Rapid kinetics of tBid-induced cytochrome c and Smac/DIABLO release and mitochondrial depolarization.
Cleavage of Bid has been shown to promote apoptosis by inducing mitochondrial membrane permeabilization with the resultant release of apoptosis-inducing proteins from the intermembrane space into the cytosol. However, direct visualization of the Bid-induced release of various proteins from the highly compartmentalized intermembrane space and the changes in the mitochondrial metabolic machinery remain elusive. Using green fluorescent protein fusion proteins and immunostaining in individual permeabilized HepG2 cells, first we demonstrated that truncated Bid (15.5-kDa C-terminal fragment, tBid) evoked a rapid and essentially complete release of cytochrome c and Smac/DIABLO from every mitochondrion. To establish at a resolution of seconds the kinetics of tBid-induced cytochrome c and Smac/DIABLO release and depolarization, we monitored the mitochondrial membrane potential (DeltaPsi(m)) fluorimetrically in permeabilized cells and applied a rapid filtration method to obtain cytosolic fractions for Western blotting. We found that subnanomolar doses of tBid were sufficient to evoke cytochrome c release and mitochondrial depolarization, whereas full-length Bid was 100-fold less effective. Bcl-x(L) prevented tBid-induced cytochrome c release and depolarization. In response to 2.5 nm tBid, cytochrome c release started after a 10 s delay, displayed rapid progression, and was complete at 50-70 s. Release of Smac/DIABLO was synchronized with cytochrome c release, whereas the loss of DeltaPsi(m) lagged slightly behind cytochrome c release. Furthermore, tBid-induced cytochrome c release was insensitive to changes in substrate composition, but tBid-induced depolarization did not occur in the presence of extramitochondrial ATP supply. Thus, tBid-induced permeabilization of the outer membrane permits rapid release of cytochrome c and Smac/DIABLO from all domains of the intermembrane space. The tBid-induced loss of DeltaPsi(m) occurs after cytochrome c release and reflects impairment of oxidative metabolism.
Madesh
Muniswamy
M
Department of Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Antonsson
Bruno
B
Srinivasula
Srinivasa M
SM
Alnemri
Emad S
ES
Hajnóczky
György
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D051023:BH3 Interacting Domain Death Agonist Protein; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D049452:Green Fluorescent Proteins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000737:chemistry*; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014162:Transfection; D051020:bcl-X Protein
D051017:Apoptosis Regulatory Proteins; C494811:BCL2L1 protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002352:Carrier Proteins; D003574:Cytochrome c Group; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D024101:Mitochondrial Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D010100:Oxygen