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

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

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

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

Respiratory and ATP hydrolysis/synthesis activities of mitochondria.

<p>Mitochondria were isolated from yeast cells grown at 28°C in rich glycerol+ethanol (YPEG) or rich galactose (YPGALA), as indicated. All cultures contained less than 5% ρ⁻/ρ⁰ cells, except that of <i>Δatp9</i> where about 50% ρ⁻/ρ⁰ cells were scored. Additions were 0.15 mg/ml proteins, 4 mM NADH, 150 mM ADP, 4 mM CCCP, and 3 µg/ml oligomycin (<i>Oligo</i>). The values reported are averages of triplicate assays ± standard deviation. Respiratory and ATP synthesis activities were measured on freshly isolated, osmotically-protected mitochondria buffered at pH 6.8. For the ATPase assays, mitochondria kept at −80°C were thawed and the reaction was performed in absence of osmotic protection at pH 8.4. <i>ND</i>, not determined.</p