Figure 4: Modulation of mitochondrial protein sorting considerably restores the bioenergetic capacity of a yeast model of ATP synthase disorders.
From: Mitochondrial protein sorting as a therapeutic target for ATP synthase disorders
(a,b) Tim21 overexpression accelerates the incorporation of subunits Qcr8 and Su h into cytochrome bc1-containing respiratory chain supercomplexes and ATP synthase, respectively, in fmc1Δ mitochondria. Radiolabelled, in vitro-synthesized proteins were imported into isolated mitochondria and their assembly into their respective complexes (indicated with arrows) was followed using BN-PAGE and autoradiography. Δψ, mitochondrial inner membrane potential; III2/IV2, III2/IV1, III2: respiratory chain supercomplexes formed by complex III (cytochrome bc1) and IV (cytochrome c oxidase, COX); V1 and V2, monomeric and dimeric F1Fo-ATP synthase (complex V), respectively. (c) Genetic modulation of mitochondrial protein sorting improves the mitochondrial inner membrane potential (Δψ) of fmc1Δ yeast. The Δψ of indicated mitochondria was monitored using a DiSC3(5) fluorescence quenching assay (see Methods). The difference in fluorescence before and after addition of valinomycin indicates the magnitude of Δψ of the analysed mitochondria. (d) Genetic modulation of mitochondrial protein sorting improves the bioenergetic capacity of fmc1Δ yeast. The following parameters were measured in isolated mitochondria: maximal/uncoupled respiratory rate (upper left), maximal/uncoupled cytochrome c oxidase (COX) activity with ascorbate/TMPD as a substrate (upper right), oligomycin-sensitive ATP hydrolysis using a colorimetric ATPase assay (lower left), and oligomycin-sensitive ATP synthesis using a luciferase assay (lower right). Bar height corresponds to median value of individual measurements displayed as points (both technical and biological replicates). Significance according to a t-test is represented as **P<0.01 or *P<0.05.
