BCL-XL directly retrotranslocates the monomeric BAK

BCL-XL, an anti-apoptotic BCL-2 family protein, potently inhibits BAK oligomerization and the formation of toxic mitochondrial pores in response to cellular stress. This report aims to explore which form of mitochondrial monomeric and oligomerized BAK can be retrotranslocated by BCL-XL. Fluorescence imaging of living cells co-expressing CFP-BCL-XL and YFP-BAK showed that BCL-XL markedly inhibited mitochondrial BAK oligomerization and resulted in partial cytosolic BAK distribution. Live-cell fluorescence resonance energy transfer (FRET) analyses showed that BAK auto-oligomerized on mitochondria and BCL-XL physically sequestrated monomeric BAK to prevent BAK oligomerization. Fluorescence loss in photobleaching (FLIP) analyses showed that BCL-XL retrotranslocated the monomeric BAK from mitochondria into cytosol, whereas monomeric BAK reduced the retrotranslocation rate of BCL-XL. Live-cell time-lapse imaging and FLIP experiments in living cells with BAK oligomers displayed that BCL-XL did not depolymerize or retrotranslocate the oligomerized BAK. Collectively, BCL-XL retrotranslocates monomeric instead of oligomerized BAK from mitochondria into cytosol.