DNA isolated by total DNA extraction contained ca. 1% of circular molecules, 31 micron in contour length; Mt DNA isolated from purified mitochondria contained 2--4% of these 31 micron circles. Analysis with Eco RI restriction endonuclease revealed that each of the four races examined, s, A, T and E had a characteristic fragment pattern. Races s and A Mt DNA differed by only one fragment after Eco RI enzymatic digestion; similarly, these two DNA differed by only one or two fragments after Hae III digestion.     

Mitochondrial DNA from Podospora anserina. II. Properties of mutant DNA and multimeric circular DNA from senescent cultures.

Summary Mitochondrial (Mt) DNA from mitochondrial mutants of race s Podospora anserina and from senescent cultures of races s and A was examined. In mutants, we observed that fewer full length circles (31 ) were present; instead, smaller circles characteristic for each mutant sudied were found. Eco Rl digestion of these mutant MtDNAs indicated that in certain mutants, although specific fragments were absent, the total molecular weight of the fragments was not much different than wild-type.The properties of senescent MtDNA was strikingly different from either wild-type or mutant Mt DNA. First, a multimeric set of circular DNA was observed for both race s and A, with a monomeric repeat size of 0.89 . These circles ranged in size from 0.89 to greater than 20 ; only one molecule out of some 200 molecules was thought to be of full length (31 ). Density gradient analysis showed that there were two density species: a majority were at the same density as wild-type (1.694 g/cm³) and a second at 1.699 g/cm³. Most of the circular molecules from MtDNA isolated by either total DNA extraction or by extraction of DNA from isolated mitochondria were contained in the heavy DNA fraction. Eco R1 enzymatic digestion indicated that the light DNA had several fragments (amounting to about 23×10⁶ daltons) missing, compared with young, wild-type MtDNA. Heavy senescent MtDNA was not cleaved by Eco R1. Analysis with Hae III restriction endonuclease showed also that light senescent MtDNA was missing certain fragments. Heavy MtDNA of average size 20×10⁶ daltons, yielded only one fragment, 2,500 bp long, by digestion with Hae III restriction endonuclease. Digestion of heavy DNA with Alu I enzyme yielded 10 fragments totalling 2,570 bp. By three criteria, electron-microscopy, Eco R1 and Hae digestion, we conclude that the heavy MtDNA isolated from senescent cultures of Podospora anserina consisted of a monomeric tandemly repeating subunit of about 2,600 bp length.These results on the properties of senescent MtDNA are discussed with regard to the published properties of the rho ^- mutation in the yeast, S. cerevisiae.     

Mitochondrial metabolism and aging in the filamentous fungus Podospora anserina

The filamentous fungus Podospora anserina has a limited lifespan. In this organism, aging is systematically associated to mitochondrial DNA instability. We recently provided evidence that the respiratory function is a key determinant of its lifespan. Loss of function of the cytochrome pathway leads to the compensatory induction of an alternative oxidase, to a decreased production of reactive oxygen species and to a striking increase in lifespan. These changes are associated to the stabilization of the mitochondrial DNA. Here we review and discuss the links between these different parameters and their implication in the control of lifespan. Since we demonstrated the central role of mitochondrial metabolism in aging, the same relationship has been evidenced in several model systems from yeast to mice, confirming the usefulness of simple organisms as P. anserina for studying lifespan regulation.     

Aminopeptidasen des Ascomyceten Podospora anserina

Zusammenfassung Aus dem Wildstamm des Ascomyceten Podospora anserina wurden zwei Aminopeptidasen isoliert, die immunologisch nicht verwandt sind: P1 hat ein MG von ung. 140000, sie kann durch Hitzebehandlung aktiviert werden (z. B. 8 min bei 80° C um 100%) und wird von DFP (5 mM) vollständig gehemmt. Wie Hydrolyseversuche mit über 30 Oligopeptiden zeigten, hat sie eine ziemlich enge Substratspezifität (z. B. für Ala-Pro, Gly-Pro-Ala, Glu-Gly-Phe und Valinpeptide). P1 wird durch Behandlung mit 0,1% SDS nicht in Untereinheiten gespalten.P2, eine typische (Leucin) aminopeptidase, hat ein MG von etwa 70000, sie wird durch Hitze nicht aktiviert und durch DFP kaum gehemmt. P2 ist sehr unspezifisch und hydrolysiert alle getesteten Oligopeptide, wie z. B. Di- und Triglycin, Pro-, Asp-, Glu- und Trp-Peptide, die oxydierten Insulin-A- und (z. T.)-B-Ketten, jedoch nicht das native Protein. P2 wird durch 0,1% SDS in inaktive Untereinheiten (MG um 15000) gespalten, die reaggregieren können, wobei aktive Proteine von etwa 35000 bzw. 150000 entstehen, jedoch keine P2 (70000) mehr. Daraus und aus Untersuchungen mit der Immundiskelektrophorese wird geschlossen, daß die (Leucin)aminopeptidase P2 von P. anserina aus Untereinheiten besteht, die abhängig von noch unbekannten Bedingungen mehr oder weniger aggregiert vorliegen können.Dies wird auch durch die Verhältnisse bei der rhythmischen Mutante zonata bestätigt: Hier konnte P1 nicht nachgewiesen werden, dagegen wurde ein Enzym (Pz) isoliert, das serologisch mit P2 verwandt ist, in verdünnter Lösung jedoch ein MG von 35000 aufwies. Pz bildet in der Diskelektrophorese eine sehr breite Bande, der K _m -Wert des Enzyms (um 10^-3) für Leu-4-NA liegt um das 10fache höher als beim Wildenzym.Morphologische und cytochemische Ähnlichkeiten zwischen zonata und solchem wild-Mycel, in dem eine heterogenische (vegetative) Inkompatibilitätsreaktion abläuft (z. B. anomaler Wuchs, freie AP-Aktivität im Cytoplasma), werden diskutiert.

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Aminopeptidases from the ascomycete Podospora anserina Genetical differences between the wild strain and the mutant zonata

Summary Two immunologically unrelated aminopeptidases (P1 and P2) from the wild strain of the Ascomycete Podospora anserina were isolated and characterized. P1 has a molecular weight (MW) of about 140000, it can be activated by heat treatment (e. g. 8 min/80° C=100%) and is completely inhibited by DFP (5 mM). The enzyme specificity was tested with more than 30 oligopeptides as substrates, However, few of them were attacked significantly (Ala-Pro, Gly-Pro-Ala, Glu-Gly-Phe and two valine peptides). P1 is not cleaved into subunits by treatment with 0.1% SDS, but serological tests indicate the existence of a dimer in crude mycelial extracts.P2 (a typical leucine aminopeptidase) has a MW of about 70000. There is no heat activation and no inhibition by DFP. It has a large substrate range, hydrolyzing all oligopeptides tested, including di- and triglycine, pro-, asp-, glu-, and trp-peptides. Oxydized A- and B-chains of insuline are also partly digested but not the native protein. P2 is cleaved by 0.1% SDS into inactive subunits (MW about 15000) which may be reaggregated yielding active proteins with MWs of approximately 35000 and 150000 but no aggregates of the original MW of 70000.From these facts and from immunoelectrophoretical investigations it is concluded that the (leucine) aminopeptidase P2 is composed of subunits, which appear more or less aggregated, depending on conditions still unknown.This is corroborated by analyses of the clock mutant zonata: In this strain P1 could not be detected. An enzyme (Pz) was isolated which is serologically related to P2 but has a molecular weight of only 35000. Pz yields a broad band in disc electrophoresis and its K _m -value is tenfold increased as compared with the wildtype enzyme.Morphological and cytochemical similarities between the mutant zonata and wildtype mycelia displaying the action of heterogenic (vegetative) incompatibility (e. g. abnormal growth, free AP-activity in the cytoplasm) are discussed.

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Abkürzungen AP Aminopeptidase - DE Diskelektrophorese - DFP Diisopropylfluorphosphat - IEF Isoelektrische Fokussierung - Hb Hämoglobin - Leu-4-NA Leucin-4-nitroanilid - Leu--NA Leucin--naphthylamid - MG Molekulargewicht - RSA Rinderserumalbumin - SDS Natriumdodecylsulfat Mit Unterstützung der Deutschen Forschungsgemeinschaft und des Landesamtes für Forschung (NRW).     

Rhythmisches Mycelwachstum bei Podospora anserina

The correlation between membrane structure and morphogenesis in fungi was examined by studying the effect of surface-active and membrane-altering substances upon the colonial shape of Podospora anserina. The following results were obtained:

1. The alcohols 1-propanol, 1-butanol and 1-octanol inhibited hyphal elongation and thus caused zonations in the wild strain. In the clock-mutant zonata, however, the zonations disappeared at high concentrations of the alcohols.
2. The detergents sodium-dodecylsulfate, sodium-desoxycholate, triton X-100 and brij-35 also induced zonations, i.e. rhythmic growth. Growing on complete media, the sensibility of the strains used was markedly reduced.
3. The antibiotics nonactin, nigericin and monensin, too, inhibited growth and caused zonations, which, however, were light dependent. In contrast, valinomycin inhibited the elongation of the hyphae only.

The results are discussed as consequences of alterations in structure and features of the membranes.     

Suppression of Mitochondrial DNA Instability of Autosomal Dominant Forms of Progressive External Ophthalmoplegia-Associated ANT1 Mutations in Podospora anserina

Maintenance and expression of mitochondrial DNA (mtDNA) are essential for the cell and the organism. In humans, several mutations in the adenine nucleotide translocase gene ANT1 are associated with multiple mtDNA deletions and autosomal dominant forms of progressive external ophthalmoplegia (adPEO). The mechanisms underlying the mtDNA instability are still obscure. A current hypothesis proposes that these pathogenic mutations primarily uncouple the mitochondrial inner membrane, which secondarily causes mtDNA instability. Here we show that the three adPEO-associated mutations equivalent to A114P, L98P, and V289M introduced into the Podospora anserina ANT1 ortholog dominantly cause severe growth defects, decreased reactive oxygen species production (ROS), decreased mitochondrial inner membrane potential (Δψ), and accumulation of large-scale mtDNA deletions leading to premature death. Interestingly, we show that, at least for the adPEO-type M106P and A121P mutant alleles, the associated mtDNA instability cannot be attributed only to a reduced membrane potential or to an increased ROS level since it can be suppressed without restoration of the Δψ or modification of the ROS production. Suppression of mtDNA instability due to the M106P and A121P mutations was obtained by an allele of the rmp1 gene involved in nucleo-mitochondrial cross- talk and also by an allele of the AS1 gene encoding a cytosolic ribosomal protein. In contrast, the mtDNA instability caused by the S296M mutation was not suppressed by these alleles.THE maintenance and expression of mitochondrial DNA (mtDNA) depend on many nuclear-encoded gene products. Recent studies have shown that defects in this maintenance can have devastating consequences for the cell and the organism. In humans, these defects are an important cause of neurological diseases including autosomal dominant (or recessive) progressive external ophthalmoplegia (adPEO) (Chinnery 2003; Copeland 2008). These disorders are characterized by multiple large-scale deletions of mtDNA. Three different genes that can cause PEO with multiple mtDNA deletions have been identified: the mtDNA polymerase (POLG), the heart/muscle isoform of the adenine nucleotide translocator (ANT1), and the mitochondrial DNA helicase, Twinkle.The adenine nucleotide translocator (ANT), also known as the ADP/ATP mitochondrial translocator, is the most abundant protein in the inner mitochondrial membrane (Riccio et al. 1975; Nury et al. 2006; Klingenberg 2008). It exports ATP produced by mitochondrial oxidative phosphorylation toward the cytosol to meet the energy requirements of the cell; in exchange, it transports ADP into the mitochondrial matrix to fuel the conversion of ADP to ATP by the F₁F_O-ATP synthase. In humans, four isoforms of the ANT protein exist, and they are differently expressed in a tissue-specific manner (Stepien et al. 1992; Palmieri 2004; Dolce et al. 2005). The human ANT1 isoform is predominantly expressed in skeletal and cardiac muscle, and specific ANT1 mutations are associated with adPEO characterized by mtDNA instability (Kaukonen et al. 1999, 2000; Napoli et al. 2001; Komaki et al. 2002; Siciliano et al. 2003). In mice, Ant1 knockout induces mitochondrial myopathy (Graham et al. 1997), increased H₂O₂ production, and mtDNA damage and inhibits oxidative phosphorylation (Esposito et al. 1999). Some of these mutations were introduced in the AAC2 gene of Saccharomyces cerevisiae that encodes the major ADP/ATP mitochondrial translocator isoform in this organism. Numerous and sometimes contradictory effects have been reported depending in particular on the yeast laboratory strains examined (Kaukonen et al. 2000; Chen 2002, 2004; Fontanesi et al. 2004; Palmieri et al. 2005; Wang et al. 2008b).In an attempt to better understand how these mutations affect mitochondrial DNA stability and their functional consequences on mitochondrial metabolism, we decided to introduce them in the unique ADP/ATP translocator gene of Podospora anserina, PaAnt. Like S. cerevisiae, the filamentous fungus P. anserina is an excellent system for genetic and molecular analyses. In contrast to S. cerevisiae, it is a strict multicellular aerobe that can display heteroplasmic states in which intact and rearranged mitochondrial genomes coexist. In this organism, life span is a reflection of mtDNA stability, and death is always associated with large mtDNA rearrangements. “Natural death” or aging is accompanied by large-scale reorganizations of the mtDNA whereas a nuclear-controlled premature death syndrome is accompanied by the accumulation of site-specific mtDNA deletions (Belcour et al. 1999; Silar et al. 2001 for reviews). P. anserina therefore occupies an interesting position among model systems for studying the cellular consequences of mutations in the ADP/ATP translocase gene.We show here that the mutations M106P, A121P, and S296M, equivalent to the L98P, A114P (familial), and V289M (sporadic) human mutations, severely impair the vegetative and sexual development of the fungus and are responsible for decreased ROS production and for decreased inner membrane potential (Δψ). The severity of the phenotypes differs according to the mutation. The three mutations show mtDNA instability, which leads to premature death. All these mutated traits are dominant. Interestingly, the mtDNA instability associated with the M106P and A121P mutations depends on the rmp1 gene. This gene exists under two naturally occurring alleles, rmp1-1 and rmp1-2, which control mtDNA integrity in some genetic contexts (Belcour et al. 1991; Contamine et al. 1996, 2004). When associated with the rmp1-1 allele, the M106P and A121P mutations lead to rapid mtDNA instability whereas, in the presence of the rmp1-2 allele, mtDNA instability is suppressed, and life span is considerably increased. Surprisingly, suppression is not accompanied by a restoration of the Δψ or a modification in the ROS level, demonstrating that these parameters are not sufficient to explain the M106P and A121P mtDNA instability. Mitochondrial DNA instability due to the M106P and A121P mutations is also suppressed by a mutation in the AS1 gene encoding a ribosomal protein. The suppressor effects are not observed for the S296M mutation.     

DNA sequence of the excision sites of a mitochondrial plasmid from senescent Podospora anserina.

During senescence in Podospora anserina, specific gene regions of the mitochondrial genome are excised and amplified. The most prevalent, termed alpha-event senDNA, is a 2600 bp circular molecule which is excised from the contiguous Hae III fragments 23,14 region of the mitochondrial DNA restriction map. We have cloned alpha-DNA plasmid from races s+ and A+ as well as the genomic fragments Hae III 23,14 and have sequenced those regions which constitute the alpha-junction sites. We have found that one excision site (J1) is located 24 bp from the proximal Hae III 23 restriction site and the other (J2) 172 bp from the distal Hae III 14 site. Flanking the alpha-DNA sequences on the mitochondrial genome, there are 10 bp palindromic sequences: CAATATATTG, ending 3 bases from the J1 site, and ATTATATAAT which starts 8 bases from the J2 site. Neither of these 10 bp palindromes are present on the alpha-DNA plasmid. Abutting the J1 site on the alpha-DNA there is a 5 bp sequence (GTGCT) which is repeated 8 bp downstream. In joining the two distal J1 and J2 sites, a 7 bp repeat (ACGTGCG) is produced. These results are discussed within the context of site-specific recombination.     

Mitochondrial Group II Introns, Cytochrome c Oxidase, and Senescence in Podospora anserina

Podospora anserina is a filamentous fungus with a limited life span. It expresses a degenerative syndrome called senescence, which is always associated with the accumulation of circular molecules (senDNAs) containing specific regions of the mitochondrial chromosome. A mobile group II intron (alpha) has been thought to play a prominent role in this syndrome. Intron alpha is the first intron of the cytochrome c oxidase subunit I gene (COX1). Mitochondrial mutants that escape the senescence process are missing this intron, as well as the first exon of the COX1 gene. We describe here the first mutant of P. anserina that has the alpha sequence precisely deleted and whose cytochrome c oxidase activity is identical to that of wild-type cells. The integration site of the intron is slightly modified, and this change prevents efficient homing of intron alpha. We show here that this mutant displays a senescence syndrome similar to that of the wild type and that its life span is increased about twofold. The introduction of a related group II intron into the mitochondrial genome of the mutant does not restore the wild-type life span. These data clearly demonstrate that intron alpha is not the specific senescence factor but rather an accelerator or amplifier of the senescence process. They emphasize the role that intron alpha plays in the instability of the mitochondrial chromosome and the link between this instability and longevity. Our results strongly support the idea that in Podospora, "immortality" can be acquired not by the absence of intron alpha but rather by the lack of active cytochrome c oxidase.