Copper-modulated gene expression and senescence in the filamentous fungus Podospora anserina

Abstract

We have previously shown that the control of cellular copper homeostasis by the copper-modulated transcription factor GRISEA has an important impact on the phenotype and lifespan of Podospora anserina. Here we demonstrate that copper depletion leads to the induction of an alternative respiratory pathway and to an increase in lifespan. This response compensates mitochondrial dysfunctions via the expression of PaAox, a nuclear gene coding for an alternative oxidase. It resembles the retrograde response in Saccharomyces cerevisiae. In P. anserina, this pathway appears to be induced by specific impairments of the copper-dependent cytochrome c oxidase. It is not induced as the result of a general decline of mitochondrial functions during senescence. We cloned and characterized PaAox. Transcript levels are decreased when cellular copper, superoxide, and hydrogen peroxide levels are raised. Copper also controls transcript levels of PaSod2, the gene encoding the mitochondrial manganese superoxide dismutase (PaSOD2). PaSod2 is a target of transcription factor GRISEA. During the senescence of wild-type strain s, the activity of PaSOD2 decreases, whereas the activity of the cytoplasmic copper/zinc superoxide dismutase (PaSOD1) increases. Collectively, the data explain the postponed senescence of mutant grisea as a defined consequence of copper depletion, ultimately leading to a reduction of oxidative stress. Moreover, they suggest that during senescence of the wild-type strain, copper is released from mitochondria. The involved mechanism is unknown. However, it is striking that the permeability of mitochondrial membranes in animal systems changes during apoptosis and that mitochondrial proteins with an important impact on this type of cellular death are released.

FIG. 1

Copper depletion leads to an extension of life span. Fifteen P. anserina cultures of the wild-type strain derived from independent mononucleate ascospores were grown to senescence on cornmeal agar containing 10 μM BCS and 0.33 mM ascorbic acid or containing 30 μM BCS and 1 mM ascorbic acid or without supplements. Accordingly, 15 cultures of mutant grisea were grown on cornmeal agar. All cultures were grown in race tubes at 27°C in the light. A single culture of mutant ex1 has been growing in the laboratory on cornmeal agar plates for over 12 years without signs of senescence.

FIG. 2

Copper depletion leads to reduced activity of COX and to the induction of the alternative oxidase. (A) Photometric determination of COX activity in the wild-type strain and mutant grisea. Three micrograms of mitochondrial protein was incubated with reduced cytochrome c as described in Materials and Methods. The protein preparation and measurements were repeated three times. (B) Oxygen uptake measurements of the wild-type strain, mutant grisea, and mutant ex1, without addition of respiratory inhibitors, with addition of 1 mM KCN, or with addition of 4 mM SHAM. (C) Oxygen uptake measurements of the wild-type strain grown on 30 μM BCS and 1 mM ascorbic acid and of mutant grisea grown on 100 μM CuSO₄. Experiments described for panels B and C were carried out at least in triplicate with two or three independent isolates of each strain.

FIG. 3

Multiple protein alignment of the deduced amino acid sequences of P. anserina PaAOX with N. crassa AOD-1 (29), M. grisea MgAOX (61), Hansenula anomala (49), Arabidopsis thaliana AOX1B (48), and S. guttatum AOX1 (46). The area with black background corresponds to residues completely conserved between all species, while the area with gray background displays homology of PaAOX with the AOX sequence of several but not all examples (indicated in percent at the end of the sequence). The amino acids indicated by arrows were proposed to form the binuclear iron center on the matrix side of the mitochondrial inner membrane, whereas Glu₂₇₀ (∗) was found to be essential for the catalytic activity of the alternative oxidase from S. guttatum (1, 4, 53). These amino acids are also conserved in PaAOX. The positions of two introns in the nucleotide sequence of PaAox are indicated by black triangles.

FIG. 4

Transcription of the PaAox gene is dependent on the cellular copper concentration. (A) Transcript level of PaAox for the wild-type strain (s) compared to that for mutant grisea (gr). (B and C) Northern blot analysis of PaAox in wild-type strain s (B) and mutant grisea (C) at low or high copper concentrations in the medium. The copper concentration was reduced by the addition of 10 μM BCS and 0.33 mM ascorbic acid or 30 μM BCS and 1 mM ascorbic acid or 50 μM BCS and 1.7 mM ascorbic acid. Copper levels were increased by the addition of 0.1, 1.0, or 10 μM CuSO₄ to the medium. In the lower part of the figure, the ethidium bromide-stained RNA is shown as loading control.

FIG. 5

PaAOX-dependent respiration is not induced in senescent cultures of the wild-type strain. (A) Oxygen uptake measurements of juvenile (10 days), middle-aged (15 days), and senescent cultures of the wild-type strain. Respiration was measured without addition of respiratory inhibitors, after the addition of 1 mM KCN, or after the addition of 4 mM SHAM. (B) Western blot analysis of PaAOX proteins of juvenile (juv.), middle-aged (m.a.), and senescent (sen.) cultures of the wild-type strain and of mutant grisea. In addition, mitochondrial protein preparations of mutant ex1 were analyzed. PaAOX was detected with AOA monoclonal antibodies directed against the AOX of S. guttatum strain Schott and visualized by enhanced chemiluminescence. An antibody directed against the β subunit of the ATPase complex was used as a loading control. (C) Northern blot analysis of PaAox transcription in juvenile and senescent cultures of the wild-type strain and of mutant grisea. The ethidium bromide-stained RNA is shown as a loading control.

FIG. 6

Transcription of PaAox is reduced by the addition of paraquat and hydrogen peroxide. (A and B) Northern blot analysis of PaAox transcripts in the wild-type strain and in mutant grisea. Paraquat (Pq, 100 μM) was added to the medium 15, 30, 60, and 240 min before RNA preparation. (C and D) H₂O₂ (0.01%) was added to the medium 15, 30, 60, 240, and 960 min before RNA preparation. These cultures were grown in the dark.

FIG. 7

PaSOD2 is active in the wild-type strain and is dependent upon cellular copper. (A) SOD activity assay. Proteins were prepared from the wild-type strain and from mutant grisea after cultivation of these strains in CM without supplements (−) or in CM containing 100 μM MnCl₂ (Mn²⁺) or 100 μM CuSO₄ (Cu²⁺), run on a native polyacrylamide gel, and stained for SOD activity. (B) Northern blot analysis of PaSod2 transcription. Different metals (100 μM CuSO₄ [Cu²⁺], 100 μM AgCl [Ag⁺], 100 μM MnCl₂ [Mn²⁺], 100 μM FeSO₄ [Fe²⁺], 100 μM FeCl₃ [Fe³⁺]) were added to the medium. −, control. (C) Northern blot analysis of PaSod2 transcription at different copper concentrations. To reduce the copper concentration, cultures were grown in medium containing 1 μM BCS and 33 μM ascorbic acid or 10 μM BCS and 0.33 mM ascorbic acid or 50 μM BCS and 1.7 mM ascorbic acid. Copper levels were increased by the addition of 0.1 or 10 μM CuSO₄ to the medium. −, no addition of CuSO₄ (control).

FIG. 8

PaSod2 is a putative target gene of transcription factor GRISEA. RNA was isolated from wild-type and grisea cultures grown with 100 μM CuSO₄ or without supplements. RNA was treated with DNase I to reduce DNA contamination and subsequently reverse transcribed by using an oligo(dT) primer. Reverse-transcribed RNA samples (cDNA) were used as a template. The PaSod2 transcripts were amplified by using primers MnSOD1 and MnSOD2 (see Materials and Methods). The Gpd gene was amplified in the same reaction and served as an internal control (positive control). DNase I-treated RNA (RNA) was included to exclude amplification of DNA (negative control). Lane m, 100-bp size standard. Numbers on the left indicate fragment sizes in kilobase pairs.

FIG. 9

The activity of PaSOD2 is reduced in senescent cultures of the wild-type strain. Proteins of two independent juvenile (juv.) and two senescent (sen.) cultures were isolated, as well as proteins from a juvenile strain grown for 4 h on 100 μM paraquat (+ Pq) or for 4 days on 100 μM MnCl₂ (+ Mn²⁺). These proteins were subjected to native polyacrylamide gel electrophoresis. SOD activity was determined by staining of the gel.