SummaryTreatment of Arabidopsis cell culture for 16 h with H 2 O 2 , menadione or antimycin A induced an oxidative stress decreasing growth rate and increasing DCF fluorescence and lipid peroxidation products. Treated cells remained viable and maintained significant respiratory rates. Mitochondrial integrity was maintained, but accumulation of alternative oxidase and decreased abundance of lipoic acid-containing components during several of the treatments indicated oxidative stress. Analysis of the treatments was undertaken by IEF/SDS-PAGE, comparison of protein spot abundances and tandem mass spectrometry. A set of 25 protein spots increased >3-fold in H 2 O 2 /menadione treatments, a subset of these increased in antimycin A-treated samples. A set of 10 protein spots decreased significantly during stress treatments. A specific set of mitochondrial proteins were degraded by stress treatments. These damaged components included subunits of ATP synthase, complex I, succinyl CoA ligase, aconitase, and pyruvate and 2-oxoglutarate dehydrogenase complexes. Nine increased proteins represented products of different genes not found in control mitochondria. One is directly involved in antioxidant defense, a mitochondrial thioredoxin-dependent peroxidase, while another, a thioredoxin reductase-dependent protein disulphide isomerase, is required for protein disulfide redox homeostasis. Several others are generally considered to be extramitochondrial but are clearly present in a highly purified mitochondrial fraction used in this study and are known to play roles in stress response. Using H 2 O 2 as a model stress, further work revealed that this treatment induced a protease activity in isolated mitochondria, putatively responsible for the degradation of oxidatively damaged mitochondrial proteins and that O 2 consumption by mitochondria was significantly decreased by H 2 O 2 treatment.Abbreviations: DCF-DA, 2 0 ,7 0 -dichlorofluorescin diacetate; FDA, fluorescein diacetate; MDA, malondialdehyde; TCA, tricarboxylic acid; SOD, superoxide dismutase; ROS, reactive oxygen species; PDI, protein disulphide isomerase.
Divalent metal binding proteins in the Arabidopsis mitochondrial proteome were analysed by mobility shifts in the presence of divalent cations during two-dimensional diagonal sodium dodecyl sulphate^polyacrylamide gel electrophoresis. Tandem mass spectrometry and searches of the predicted Arabidopsis protein dataset were used in an attempt to identify 34 of the proteins which shifted. This analysis identi¢ed a total of 23 distinct protein spots as the products of at least 11 di¡erent Arabidopsis genes. A series of proteins known to be divalent cation-binding proteins, or to catalyse divalent cation-dependent reactions, were identi¢ed. These included: succinyl CoA ligase L L subunit, Mn-superoxide dismutase (SOD), an Fe^S centred component of complex I and the REISKE iron^sulphur protein of the b/c 1 complex. A further set of four proteins of known function but without known divalent binding properties were also identi¢ed: the Vb subunit of cytochrome c oxidase, a subunit of ATP synthase (orfB), the acyl carrier protein, and the translocase of the outer membrane (TOM20). Three other proteins, of unknown function, were also found to shift in the presence of divalent cations. This approach has broad application for the identi¢cation of sub-proteomes based on the metal interaction of polypeptides. ß
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