Abstract Apoptosis is a
tightly controlled form of programmed cell death that is essential
to proper tissue development and maintenance. Most apoptotic pathways
converge towards the permeabilisation of the outer mitochondrial
membrane. This crucial step involves a cytosolic protein, Bax, which
relocates to the mitochondrial membrane in response to apoptotic
signals and contributes to its permeabilization through pore formation.
This allows the release of cytochrome c, which then activates caspases
to cause cell death. To investigate the interaction of Bax with lipid
membranes, as well as the oligomerization process associated with
pore formation, we have developed an in vitro system containing purified
EGFP-Bax and large unilamellar vesicles containing a small fraction
of fluorescent lipids. Insertion of the protein in the lipid membrane
is triggered by the addition of a second protein, tBid, which can
be fluorescently labeled as well through maleimide chemistry. We
have examined the associations between the different components of
our system by fluorescence cross-correlation spectroscopy (XCS),
and the oligomer stoichiometry of these associations by fluorescence
intensity distribution analysis (FIDA). Our results indicate that
Bax forms large oligomers of defined size in lipid membranes after
transient interaction with tBid. |