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

Detection of PaLON1 mutant proteins.

<p>Mitochondrial extracts (70 μg) purified from the indicated strains were resolved on an SDS-polyacrylamide gel and subjected to immunoblotting. The PaLON1 protein and the β-subunit of mitochondrial ATPase were detected by a <i>P. anserina</i> anti-PaLON antibody and an <i>S. cerevisiae</i> anti-Atp2 antibody, respectively. Mitochondrial extraction and western blotting were repeated at least twice for each strain. The left and right panels correspond to two independent membranes.</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

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

MtDNA profile of the

<p>Δ<b><i>Lon1</i></b><b> strain.</b> MtDNA profiles were determined by <i>Hae</i>III digestion of total DNA extracted from mycelium for the indicated dying strains. <i>Hae</i>III digestion of mtDNA from a young wild-type mycelium was used as a control. The arrows indicate the senDNAα multimeric subgenomic molecules present in dying strains (right and left panels). The identity of the senDNAα molecules was assessed by Southern blot analysis using an intron α specific probe (right panel). In addition to senDNAα (2.5 kb), hybridization revealed two <i>Hae</i>III fragments on the intact mitochondrial genome (1.9 and 0.8 kb). Note that these two bands were only detected in young wild-type (Control) and dying Δ<i>Lon1</i> strains.</p

Model of how a reduction in the hydrophobicity of subunit 9 permits its functional expression from nuclear DNA.

<p>When the hydrophobicity of subunit 9 is too high, the protein cannot cross the inner mitochondrial membrane (IM) and is degraded in the intermembrane space by the i-AAA protease. With reduced hydrophobicity, subunit 9 can cross the IM and is processed by the matrix processing peptidase (MPP), properly inserted into the IM, and assembled into ATP synthase (see text for details). OM, outer mitochondrial membrane; MTS, mitochondrial targeting sequence, TMH, transmembrane segment; TOM, translocase of the OM; TIM, translocase of the IM.</p

The <i>P. anserina Atp9</i> proteins are less hydrophobic than yeast Atp9p.

<p>A) Hydropathy profiles of the <i>PaAtp9-7</i> and <i>PaAtp9-5</i> proteins and yeast Atp9p, generated according to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002876#pgen.1002876-Kyte1" target="_blank">[54]</a> with a window size of 13. B) <i>P. anserina</i> strains expressing exclusively either <i>PaAtp9-7</i> (PaΔAtp9-5) or <i>PaAtp9-5</i> (PaΔAtp9-7) were constructed and ATP synthase was enriched from their mitochondrial extracts, separated by SDS-PAGE and silver-stained along with <i>WT</i> yeast ATP synthase. Positions of some ATP synthase subunits are indicated. The <i>PaAtp9</i>-5 protein is stained much more strongly than the <i>PaAtp9-7</i> protein, which may be due to the differences in their amino acid sequences.</p

A nuclear version of the yeast mitochondrial <i>ATP9</i> gene fails to complement the <i>Δatp9</i> yeast.

<p>We engineered a nuclear version of the yeast <i>ATP9</i> gene (yAtp9-Nuc) by adding a mitochondrial targeting sequence (derived from the <i>P. anserina Atp9-7</i> gene) and adjusting the genetic code for nuclear expression of the endogenous gene (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002876#pgen.1002876.s003" target="_blank">Figure S2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002876#pgen.1002876.s004" target="_blank">S3A</a> for amino acid and nucleotide sequences). yAtp9-Nuc was tested for its capacity to complement <i>Δatp9</i> yeast with respect to respiratory capacity. A) Growth on rich glucose (YPGA) and glycerol (N3) media of serial dilutions of <i>WT</i>, <i>Δatp9</i>, and <i>Δatp9</i> transformed with yAtp9-Nuc. B) Total cellular (<i>T</i>), mitochondrial (<i>M</i>) and post-mitochondrial supernatant (<i>C</i>) protein extracts were prepared from <i>WT</i> and <i>Δatp9</i>+yAtp9-Nuc strains. Samples were separated via SDS-PAGE and probed with antibodies against yeast Atp9p and the cytosolic protein Pgk1p (phosphoglycerate kinase). C) Western blot of total proteins prepared from <i>WT</i> and <i>Δatp9</i> yeast transformed with yAtp9-Nuc with or without the <i>YME1</i> gene (<i>Δyme1</i>) reveals that the yAtp9-Nuc protein is degraded by the i-AAA protease (an oligomer of Yme1p).</p

Transcriptome profiles of yeast strains expressing <i>P. anserina Atp9</i> genes indicate functional OXPHOS and regulatory responses to the nuclear relocation of <i>ATP9</i>.

<p>For all genes in the yeast genome, the expression levels in AMY11 (expressing <i>PaAtp9-7</i>) are plotted against those of AMY10 <i>(PaAtp9-5)</i>, both displayed as log₂ ratios to <i>WT</i> expression levels; differentially expressed genes in the main functionally relevant categories are indicated by colours. The square formed by the grey lines delineates the boundaries of statistically significant expression differences (see <i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002876#pgen.1002876.s007" target="_blank">Text S1</a></i>) between either strain and the <i>WT</i>; genes beyond the diagonal grey lines are differentially expressed between AMY10 and AMY11. For clarity, genes in the categories listed are only indicated if they were differentially expressed relative to <i>WT</i> in at least one strain. Categories were defined as follows: OXPHOS pathway - subunits (1/35 differentially expressed) and biogenesis factors (1/42); Retrograde pathway – transcriptional targets of the factors Gcn4p (29/126) and Rtg3p (6/31), plus <i>CIT2</i> and <i>CIT3</i>; Heat response - Gene Ontology (GO)-annotated “response to heat” genes (28/199); Morphology - Phd1p targets (23/81), plus GO “cell-cell adhesion” (2/4) and “cytokinesis, completion of separation” genes (6/11). All categories except OXPHOS were significantly enriched among differentially expressed genes (according to Fisher's exact test with multiple hypothesis testing correction, or to Model Gene Set Analysis (MGSA); see <i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002876#pgen.1002876.s007" target="_blank">Text S1</a></i>).</p

Deletion of the yeast mitochondrial <i>ATP9</i> gene and resulting phenotypes.

<p>A) The mitochondrial <i>ATP9</i> gene was deleted and replaced with <i>ARG8^m</i> (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002876#pgen.1002876.s002" target="_blank">Figure S1</a> for details) in a wild-type strain lacking the nuclear <i>ARG8</i> gene. As a result, the <i>Δatp9</i> yeast grow on glucose (Glu) media lacking arginine (Arg) whereas the parental strain (<i>WT</i>) does not; in addition, <i>Δatp9</i> yeast cannot grow on glycerol (Gly). B) ATP synthase levels in <i>WT</i> and <i>Δatp9</i>. Isolated mitochondria were separated by BN-PAGE and western blotted with antibodies against Atp4p; <i>V</i>₁ and <i>V</i>_n respectively indicate monomeric and oligomeric forms of ATP synthase. C) Pulse labelling of proteins translated in mitochondria. Total proteins were prepared from cells incubated in the presence of ³⁵S methionine and cysteine as well as cycloheximide to inhibit cytosolic protein synthesis. Proteins (40,000 cpm per lane) were separated on 12% (Cox3p and Atp6p) or 17% (Atp9p and Atp8p) SDS-PAGE containing 6 M urea. D) Electron microscopy of <i>WT</i> (<i>a</i>) and <i>Δatp9</i> (<i>b–d</i>) cells grown in galactose (80 nm-thin sections); <i>m</i>, mitochondria; <i>Cr</i>, cristae; <i>Ib</i>, inclusion bodies; arrowheads in (<i>a</i>) point to <i>Cr</i>, to outer mitochondrial membrane in (<i>c</i>), and to septae in (<i>d</i>); <i>bars</i>, 0.2 µm.</p