Genetics of Apoptosis

(Barry) #1

CsA has mitochondrial effects beyond inhibition of PTP cannot be excluded
(Jurgensmeier et al., 1998).
Not all reports agree that PTP activation is necessary for Bax-induced cytochrome
c release. A Mg2+-sensitive mechanism resistant to the PTP inhibitors CsA, bongkrekic
acid, ADP, and EGTA has been reported (Eskes et al., 1998). CsA-resistant induction
of cytochrome c release has also been described for Bax, Bid, and Bik (Shimizu and
Tsujimoto, 2000; von Ahsen et al., 2000a). Furthermore, studies of Bax in mutant
yeast backgrounds have disputed that VDAC is required for Bax-mediated
translocation of cytochrome c, growth arrest, or lethality (Priault et al., 1999; Gross
et al., 2000; Harris et al., 2000).


2.6

Proapoptotic functions: homooligomers and pores

Pores formed by homooligomers of Bax are also candidates for the mechanism of
cytochrome c escape from mitochondria. Recombinant Bax forms high-conductance
channels in artificial membranes with predominant conductance of 0.5–1.5 nS
(Antonsson et al., 1997; Schlesinger et al., 1997). Pore sizing with dextran molecules
of different Stokes diameters indicates Bax pore diameters of up to 22–30 A, large
enough for cytochrome c passage (Saito et al., 2000). Analysis of the transport as a
function of Bax concentration by Hill plots suggested that four Bax molecules were
required for cytochrome c transport. Cross-linker studies and CHAPS extraction of
endogenous Bax recruited to mitochondria following apoptotic stimuli demonstrate
Bax complexes in the range of 41–260 kDa, consistent with homodimers and larger
oligomers (Gross et al., 1998; Antonsson et al., 2001; Mikhailov et al., 2001).
Recently, patch-clamping studies of mitochondria and proteoliposomes
incorporating mitochondrial outer membranes identified a novel, high-conductance
channel (mitochondrial apoptosis-induced channel [MAC]) that appeared within 12
h of removal of IL-3 from FL5.12 prolymphoid cells (Pavlov et al., 2001). A channel
with similar properties was formed in yeast outer mitochondrial membranes within
16 h of Bax induction. MAC activity was not observed in IL-3-deprived FL5.12 cells
previously transfected with Bcl-2. The maximal conductance of MAC in mammalian
cells corresponds to a pore diameter of 40 A, suitable for passage of cytochrome c.
Bax has also been reported to decrease the stability of planar lipid bilayers in a
concentration-dependent manner (Basañez et al., 1999). The mechanism involved a
decrease in linear tension, which is expected to result in hydrophilic pores within the
lipid membrane itself. This effect was not observed with Bcl-xL, nor could Bcl-xL
protect membrane stability when added to membranes concurrently or sequentially
with Bax.


MAKING SENSE OF THE BCL-2 FAMILY OF APOPTOSIS REGULATORS 55
Free download pdf