Heterodimerization-independent Functions of Cell Death Regulatory Proteins Bax and Bcl-2 in Yeast and Mammalian Cells

The pro-apoptotic protein Bax can homodimerize with itself and heterodimerize with the anti-apoptotic protein Bcl-2, but the significance of these protein-protein interactions remains unclear. Alanine substitution mutations were created in a well conserved IGDE motif found within the BH3 domain of Bax (residues 66–69) and the resulting mutant Bax proteins were tested for ability to homodimerize with themselves and to heterodimerize with Bcl-2. Correlations were made with cell death induction by these mutants of Bax both in mammalian cells where Bax may function through several mechanisms, and in yeast where Bax may exert its lethal actions through a more limited repertoire of mechanisms perhaps related to its ability to form ion channels in intracellular membranes. Two of the mutants, Bax(D68A) and Bax(E69A), retained the ability to homodimerize but failed to interact with Bcl-2 as determined by yeast two-hybrid assays and co-immunoprecipitation analysis using transfected mammalian cells. The Bax(E69A) protein exhibited a lethal phenotype in yeast, which could be specifically suppressed by co-expression of Bcl-2, despite its failure to dimerize with Bcl-2. Both the Bax(D68A) and Bax(E69A) proteins induced apoptosis when overexpressed in human 293 cells, despite an inability to bind to Bcl-2. Moreover, co-expression of Bcl-2 with Bax(D68A) and Bax(E69A) rescued mammalian cells from apoptosis. In contrast, a mutant of Bax lacking the IGDE motif, Bax(ΔIGDE), was incapable of either homodimerizing with itself or heterodimerizing with Bcl-2 and was inactive at promoting cell death in either yeast or mammalian cells. Although failing to interact with Bcl-2, the Bax(D68A) and Bax(E69A) mutants retained the ability to bind to Bid, a putative Bax-activating member of the Bcl-2 family, and collaborated with Bid in inducing apoptosis. When taken together with previous observations, these findings indicate that (i) Bax can induce apoptosis in mammalian cells irrespective of heterodimerization with Bcl-2 and (ii) Bcl-2 can rescue both mammalian cells and yeast from the lethal effects of Bax without heterodimerizing with it. However, these results do not exclude the possibility that BH3-dependent homodimerization of Bax or interactions with Bax activators such as Bid may either assist or be required for the cell death-inducing mechanism of this protein. The pro-apoptotic protein Bax can homodimerize with itself and heterodimerize with the anti-apoptotic protein Bcl-2, but the significance of these protein-protein interactions remains unclear. Alanine substitution mutations were created in a well conserved IGDE motif found within the BH3 domain of Bax (residues 66–69) and the resulting mutant Bax proteins were tested for ability to homodimerize with themselves and to heterodimerize with Bcl-2. Correlations were made with cell death induction by these mutants of Bax both in mammalian cells where Bax may function through several mechanisms, and in yeast where Bax may exert its lethal actions through a more limited repertoire of mechanisms perhaps related to its ability to form ion channels in intracellular membranes. Two of the mutants, Bax(D68A) and Bax(E69A), retained the ability to homodimerize but failed to interact with Bcl-2 as determined by yeast two-hybrid assays and co-immunoprecipitation analysis using transfected mammalian cells. The Bax(E69A) protein exhibited a lethal phenotype in yeast, which could be specifically suppressed by co-expression of Bcl-2, despite its failure to dimerize with Bcl-2. Both the Bax(D68A) and Bax(E69A) proteins induced apoptosis when overexpressed in human 293 cells, despite an inability to bind to Bcl-2. Moreover, co-expression of Bcl-2 with Bax(D68A) and Bax(E69A) rescued mammalian cells from apoptosis. In contrast, a mutant of Bax lacking the IGDE motif, Bax(ΔIGDE), was incapable of either homodimerizing with itself or heterodimerizing with Bcl-2 and was inactive at promoting cell death in either yeast or mammalian cells. Although failing to interact with Bcl-2, the Bax(D68A) and Bax(E69A) mutants retained the ability to bind to Bid, a putative Bax-activating member of the Bcl-2 family, and collaborated with Bid in inducing apoptosis. When taken together with previous observations, these findings indicate that (i) Bax can induce apoptosis in mammalian cells irrespective of heterodimerization with Bcl-2 and (ii) Bcl-2 can rescue both mammalian cells and yeast from the lethal effects of Bax without heterodimerizing with it. However, these results do not exclude the possibility that BH3-dependent homodimerization of Bax or interactions with Bax activators such as Bid may either assist or be required for the cell death-inducing mechanism of this protein. Bcl-2 family proteins play an important role as regulators of programmed cell death and apoptosis (1Reed J.C. J. Cell Biol. 1994; 124: 1-6Crossref PubMed Scopus (2399) Google Scholar, 2Yang E. Korsmeyer S.J. Blood. 1996; 88: 386-401Crossref PubMed Google Scholar, 3Kroemer G. Nature Med. 1997; 3: 614-620Crossref PubMed Scopus (1722) Google Scholar). Many Bcl-2 family proteins including the anti-apoptotic proteins Bcl-2 and Bcl-XL and the pro-apoptotic proteins Bax and Bak can physically interact with each other, forming a complex network of homo- and heterodimers (1Reed J.C. J. Cell Biol. 1994; 124: 1-6Crossref PubMed Scopus (2399) Google Scholar, 2Yang E. Korsmeyer S.J. Blood. 1996; 88: 386-401Crossref PubMed Google Scholar). 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The of these findings with to mechanisms of Bcl-2 and Bax function T. Krajewski S. M. B. Reed J.C. 1994; Google was as the for were created using a H. Aime-Sempe C. Sato T. Reed J.C. J. Biol. Chem. 1996; 271: 7440-7444Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, H. B. Reed J.C. Biol. 1996; PubMed Scopus Google Scholar). mutants were in the two-hybrid in with the domain H. Aime-Sempe C. Sato T. Reed J.C. J. Biol. Chem. 1996; 271: 7440-7444Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, H. B. Reed J.C. Biol. 1996; PubMed Scopus Google Scholar). The were in with the Bax and and and and and mutants were from S. Reed J.C. S. 1996; PubMed Scopus Google by with and for studies in mammalian cells. and were using the the Bax for the which a of the mutants were from the yeast two-hybrid by and a Bax in which the BH3 domain (residues was with the BH3 domain of human Bcl-2 (residues was using the human Bcl-2 S. Sato T. Krajewski S. S. Reed J.C. 1995; Full Text PDF PubMed Scopus Google as of the with and and Bax T. Krajewski S. M. B. Reed J.C. 1994; Google as the with and in with the Bax and lacking the the was of Bax were from by with Bid protein was by from a using the and and the resulting was and to and by with and The of was by interactions were by yeast two-hybrid as in using either cells for assays or cells for in with and M. Aimé-Sempé C. Sato T. Reed J.C. J. Biol. Chem. 1995; 270: 11962-11968Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 7Zha H. Aime-Sempe C. Sato T. Reed J.C. J. Biol. Chem. 1996; 271: 7440-7444Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, H. B. Reed J.C. Biol. 1996; PubMed Scopus Google Scholar, T. Hanada M. S. S. E. Reed J.C. 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Biol. 1996; PubMed Scopus Google Scholar). 293 cells were in in of The cells were transfected with of or mutants of Bax by a the effects of Bcl-2 or Bid of Bax mutants, each of Bax mutant was with of or the without The was with and the cells were for The and cells were and to or with a 293 cells were in of The cells were with of and each of and or by a the cells were in of and were by with of protein with of at for in of were to and analysis using or Bcl-2 S. C. M. E. Reed J.C. 1995; Google Scholar). was using and by an with to x-ray cells with were to an of were by the and for protein H. B. Reed J.C. Biol. 1996; PubMed Scopus Google Scholar). of in and to was using as H. Aime-Sempe C. Sato T. Reed J.C. J. Biol. Chem. 1996; 271: 7440-7444Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, H. B. Reed J.C. Biol. 1996; PubMed Scopus Google Scholar). by deletional analysis that a well conserved motif, IGDE (residues within the BH3 domain of the Bax protein required for Bax heterodimerization with Bcl-2, and death promoting in both yeast and mammalian cells H. B. Reed J.C. Biol. 1996; PubMed Scopus Google Scholar). substitution mutants of each of these and tested for ability to interact with or Bcl-2 in yeast two-hybrid assays where proteins with either a domain or domain to In a mutant of Bax was in which the BH3 domain of Bcl-2 was for the BH3 domain of these proteins were without the domain with to the The domain required for in yeast H. B. Reed J.C. Biol. 1996; PubMed Scopus Google its for yeast two-hybrid The of of these proteins at in yeast was by in the and proteins to the Bax protein in yeast two-hybrid the ability to interact with Bax and Bcl-2 as well as to homodimerize with Although assays were were in the of by the and mutants with In contrast, the Bax(D68A) and Bax(E69A) mutants failed to interact with Bcl-2 but retained the ability to bind Bax and to homodimerize with themselves Bax the BH3 domain of Bcl-2 was of with Bax and Bcl-2 but not homodimerize with The of these protein interactions by yeast two-hybrid assays was by of lacking or two-hybrid proteins such as domain and not two-hybrid analysis of Bax BH3 domain assays were using cells with the domain or domain proteins and to induce of the in the In Bax and Bcl-2 proteins the The results of assays where of a within by In where cells were was in a assays were using cells with the domain or domain proteins and to induce of the in the In Bax and Bcl-2 proteins the The results of assays where of a within by In where cells were was the effects of BH3 domain mutants the death inducing of Bax in yeast, the proteins were with with the and Bax(E69A) protein when for of the these proteins In contrast, of were when yeast were with and Bax(D68A) proteins or when proteins such as were in these cells and with studies H. B. Reed J.C. Biol. 1996; PubMed Scopus Google a mutant in which the IGDE motif (residues 66–69) was failed to when in the and Bax(E69A) mutants in yeast, the Bax(D68A) and mutants do Bcl-2 can rescue yeast from the lethal effects of Bax protein M. Aimé-Sempé C. Sato T. Reed J.C. J. Biol. Chem. 1995; 270: 11962-11968Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, H. B. Reed J.C. Biol. 1996; PubMed Scopus Google Scholar, T. Hanada M. S. S. E. Reed J.C. S. 1994; PubMed Scopus Google Scholar). the Bax(E69A) mutant retained but failed to heterodimerize with Bcl-2, to the effects of Bcl-2 this these the were for which or mutant of Bax as with domains were with a Bcl-2 was by ability to heterodimerize with Bcl-2, by yeast with Bcl-2 and or was not with cells which been with the or such as In Bcl-2 rescued yeast the Bax(E69A) mutant which to interact with Bcl-2 in yeast two-hybrid The of these results was by of mutants of Bax including and the of which the domain required for in yeast H. B. Reed J.C. Biol. 1996; PubMed Scopus Google Scholar). observations, that heterodimerization not required for of Bax in The mutant failed to yeast, but to yeast two-hybrid can bind to was that this protein which the BH3 domain of Bcl-2 function to Bcl-2, forming a with Bax and its in However, when with Bax in yeast, the protein failed to rescue that heterodimerization for the lethal effects of Bax in The in yeast of of the mutants of Bax tested was by analysis of the Bax proteins were was to cells with a Bcl-2 that of cells could be for of protein of the Bax and proteins were from the with which to for these studies and which were to the of mutant in of with an that Bax mutants were at to or in of the Bax protein. be the mutants of Bax which failed to to to in yeast, that cells could of these proteins with Bax the function of Bax mutants in mammalian cells, the protein results in yeast two-hybrid could be by co-immunoprecipitation assays where Bax mutants were with Bcl-2 in human cells. these the and mutant Bax proteins were with Two of cells, cell were for protein and with by using in Bcl-2 with the and In contrast, Bcl-2 not with the protein and with the protein of Bcl-2 was in with from cells transfected with a the of the was not to the and mutants these proteins were in mammalian cells and could not be In yeast two-hybrid of the Bax mutants created retained the ability to bind to the of human Bax protein in cells to the ability of Bax mutants to with the with an for the protein that of protein were with the and mutants retained the ability to bind to Bax in mammalian cells. the with of of the and mutant Bax these co-immunoprecipitation assays the results of yeast two-hybrid that the mutant the ability to dimerize with Bcl-2 and the Bax(D68A) and Bax(E69A) mutants can bind to Bax but have ability to bind Bcl-2. The effects of these Bax mutants were with Bax by in cells, the of cells with and by with the in the Bax protein and the and Bax(E69A) mutant proteins induced more apoptosis in cells when with transfected cells. to the results in yeast, the and Bax(E69A) protein cell death in mammalian cells. However, the results in yeast, the Bax(D68A) protein was in mammalian cells. indicate that Bax mutants which to heterodimerize with Bax(D68A) and Bax(E69A), pro-apoptotic in mammalian cells. Bcl-2 could apoptosis induction by mutants of Bax which do not heterodimerize with cells were with of Bax and Bcl-2 and the of cells was determined by in Bcl-2 apoptosis induced by and the of Bcl-2 and that Bcl-2 not the of and mutant Bax this a for the of cells the ability of Bcl-2 to cell death induction by mutants of Bax with heterodimerization a of Bax or Bax(E69A) was with of Bcl-2 and Bcl-2 cell death induction by the Bax and with the that the of Bcl-2 as a of apoptosis by of heterodimerization results were with the Bax(D68A) mutant these that or the ability of Bcl-2 to heterodimerize with Bax and its ability to cells from cell The Bid protein the of Bcl-2 family proteins in that can heterodimerize with Bax and apoptosis Korsmeyer S.J. 1996; PubMed Scopus Google Scholar). of the BH3 domain of Bid that its ability to heterodimerize with Bax with Bcl-2 or with apoptosis Korsmeyer S.J. 1996; PubMed Scopus Google Scholar). the Bax mutants could bind to Bid and with Bid in inducing apoptosis. a protein was in and tested for to in Bax and Bax mutant in the mutant and Bax(E69A) proteins to at as as that Bid these mutants of Bax to bind to Bax In contrast, of these Bax proteins with the of the cells were with Bid and Bax mutants, using a of that was by itself to induce of cell When with either Bax or the mutants of Bid the of cells, that Bid can the of Bax and the Bax mutants of the and proteins and that Bid not the of or mutant proteins in cells the Bax(D68A) and Bax(E69A) mutants or Bcl-2 the ability to bind and with that the substitution mutations created within Bax have not the function of these proteins or ability to interact with Bax cell death in mammalian cells and yeast mechanisms can be the BH3 domain of Bax can bind to Bcl-2 and related anti-apoptotic mechanism to the of pro-apoptotic Bcl-2 family proteins which BH3 domains Gallo G.J. Elangovan B. Houghton A.B. S. Ebb R.G. T. T. Lutz R.J. Chinnadurai G. 1995; Google Scholar, S. G. EMBO J. 1997; PubMed Scopus Google Scholar). Bax may form channels for or in the intracellular where B. S. S. G. 1997; PubMed Scopus Google Scholar). have that the lethal phenotype by pro-apoptotic of the Bcl-2 family such as Bax and Bak in and yeast a of this yeast Bcl-2 H. B. Reed J.C. Biol. 1996; PubMed Scopus Google Scholar). with these been that of a of Bak of its BH3 domain to a domain for inducing apoptosis in mammalian cells in which anti-apoptotic proteins such as Bcl-2 and Bcl-XL such BH3 mutants of Bak which the and inactive in yeast T. Flemington C. Houghton A.B. Ebb R.G. Gallo G.J. Elangovan B. Chinnadurai G. Lutz R.J. EMBO J. 1995; 14: 5589-5596Crossref PubMed Scopus (441) Google Scholar, B. M. B. C. T. G. Biol. 1997; PubMed Scopus Google Scholar). In mammalian cells, the of these mechanisms for the pro-apoptotic of Bax and Bak to be but to play a the mutants of Bax that BH3-dependent heterodimerization with Bcl-2 not required for the pro-apoptotic function of Bax in mammalian cells. was specifically by the ability of the Bax(D68A) and Bax(E69A) mutants to induce apoptosis when overexpressed in cells, without forming heterodimers with Bcl-2. Although to exclude the possibility that these mutants of Bax the ability to heterodimerize with anti-apoptotic of the Bcl-2 family that may be in mammalian cells, these mutants do not interact with Bcl-XL not Bax not heterodimerize with Bcl-2 or Bcl-XL to apoptosis in mammalian cells. Moreover, the that Bcl-2 can human cells from cell death induced by of the Bax(D68A) and Bax(E69A) mutants that for Bcl-2 to dimerize with Bax for apoptosis induced by of this protein. results have been by G. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, B. G. J. Biol. 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Although studies have the effects of BH3 domain mutations the ability of Bax and Bak to interact with Bcl-XL or to bind the Bax protein T. Flemington C. Houghton A.B. Ebb R.G. Gallo G.J. Elangovan B. Chinnadurai G. Lutz R.J. EMBO J. 1995; 14: 5589-5596Crossref PubMed Scopus (441) Google Scholar, G. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, B. G. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google have not the of these mutant Bax proteins could homodimerize with themselves and this related to function as death-inducing of the mutants of the IGDE motif within Bax BH3 domain with as determined by yeast two-hybrid In human cells, these mutants of Bax retained the ability to induce that homodimerization and cell death may be In the in which the BH3 domain from Bcl-2 was for that of failed to homodimerize and was inactive at inducing cell death in yeast, the that homodimerization may be with Bax this protein was in mammalian cells, in that However, of the mutants, its cell death in yeast, and of homodimerizing with homodimerization required for the function of can be the Bax(D68A) mutant was in mammalian cells but in its function in yeast remains to be but can be to the of family protein and function in mammalian cells, where a of and proteins as well as can that not found in yeast G. 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