A matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) analysis of proteins released from isolated liver mitochondria treated with recombinant truncated Bid.
A crucial event in the process of apoptosis is caspase-dependent generation of truncated Bid (tBid), inducing release of cytochrome c. In an in vitro reconstitution system we combined purified recombinant tBid with isolated liver mitochondria and identified the released proteins using a proteomic matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) approach. In order to meet physiological conditions, the concentration of tBid was chosen such that it was unable to induce cytochrome c release in mitochondria derived from liver-specific Bcl-2-transgenic mice. Several mitochondrial proteins were identified to be released in a tBid-dependent way, among which cytochrome c, DIABLO/Smac, adenylate kinase 2, acyl-CoA-binding protein, endonuclease G, polypyrimidine tract-binding protein, a type-I RNA helicase, a WD-40 repeat-containing protein and the serine protease Omi. Western blotting confirmed the absence of adenylate kinase 3, a matrix mitochondrial protein. These results demonstrate that a physiologically relevant concentration of tBid is sufficient to induce release of particular intermembrane mitochondrial proteins belonging to a broad molecular-mass range.
Van Loo
G
G
Flanders Interuniversity Institute for Biotechnology and Ghent University, Department of Molecular Biology, Unit of Molecular Signaling and Cell Death, KL Ledeganckstraat 35, B-9000 Gent, Belgium.
Demol
H
H
van Gurp
M
M
Hoorelbeke
B
B
Schotte
P
P
Beyaert
R
R
Zhivotovsky
B
B
Gevaert
K
K
Declercq
W
W
Vandekerckhove
J
J
Vandenabeele
P
P
D000263:Adenylate Kinase / Q000032:analysis / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology*; D003574:Cytochrome c Group / Q000032:analysis / Q000378:metabolism; D026261:Diazepam Binding Inhibitor / Q000032:analysis; D004706:Endodeoxyribonucleases / Q000032:analysis / Q000378:metabolism; D000074923:High-Temperature Requirement A Serine Peptidase 2; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000032:analysis / Q000378:metabolism*; D038941:Polypyrimidine Tract-Binding Protein; D016601:RNA-Binding Proteins / Q000032:analysis / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D012261:Ribonucleoproteins / Q000032:analysis / Q000378:metabolism; D012697:Serine Endopeptidases / Q000032:analysis / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D051017:Apoptosis Regulatory Proteins; D051023:BH3 Interacting Domain Death Agonist Protein; C494815:Bid protein, mouse; D002352:Carrier Proteins; D003574:Cytochrome c Group; C499605:Diablo protein, mouse; D026261:Diazepam Binding Inhibitor; D007527:Isoenzymes; D024101:Mitochondrial Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D038941:Polypyrimidine Tract-Binding Protein; D000263:Adenylate Kinase; C110938:adenylate kinase 2; D004706:Endodeoxyribonucleases; C052391:endonuclease G; D012697:Serine Endopeptidases; D000074923:High-Temperature Requirement A Serine Peptidase 2; C000615915:Htra2 protein, mouse