Search tips
Search criteria 


Logo of procellspringeropen.comspringeropen.comThis articleSubmit a manuscriptRegisterThis journal
Protein Cell. 2010 February; 1(2): 161–173.
Published online 2010 February 6. doi:  10.1007/s13238-010-0017-y
PMCID: PMC2982194

Metaxin deficiency alters mitochondrialmembrane permeability and leads to resistance to TNF-induced cell killing


Metaxin, a mitochondrial outer membrane protein, is critical for TNF-induced cell death in L929 cells. Its deficiency, caused by retroviral insertion-mediated mutagenesis, renders L929 cells resistance to TNF killing. In this study, we further characterized metaxin deficiency-caused TNF resistance in parallel with Bcl-XL overexpression-mediated death resistance. We did not find obvious change in mitochondria membrane potential in metaxindeficient (Metmut) and Bcl-XL-overexpressing cells, but we did find an increase in the release rate of the mitochondrial membrane potential probe rhodamine 123 (Rh123) that was preloaded into mitochondria. In addition, overexpression of a function-interfering mutant of metaxin (MetaΔTM/C) or Bcl-XL in MCF-7.3.28 cells also resulted in an acquired resistance to TNF killing and a faster rate of Rh123 release, indicating a close correlation between TNF resistance and higher rates of the dye release from the mitochondria. The release of Rh123 can be controlled by the mitochondrial membrane permeability transition (PT) pore, as targeting an inner membrane component of the PT pore by cyclosporin A (CsA) inhibited Rh123 release. However, metaxin deficiency and Bcl-XL overexpression apparently affect Rh123 release from a site(s) different from that of CsA, as CsA can overcome their effect. Though both metaxin and Bcl-XL appear to function on the outer mitochondrial membrane, they do not interact with each other. They may use different mechanisms to increase the permeability of Rh123, since previous studies have suggested that metaxin may influence certain outer membrane porins while Bcl-XL may form pores on the outer membrane. The alteration of the mitochondrial outer membrane properties by metaxin deficiency and Bcl-XL overespression, as indicated by a quicker Rh123 release, may be helpful in maintaining mitochondrial integrity.

Keywords: metaxin, apoptosis, necrosis, mitochondria


An erratum to this article can be found at


  • Adams J.M., Cory S. The Bcl-2 protein family: arbiters of cell survival. Science. 1998;281:1322–1326. doi: 10.1126/science.281.5381.1322. [PubMed] [Cross Ref]
  • Antos N., Budzinska M., Kmita H. An interplay between the TOM complex and porin isoforms in the yeast Saccharomy cescerevisiae mitochondria. FEBS Lett. 2001;500:12–16. doi: 10.1016/S0014-5793(01)02575-3. [PubMed] [Cross Ref]
  • Armstrong L.C., Komiya T., Bergman B.E., Mihara K., Bornstein P. Metaxin is a component of a preprotein import complexin the outer membrane of the mammalian mitochondrion. J Biol Chem. 1997;272:6510–6518. doi: 10.1074/jbc.272.10.6510. [PubMed] [Cross Ref]
  • Bernal S.D., Lampidis T.J., Summerhayes I.C., Chen L.B. Rhodamine-123 selectively reduces clonal growth of carcinoma cells in vitro. Science. 1982;218:1117–1119. doi: 10.1126/science.7146897. [PubMed] [Cross Ref]
  • Beutler B., Cerami A. Cachectin and tumour necrosis factor as two sides of the same biological coin. Nature. 1986;320:584–588. doi: 10.1038/320584a0. [PubMed] [Cross Ref]
  • Beutler B., Cerami A. Tumor necrosis, cachexia, shock, and inflammation: A common mediator. Ann Rev Biochem. 1988;57:505–518. doi: 10.1146/ [PubMed] [Cross Ref]
  • Beyaert R., Fiers W. Molecular mechanisms of tumor necrosis factor-induced cytotoxicity. What we do understand and what we do not. FEBS. 1994;340:9–16. doi: 10.1016/0014-5793(94)80163-0. [PubMed] [Cross Ref]
  • Boldin M.P., Goncharov T.M., Goltsev Y.V., Wallach D. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1 and TNF receptor-induced cell death. Cell. 1996;85:803–815. doi: 10.1016/S0092-8674(00)81265-9. [PubMed] [Cross Ref]
  • Bornstein P., McKinney C.E., LaMarca M.E., Winfield S., Shingu T., Devarayalu S., Vos H.L., Ginns E.I. Metaxin, a gene contiguous to both thrombospondin 3 and glucocerebrosidase, is required for embryonic development in the mouse: implications for Gaucher disease. Proc Natl Acad Sci U S A. 1995;92:4547–4551. doi: 10.1073/pnas.92.10.4547. [PubMed] [Cross Ref]
  • Brekke O.L., Shalaby M.R., Sundan A., Espevik T., Bjerve K. S. Butylated hydroxyanisole specifically inhibits tumornecrosis factor-induced cytotoxicity and growth enhancement. Cytokine. 1992;4:269–280. doi: 10.1016/1043-4666(92)90067-2. [PubMed] [Cross Ref]
  • Carswell E.A., Old L.J., Kassel R.L., Green S., Fiore N., Williamson B. An endotoxin-induced serum factor that causes necrosis of tumors. Proc Natl Acad Sci U S A. 1975;72:3666–3670. doi: 10.1073/pnas.72.9.3666. [PubMed] [Cross Ref]
  • Cho Y.S., Challa S., Moquin D., Genga R., Ray T.D., Guildford M., Chan F.K. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virusinduced inflammation. Cell. 2009;137:1112–1123. doi: 10.1016/j.cell.2009.05.037. [PMC free article] [PubMed] [Cross Ref]
  • Fiers W., Beyaert R., Declercq W., Vandenabeele P. More than one way to die: apoptosis, necrosis and reactive oxygen damage. Oncogene. 1999;18:7719–7730. doi: 10.1038/sj.onc.1203249. [PubMed] [Cross Ref]
  • Galluzzi L., Kepp O., Kroemer G. RIP kinases initiate programmed necrosis. J Mol Cell Biol. 2009;1:8–10. doi: 10.1093/jmcb/mjp007. [PubMed] [Cross Ref]
  • Gardner T. S., Collins J. J. Neutralizing noise in gene networks. Nature. 2000;405:520–521. doi: 10.1038/35014708. [PubMed] [Cross Ref]
  • Golstein P., Ojcius D.M., Young J.D. Cell death mechanisms and the immune system. Immunol Rev. 1991;121:29–65. doi: 10.1111/j.1600-065X.1991.tb00822.x. [PubMed] [Cross Ref]
  • Goossens V., De Vos K., Vercammen D., Steemans M., Vancompernolle K., Fiers W., Vandenabeele P., Grooten J. Redox regulation of TNF signaling. Biofactors. 1999;10:145–156. doi: 10.1002/biof.5520100210. [PubMed] [Cross Ref]
  • Goossens V., Grooten J., De Vos K., Fiers W. Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity. Proc Natl Acad Sci U S A. 1995;92:8115–8119. doi: 10.1073/pnas.92.18.8115. [PubMed] [Cross Ref]
  • Gross A., Pilcher K., Blachly-Dyson E., Basso E., Jockel J., Bassik M.C., Korsmeyer S.J., Forte M. Biochemical and genetic analysis of the mitochondrial response of yeast to BAX and BCL-X(L) Mol Cell Biol. 2000;20:3125–3136. doi: 10.1128/MCB.20.9.3125-3136.2000. [PMC free article] [PubMed] [Cross Ref]
  • Harris M.H., Vander Heiden M.G., Kron S.J., Thompson C.B. Role of oxidative phosphorylation in Bax toxicity. Mol Cell Biol. 2000;20:3590–3596. doi: 10.1128/MCB.20.10.3590-3596.2000. [PMC free article] [PubMed] [Cross Ref]
  • He S., Wang L., Miao L., Wang T., Du F., Zhao L., Wang X. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell. 2009;137:1100–1111. doi: 10.1016/j.cell.2009.05.021. [PubMed] [Cross Ref]
  • Holler N., Zaru R., Micheau O., Thome M., Attinger A., Valitutti S., Bodmer J.L., Schneider P., Seed B., Tschopp J. Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol. 2000;1:489–495. doi: 10.1038/82732. [PubMed] [Cross Ref]
  • Janicke R.U., Sprengart M.L., Wati M.R., Porter A.G. Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem. 1998;273:9357–9360. doi: 10.1074/jbc.273.16.9357. [PubMed] [Cross Ref]
  • Kmita H., Budzinska M. Involvement of the TOM complex in external NADH transport into yeast mitochondria depleted of mitochondrial porin1. Biochim Biophys Acta. 2000;1509:86–94. doi: 10.1016/S0005-2736(00)00284-4. [PubMed] [Cross Ref]
  • Kroemer G., Dallaporta B., Resche-Rigon M. Themitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol. 1998;60:619–642. doi: 10.1146/annurev.physiol.60.1.619. [PubMed] [Cross Ref]
  • Kroemer G., Reed J.C. Mitochondrial control of cell death. Nat Med. 2000;6:513–519. doi: 10.1038/74994. [PubMed] [Cross Ref]
  • Kunz J., Hall M.N. Cyclosporin A, FK506 and rapamycin: more than just immunosuppression. Trends Biochem Sci. 1993;18:334–338. doi: 10.1016/0968-0004(93)90069-Y. [PubMed] [Cross Ref]
  • Li H., Yuan J. Deciphering the pathways of life and death. Curr Opin Cell Biol. 1999;11:261–266. doi: 10.1016/S0955-0674(99)80035-0. [PubMed] [Cross Ref]
  • Lin Y., Choksi S., Shen H.M., Yang Q.F., Hur G.M., Kim Y.S., Tran J.H., Nedospasov S.A., Liu Z.G. Tumor necrosis factor-induced nonapoptotic cell death requires receptor-interacting protein-mediated cellular reactive oxygen species accumulation. J Biol Chem. 2004;279:10822–10828. doi: 10.1074/jbc.M313141200. [PubMed] [Cross Ref]
  • Liu M.Y., Colombini M. Regulation of mitochondrial respiration by controlling the permeability of the outer membrane through the mitochondrial channel, VDAC. Biochim Biophys Acta. 1992;1098:255–260. doi: 10.1016/S0005-2728(05)80344-5. [PubMed] [Cross Ref]
  • Lotem J., Kama R., Sachs L. Suppression or induction of apoptosis by opposing pathways downstream from calciumactivated calcineurin. Proc Natl Acad Sci U S A. 1999;96:12016–12020. doi: 10.1073/pnas.96.21.12016. [PubMed] [Cross Ref]
  • Marzo I., Brenner C., Zamzami N., Jurgensmeier J.M., Susin S.A., Vieira H.L., Prevost M.C., Xie Z., Matsuyama S., Reed J.C., Kroemer G. Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science. 1998;281:2027–2031. doi: 10.1126/science.281.5385.2027. [PubMed] [Cross Ref]
  • Minn A.J., Velez P., Schendel S.L., Liang H., Muchmore S.W., Fesik S.W., Fill M., Thompson C.B. Bcl-x(L) forms an ion channel in synthetic lipid membranes. Nature. 1997;385:353–357. doi: 10.1038/385353a0. [PubMed] [Cross Ref]
  • Muchmore S.W., Sattler M., Liang H., Meadows R.P., Harlan J.E., Yoon H.S., Nettesheim D., Chang B.S., Thompson C.B., Wong S.L., et al. X-ray and NMR structureof human Bcl-xL, an inhibitor of programmed cell death. Nature. 1996;381:335–341. doi: 10.1038/381335a0. [PubMed] [Cross Ref]
  • Nadakavukaren K.K., Nadakavukaren J.J., Chen L.B. Increased rhodamine 123 uptake by carcinoma cells. Cancer Res. 1985;45:6093–6039. [PubMed]
  • Neale M.L., Fiera R.A., Matthews N. Involvement of phospholipase A2 activation in tumour cell killing by tumour necrosis factor. Immunology. 1988;64:81–85. [PubMed]
  • Old L.J. Tumor necrosis factor (TNF) Science. 1985;230:630–632. doi: 10.1126/science.2413547. [PubMed] [Cross Ref]
  • Ono K., Wang X., Han J. Resistance to tumornecrosis factor-induced cell death mediated by PMCA4 deficiency. Mol Cell Biol. 2001;21:8276–88. doi: 10.1128/MCB.21.24.8276-8288.2001. [PMC free article] [PubMed] [Cross Ref]
  • Pastorino J.G., Simbula G., Yamamoto K., Glascott P.A.J., Rothman R.J., Farber J.L. The cytotoxicity of tumor necrosis factor depends on induction of the mitochondrial permeability transition. J Biol Chem. 1996;271:29792–29798. doi: 10.1074/jbc.271.47.29792. [PubMed] [Cross Ref]
  • Pfanner N., Geissler A. Versatility of the mitochondrial protein import machinery. Nat Rev Mol Cell Biol. 2001;2:339–349. doi: 10.1038/35073006. [PubMed] [Cross Ref]
  • Ravagnan L., Marzo I., Costantini P., Susin S.A., Zamzami N., Petit P.X., Hirsch F., Goulbern M., Poupon M.F., Miccoli L., et al. Lonidamine triggers apoptosis via a direct, Bcl-2-inhibited effect on the mitochondrial permeability transition pore. Oncogene. 1999;18:2537–2546. doi: 10.1038/sj.onc.1202625. [PubMed] [Cross Ref]
  • Rothe J., Gehr G., Loetscher H., Lesslauer W. Tumornecrosis factor receptors-structure and function. Immunol Res. 1992;11:81–90. doi: 10.1007/BF02918612. [PubMed] [Cross Ref]
  • Schnaitman C., Greenawalt J.W. Enzymatic properties of the inner and outer membranes of rat liver mitochondria. J Cell Biol. 1968;38:158–175. doi: 10.1083/jcb.38.1.158. [PMC free article] [PubMed] [Cross Ref]
  • Schulze-Osthoff K., Bakker A.C., Vanhaesebroeck B., Beyaert R., Jacob W.A., Fiers W. Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation. J Biol Chem. 1992;267:5317–5323. [PubMed]
  • Shimizu S., Narita M., Tsujimoto Y. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature. 1999;399:483–487. doi: 10.1038/20959. [PubMed] [Cross Ref]
  • Strasser A., O’Connor L., Dixit V.M. Apoptosis signaling. Annu Rev Biochem. 2000;69:217–245. doi: 10.1146/annurev.biochem.69.1.217. [PubMed] [Cross Ref]
  • Tartaglia L.A., Ayres T.M., Wong G.H., Goeddel D.V. A novel domain within the 55 kD TNF receptor signals cell death. Cell. 1993;74:845–853. doi: 10.1016/0092-8674(93)90464-2. [PubMed] [Cross Ref]
  • Tartaglia L.A., Goeddel D.V. Two TNF receptors. Immunol Today. 1992;13:151–153. doi: 10.1016/0167-5699(92)90116-O. [PubMed] [Cross Ref]
  • Tewari M., Dixit V.M. Fas- and tumor necrosis factor-induced apoptosis ininhibited by the poxvirus crmA gene product. J Biol Chem. 1995;270:3255–3260. doi: 10.1074/jbc.270.39.22705. [PubMed] [Cross Ref]
  • Tracey K.J., Cerami A. Tumor necrosis factor, other cytokines and disease. Annu Rev Cell Biol. 1993;9:317–343. doi: 10.1146/annurev.cb.09.110193.001533. [PubMed] [Cross Ref]
  • Vander Heiden M.G., Chandel N.S., Li X.X., Schumacker P.T., Colombini M., Thompson C.B. Outer mitochondrial membrane permeability can regulate coupled respiration and cell survival. Proc Natl Acad Sci US A. 2000;97:4666–4671. doi: 10.1073/pnas.090082297. [PubMed] [Cross Ref]
  • Vander Heiden M.G., Thompson C.B. Bcl-2 proteins: regulators of apoptosis or of mitochondrial homeostasis? Nat Cell Biol. 1999;1:E209–216. doi: 10.1038/70237. [PubMed] [Cross Ref]
  • Vercammen D., Beyaert R., Denecker G., Goossens V., Van Loo G., Declercq W., Grooten J., Fiers W., Vandenabeele P. Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor. J Exp Med. 1998;187:1477–1485. doi: 10.1084/jem.187.9.1477. [PMC free article] [PubMed] [Cross Ref]
  • Wang X., Ono K., Kim S.O., Kravchenko V., Lin S.C., Han J. Metaxin is required for tumor necrosis factor-induced cell death. EMBO Rep. 2001;2:628–633. doi: 10.1093/embo-reports/kve135. [PubMed] [Cross Ref]
  • Yang J., Liu X., Bhalla K., Kim C.N., Ibrado A.M., Cai J., Peng T.I., Jones D.P., Wang X. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science. 1997;275:1129–1132. doi: 10.1126/science.275.5303.1129. [PubMed] [Cross Ref]
  • Zamzami N., Kroemer G. The mitochondrion in apoptosis: how Pandora’s box opens. Nat Rev Mol Cell Biol. 2001;2:67–71. doi: 10.1038/35048073. [PubMed] [Cross Ref]
  • Zhang D.W., Shao J., Lin J., Zhang N., Lu B.J., Lin S.C., Dong M. Q., Han J. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science. 2009;325:332–336. doi: 10.1126/science.1172308. [PubMed] [Cross Ref]

Articles from Protein & Cell are provided here courtesy of Springer