Bcl-2 family proteins are the main regulators of the mitochondrial apoptosis
pathway. In response to cellular stress, the pro-apoptotic Bcl-2 family members Bax
and Bak undergo major conformational changes on the mitochondria and form
oligomers. Bax/Bak activation leads to the permeabilization of the outer
mitochondrial membrane and the release of mitochondrial intermembrane space
proteins, such as cytochrome c into the cytosol. Cytosolic cytochrome c induces the
caspase cascade and thereby, the cell removes itself in a coordinated manner. Bax
and Bak are tightly regulated by other Bcl-2 proteins to prevent spontaneous cell
death. Anti-apoptotic Bcl-2 proteins, for example, block Bax and Bak activation by
the transient interactions on the mitochondria. Bax resides in the cytosol and
constantly translocates to the mitochondria. Anti-apoptotic Bcl-2 proteins with the
transient interactions retrotranslocate Bax into the cytosol and thus, inactive Bax is
stabilized in the cytosol. We showed that Bax retrotranslocation is not only
dependent on interactions between Bax BH3 domain and the hydrophobic grooves,
but also requires the Bcl-xL C-terminus. Deletions or substitutions in the Bcl-xL
membrane anchor cause the deceleration of Bax retrotranslocation measured by
fluorescence loss in photobleaching (FLIP). Moreover, the Bax population on the
mitochondria is increased when the Bax retrotranslocation is diminished. However,
mitochondrial Bax is not per se active at reduced Bax retrotranslocation. Only in
response to apoptotic stimuli, cells with increased mitochondrial Bax pools are
sensitized to death. We discovered that mitochondrial Bak is also retrotranslocated,
but Bak retrotranslocation is slower than Bax shuttling. Therefore, the increased
mitochondrial Bak localization is likely caused by the lower Bak retrotranslocation
rate. Generation of chimeras with exchanged C-terminal domains of Bax and Bak
cause inverse subcellular localization of these proteins. The mitochondrial Bax with
the Bak membrane anchor has lower shuttling rates and shows more toxicity in the
absence of apoptotic stimuli compared to wildtype Bax. In contrast, Bak with the Bax
membrane anchor has great similarities in terms of the localization patterns and
retrotranslocation rates compared to wildtype Bax. In other words, interchanging
the membrane anchors between Bax and Bak shows that retrotranslocation rates are highly dependent on the membrane anchor hydrophobicities. Ultimately, this
determines the Bax and Bak localization and their shuttling rates.
Bax undergoes conformational changes during translocation to the mitochondria
where Bax association is mediated by the Bax C-terminus. Recent structural evidence
suggests that in order to expose Bax C-terminus residing within the Bax globular
protein, the latch (helices 6-8) domain has to separate from the core (helices 1-
5) domain. However, we found that the Bax helix 6 is important for the interaction
with VDAC2 and thus, Bax and Bcl-xL could transiently interact on VDAC2 to induce
Bax retrotranslocation. However, the failure of Bax retrotranslocation from the
mitochondria could lead to Bax oligomerization and cell death. Hindering the
conformational changes involving helix 5 and helix 6 in Bax, generating Bax 5-6,
enabled us to analyze the consequences of failed Bax retrotranslocation in cell. Bax
5-6 does not participate to VDAC2 complex on the mitochondria, as shown by bluenative
PAGE. Furthermore, under stress conditions Bax 5-6 has higher apoptotic
activities in cells. We discovered misregulated Bax can be ubiquitinated in the cell by
the E3 ligase Parkin leading to proteasomal degradation of targeted Bax to protect
cells from stimulus-independent cell death. Proteasome-dependent Bax degradation
could represent a quality control mechanism for Bax retrotranslocation. |