Figure 6.

Cytosol (PEF) increases both complex III and IV accessibility after tBid-induced cytochrome c release. Mitochondria were incubated with or without tBid in either buffer B or cytosol, at 0 or 22°C. At suitable times (1.7, 2.7, or 3.7 h), mitochondria were pelleted and analyzed by Western blot for cytochrome c translocation (bottom) or washed and resuspended in buffer E for measurement of complex III (top) and IV (middle) accessibility (arbitrary units). For comparison between assays, mitochondria were lysed by incubation in water for 30 min on ice. These osmotically lysed mitochondria retain their cytochrome c due to the low salt content (see Materials and Methods). Data are mean and SD for duplicate measurements and are representative of three experiments. Note: The time course of permeabilization by PEF is slower in Fig. 6 than Fig. 5; this merely reflects a normal variation between Xenopus extracts in time of onset of apoptotic changes ( Newmeyer et al. 1994; Farschon et al. 1997).