Identification of intra- and intermolecular disulphide bonding in the plant mitochondrial proteome by diagonal gel electrophoresis.
Redox active proteins in plant mitochondria were examined using 2-D oxidant/reductant diagonal-SDS-PAGE to separate and identify proteins with intermolecular or intramolecular disulphide bonds using diamide in the first dimension and DTT in the second dimension. Eighteen proteins spots were resolved either above or below the diagonal and these were in-gel digested and identified by MS/MS. This analysis revealed intermolecular disulphide bonds in alternative oxidase, O-acetylserine (thiol) lyase, citrate synthase and between subunits of the ATP synthase. Intramolecular disulphide bonds were observed in a range of mitochondrial dehydrogenases, elongation factor Tu, adenylate kinase and the phosphate translocator. Many of the soluble proteins found were known glutaredoxin/thioredoxin targets in other plants, but the membrane proteins were not found by these methods nor were the nature of the disulphides able to be investigated. The accessibility of thiols involved in disulphide bonds to modification by a lipid derived aldehyde gave an insight into the potential impact of Cys modification on redox-functions in mitochondria during lipid peroxidation. Comparison of the protein sequences of the identified proteins with homologs from other species has identified specific Cys residues that may be responsible for plant-specific redox modulations of mitochondrial proteins.
Winger
Alison M
AM
ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, Crawley, WA, Australia.
Taylor
Nicolas L
NL
Heazlewood
Joshua L
JL
Day
David A
DA
Millar
A Harvey
AH
D017360:Arabidopsis / Q000378:metabolism / Q000648:ultrastructure*; D001665:Binding Sites; D002478:Cells, Cultured; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000032:analysis* / Q000378:metabolism*; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
D004220:Disulfides; D010940:Plant Proteins; D020543:Proteome