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. 1996 Aug 15;15(16):4130–4141.

Bcl-2 mutants with restricted subcellular location reveal spatially distinct pathways for apoptosis in different cell types.

W Zhu 1, A Cowie 1, G W Wasfy 1, L Z Penn 1, B Leber 1, D W Andrews 1
PMCID: PMC452136  PMID: 8861942

Abstract

Human Bcl-2 is located in multiple intracellular membranes when expressed in MDCK and Rat-1/myc cells. We restricted expression to the endoplasmic reticulum or mitochondria by exchanging the Bcl-2 carboxy-terminal insertion sequence for an equivalent sequence from cytochrome b5 or ActA, respectively. MDCK cells are protected from serum deprivation-induced apoptosis by both wild-type Bcl-2 and the mutant targeted to mitochondria but not by the mutant targeted to endoplasmic reticulum. In contrast, when expressed in Rat-1/myc cells, the Bcl-2 mutant located at the endoplasmic reticulum is more effective than that targeted to mitochondria. In MDCK cells both mutants bind Bax as effectively as wild-type, demonstrating that Bax binding is not sufficient to prevent apoptosis.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akao Y., Otsuki Y., Kataoka S., Ito Y., Tsujimoto Y. Multiple subcellular localization of bcl-2: detection in nuclear outer membrane, endoplasmic reticulum membrane, and mitochondrial membranes. Cancer Res. 1994 May 1;54(9):2468–2471. [PubMed] [Google Scholar]
  2. Allday M. J., Inman G. J., Crawford D. H., Farrell P. J. DNA damage in human B cells can induce apoptosis, proceeding from G1/S when p53 is transactivation competent and G2/M when it is transactivation defective. EMBO J. 1995 Oct 16;14(20):4994–5005. doi: 10.1002/j.1460-2075.1995.tb00182.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Andrews D. W., Gupta J., Abisdris G. Evidence that the middle T antigen of polyomavirus interacts with the membrane skeleton. Mol Cell Biol. 1993 Aug;13(8):4703–4713. doi: 10.1128/mcb.13.8.4703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bargou R. C., Daniel P. T., Mapara M. Y., Bommert K., Wagener C., Kallinich B., Royer H. D., Dörken B. Expression of the bcl-2 gene family in normal and malignant breast tissue: low bax-alpha expression in tumor cells correlates with resistance towards apoptosis. Int J Cancer. 1995 Mar 16;60(6):854–859. doi: 10.1002/ijc.2910600622. [DOI] [PubMed] [Google Scholar]
  5. Boise L. H., González-García M., Postema C. E., Ding L., Lindsten T., Turka L. A., Mao X., Nuñez G., Thompson C. B. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell. 1993 Aug 27;74(4):597–608. doi: 10.1016/0092-8674(93)90508-n. [DOI] [PubMed] [Google Scholar]
  6. Borner C., Martinou I., Mattmann C., Irmler M., Schaerer E., Martinou J. C., Tschopp J. The protein bcl-2 alpha does not require membrane attachment, but two conserved domains to suppress apoptosis. J Cell Biol. 1994 Aug;126(4):1059–1068. doi: 10.1083/jcb.126.4.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boyd J. M., Malstrom S., Subramanian T., Venkatesh L. K., Schaeper U., Elangovan B., D'Sa-Eipper C., Chinnadurai G. Adenovirus E1B 19 kDa and Bcl-2 proteins interact with a common set of cellular proteins. Cell. 1994 Oct 21;79(2):341–351. doi: 10.1016/0092-8674(94)90202-x. [DOI] [PubMed] [Google Scholar]
  8. Chen-Levy Z., Nourse J., Cleary M. L. The bcl-2 candidate proto-oncogene product is a 24-kilodalton integral-membrane protein highly expressed in lymphoid cell lines and lymphomas carrying the t(14;18) translocation. Mol Cell Biol. 1989 Feb;9(2):701–710. doi: 10.1128/mcb.9.2.701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cleary M. L., Smith S. D., Sklar J. Cloning and structural analysis of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t(14;18) translocation. Cell. 1986 Oct 10;47(1):19–28. doi: 10.1016/0092-8674(86)90362-4. [DOI] [PubMed] [Google Scholar]
  10. D'Arrigo A., Manera E., Longhi R., Borgese N. The specific subcellular localization of two isoforms of cytochrome b5 suggests novel targeting pathways. J Biol Chem. 1993 Feb 5;268(4):2802–2808. [PubMed] [Google Scholar]
  11. Dive C., Hickman J. A. Drug-target interactions: only the first step in the commitment to a programmed cell death? Br J Cancer. 1991 Jul;64(1):192–196. doi: 10.1038/bjc.1991.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dole M., Nuñez G., Merchant A. K., Maybaum J., Rode C. K., Bloch C. A., Castle V. P. Bcl-2 inhibits chemotherapy-induced apoptosis in neuroblastoma. Cancer Res. 1994 Jun 15;54(12):3253–3259. [PubMed] [Google Scholar]
  13. Evan G. I., Wyllie A. H., Gilbert C. S., Littlewood T. D., Land H., Brooks M., Waters C. M., Penn L. Z., Hancock D. C. Induction of apoptosis in fibroblasts by c-myc protein. Cell. 1992 Apr 3;69(1):119–128. doi: 10.1016/0092-8674(92)90123-t. [DOI] [PubMed] [Google Scholar]
  14. Fanidi A., Harrington E. A., Evan G. I. Cooperative interaction between c-myc and bcl-2 proto-oncogenes. Nature. 1992 Oct 8;359(6395):554–556. doi: 10.1038/359554a0. [DOI] [PubMed] [Google Scholar]
  15. Frisch S. M., Francis H. Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol. 1994 Feb;124(4):619–626. doi: 10.1083/jcb.124.4.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. González-García M., Pérez-Ballestero R., Ding L., Duan L., Boise L. H., Thompson C. B., Núez G. bcl-XL is the major bcl-x mRNA form expressed during murine development and its product localizes to mitochondria. Development. 1994 Oct;120(10):3033–3042. doi: 10.1242/dev.120.10.3033. [DOI] [PubMed] [Google Scholar]
  17. Henderson S., Huen D., Rowe M., Dawson C., Johnson G., Rickinson A. Epstein-Barr virus-coded BHRF1 protein, a viral homologue of Bcl-2, protects human B cells from programmed cell death. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8479–8483. doi: 10.1073/pnas.90.18.8479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hengartner M. O., Horvitz H. R. C. elegans cell survival gene ced-9 encodes a functional homolog of the mammalian proto-oncogene bcl-2. Cell. 1994 Feb 25;76(4):665–676. doi: 10.1016/0092-8674(94)90506-1. [DOI] [PubMed] [Google Scholar]
  19. Hinshaw V. S., Olsen C. W., Dybdahl-Sissoko N., Evans D. Apoptosis: a mechanism of cell killing by influenza A and B viruses. J Virol. 1994 Jun;68(6):3667–3673. doi: 10.1128/jvi.68.6.3667-3673.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hockenbery D. M., Oltvai Z. N., Yin X. M., Milliman C. L., Korsmeyer S. J. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell. 1993 Oct 22;75(2):241–251. doi: 10.1016/0092-8674(93)80066-n. [DOI] [PubMed] [Google Scholar]
  21. Hockenbery D., Nuñez G., Milliman C., Schreiber R. D., Korsmeyer S. J. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature. 1990 Nov 22;348(6299):334–336. doi: 10.1038/348334a0. [DOI] [PubMed] [Google Scholar]
  22. Jacobson M. D., Raff M. C. Programmed cell death and Bcl-2 protection in very low oxygen. Nature. 1995 Apr 27;374(6525):814–816. doi: 10.1038/374814a0. [DOI] [PubMed] [Google Scholar]
  23. Janiak F., Glover J. R., Leber B., Rachubinski R. A., Andrews D. W. Targeting of passenger protein domains to multiple intracellular membranes. Biochem J. 1994 May 15;300(Pt 1):191–199. doi: 10.1042/bj3000191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Janiak F., Leber B., Andrews D. W. Assembly of Bcl-2 into microsomal and outer mitochondrial membranes. J Biol Chem. 1994 Apr 1;269(13):9842–9849. [PubMed] [Google Scholar]
  25. Kane D. J., Sarafian T. A., Anton R., Hahn H., Gralla E. B., Valentine J. S., Ord T., Bredesen D. E. Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species. Science. 1993 Nov 19;262(5137):1274–1277. doi: 10.1126/science.8235659. [DOI] [PubMed] [Google Scholar]
  26. Korsmeyer S. J. Bcl-2 initiates a new category of oncogenes: regulators of cell death. Blood. 1992 Aug 15;80(4):879–886. [PubMed] [Google Scholar]
  27. Kutay U., Hartmann E., Rapoport T. A. A class of membrane proteins with a C-terminal anchor. Trends Cell Biol. 1993 Mar;3(3):72–75. doi: 10.1016/0962-8924(93)90066-a. [DOI] [PubMed] [Google Scholar]
  28. Lam M., Dubyak G., Chen L., Nuñez G., Miesfeld R. L., Distelhorst C. W. Evidence that BCL-2 represses apoptosis by regulating endoplasmic reticulum-associated Ca2+ fluxes. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6569–6573. doi: 10.1073/pnas.91.14.6569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lin K. I., Lee S. H., Narayanan R., Baraban J. M., Hardwick J. M., Ratan R. R. Thiol agents and Bcl-2 identify an alphavirus-induced apoptotic pathway that requires activation of the transcription factor NF-kappa B. J Cell Biol. 1995 Dec;131(5):1149–1161. doi: 10.1083/jcb.131.5.1149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mann R., Mulligan R. C., Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983 May;33(1):153–159. doi: 10.1016/0092-8674(83)90344-6. [DOI] [PubMed] [Google Scholar]
  31. Mitoma J., Ito A. The carboxy-terminal 10 amino acid residues of cytochrome b5 are necessary for its targeting to the endoplasmic reticulum. EMBO J. 1992 Nov;11(11):4197–4203. doi: 10.1002/j.1460-2075.1992.tb05513.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Miyashita T., Kitada S., Krajewski S., Horne W. A., Delia D., Reed J. C. Overexpression of the Bcl-2 protein increases the half-life of p21Bax. J Biol Chem. 1995 Nov 3;270(44):26049–26052. doi: 10.1074/jbc.270.44.26049. [DOI] [PubMed] [Google Scholar]
  33. Miyashita T., Krajewski S., Krajewska M., Wang H. G., Lin H. K., Liebermann D. A., Hoffman B., Reed J. C. Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene. 1994 Jun;9(6):1799–1805. [PubMed] [Google Scholar]
  34. Monaghan P., Robertson D., Amos T. A., Dyer M. J., Mason D. Y., Greaves M. F. Ultrastructural localization of bcl-2 protein. J Histochem Cytochem. 1992 Dec;40(12):1819–1825. doi: 10.1177/40.12.1453000. [DOI] [PubMed] [Google Scholar]
  35. Morgenstern J. P., Land H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 1990 Jun 25;18(12):3587–3596. doi: 10.1093/nar/18.12.3587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Newmeyer D. D., Farschon D. M., Reed J. C. Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria. Cell. 1994 Oct 21;79(2):353–364. doi: 10.1016/0092-8674(94)90203-8. [DOI] [PubMed] [Google Scholar]
  37. Nguyen M., Branton P. E., Walton P. A., Oltvai Z. N., Korsmeyer S. J., Shore G. C. Role of membrane anchor domain of Bcl-2 in suppression of apoptosis caused by E1B-defective adenovirus. J Biol Chem. 1994 Jun 17;269(24):16521–16524. [PubMed] [Google Scholar]
  38. Nguyen M., Millar D. G., Yong V. W., Korsmeyer S. J., Shore G. C. Targeting of Bcl-2 to the mitochondrial outer membrane by a COOH-terminal signal anchor sequence. J Biol Chem. 1993 Dec 5;268(34):25265–25268. [PubMed] [Google Scholar]
  39. Oltvai Z. N., Korsmeyer S. J. Checkpoints of dueling dimers foil death wishes. Cell. 1994 Oct 21;79(2):189–192. doi: 10.1016/0092-8674(94)90188-0. [DOI] [PubMed] [Google Scholar]
  40. Oltvai Z. N., Milliman C. L., Korsmeyer S. J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell. 1993 Aug 27;74(4):609–619. doi: 10.1016/0092-8674(93)90509-o. [DOI] [PubMed] [Google Scholar]
  41. Opas M., Dziak E., Fliegel L., Michalak M. Regulation of expression and intracellular distribution of calreticulin, a major calcium binding protein of nonmuscle cells. J Cell Physiol. 1991 Oct;149(1):160–171. doi: 10.1002/jcp.1041490120. [DOI] [PubMed] [Google Scholar]
  42. Penn L. J., Brooks M. W., Laufer E. M., Land H. Negative autoregulation of c-myc transcription. EMBO J. 1990 Apr;9(4):1113–1121. doi: 10.1002/j.1460-2075.1990.tb08217.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sedlak T. W., Oltvai Z. N., Yang E., Wang K., Boise L. H., Thompson C. B., Korsmeyer S. J. Multiple Bcl-2 family members demonstrate selective dimerizations with Bax. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7834–7838. doi: 10.1073/pnas.92.17.7834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Soltys B. J., Gupta R. S. Interrelationships of endoplasmic reticulum, mitochondria, intermediate filaments, and microtubules--a quadruple fluorescence labeling study. Biochem Cell Biol. 1992 Oct-Nov;70(10-11):1174–1186. doi: 10.1139/o92-163. [DOI] [PubMed] [Google Scholar]
  45. Strasser A., Harris A. W., Jacks T., Cory S. DNA damage can induce apoptosis in proliferating lymphoid cells via p53-independent mechanisms inhibitable by Bcl-2. Cell. 1994 Oct 21;79(2):329–339. doi: 10.1016/0092-8674(94)90201-1. [DOI] [PubMed] [Google Scholar]
  46. Tanaka S., Saito K., Reed J. C. Structure-function analysis of the Bcl-2 oncoprotein. Addition of a heterologous transmembrane domain to portions of the Bcl-2 beta protein restores function as a regulator of cell survival. J Biol Chem. 1993 May 25;268(15):10920–10926. [PubMed] [Google Scholar]
  47. Veis D. J., Sorenson C. M., Shutter J. R., Korsmeyer S. J. Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell. 1993 Oct 22;75(2):229–240. doi: 10.1016/0092-8674(93)80065-m. [DOI] [PubMed] [Google Scholar]
  48. Wang H. G., Millan J. A., Cox A. D., Der C. J., Rapp U. R., Beck T., Zha H., Reed J. C. R-Ras promotes apoptosis caused by growth factor deprivation via a Bcl-2 suppressible mechanism. J Cell Biol. 1995 May;129(4):1103–1114. doi: 10.1083/jcb.129.4.1103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yang E., Zha J., Jockel J., Boise L. H., Thompson C. B., Korsmeyer S. J. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell. 1995 Jan 27;80(2):285–291. doi: 10.1016/0092-8674(95)90411-5. [DOI] [PubMed] [Google Scholar]
  50. Yang T., Kozopas K. M., Craig R. W. The intracellular distribution and pattern of expression of Mcl-1 overlap with, but are not identical to, those of Bcl-2. J Cell Biol. 1995 Mar;128(6):1173–1184. doi: 10.1083/jcb.128.6.1173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yin X. M., Oltvai Z. N., Korsmeyer S. J. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature. 1994 May 26;369(6478):321–323. doi: 10.1038/369321a0. [DOI] [PubMed] [Google Scholar]
  52. Zamzami N., Marchetti P., Castedo M., Zanin C., Vayssière J. L., Petit P. X., Kroemer G. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J Exp Med. 1995 May 1;181(5):1661–1672. doi: 10.1084/jem.181.5.1661. [DOI] [PMC free article] [PubMed] [Google Scholar]

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