Podospora anserina AS6 gene encodes the cytosolic ribosomal protein of the E. coli S12 family

The ribosomal proteins of the E. coli S4, S5 and S12 families that are part of the ribosome accuracy center control translation accuracy both in prokaryotes and eukaryotes. In Podospora anserina, genes coding for S4 and S5 have already been identified. Here, we identify the gene coding for the S12 protein homologue and show that it is identical to the genetically known AS6 gene

Efficient tools to target DNA to Podospora anserina

Here we report the construction of two plasmids designed to target DNA sequences to two specific loci of Podospora anserina

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

Two Copies of mthmg1, Encoding a Novel Mitochondrial HMG-Like Protein, Delay Accumulation of Mitochondrial DNA Deletions in Podospora anserina

In the filamentous fungus Podospora anserina, two degenerative processes which result in growth arrest are associated with mitochondrial genome (mitochondrial DNA [mtDNA]) instability. Senescence is correlated with mtDNA rearrangements and amplification of specific regions (senDNAs). Premature death syndrome is characterized by the accumulation of specific mtDNA deletions. This accumulation is due to indirect effects of the AS1-4 mutation, which alters a cytosolic ribosomal protein gene. The mthmg1 gene has been identified as a double-copy suppressor of premature death. It greatly delays premature death and the accumulation of deletions when it is present in two copies in an AS1-4 context. The duplication of mthmg1 has no significant effect on the wild-type life span or on senDNA patterns. In an AS1(+) context, deletion of the mthmg1 gene alters germination, growth, and fertility and reduces the life span. The Δmthmg1 senescent strains display a particular senDNA pattern. This deletion is lethal in an AS1-4 context. According to its physical properties (very basic protein with putative mitochondrial targeting sequence and HMG-type DNA-binding domains) and the cellular localization of an mtHMG1-green fluorescent protein fusion, mtHMG1 appears to be a mitochondrial protein possibly associated with mtDNA. It is noteworthy that it is the first example of a protein combining the two DNA-binding domains, AT-hook motif and HMG-1 boxes. It may be involved in the stability and/or transmission of the mitochondrial genome. To date, no structural homologues have been found in other organisms. However, mtHMG1 displays functional similarities with the Saccharomyces cerevisiae mitochondrial HMG-box protein Abf2

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 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

Mitochondrial proteolytic activity of <i>PaLon1</i> mutants.

<p>Values represent the average ratio of mutant/wild type ± standard deviation of three independent experiments. Three incubation times were used for each experiment.</p

Phenotypic characteristics of <i>PaLon1</i> mutants.

<p>(A) Mycelium phenotype of <i>PaLon1-1</i> germinating ascospores on germination medium at 27°C after 2 days of growth. A cross between <i>PaLon1-1</i> and the wild-type strain gave rise to a progeny of <i>PaLon1-1</i> germinating ascospores with a less dense mycelium (letters: b, c, e, f, h, i, k) than that of the wild type (letters: a, d, g, j, l). (B) Growth phenotype exhibited by the <i>PaLon1</i> mutants. Strains were grown on M2 standard medium for 2 days (27°C and 36°C), 3 days (18°C), or 7 days (11°C). The genotype of each strain is shown in the table, except for the <i>rmp1-1</i> (<i>mat</i>−) and <i>rmp1-2</i> (<i>mat</i>+) alleles that are represented by a gray and white tone, respectively. (C) DASPMI staining of mitochondria. Mitochondria of growing strains (2 days at 27°C on M2) were stained with DASPMI, a vital mitochondrion-specific dye. For each indicated strain, filaments were gently mixed with a drop of DASPMI (25 mg/ml) directly on microscope slides and observed immediately with a fluorescence microscope (450–490/500–550 nm). All panels are at the same magnification and the scale bar corresponds to 5 μm. (D) Hydrogen peroxide (H₂O₂) sensitivity. Nine subcultures of each strain were inoculated in M2 or M2 supplemented with 0.005% (1.47 mM) or 0.01% (2.94 mM) H₂O₂. Growth (cm) was determined by measuring the radius of each thallus after 2 days at 27°C in the dark. Error bars indicate standard deviation. The statistically significant increase in the sensitivity of the <i>PaLon1</i>-f and Δ<i>Lon1</i> strains to 2.94 mM H₂O₂ is marked by an asterisk. In each case, the <i>p</i>-value (0.001) is below 0.05, as determined by the Mann-Whitney test.</p