Alternative Oxidase Dependent Respiration Leads to an Increased Mitochondrial Content in Two Long-Lived Mutants of the Ageing Model Podospora anserina

The retrograde response constitutes an important signalling pathway from mitochondria to the nucleus which induces several genes to allow compensation of mitochondrial impairments. In the filamentous ascomycete Podospora anserina, an example for such a response is the induction of a nuclear-encoded and iron-dependent alternative oxidase (AOX) occurring when cytochrome-c oxidase (COX) dependent respiration is affected. Several long-lived mutants are known which predominantly or exclusively respire via AOX. Here we show that two AOX-utilising mutants, grisea and PaCox17::ble, are able to compensate partially for lowered OXPHOS efficiency resulting from AOX-dependent respiration by increasing mitochondrial content. At the physiological level this is demonstrated by an elevated oxygen consumption and increased heat production. However, in the two mutants, ATP levels do not reach WT levels. Interestingly, mutant PaCox17::ble is characterized by a highly increased release of the reactive oxygen species (ROS) hydrogen peroxide. Both grisea and PaCox17::ble contain elevated levels of mitochondrial proteins involved in quality control, i. e. LON protease and the molecular chaperone HSP60. Taken together, our work demonstrates that AOX-dependent respiration in two mutants of the ageing model P. anserina is linked to a novel mechanism involved in the retrograde response pathway, mitochondrial biogenesis, which might also play an important role for cellular maintenance in other organisms

Western blot analysis of mitochondrial PaLON protease and the molecular chaperone HSP60.

<p><b>A</b> Mitochondrial proteins in WT and mutants grisea and <i>PaCox17</i>::ble were analysed with antibodies against HSP60 after transfer to a PVDF membrane. HSP60 levels are increased in the two mutants. <b>B</b> Protein levels of LON protease (PaLON) are moderately increased in the mutants compared to the WT. Below each immunodetection a densitometric analysis of signal intensities (x-fold level compared to the WT) is shown. Intensities of the PaPORIN signals were used for normalisation. UniProt accession numbers: PaLON: B2AZ54; PaHSP60: B2B270 and PaPORIN: B2B736.</p

Metabolic rates of live mycelia.

<p><b>A</b> Respirometry reveals that mutants grisea and <i>PaCox17</i>::ble are characterized by elevated oxygen consumption compared to the WT. <b>B</b> Assessment of heat production by calorimetry is also increased in the two mutants. <b>C</b> However, the mutant genotype does not influence the calorimetric/respirometric (CR) ratio. Data represent mean ± standard error. *: p<0.05; **: p<0.01; n. s.: not significant.</p

Total superoxide dismutase and catalase activity.

<p><b>A</b> The measurement of SOD activity shows a significant increase in <i>PaCox17</i>::ble compared to the WT. <b>B</b> Catalase activity is not significantly changed between the WT and mutants grisea and <i>PaCox17</i>::ble, respectively. Data represent mean ± standard error. **: p<0.01; n. s.: not significant.</p

Mycelial hydrogen peroxide production.

<p>H₂O₂ production in WT and mutants grisea and <i>PaCox17</i>::ble as measured by mycelial DAB precipitation. While the amount of H₂O₂ is slightly reduced in mutant grisea compared to the WT, it is strongly increased in <i>PaCox17</i>::ble.</p

Mitochondrial content.

<p><b>A</b> Mycelia were stained with Mitotracker Green and analysed by fluorescence microscopy. Representative hyphae are shown. Scale bar: 2 µm. <b>B</b> Protoplasts prepared from mutants grisea and <i>PaCox17</i>::ble contain significantly more mitochondria than the WT as revealed by NAO staining. Mitochondrial content in the WT was set to 100%. <b>C</b> Determination of the mtDNA/nuclear DNA ratio as a marker for mitochondrial quantity by PCR. <i>left</i>: representative 1% agarose gel showing separated <i>PaGpd</i> (nuclear DNA) and <i>PaLsu</i> (mtDNA) amplification products stained with ethidiumbromide, NC: negative control, pd: primer dimers. <i>right</i>: densitometric analysis of band intensities. The mtDNA/nuclear DNA ratio in the WT was set to 1. <b>D</b> Western blot analysis to detect PaPORIN levels in total protein extracts from the wild type strain and the two mutants. As a loading control the Coomassie-stained transfer membrane is shown. Data represent mean ± standard error. *: p<0.05; ***: p<0.001, Student's <i>t</i> test, two-tailed.</p

Determination of ATP content in mycelial homogenates.

<p>ATP levels were measured by a luminescence based assay. Mutants grisea and <i>PaCox17</i>::ble contain significantly less ATP than the WT. The age of the mycelia from which the homogenates were prepared is 10 d. Data represent mean ± standard error. *: p<0.05.</p