Interaction between the oxa1 and rmp1 genes modulates respiratory complex assembly and life span in Podospora anserina

Abstract

A causal link between deficiency of the cytochrome respiratory pathway and life span was previously shown in the filamentous fungus Podospora anserina. To gain more insight into the relationship between mitochondrial function and life span, we have constructed a strain carrying a thermosensitive mutation of the gene oxa1. OXA1 is a membrane protein conserved from bacteria to human. The mitochondrial OXA1 protein is involved in the assembly/insertion of several respiratory complexes. We show here that oxa1 is an essential gene in P. anserina. The oxa1(ts) mutant exhibits severe defects in the respiratory complexes I and IV, which are correlated with an increased life span, a strong induction of the alternative oxidase, and a reduction in ROS production. However, there is no causal link between alternative oxidase level and life span. We also show that in the oxa1(ts) mutant, the extent of the defects in complexes I and IV and the life-span increase depends on the essential gene rmp1. The RMP1 protein, whose function is still unknown, can be localized in the mitochondria and/or the cytosolic compartment, depending on the developmental stage. We propose that the RMP1 protein could be involved in the process of OXA1-dependent protein insertion.

Figure 1.—

Cloning and inactivation of the oxa1 gene. (A) Partial restriction map of the P. anserina wild-type oxa1 gene. Nomenclature is according to the submitted sequence (no. AF 382189). The arrows represent the oligonucleotides used for further PCR experiments with their 5′–3′orientation. (B) Partial restriction map of the pBCoxa1^ts vector. The XhoI-NarI fragment of the P. anserina wild-type oxa1 gene was cloned in the pBC-hygro vector (pBCoxa1). An NruI-PpuMI modified fragment obtained in a two-step PCR amplification replaced the NruI-PpuMI wild-type fragment. The first step was the amplification of two fragments using two couples of primers (NruI/C2 and C1/PpuMI), including two complementary oligonucleotides (C1 and C2) designed with the desired T-to-C substitution. In the second step, the NruI/PpuMI fragment was amplified using the two preceding amplified fragments as matrixes. (C) Partial restriction map of the pULoxa1^ts vector. The MluI-SpeI fragment of pBCoxa1^ts was cloned in a pUL vector carrying the leu1 gene. (D) Partial restriction map of the pBCΔoxa1::ble vector. The NruI-PpuMI wild-type fragment of pBCoxa1 was replaced by an Ecl136II-HindIII DNA fragment carrying the ble gene using a HindIII/PpuMI linker. The shaded boxes correspond to the oxa1 coding sequence. Dashed and dotted lines correspond to plasmid sequences. Ap, Cm, and hygro are bacterial genes conferring, respectively, the ampicillin, chloramphenicol, and hygromycin resistance to the pUC- and pBC-hygro-derived vectors.

Figure 2.—

In-gel detection of complex I, IV, and V activities in various strains grown at 30°. Sample preparation and blue-native polyacrylamide gel electrophoresis (BN-PAGE) were carried out as described (Schagger and von Jagow 1991). Mitochondria from P. anserina were rendered soluble in the presence of 2% (w/v) N-dodecyl maltoside. In-gel activity assays of complexes I, IV, and V were performed as described (Nijtmans et al. 2002). Genotypes of the strains are detailed below the gel. The oxa1 gene is either present (+) or inactivated (Δ). + or − indicates the presence or the absence of the (oxa1^ts) and (rmp1-1) transgenes. The two mat alleles, mat+ and mat−, are indicated by + and −, respectively. The specific activity of cytochrome c oxidase (COX) is expressed as the percentage of the wild type (wild type is 856 ± 54 nmol of oxidized cytochrome c per minute per milligram of mitochondrial protein). The wild-type strain (lane 1) gave the same results irrespective of mat and rmp1 haplotype.

Figure 3.—

Immunochemical detection of AOX by Western blot analysis. Twenty micrograms of mitochondrial proteins extracted from cultures grown at 30° were fractionated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed first with an anti-AOX mouse monoclonal antibody generated against the AOX of Sauromatum guttatum (Elthon et al. 1989). Blots were then reprobed with an anti-βATPase rabbit antibody as a standardization control. The bound antibodies were detected using an enhanced chemiluminescence detection system (Pierce, Rockford, IL). For genotypes see legend of Figure 2. The percentage of respiration sensitive to cyanide (KCN, 1 mm) and SHAM (1 mg/ml) reflects the engagement of electrons in the cytochrome and alternative pathway, respectively. Oxygen uptake measurements were performed on protoplast suspensions (10⁸ protoplasts/ml in 0.6 m sorbitol) in an oxytherm chamber with a Clark-type O₂ electrode (Hansatech). Wild-type strains used as controls (lane 1) gave the same results irrespective of their mat and rmp1 haplotype.

Figure 4.—

ROS measurements. (A) ROS production estimated as the kinetics of dichorofluorescin (DCF) production resulting from the oxidation of the diacetate form (H₂DCF-DA) as described previously (Dufour et al. 2000). For each strain grown at 30°, ∼10⁷ protoplasts were incubated with H₂DCF-DA (80 μm in 0.6 m sortitol/10 mm Tris-HCl pH 7.5) at 30° and ROS production was analyzed by measuring, every 10 min, the apparition of the fluorescent DCF in a XL3C flow cytofluometer (Coulter, France). (B) Histochemical detection of superoxide radicals on mycelium (Munkres 1990). Cultures were grown at 27° or 30° on M2 medium until a diameter of 3 cm was reached: the petri dishes were flooded with 3–5 ml of staining solution containing 2.5 mm NBT diluted in 5 mm 3-(N-morpholino) propane sulfonate-NaOH (MOPS) buffer, pH 7.6. After an incubation of 60 min at 30°, the solution was discarded and plates kept in the dark. A blue, water-insoluble formazan precipitate, obtained from the reduction of the water-soluble NBT, visualized superoxide radicals. rmp1-1, rmp1-2, Δcox5, and wt, respectively, correspond to Δoxa1 (oxa1^ts) rmp1-1 mat−, Δoxa1 (oxa1^ts) rmp1-2 mat+, Δcox5 mat+, and the wild-type mat+ strains. These last two strains gave the same results irrespective of their mat and rmp1 haplotype.