Figure 2
Loss of TbMrf1 affects the structural integrity of protein complexes that contain mt encoded subunits. (a) The sedimentation pattern of 12S and 9S rRNAs in BF 427 and dKO TbMrf1 1wk and 7wk cell lines. Whole cell lysates from 5 × 108 parasites were resolved on a 10–30% glycerol gradient. RNA was extracted from each fraction and separated on 5% polyacrylamide/8 M urea gels that were blotted and probed for 12S and 9S mitochondrial rRNAs and 18S cytosolic rRNA (sedimentation control). BF 427–8 μg of total RNA from BF 427. Input – 3 μg of total RNA isolated from the remaining material that was loaded on a gradient. (b) After normalization to BF 427 RNA, the relative intensities of 9S and 12S rRNA signals from each sample were plotted. (c) The native F1- and FoF1-ATPase complexes were visualized using light blue native electrophoresis. Purified mitochondria from BF 427 and dKO TbMrf1 1wk and 7wk cultures were lysed with dodecyl maltoside, fractionated on 3–12% BisTris gel and blotted on a PVDF membrane. The F1-ATPase (F1) and the FoF1-ATPase monomer and dimer were all visualized using specific polyclonal antibodies against F1-ATPase subunit β and Fo-ATPase subunit OSCP. (d) SDS-PAGE Western blot analyses of the same mitochondrial lysates as in (c). The steady state abundance of mt hsp70, TbAAC, F1-ATPase subunits β and p18 and Fo-ATPase subunits OSCP and ATPaseTb2 were determined using specific antibodies.
