Bax Is Present as a High Molecular Weight Oligomer/Complex in the Mitochondrial Membrane of Apoptotic Cells

Bax is a Bcl-2 family protein with proapoptotic activity, which has been shown to trigger cytochrome crelease from mitochondria both in vitro and in vivo. In control HeLa cells, Bax is present in the cytosol and weakly associated with mitochondria as a monomer with an apparent molecular mass of 20,000 Da. After treatment of the HeLa cells with the apoptosis inducer staurosporine or UV irradiation, Bax associated with mitochondria is present as two large molecular weight oligomers/complexes of 96,000 and 260,000 Da, which are integrated into the mitochondrial membrane. Bcl-2 prevents Bax oligomerization and insertion into the mitochondrial membrane. The outer mitochondrial membrane protein voltage-dependent anion channel and the inner mitochondrial membrane protein adenosine nucleotide translocator do not coelute with the large molecular weight Bax oligomers/complexes on gel filtration. Bax oligomerization appears to be required for its proapoptotic activity, and the Bax oligomer/complex might constitute the structural entirety of the cytochromec-conducting channel in the outer mitochondrial membrane. Bax is a Bcl-2 family protein with proapoptotic activity, which has been shown to trigger cytochrome crelease from mitochondria both in vitro and in vivo. In control HeLa cells, Bax is present in the cytosol and weakly associated with mitochondria as a monomer with an apparent molecular mass of 20,000 Da. After treatment of the HeLa cells with the apoptosis inducer staurosporine or UV irradiation, Bax associated with mitochondria is present as two large molecular weight oligomers/complexes of 96,000 and 260,000 Da, which are integrated into the mitochondrial membrane. Bcl-2 prevents Bax oligomerization and insertion into the mitochondrial membrane. The outer mitochondrial membrane protein voltage-dependent anion channel and the inner mitochondrial membrane protein adenosine nucleotide translocator do not coelute with the large molecular weight Bax oligomers/complexes on gel filtration. Bax oligomerization appears to be required for its proapoptotic activity, and the Bax oligomer/complex might constitute the structural entirety of the cytochromec-conducting channel in the outer mitochondrial membrane. Apoptosis is mediated through two major pathways, the death receptor pathway and the mitochondrial pathway (1Hengartner M.O. Nature. 2000; 407: 770-776Crossref PubMed Scopus (6296) Google Scholar). The mitochondrial pathway is controlled and regulated by the Bcl-2 family of proteins (2Yang E. Korsmeyer S.J. Blood. 1996; 88: 386-401Crossref PubMed Google Scholar, 3Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 4Kelekar A. Thompson C.B. 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Cell Death 1997; PubMed Scopus Google in with and Apoptosis by the cells in staurosporine for the cells UV and for cells in the of staurosporine or UV the of the the cells in by at for and in mitochondrial The cells in with by through a The through the and at for The and the in The The at for to and The at for This and at for to the cell The to the mitochondrial in and at for the mitochondrial in of and on of a of of on of of The at in a for at at the of and and in at mitochondria in of with caspase for at the of the the at for and the mitochondria with The mitochondrial in or on for and at for The to the mitochondrial the protein The mitochondrial in to a protein of and on for the of the the at The which proteins, and to a of The in on for and at for The which membrane proteins, at on a in and at a of by The with gel proteins from the The of the by that at the and the at After of and from the by The on and to The proteins with the as in the and the with the from Bax with and at the and the into the cells with the Bax The between and to the addition of of of the Bax and the cells for the of the the cells and mitochondria isolated by as isolated from cells to of mitochondrial protein with caspase in for at the of the the mitochondria by and in of The and to of and the for at The by the addition of to a of and for at The mitochondria by and by in for at The at for and the with of protein for at by and of and of protein and on for at by and with of The in and the on in a to membranes and by with and at for and with and as Bax with a at the in and purified as S. Mazzei G. E. Antonsson B. 1999; PubMed Scopus Google Scholar). by the in the for The purified protein in at with a at the and purified as S. Osen-Sand Nichols A. R. Montessuit S. S. K. Antonsson B. Martinou J. Cell Biol. 1999; PubMed Scopus Google Scholar). The purified in at by of with of caspase and of caspase and the for at of as by from caspase by to by into and at HeLa cells have been to the of Bax in apoptotic Apoptosis by the cells in the of staurosporine for or by UV with in HeLa cells, Bax both in the cytosol and associated with the mitochondria In apoptotic cells, Bax from the Although a in Bax associated with the mitochondria in the apoptotic cells, not for the Bax that Bax that is not to the mitochondria is in apoptotic cells, through A. Y. Reed J.C. 1998; 17: PubMed Scopus Google Scholar). the of Bax associated with mitochondria from control and apoptotic HeLa cells, mitochondrial proteins with as and The mitochondrial on gel and the from the by with an to the and an of their molecular and of their Bax from control or apoptotic HeLa cell mitochondria as a at to a molecular mass of 260,000 Bax in control apoptotic mitochondrial of other mitochondrial membrane proteins that have been to with including Bak, Bcl-XL, and with Bax as molecular weight in between mitochondrial from control and apoptotic cells the mitochondrial membrane protein with a in and a in The of the at has been reported to of Bax and other Bcl-2 family members Y.-T. Youle R.J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In a reported that to trigger oligomerization of Bax B. Montessuit S. S. R. Martinou J.-C. 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In the mitochondrial from control cells, Bax in This to a molecular mass of 20,000 Da, which is to the molecular mass of In in the mitochondrial from cells apoptosis been by staurosporine Bax as large in with molecular of 260,000 and 96,000 The of Bax in between and on the of cells that apoptosis at the of with a that the protein Bax not with for that the mitochondria isolated by by that the Bax associated with the the mitochondrial purified on a the of the The mitochondria the Bax in the mitochondrial from apoptotic cells, Bax present in both apoptotic and control mitochondrial In apoptosis by HeLa cells to UV oligomerization of Bax associated with the mitochondria. to mitochondrial from cells, two of Bax at and on gel isolated HeLa mitochondria with caspase for The from the mitochondria the Bax as mitochondria from or HeLa cells Thus, apoptosis by in Bax In addition to its with Bcl-2 family Bax has been reported to with other mitochondrial membrane with the outer mitochondrial membrane protein and the inner membrane protein both of the have been reported S. M. Y. Nature. 1999; PubMed Scopus Google Scholar, I. C. Susin S.A. S. Reed J.C. Kroemer G. Science. 1998; 281: PubMed Scopus Google Scholar). the Bax these proteins, the from the gel for Bak, Bcl-XL, and major of in and present a molecular weight which forms of in the between control and apoptotic cells Thus, the Bax are not present in the mitochondrial membrane of cells, between and the Bax are a large with a major in for Bak, between control and apoptotic cells the Bax as a at in the mitochondrial from both control and apoptotic the Bax at between the control and apoptotic mitochondrial that is of the Bax from mitochondria of apoptotic The of recombinant Bax from the gel two at and This that recombinant Bax forms two of It appears that the be a of the In the mitochondrial from apoptotic HeLa cells, two Bax the at and the at and The Bax from apoptotic mitochondrial at molecular with the recombinant This in apparent molecular weight might that in the mitochondrial membrane the Bax with as the of the Bax Bax to and in of Bax the cells in the of the caspase to cells from and for by that the Bax both in the cytosol and the mitochondria isolated from and cells, membrane proteins with and the on gel filtration. the cytosol from the cells, and the on gel filtration. In the mitochondrial from cells and in the cytosol from cells, Bax in the mitochondrial from cells Bax to in staurosporine or HeLa cells The of Bax in the with the not the to and that the proteins of the the of the mitochondria from the cells for the of Although the of the proteins low, in the Bax the of the Bax on isolated mitochondria from the cells as and Bax with and the on with and in Bax with to and the with that the Bax from mitochondria of apoptotic cells are of Bax and are not Bax in complex with other Bax not in the cytosol It has been shown that in Bax the is not and not S. Osen-Sand Nichols A. R. Montessuit S. S. K. Antonsson B. Martinou J. Cell Biol. 1999; PubMed Scopus Google not the Bax in the The molecular of the in the mitochondrial and the of Bax in the to Bax and Although molecular weight can be the molecular be in of the between a Bax and to a complex with an protein of of mitochondrial from cells by gel on from cells and cells with and Bax isolated and with proteins by at for of the of protein on a in and at a of of and by and with The from the mitochondrial in addition by and the to and and the to and The cytosol from cells with and as the mitochondrial with have shown that treatment of isolated HeLa mitochondria in the insertion of Bax into the outer mitochondrial membrane R. S. Antonsson B. Martinou J.-C. Cell. Biol. 2000; PubMed Scopus Google Scholar). the in Bax required for into the mitochondrial membrane mitochondria from control and apoptotic cells with which proteins to the proteins integrated into the membrane associated with the membrane After the and the membrane by the by R. S. Antonsson B. 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This Bax in mitochondrial from HeLa cells apoptosis by staurosporine or UV that the addition of caspase Bid, a Bax to mitochondria isolated from HeLa cells to trigger the formation of large molecular weight Bax that oligomerization of Bax and its to large be a to apoptotic The Bax in the mitochondrial and they in the cytosol, that they at or within the outer mitochondrial membrane. the that these large Bax of mitochondria into the outer mitochondrial their This be with that Bax from the cytosol to mitochondria apoptosis Y.-T. Youle R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, C.L. A. Youle R.J. J. Cell Biol. 1997; PubMed Scopus Google Scholar, A. M. R. K. Korsmeyer S.J. J. Cell Biol. 1998; 143: PubMed Scopus Google and that of Bax is by its insertion in the mitochondrial membrane A. Jockel J. 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