Rapid kinetics of tBid-induced cytochrome c and Smac/DIABLO release and mitochondrial depolarization

Muniswamy Madesh, Bruno Antonsson, Srinivasa M. Srinivasula, Emad S. Alnemri, György Hajnóczky

Research output: Contribution to journalArticle

151 Scopus citations

Abstract

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 (Δψm) 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 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 Δψm 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 Δψm occurs after cytochrome c release and reflects impairment of oxidative metabolism.

Original languageEnglish (US)
Pages (from-to)5651-5659
Number of pages9
JournalJournal of Biological Chemistry
Volume277
Issue number7
DOIs
StatePublished - Feb 15 2002

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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