Abstract
Modification with arginine-specific glyoxals modulates the permeability transition (PT) of rat liver mitochondria, with inhibitory or inducing effects that depend on the net charge of the adduct(s). Here, we show that phenylglyoxal (PGO) affects the PT in a species-specific manner (inhibition in mouse and yeast, induction in human and Drosophila mitochondria). Following the hypotheses (i) that the effects are mediated by conserved arginine(s) and (ii) that the PT is mediated by the F-ATP synthase, we have narrowed the search to 60 arginines. Most of these residues are located in subunits α, β, γ, ϵ, a, and c and were excluded because PGO modification did not significantly affect enzyme catalysis. On the other hand, yeast mitochondria lacking subunit g or bearing a subunit g R107A mutation were totally resistant to PT inhibition by PGO. Thus, the effect of PGO on the PT is specifically mediated by Arg-107, the only subunit g arginine that has been conserved across species. These findings are evidence that the PT is mediated by F-ATP synthase.
Highlights
Modification with arginine-specific glyoxals modulates the permeability transition (PT) of rat liver mitochondria, with inhibitory or inducing effects that depend on the net charge of the adduct(s)
Following the hypotheses (i) that the effects are mediated by conserved arginine(s) and (ii) that the PT is mediated by the F-ATP synthase, we have narrowed the search to 60 arginines
The PT is mediated by opening of a regulated channel, the permeability transition pore (PTP), which coincides with the mitochondrial megachannel (MMC) identified by electrophysiology [2, 3]
Summary
The effects of PGO on the PTP have been conserved but differ between species Consistent with the effect of PGO on the PTP of rat liver mitochondria, PGO desensitized the PTP to Ca2ϩ in mouse liver mitochondria (MLM) (Fig. 1A) as well as in mitochondria from Saccharomyces cerevisiae in the presence of ETH129, a selective ionophore that allows Ca2ϩ equilibration across the inner membrane in yeast mitochondria (Fig. 1B). Sensitization of PTP opening by PGO in human and Drosophila cells was not due to digitonin permeabilization, as it was observed in mitochondria isolated from these cells (results not shown). These findings indicate that the ability to modulate the PTP by PGO has been conserved across species. Blue native gel separation and in-gel activity staining after digitonin extraction did not reveal any major effect of PGO on F-ATP synthase oligomeric state in MLM (Fig. 2E). In these experiments, we used MLM because they are easy to purify and because the mouse enzyme is virtu-. Number and positions of arginine residues conserved between F-ATP synthase of S. cerevisiae, H. sapiens, and M. musculus (in parentheses when different)
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