Analysis of endogenous Bax complexes during apoptosis using blue native PAGE: Implications for Bax activation and oligomerization

Anthony J. Valentijn, John Paul Upton, Andrew P. Gilmore

    Research output: Contribution to journalArticlepeer-review


    Bax, a pro-apoptotic Bcl-2 family protein, translocates to mitochondria during apoptosis, where it causes MOMP (mitochondrial outer membrane permeabilization). MOMP releases proapoptotic factors, such as cytochrome c and SMAC (second mitochondrial activator of caspases)/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 causing its translocation and oligomerization into high-molecular-mass 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. In the present study, 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 ECM (extracellular matrix). Using BN-PAGE (blue native 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 oligomerization is an event that must be interpreted differently from the currently held view that it represents the apoptotic pore. © The Authors.
    Original languageEnglish
    Pages (from-to)347-357
    Number of pages10
    JournalBiochemical Journal
    Issue number2
    Publication statusPublished - 1 Jun 2008


    • Anoikis
    • Apoptosis
    • Bax
    • Blue native PAGE
    • Mitochondrion


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