Analysis of endogenous Bax complexes during apoptosis using blue native PAGE - implications for Bax activation and oligomerisation

Anthony J Valentijn , John-Paul Upton , Andrew P Gilmore (1)

1 Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences

Bax, a pro-apoptotic Bcl-2 family protein, translocates to mitochondria during apoptosis, where it brings about mitochondrial outer membrane permeabilisation (MOMP). MOMP releases pro-apoptotic factors, such as cytochrome c and SMAC/Diablo, into the cytosol where they activate caspases. It is often inferred that Bax activation occurs in a single step, a conformational change in the protein bringing about its translocation and oligomerisation into high molecular weight membrane pores. However, a number of studies have shown that Bax translocation to mitochondria does not necessarily induce MOMP. Indeed, Bax translocation can occur several hours prior to release of cytochrome c, indicating that its regulation may be a complex series of events, some of which occur following its association with mitochondria. We have examined endogenous Bax in epithelial cells undergoing anoikis, a physiologically relevant form of apoptosis that occurs when normal cells lose contact with the extracellular matrix (ECM). Using blue native polyacrylamide gel electrophoresis (BN-PAGE) we show that Bax forms a 200 kDa complex before caspase activation. Furthermore, Bax in this 200 kDa complex is not in the active conformation, as determined by exposure of N-terminal epitopes. These results indicate that Bax oligomerisation is an event that must be interpreted differently from the currently held view that it represents the apoptotic pore.

Mots clés: Life Sciences

Anthony J Valentijn, John-Paul Upton, Andrew P Gilmore. Analysis of endogenous Bax complexes during apoptosis using blue native PAGE - implications for Bax activation and oligomerisation. Biochemical Journal, 2008, 412 (2), pp.347-357. ⟨10.1042/BJ20071548⟩. ⟨hal-00478915⟩