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

The copper-binding metallothionein PaMT1 is successfully targeted into the mitochondrial matrix.

<p>(A) The expression-level in the transformants T4 and T5, both express PaMT1 directed into the matrix, is comparable to wild-type s grown under pronounced copper-stress (+250 µM CuSO₄) (upper panel). Porin was used as control for equal loading (middle panel) and absence of genomic DNA in RNA samples (lower panel). (B) <i>In situ</i> immunodetection of PaMT1-His verifies its mitochondrial localization in T4 (Bf: brightfield images) Scale bar: 10 µm. (C) Western blot analysis shows that his-tagged PaMT1 in transformant T4 is targeted to mitochondria. α-Porin: mitochondrial loading control. (D) Total copper content of mitochondria is unaffected in the matrix-PaMT1 transformant T4 (filled bar) compared to wild-type s (open bar) as measured by TXRF.</p

The abundance of mRNA of the copper-regulated genes (<i>MT2A</i>, <i>Hsp70</i> and <i>PrP</i>) is increased in WI-38 human diploid fibroblasts (HDFs) incubated with copper salts (CuSO₄ or CuCl₂) at 500 µM for 16 h.

<p>Sodium sulfate (Na₂SO₄) and sodium chloride (NaCl) were used as control salts. CTL represented the same treatment without copper or sodium salts. The results of real-time PCR are expressed as fold induction compared to controls (CTL). RPL13A, ribosomal protein L13A, was used as housekeeping gene. The presented graph is representative of two independent experiments.</p

Targeting of PaMT1-his to mitochondria influences respiration and lifespan in <i>P. anserina</i>.

<p>(A) Cu-binding of PaMT1 reduces COX activity and induces AOX. Oxygen-consumption measurements of wild-type, two independent PaMT1 transformants (T4 and T5) and a control, overexpressing PaMT1 in the cytosol (T9-PaMT1 cyto) are shown. The percentage of respiration via COX or AOX, respectively, was determined by inhibition with 10 mM KCN or 4 mM SHAM, respectively. The values represent mean±SE. (B) Western blot analysis of isolated mitochondria of wild-type and the two PaMT1 transformants T4 and T5 probed with an antibody against the AOX of <i>S. guttatum</i>. (C) The mean lifespan of PaMT1 transformants T4 (n = 31) and T5 (n = 30) is doubled compared to the wild-type s (n = 12) (73 d and 75 d vs. 37 d) as determined in race tubes on standard cornmeal medium.</p

Detection of copper in WI-38 human diploid fibroblasts (HDFs) incubated with copper sulfate (CuSO₄).

<p>Cells were incubated with 500 µM of CuSO₄ for 16 h and were fixed just thereafter. Cells were treated for detection of copper by electron microscopy and Energy Dispersive X-ray (EDX) analysis. (A) Micrograph of a fibroblast incubated with CuSO₄, magnification 7,500×. (B) Micrograph of a fibroblast incubated with CuSO₄, magnification 33,000×. (C) Area selected inside a granule within a fibroblast incubated with CuSO₄ and results of EDX analysis. (D) Area selected outside a granule within a fibroblast incubated with CuSO₄ and results of EDX analysis.</p