Drosophila adPEO Mutations Figure 4. OXPHOS enzymatic activities and ROS levels in Drosophila lines expressing mutant d-mtDNA helicase. (A) Activities of complex IV were measured in mitochondrial protein extracts obtained from third instar larvae of each genotype expressing the different variants with da-GAL4. Data represent the mean 6 s.d., *P#0.05, as compared with wild-type (w1118), of at least three independent determinations. (B) Imaginal discs from third instar larvae were dissected and digested with trypsin prior to incubation with MitoSox (5 mM). The cell suspension was analysed using a BD Bioscience FACS Vantage SE instrument, and the data were processed with Cell Quest Pro Software. Data represent the mean 6 s.d. as compared with wild-type (w1118). (C–F) Overexpression of d-mtDNA helicase with the engrailed driver (en-GAL4) (posterior compartment, P) in wing imaginal discs, stained with dihydroethidium (DHE 30 mM) that binds superoxide anions. C, w;en-GAL4;UAS helicaseWT; D, w;en-GAL4;UAS helicaseK388A; E, w;en- GAL4;UAS helicaseW441C; F, w;en-GAL4;UAS helicaseA442P. doi:10.1371/journal.pone.0043954.g004 hexamer formation [37,38]. Thus, the mutant K338A likely forms hexamers with the same efficiency as the wild type polypeptides. However, in the T7 gp4 enzyme, analogous residue A475 in the helicase domain is in close contact with analogous residues R374 and F370 in the linker region, suggesting that it may be required to form stable multimers. The finding that the A475P adPEO variant fractionates mainly as a monomer in gel filtration supports this interpretation for the human enzyme [39]. Likewise, the loss of helicase activity due to hexamer instability could thus explain the phenotype observed in this work in the overexpression of the A442P mutation in flies; whereas hexameric proteins were detected in mitochondrial extracts from Schneider cells, the conformation and/or stability of the oligomers may be compro- mised. We found that the overexpression of the wild type form of d- mtDNA helicase slightly increases mtDNA copy number in adults, as it does both in Schneider cells and in mice [11,24], indicating that d-mtDNA helicase is modestly limiting for mtDNA replication under physiological conditions. Overexpression of the W441C mutant reduces the mtDNA content in the larval stages, but is recovered in adult cells. This suggests that mtDNA depletion occurs during embryonic and/or larval development but that replacement of mtDNA in postmitotic cells is relatively slow and may not be affected significantly by the W441C mutation, at least during the relatively short life of Drosophila. Interestingly, the life PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e43954