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Papers In Press, published online ahead of print December 6, 2001
Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107
Corresponding Author: gyorgy.hajnoczky{at}mail.tju.edu
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 GFP 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 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-xL prevented tBID-induced cytochrome c release and depolarization. In response to 2.5 nM tBid, cytochrome c release started after 10s delay, displayed rapid progression and was complete at 50-70s. Release of Smac/DIABLO was synchronized with cytochrome c release, whereas the loss of the mitochondrial membrane potential 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 mitochondrial membrane potential occurs after cytochrome c release and reflects impairment of oxidative metabolism.
J. Biol. Chem, 10.1074/jbc.M108171200
Submitted on August 23, 2001
Revised on December 6, 2001
Accepted on December 6, 2001
Rapid kinetics of tBid-induced cytochrome c and Smac/DIABLO release and mitochondrial depolarization
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