Impact of a disruption of a pathway delivering copper to mitochondria on Podospora anserina metabolism and life span

Abstract

A global depletion of cellular copper as the result of a deficiency in high-affinity copper uptake was previously shown to affect the phenotype and life span of the filamentous fungus Podospora anserina. We report here the construction of a strain in which the delivery of copper to complex IV of the mitochondrial respiratory chain is affected. This strain, PaCox17::ble, is a PaCox17-null mutant that does not synthesize the molecular chaperone targeting copper to cytochrome c oxidase subunit II. PaCox17::ble is characterized by a decreased growth rate, a reduction in aerial hyphae formation, reduced female fertility, and a dramatic increase in life span. The mutant respires via a cyanide-resistant alternative pathway, displays superoxide dismutase (SOD) activity profiles significantly differing from those of the wild-type strain and is characterized by a stabilization of the mitochondrial DNA. Collectively, the presented data define individual components of a molecular network effective in life span modulation and copper as an element with a dual effect. As a cofactor of complex IV of the respiratory chain, it is indirectly involved in the generation of reactive oxygen species (ROS) and thereby plays a life span-limiting role. In contrast, Cu/Zn SOD as a ROS-scavenging enzyme lowers molecular damage and thus positively affects life span. Such considerations explain the reported differences in life span of independent mutants and spread more light on the delicate tuning of the molecular network influencing biological ageing.

FIG. 1.

Construction of knockout plasmid pBHP-9. In the first step, parts of the plasmid upstream and downstream of the PaCox17 ORF were amplified by PCR and ligated into pBSSK in the presence of a short BamHI/NheI linker, resulting in plasmid pPaΔCox17. The genomic sequence located upstream of the PaCox17 ORF which turned out to be discontinuous in pPaΔCox17 was replaced by a continuous genomic fragment. The BamHI/NheI linker was replaced by a ble resistance cassette, and then a hygromycin B resistance cassette (hph) was integrated in the bacterial backbone. The resulting plasmid is pBHP-9.

FIG. 2.

Complementation of the yeast mutant strain W303ΔCOX17 by pAD4 carrying the P. anserina cDNA of PaCox17. The yeast strains are as follows (both plates): first line, wild-type W303; second line, respiratory-deficient Cox17 mutant strain W303ΔCOX17; third line, mutant W303ΔCOX17 transformed with empty pAD4; fourth line, mutant W303ΔCOX17 transformed with PaCox17 cDNA cloned in yeast expression plasmid pAD4. Strains were grown to an optical density at 600 nm of 1.0. Serial 10-fold dilutions were prepared, and 5 μl was spotted onto YPD or YPG plates. These strains were grown for 2 days (30°C) in YPG (right plate) or in YPD (left plate) medium.

FIG. 3.

(A) Sequence of the PaCox17 locus and of the derived amino acid sequence. The coding sequence is shown in capital letters. Two introns (58 and 71 bp, respectively) disrupt the ORF. The 5′ splice sites GTATGT and GTCCGT resemble the consensus (GTANGT) for filamentous fungi, and the two 3′ splice sites ATAG match with the consensus (A/C)(C/T)AG. A putative polyadenylation site AATAGA (consensus, AATAAA) is found at position 495 (4). Protein-binding sites are described in the text. PaCox17 codes for a copper chaperone of 80 amino acids. The sequence of the cDNA is underlined. Putative promoter binding sites are shown in boldface, with the name of the binding protein below. (B) Amino acid comparison between PaCOX17 and COX17 from other species (Neurospora crassa; S. cerevisiae, accession number NP_013092; Mus musculus, accession number BAB32486; Chlamydomonas reinhardtii, accession number AAF82382; and Arabidopsis thaliana, accession numbers AAK73496 and AAK73497). Identical amino acids are shown by inverted colors. In the first lane, positions which were recently shown to be critical for restoring the respiratory competence of a Cox17-null mutant are indicated by “#” (53). The four essential cysteinyl residues are marked by asterisks.

FIG. 4.

(A) Plasmid pBHP-9 containing the PaCox17 locus in which the PaCox17 ORF is replaced by a bleocin resistance cassette (ble^R). Further, the backbone of pBHP-9 contains a hygromycin resistance cassette (hph^R) as a second marker. Crossing over on both sides of the genomic PaCox17 ORF lead to its replacement by the bleocin resistance cassette of plasmid pBHP-9, resulting in the formation of transgenic strain PaCox17::ble. Distances in the schematic drawings are not drawn to scale. (B) Southern analysis of wild-type strain s and mutant strains PaCox17::ble-12 (PaCox17::ble, lane 1) and PaCox17::ble-37312 (PaCox17::ble, lane 2). Isolated genomic DNA was cut with PvuII (positions shown in panel A) and hybridized to the DIG-labeled PaCox17 S1*S2 probe, which encompasses the second half of the PaCox17-ORF and a 122-bp 3′ region (black bars in panel A). The first two lanes contain DNA of knockout plasmid pBHP-9 and DNA from wild-type strain s⁻ (monokaryotic) as controls; the third lane contains DNA of the heterokaryotic primary transformant T28. The fourth and fifth lanes contain DNA from the two examples of progeny (mutants PaCox17::ble) derived from monokaryotic ascospores of the primary transformant and wild-type strain s, respectively. DIG-labeled DNA of phage lambda was cut with HindIII and used as length standard (sixth lane at the right). (C) Phenotype of wild-type and mutant PaCox17::ble. Wild-type strain s is shown on the left; the mutant PaCox17::ble-37312 strain is shown on the right side on one agar plate. In contrast to the wild type, the mutant shows slower growth and mycelia lacking aerial hyphae. (D) Northern analysis of wild-type strain s (lane 1), mutant PaCox17::ble-37312 (lane 2), and mutant grisea (lane 3). An rDNA probe (plasmid pMY60 [67] containing a HindIII fragment of the rDNA unit of S. carlsbergensis) and an ethidium bromide-stained gel were used as loading controls.

FIG. 5.

Western blot analysis of AOX proteins in PaCox17::ble strains grown in rich medium or rich medium with 250 μM copper sulfate. The juvenile wild-type (WT) strains, mutant ex1, mutant grisea on rich medium, or on mutant grisea on medium containing 250 μM additional copper serve as controls. Mitochondria were isolated, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and blotted onto nitrocellulose. PaAOX was detected with monoclonal mouse antibodies against the AOX of Sauromatum guttatum strain Schott (20). An antibody against mitochondrial ATPase subunit β was used as loading control.

FIG. 6.

Oxygen uptake in the wild-type strain s, mutant grisea, and the transgenic PaCox17::ble strain. The type of respiration was discriminated by the addition of respiratory inhibitors (either 1 mM KCN or 4 mM SHAM). PaCox17::ble strains were compared to wild-type strain s and mutant grisea. Strains were grown in rich medium or in rich medium with an additional 10 μM copper sulfate. These experiments were each carried out at least five times with two or more independent isolates.

FIG. 7.

Activity of SODs and expression level of PaMt1. (A) Native proteins were isolated from PaCox17::ble strains that were juvenile (7 days) and middle aged (m.a.; 36 days), grown with or without 250 μM copper sulfate; wild-type strain s (juvenile [7 days] or senescent [36 days]); and mutant ex1 (ex) and mutant grisea (with or without 250 μM copper sulfate) as indicated. Subsequently, the proteins were separated by native polyacrylamide gel electrophoresis (see Materials and Methods). (B) Northern analysis of mutant grisea, wild-type strain s, and mutant PaCox17::ble-37312. PaCtr3 was used as probe; therefore, plasmid pAD4-PaCtr3 (12) was radioactively labeled. An rDNA probe (plasmid pMY60 [67] containing a HindIII fragment of the rDNA unit of S. carlsbergensis) was used as a loading control.

FIG. 8.

Southern blot analysis of the mitochondrial PaCoxI region in two wild-type strains (B) and in two PaCox17::ble strains (C). (A) BglII restriction map of the PaCoxI and PaCyt b region of the mtDNA. The position of the hybridizing pl-intron and BglII fragments 17 and 5 are indicated. (B and C) Strains were grown on complex medium (left half of panels B and C) or with an additional 250 μM CuSO₄ (right half of panels B and C). DNA samples were digested with BglII, separated on an 1% agarose gel, blotted, and hybridized to a plDNA specific probe. Signals corresponding to the mtDNA fragments 17 (1.9 kbp) and 5 (4.5 kbp) and the amplified plDNA are indicated by arrows. (B) DNA of two wild-type strains (7, 14, 21, and 28 days and senescent [ca. 36 days]) was used. (C) DNA of two independent PaCox17-null mutants was used (aged 7, 21, 96, 157, and 300 days).

FIG. 9.

Life span determination of transgenic strain PaCox17::ble. A total of 60 cultures derived from monokaryotic ascospores were grown on BMM agar in race tubes at 27°C in the light. After 320 days, 40 cultures were still alive, displaying no symptoms of aging.