ATM1 second copy complementation and mitochondrial function.

<p>(<b>A</b>) Scheme visualizing the strategy to insert second copies of myc-tagged ATM1s from <i>Toxoplasma</i>, <i>Plasmodium</i> and yeast (<i>Tg</i>, <i>Pf</i> and <i>Sc</i>, respectively) into the uracil phosphoribsyltransferase (UPRT) locus of <i>T</i>. <i>gondii</i>. (<b>B</b>) Modification of the UPRT locus was validated by genomic PCR. The approximate binding sites of the employed primers are shown in scheme A and sequences listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1012593#ppat.1012593.s011" target="_blank">S2 Table</a>. (<b>C</b>) Immunofluorescence assay of <i>Tg</i>ATM1-Ty-U1 parasites and <i>Tg</i>ATM1-Ty-U1 parasites expressing a second copy of myc-tagged <i>Toxoplasma</i>- (<i>Tg</i>ATM1-Ty-U1/c<i>Tg</i>ATM1-myc), <i>Plasmodium</i>- (<i>Tg</i>ATM1-Ty-U1/c<i>Pf</i>ATM1-myc) or yeast ATM1 (<i>Tg</i>ATM1-Ty-U1/c<i>Sc</i>ATM1-myc). Parasites were stained with anti-Ty and anti-myc antibodies and merged signals are shown. Images are representative of three independent biological replicates. (<b>D</b> and <b>E</b>) Representative traces from an extracellular flux analysis following 0 or 72 hours of Rapa treatment of parental (DiCreU1), uncomplemented <i>Tg</i>ATM1-Ty-U1 parasites or <i>Tg</i>ATM1-Ty-U1 parasites expressing a second copy of myc-tagged yeast ATM1 (<i>Tg</i>ATM1-Ty-U1/c<i>Sc</i>ATM1-myc). The oxygen consumption rate (OCR, D) and extracellular media acidification rate (ECAR, E) are shown. The legend shown in D applies to both panels. Other overexpressing strains behaved similarly–see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1012593#ppat.1012593.s012" target="_blank">S3 Table</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1012593#ppat.1012593.g004" target="_blank">Fig 4E and 4F</a>.</p> <p>(PDF)</p>