Biological Roles of the Podospora anserina Mitochondrial Lon Protease and the Importance of Its N-Domain

Mitochondria have their own ATP-dependent proteases that maintain the functional state of the organelle. All multicellular eukaryotes, including filamentous fungi, possess the same set of mitochondrial proteases, unlike in unicellular yeasts, where ClpXP, one of the two matricial proteases, is absent. Despite the presence of ClpXP in the filamentous fungus Podospora anserina, deletion of the gene encoding the other matricial protease, PaLon1, leads to lethality at high and low temperatures, indicating that PaLON1 plays a main role in protein quality control. Under normal physiological conditions, the PaLon1 deletion is viable but decreases life span. PaLon1 deletion also leads to defects in two steps during development, ascospore germination and sexual reproduction, which suggests that PaLON1 ensures important regulatory functions during fungal development. Mitochondrial Lon proteases are composed of a central ATPase domain flanked by a large non-catalytic N-domain and a C-terminal protease domain. We found that three mutations in the N-domain of PaLON1 affected fungal life cycle, PaLON1 protein expression and mitochondrial proteolytic activity, which reveals the functional importance of the N-domain of the mitochondrial Lon protease. All PaLon1 mutations affected the C-terminal part of the N-domain. Considering that the C-terminal part is predicted to have an α helical arrangement in which the number, length and position of the helices are conserved with the solved structure of its bacterial homologs, we propose that this all-helical structure participates in Lon substrate interaction

Interaction Between the oxa1 and rmp1 Genes Modulates Respiratory Complex Assembly and Life Span in Podospora anserina

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

Regulation of Aerobic Energy Metabolism in Podospora anserina by Two Paralogous Genes Encoding Structurally Different c-Subunits of ATP Synthase

International audienceMost of the ATP in living cells is produced by an F-type ATP synthase. This enzyme uses the energy of a transmembrane electrochemical proton gradient to synthesize ATP from ADP and inorganic phosphate. Proton movements across the membrane domain (FO) of the ATP synthase drive the rotation of a ring of 8-15 c-subunits, which induces conformational changes in the catalytic part (F1) of the enzyme that ultimately promote ATP synthesis. Two paralogous nuclear genes, called Atp9-5 and Atp9-7, encode structurally different c-subunits in the filamentous fungus Podospora anserina. We have in this study identified differences in the expression pattern for the two genes that correlate with the mitotic activity of cells in vegetative mycelia: Atp9-7 is transcriptionally active in non-proliferating (stationary) cells while Atp9-5 is expressed in the cells at the extremity (apex) of filaments that divide and are responsible for mycelium growth. When active, the Atp9-5 gene sustains a much higher rate of c-subunit synthesis than Atp9-7. We further show that the ATP9-7 and ATP9-5 proteins have antagonist effects on the longevity of P. anserina. Finally, we provide evidence that the ATP9-5 protein sustains a higher rate of mitochondrial ATP synthesis and yield in ATP molecules per electron transferred to oxygen than the c-subunit encoded by Atp9-7. These findings reveal that the c-subunit genes play a key role in the modulation of ATP synthase production and activity along the life cycle of P. anserina. Such a degree of sophistication for regulating aerobic energy metabolism has not been described before

Caenorhabditis elegans expressing the Saccharomyces cerevisiae NADH alternative dehydrogenase Ndi1p, as a tool to identify new genes involved in complex I related diseases

Isolated complex I deficiencies are one of the most commonly observed biochemical features in patients suffering from mitochondrial disorders. In the majority of these clinical cases the molecular bases of the diseases remain unknown suggesting the involvement of unidentified factors that are critical for complex I function.The Saccharomyces cerevisiae NDI1 gene, encoding the mitochondrial internal NADH dehydrogenase was previously shown to complement a complex I deficient strain in Caenorhabitis elegans with notable improvements in reproduction, whole organism respiration. These features indicate that Ndi1p can functionally integrate the respiratory chain, allowing complex I deficiency complementation. Taking into account the Ndi1p ability to bypass complex I, we evaluate the possibility to extend the range of defects/mutations causing complex I deficiencies that can be alleviated by NDI1 expression.We report here that NDI1 expressing animals unexpectedly exhibit a slightly shortened lifespan, a reduction in the progeny and a depletion of the mitochondrial genome. However, Ndi1p is expressed and targeted to the mitochondria as a functional protein that confers rotenone resistance to those animals and without affecting their respiration rate and ATP content.We show that the severe embryonic lethality level caused by the RNAi knockdowns of complex I structural subunit encoding genes (e.g. NDUFV1, NDUFS1, NDUFS6, NDUFS8 or GRIM-19 human orthologs) in wild type animals is significantly reduced in the Ndi1p expressing worm.All together these results open up the perspective to identify new genes involved in complex I function, assembly or regulation by screening an RNAi library of genes leading to embryonic lethality that should be rescued by NDI1 expression

Experimental Relocation of the Mitochondrial ATP9 Gene to the Nucleus Reveals Forces Underlying Mitochondrial Genome Evolution

Only a few genes remain in the mitochondrial genome retained by every eukaryotic organism that carry out essential functions and are implicated in severe diseases. Experimentally relocating these few genes to the nucleus therefore has both therapeutic and evolutionary implications. Numerous unproductive attempts have been made to do so, with a total of only 5 successes across all organisms. We have taken a novel approach to relocating mitochondrial genes that utilizes naturally nuclear versions from other organisms. We demonstrate this approach on subunit 9/c of ATP synthase, successfully relocating this gene for the first time in any organism by expressing the ATP9 genes from Podospora anserina in Saccharomyces cerevisiae. This study substantiates the role of protein structure in mitochondrial gene transfer: expression of chimeric constructs reveals that the P. anserina proteins can be correctly imported into mitochondria due to reduced hydrophobicity of the first transmembrane segment. Nuclear expression of ATP9, while permitting almost fully functional oxidative phosphorylation, perturbs many cellular properties, including cellular morphology, and activates the heat shock response. Altogether, our study establishes a novel strategy for allotopic expression of mitochondrial genes, demonstrates the complex adaptations required to relocate ATP9, and indicates a reason that this gene was only transferred to the nucleus during the evolution of multicellular organisms

The ATP9-5 and ATP9-7 proteins confer different properties to the F-ATP synthase in <i>P</i>. <i>anserina</i>.

<p>All of these experiments were performed using mitochondria isolated from the apical cells of strains <i>[</i>^<i>5</i><i>7]</i> and ^<i>5</i><i>5</i>, which express either <i>Atp9-5</i> or <i>Atp9-7</i> both from the regulatory sequences of <i>Atp9-</i>5, except in panel (C) where protoplasts prepared from these strains were used. For simplicity, the mitochondrial samples are referred to as MitoATP9-5 and MitoATP9-7, whereas the protoplasts are named ProtoATP9-5 and ProtoATP9-7, respectively, to denote which <i>c</i>-subunit isomer was produced in the cells of origin. (A) BN-PAGE analysis of ATP synthase. <i>On the left</i>: Mitochondrial proteins were extracted with 2% digitonin, separated by BN-PAGE (50 μg per lane) and transferred to nitrocellulose membranes for Western blotting with antibodies against the yeast α-F₁ protein. In the conditions used, ATP synthase was detected as dimeric (V₂) and monomeric (V₁) units. <i>On the right</i>: Quantification of the immunological signals (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006161#sec010" target="_blank">Materials and Methods</a>). The values correspond to the surface areas below the V₂ and V₁ peaks. The two protein samples loaded on the BN-gel contained similar amounts of porin, as shown in panel B. A second, independent, BN-PAGE analysis is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006161#pgen.1006161.s010" target="_blank">S4 Fig</a> (B) Steady state levels of porin and AOX. 50 μg of proteins from MitoATP9-5 and MitoATP9-5 were separated <i>via</i> SDS-PAGE, transferred to a nitrocellulose membrane and probed with antibodies against porin and AOX. ^<i>Gpd</i><i>Aox</i> mitochondria were isolated from a strain that constitutively expresses AOX [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006161#pgen.1006161.ref035" target="_blank">35</a>]. C. Sensitivity of oxygen uptake to SHAM. 2x10⁸ protoplasts prepared from strains <i>[</i>^<i>5</i><i>7]</i> (ProtoATP9-7) and ^<i>5</i><i>5</i> (ProtoATP9-5) were pre-incubated for 30 min in the respiration buffer and their oxygen consumption activities were then measured using a Clark electrode. SHAM and KCN inhibitors were used at 1mM. The respiration value in the absence of inhibitor is set-up as 100%. The residual respirations after adding the adding the inhibitors are indicated. (D) Oxygen uptake by MitoATP9-5 and MitoATP9-7. Measurements with a Clark electrode were made with mitochondria at a protein concentration of 0.15 mg/ml, in the presence of NADH alone (basal respiration), and after subsequent additions of 150 μM ADP (state 3) or 4 μM CCCP (uncoupled respiration). (E) ATP synthesis. This activity was evaluated in the conditions used to measure state 3 respiration except that 1 mM instead of 150 μM ADP was added to the mitochondrial samples. (F) The histogram reports the ATP/O values calculated for MitoATP9-5 and MitoATP9-7, which is the number of ATP molecules produced per pair of electrons transferred to oxygen (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006161#pgen.1006161.s005" target="_blank">S4 Table</a> for details; ** is for p<0.01%). The data reported in panels C, D and E are the mean values ± standard deviation obtained in at least three independent experiments (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006161#pgen.1006161.s005" target="_blank">S4 Table</a> for details).</p

Transcription profiling of <i>Atp9-7</i> and <i>Atp9-5</i> in vegetative cultures of <i>P</i>. <i>anserina</i>.

<p>The relative abundance of <i>Atp9-5</i> and <i>Atp9-7</i> mRNA transcripts in vegetative cultures of <i>P</i>. <i>anserina</i>, <i>versus</i> a constitutively expressed gene (<i>Gpd</i>), was determined by real-time quantitative reverse transcription PCR. The analyzed RNA extracts were prepared from: (i) whole cultures grown on solid medium for 1 (w-1d), 2 (w-2d) and 5 (w-5d) days; (ii) the central (c-5d) and peripheral (p-5d) regions of w-5d mycelium as delineated by dotted lines; and (iii) protoplasts (apical cells) obtained from 2 (a-2d) and 5 days (a-5d) aged liquid cultures. The results are presented as histograms with an arbitrary value of 1 for <i>Atp9-7</i> transcripts in w-1d. The error bars indicate standard error (SEM) in at least three independent experiments.</p

The ATP9-5 and ATP9-7 proteins have antagonist effects on the longevity of <i>P</i>. <i>anserina</i>.

<p>This figure reports the life span values for strains in which the expression of <i>Atp9-5</i> and/or <i>Atp9-7</i> is regulated differently than in wild type, as evaluated by the linear length (in cm) the mycelium reaches before dying (Panel A), or by the estimation of the half-life (in days), which is the number of days by which 50% of the cultures were still alive (panel B) (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006161#pgen.1006161.s004" target="_blank">S3 Table</a> for details). The reported values and standard deviations were established by testing at least 32 cultures for each genotype obtained from several independent crosses (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006161#pgen.1006161.s004" target="_blank">S3 Table</a>). Statistically significant changes in longevity between strains, according to t-student and log rank tests are indicated by the bars and stars (* corresponds to a <i>P</i>-value <95%, ** to a <i>P</i>-value <0.01). The short-hand nomenclature of the analyzed strains is explained in the legend of <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006161#pgen.1006161.g002" target="_blank">Fig 2</a> (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006161#pgen.1006161.s002" target="_blank">S1 Table</a> for complete genotypes).</p

The mitochondrial PaLON1 protein.

<p>(A) Schematic representation of the PaLON1 protein outlining the three domains present in both prokaryotes and eukaryotes. The N-domain, which is the most divergent domain between Lon proteins, is followed by the highly conserved ATPase and protease domains. Within the N-domain, the most conserved region is within the C-terminal part (hatched). The line referring to residues 382 to 619 indicates the part of the protein presented in (B). Diamond (S423L), point (L430P), and inverted triangles (Δ514–567) mark changes induced by <i>PaLon1-31</i>, <i>PaLon1-1</i> and <i>PaLon1-f</i>, respectively. (B). Primary sequence and secondary structure of the C-terminal part of the N-domain of <i>B. subtilis</i>, <i>E. coli,</i> and <i>P. anserina</i> Lon proteases. Sequences were aligned using the Clustal W program. Conserved amino acids are boxed in black (identical) and gray (similar). For the <i>P. anserina</i> sequence (PODAN), changes induced by <i>PaLon1</i> mutations are represented by the same symbols as in (A). The GenBank accession numbers for <i>B. subtilis</i> (BACSU) and <i>E. coli</i> (ESCCO) proteins are CAA99540.1 and AAC36871.1, respectively. The Walker A motif of the central ATPase domain is boxed and begins at position 607, 356, and 354 in <i>P. anserina</i>, <i>E. coli</i> and <i>B. subtilis</i> proteins, respectively. The predicted consensus secondary structure of the PaLON1 region was determined on the <a href="mailto:NPS@" target="_blank">NPS@</a> Web server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038138#pone.0038138-Combet1" target="_blank">[42]</a> using a combination of available methods. For the same region, the secondary structure information available for <i>E. coli</i> and <i>B. subtilis</i> proteins ends at residue 245 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038138#pone.0038138-Li2" target="_blank">[32]</a> or contains a gap of 36 amino acids (dotted line), respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038138#pone.0038138-Duman1" target="_blank">[33]</a>. For the <i>B. subtilis</i> protein, structure information was not available after the last α helix just before the Walker A motif. Secondary structures are indicated above each sequence as follows: lines, α helices; c letter, random coil (no secondary structure); and question mark (?), ambiguous state.</p