Proteomic identification of divalent metal cation binding proteins in plant mitochondria

Abstract

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

Experimental Analysis of the Arabidopsis Mitochondrial Proteome Highlights Signaling and Regulatory Components, Provides Assessment of Targeting Prediction Programs, and Indicates Plant-Specific Mitochondrial Proteins [W]

“…A variety of more targeted studies of protein fractions and protein complexes partially purified from Arabidopsis mitochondria also have been undertaken (Sweetlove et al, 2002;Heazlewood et al, 2003aHeazlewood et al, , 2003cHerald et al, 2003;Millar and Heazlewood, 2003;Werhahn et al, 2003). Combining the data presented in all of these reports produces a nonredundant protein set of 145 experimentally determined Arabidopsis mitochondrial proteins.…”

A Periplasmic Iron-binding Protein Contributes toward Inward Copper Supply

“…Phenotypes of ⌬futA2-In an attempt to screen for proteins with readily exchangeable copper sites in Synechocystis PCC 6803, cell extracts were resolved using diagonal, chelate, native two-dimensional polyacrylamide gel electrophoresis (two-dimensional PAGE) similar to that used by others to identify calcium-binding proteins (34). Separate aliquots of extracts were resolved by native PAGE with and without the copper chelator BCS in the second dimension.…”

“…Separate aliquots of extracts were resolved by native PAGE with and without the copper chelator BCS in the second dimension. In the screen for calcium-binding proteins, calcium was included in sample buffers (34), and therefore, extracts were prepared both with and without added copper (0.5 mM). A section of native two-dimensional PAGE (Fig.…”

“…Iron is a minor contaminant of urea (Յ0.0005%), but at 6 M urea this represents one plausible iron source. The affinity of FutA2 for Fe(III) was estimated, by analogy to other ferric binding proteins (34), via competition with citrate. A citrate concentration of 20.8 mM (620-fold molar excess relative to FutA2) removed 50% of the Fe(III) (Fig.…”

MetalloPred: A tool for hierarchical prediction of metal ion binding proteins using cluster of neural networks and sequence derived features

“…Traditionally the metalloproteins have been identified, based on experimental techniques such as absorbance spectroscopy [21], gel electrophoresis [22], metal-affinity columns and shift assay [23], chromatography [24], mass spectroscopy [22], NMR [9] and combined spectroscopic studies [25]. These techniques which require purified or semi-purified proteins of interest, do not facilitate identification of unknown proteins from a complex mixture, or require multi-step processes and very specialized equipment which limit their application ranges.…”