Specific modification of mitochondrial protein thiols in response to oxidative stress: a proteomics approach.
Mitochondria play a central role in redox-linked processes in the cell through mechanisms that are thought to involve modification of specific protein thiols, but this has proved difficult to assess. In particular, specific labeling and quantitation of mitochondrial protein cysteine residues have not been achieved due to the lack of reagents available that can be applied to the intact organelle or cell. To overcome these problems we have used a combination of mitochondrial proteomics and targeted labeling of mitochondrial thiols using a novel compound, (4-iodobutyl)triphenylphosphonium (IBTP). This lipophilic cation is accumulated by mitochondria and yields stable thioether adducts in a thiol-specific reaction. The selective uptake into mitochondria, due to the large membrane potential across the inner membrane, and the high pH of the matrix results in specific labeling of mitochondrial protein thiols by IBTP. Individual mitochondrial proteins that changed thiol redox state following oxidative stress could then be identified by their decreased reaction with IBTP and isolated by two-dimensional electrophoresis. We demonstrate the selectivity of IBTP labeling and use it to show that glutathione oxidation and exposure to an S-nitrosothiol or to peroxynitrite cause extensive redox changes to mitochondrial thiol proteins. In conjunction with blue native gel electrophoresis, we used IBTP labeling to demonstrate that thiols are exposed on the matrix faces of respiratory Complexes I, II, and IV. This novel approach enables measurement of the thiol redox state of individual mitochondrial proteins during oxidative stress and cell death. In addition the methodology has the potential to identify novel redox-dependent modulation of mitochondrial proteins.
Lin
Tsu-Kung
TK
Department of Biochemistry, University of Otago, Box 56, Dunedin, New Zealand.
Hughes
Gillian
G
Muratovska
Aleksandra
A
Blaikie
Frances H
FH
Brookes
Paul S
PS
Darley-Usmar
Victor
V
Smith
Robin A J
RA
Murphy
Michael P
MP
D000818:Animals; D002412:Cations; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007150:Immunohistochemistry; D019169:Jurkat Cells; D009682:Magnetic Resonance Spectroscopy; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D008956:Models, Chemical; D009943:Organophosphorus Compounds / Q000737:chemistry* / Q000494:pharmacology*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D030421:Peroxynitrous Acid / Q000378:metabolism; D010766:Phosphorylation; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D026403:S-Nitrosothiols / Q000378:metabolism
C456341:(4-iodobutyl)triphenylphosphonium; D002412:Cations; D009943:Organophosphorus Compounds; D010975:Platelet Aggregation Inhibitors; D026403:S-Nitrosothiols; D030421:Peroxynitrous Acid