(Ayllon et al., 2000), PP2A (Chiang, C.W. et al., 2001) and PP2B, or calcineurin
(Wang, H.G. et al., 1999).
Other BH3 proteins interact with distinct extra-mitochondrial targets, perhaps to
sequester them from sites of action (mitochondria and ER) in healthy cells. Bim is
localized to the microtubule dynein motor complex by an interaction with the dynein
light chain, DLC 1 (Puthalakath et al., 1999), and Bmf associates with dynein light
chain 2 (DLC2) in the myosin V actin motor complex (Puthalakath et al., 2001).
Several apoptotic stimuli, including paclitaxel (Bim) and anoikis (Bmf), result in
translocation of these BH3 proteins from their cytoskeletal sites to mitochondria.
DLC1 and DLC2 appear to be released together with their BH binding partners from
cytoskeletal attachments during apoptosis.
Despite its status as the first proapoptotic BH protein to be identified, we
understand relatively little about mechanisms of Bax cytoplasmic retention in healthy
cells and translocation during apoptosis (Pawlowski et al., 2000). By analogy with
Bcl-2, the COOH-terminal hydrophobic sequence in Bax was initially believed to
function as a mitochondrial signal/anchor. Montessuit et al. (1999) demonstrated
that, unlike Bcl-2, recombinant full-length Bax could be expressed as a soluble
protein. Solution of the three-dimensional structure of full-length Bax by NMR
spectroscopy (Suzuki et al., 2000) revealed that the COOH-terminal hydrophobic
sequence is folded back, shielding the hydrophobic cleft in Bax formed by the BH1,
BH2, and BH3 domains. Soon after stimulation of apoptosis, cytoplasmic Bax
undergoes a conformational change affecting an NH 2 -terminal epitope, forms
homodimers/oligomers, and becomes an integral mitochondrial membrane protein
(Goping et al., 1998; Nechustan et al., 1999). The structure of a homodimer of Bax
has yet to be solved, and it is not clear whether proapoptotic homodimers utilize a
BH3-hydrophobic groove interaction similar to the proposed mechanism of
interaction for heterodimers. The exact sequence of these apoptotic changes in Bax
is disputed (Eskes et al., 2000; Murphy et al., 2000). Mutations in the Bax COOH-
terminal hydrophobic tail resulting in constitutive mitochondrial localization
(∆Ser184 and Ser184Val) require an additional apoptotic stimulus for the NH 2 -
terminal conformational change (Nechushtan et al., 1999). Wolter et al. (1997)
demonstrated the requirement of the 21-residue COOH-terminal tail for
mitochondrial translocation. Substitution of the Bcl-2 COOH-terminal tail for the
Bax COOH-terminal tail directs Bax constitutively to mitochondria (Goping et al.,
1998). However, the Bax COOH-terminal tail acts as a mitochondrial signal/anchor
sequence when fused with dihydrofolate reductase, at least in vitro (Goping et al.,
1998; Nechushtan et al., 1999). Goping et al. (1998) identified the NH 2 -terminal
region (ART) of Bax as an inhibitory domain for mitochondrial membrane targeting
and insertion. Deletion of amino acids 1–19 from Bax resulted in mitochondrial
localization of Bax in the absence of an apoptotic signal.
Bax translocation from cytosol to mitochondria appears to be a general
phenomenon following diverse apoptotic stimuli. Two reports have identified general
inducers of Bax mitochondrial targeting that may trigger this event more broadly in
apoptosis. Saikumar et al. (1998) reported that Bax translocation was dependent on
66 GENETICS OF APOPTOSIS