Superoxide Stimulates a Proton Leak in Potato Mitochondria That Is Related to the Activity of Uncoupling Protein

Michael J Considine,Megan Goodman,Karim S Echtay,Maryse Laloi,James Whelan,Martin D Brand,Lee J Sweetlove

doi:10.1074/jbc.m301075200

Abstract

The ability of plant mitochondrial uncoupling proteins to catalyze a significant proton conductance in situ is controversial. We have re-examined conditions that lead to uncoupling of mitochondria isolated from the tubers of potato (Solanum tuberosum). Specifically, we have investigated the effect of superoxide. In the absence of superoxide, linoleic acid stimulated a proton leak in mitochondria respiring NADH that was insensitive to GTP. However, when exogenous superoxide was generated by the addition of xanthine and xanthine oxidase, there was an additional linoleic acid-stimulated proton leak that was specifically inhibited by GTP. Under these conditions of assay (NADH as a respiratory substrate, in the presence of linoleic acid and xanthine/xanthine oxidase) there was a higher rate of proton conductance in mitochondria from transgenic potato tubers overexpressing the StUCP gene than those from wild type. The increased proton leak in the transgenic mitochondria was completely abolished by the addition of GTP. This suggests that superoxide and linoleic acid stimulate a proton leak in potato mitochondria that is related to the activity of uncoupling protein. Furthermore, it demonstrates that changes in the amount of StUCP can alter the rate of proton conductance of potato mitochondria.

Highlights

The ability of plant mitochondrial uncoupling proteins to catalyze a significant proton conductance in situ is controversial
We provide further evidence that this fatty acid-dependent, superoxide-stimulated, and nucleotide-sensitive proton leak is related to uncoupling protein (UCP) activity by studying proton leak in mitochondria isolated from transgenic potato plants overexpressing the potato StUCP gene
To establish whether UCP can contribute to proton conductance in situ, we investigated linoleic acid-induced proton conductance by measuring the kinetics of proton conductance of isolated potato mitochondria as membrane potential was titrated with KCN (28)

Summary

EXPERIMENTAL PROCEDURES

Chemicals—Unless otherwise indicated all chemicals were from Sigma, Poole, UK). Growth of Potato Plants—Potato The supernatant was centrifuged at 18,000 ϫ g for 10 min to recover an organelle pellet This pellet was resuspended in wash buffer (0.3 M mannitol, 20 mM Tes-KOH (pH 7.5)) and layered onto a stepped gradient of Percoll (Amersham Biosciences Ltd., Little Chalfont, UK) consisting of steps of 50, 28, and 20% (v/v) Percoll with 0.3 M mannitol as an osmoticum. Measurement of Proton Conductance—Proton conductance was determined by simultaneous measurement of oxygen consumption and mitochondrial membrane potential using electrodes sensitive to oxygen and the potential-dependent probe, TPMPϩ as described previously (28). Mitochondria (400 ␮g) were resuspended in 2 ml of assay medium (0.3 M mannitol, 1 mM MgCl2, 100 mM KCl, 10 mM KH2PO4 (pH 7.0) 0.1% (w/v) BSA (fraction V, fatty acid free, Roche Diagnostics Ltd., Lewes, UK)) containing 50 ␮M xanthine, 1 ␮M oligomycin, and 0.1 ␮M nigericin (to collapse the difference in pH across the inner membrane). Linoleic acid (final concentration, 300 ␮M), GTP (final concentration, 2 mM), xanthine oxidase (0.015 unit; Roche Diagnostics Ltd.) and superoxide dismutase (24 units, CN Biosciences (UK) Ltd., Nottingham, UK) were added as indicated

RESULTS

DISCUSSION

Linoleic acid ϩ superoxide Linoleic acid ϩ superoxide ϩ GTP