Preservation of Mitochondrial Structure and Function after Bid- or Bax-Mediated Cytochrome c Release

Proapoptotic members of the Bcl-2 protein family, including Bid and Bax, can activate apoptosis by directly interacting with mitochondria to cause cytochrome c translocation from the intermembrane space into the cytoplasm, thereby triggering Apaf-1–mediated caspase activation. Under some circumstances, when caspase activation is blocked, cells can recover from cytochrome c translocation; this suggests that apoptotic mitochondria may not always suffer catastrophic damage arising from the process of cytochrome c release. We now show that recombinant Bid and Bax cause complete cytochrome c loss from isolated mitochondria in vitro, but preserve the ultrastructure and protein import function of mitochondria, which depend on inner membrane polarization. We also demonstrate that, if caspases are inhibited, mitochondrial protein import function is retained in UV-irradiated or staurosporine-treated cells, despite the complete translocation of cytochrome c. Thus, Bid and Bax act only on the outer membrane, and lesions in the inner membrane occurring during apoptosis are shown to be secondary caspase-dependent events.

[1]  G. Kroemer,et al.  Cytofluorometric detection of mitochondrial alterations in early CD95/Fas/APO-1-triggered apoptosis of Jurkat T lymphoma cells. Comparison of seven mitochondrion-specific fluorochromes. , 1998, Immunology letters.

[2]  D. Newmeyer,et al.  Cell-free apoptosis in Xenopus egg extracts: Inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria , 1994, Cell.

[3]  Jean-Claude Martinou,et al.  Bax-induced Cytochrome C Release from Mitochondria Is Independent of the Permeability Transition Pore but Highly Dependent on Mg2+ Ions , 1998, The Journal of cell biology.

[4]  Seamus J. Martin,et al.  Cytochrome c activation of CPP32‐like proteolysis plays a critical role in a Xenopus cell‐free apoptosis system , 1997, The EMBO journal.

[5]  W. Neupert,et al.  Different transport pathways of individual precursor proteins in mitochondria. , 1981, European journal of biochemistry.

[6]  John Calvin Reed,et al.  Bcl-2 family proteins as ion-channels , 1998, Cell Death and Differentiation.

[7]  D. Green,et al.  Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD‐specific caspase activation and independently of mitochondrial transmembrane depolarization , 1998, The EMBO journal.

[8]  M. V. Heiden,et al.  Bcl-xL Regulates the Membrane Potential and Volume Homeostasis of Mitochondria , 1997, Cell.

[9]  P. Paty,et al.  Mitochondrial Proliferation and Paradoxical Membrane Depolarization during Terminal Differentiation and Apoptosis in a Human Colon Carcinoma Cell Line , 1997, The Journal of cell biology.

[10]  J C Reed,et al.  Bax directly induces release of cytochrome c from isolated mitochondria. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[11]  J. Kerr,et al.  An electron‐microscope study of the mode of cell death induced by cancer‐chemotherapeutic agents in populations of proliferating normal and neoplastic cells , 1975, The Journal of pathology.

[12]  Eugene M. Johnson,et al.  Evidence of a Novel Event during Neuronal Death Development of Competence-to-Die in Response to Cytoplasmic Cytochrome c , 1998, Neuron.

[13]  Jean-Claude Martinou,et al.  Bid-induced Conformational Change of Bax Is Responsible for Mitochondrial Cytochrome c Release during Apoptosis , 1999, The Journal of cell biology.

[14]  N. Pfanner,et al.  Mitochondrial biogenesis: The Tom and Tim machine , 1997, Current Biology.

[15]  T. Kuwana,et al.  The Pro-Apoptotic Proteins, Bid and Bax, Cause a Limited Permeabilization of the Mitochondrial Outer Membrane That Is Enhanced by Cytosol , 1999, The Journal of cell biology.

[16]  Gerard I. Evan,et al.  The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant , 2000, Nature Cell Biology.

[17]  W. Neupert,et al.  Mitochondrial protein import: Nucleoside triphosphates are involved in conferring import-competence to precursors , 1987, Cell.

[18]  D. Green,et al.  The Release of Cytochrome c from Mitochondria: A Primary Site for Bcl-2 Regulation of Apoptosis , 1997, Science.

[19]  D. Green,et al.  Bax-induced Caspase Activation and Apoptosis via Cytochromec Release from Mitochondria Is Inhibitable by Bcl-xL* , 1999, The Journal of Biological Chemistry.

[20]  E. Hartmann,et al.  Isolation of a protein that is essential for the first step of nuclear protein import , 1994, Cell.

[21]  B. Motyka,et al.  Granzyme B-induced loss of mitochondrial inner membrane potential (Delta Psi m) and cytochrome c release are caspase independent. , 1999, Journal of immunology.

[22]  J. Farber,et al.  The Overexpression of Bax Produces Cell Death upon Induction of the Mitochondrial Permeability Transition* , 1998, The Journal of Biological Chemistry.

[23]  Keisuke Kuida,et al.  Reduced Apoptosis and Cytochrome c–Mediated Caspase Activation in Mice Lacking Caspase 9 , 1998, Cell.

[24]  S. Srinivasula,et al.  Cytochrome c and dATP-Dependent Formation of Apaf-1/Caspase-9 Complex Initiates an Apoptotic Protease Cascade , 1997, Cell.

[25]  A. Halestrap,et al.  Regulation of the mitochondrial matrix volume in vivo and in vitro. The role of calcium. , 1986, The Biochemical journal.

[26]  J C Reed,et al.  Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. , 1998, Science.

[27]  Yi-Te Hsu,et al.  Movement of Bax from the Cytosol to Mitochondria during Apoptosis , 1997, The Journal of cell biology.

[28]  S J Young,et al.  Electron tomography of neuronal mitochondria: three-dimensional structure and organization of cristae and membrane contacts. , 1997, Journal of structural biology.

[29]  L. Rubin,et al.  Blocking Cytochrome c Activity within Intact Neurons Inhibits Apoptosis , 1998, The Journal of cell biology.

[30]  T. Deerinck,et al.  Electron Tomography of Mitochondria from Brown Adipocytes Reveals Crista Junctions , 1998, Journal of bioenergetics and biomembranes.

[31]  Xiaodong Wang,et al.  Bid, a Bcl2 Interacting Protein, Mediates Cytochrome c Release from Mitochondria in Response to Activation of Cell Surface Death Receptors , 1998, Cell.

[32]  K. Dietmeier,et al.  Tom40 forms the hydrophilic channel of the mitochondrial import pore for preproteins , 1998, Nature.

[33]  T. Chittenden,et al.  Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[34]  Masashi Narita,et al.  Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC , 1999, Nature.

[35]  G. Cohen,et al.  Apoptosis, in human monocytic THP.1 cells, results in the release of cytochrome c from mitochondria prior to their ultracondensation, formation of outer membrane discontinuities and reduction in inner membrane potential , 1998, Cell Death and Differentiation.

[36]  Dean P. Jones,et al.  Prevention of Apoptosis by Bcl-2: Release of Cytochrome c from Mitochondria Blocked , 1997, Science.

[37]  N. Pfanner,et al.  The Tom and Tim machine. , 1997, Current biology : CB.

[38]  Xiaodong Wang,et al.  Induction of Apoptotic Program in Cell-Free Extracts: Requirement for dATP and Cytochrome c , 1996, Cell.

[39]  José Luis de la Pompa,et al.  Differential Requirement for Caspase 9 in Apoptotic Pathways In Vivo , 1998, Cell.

[40]  The relationship between the size of mitochondria and the intensity of light that they scatter in different energetic states. , 1981, Biochimica et biophysica acta.

[41]  K. Murphy,et al.  Bcl-2 inhibits Bax translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells , 2000, Cell Death and Differentiation.

[42]  M. Prevost,et al.  Mitochondrial Release of Caspase-2 and -9 during the Apoptotic Process , 1999, The Journal of experimental medicine.

[43]  L. Scorrano,et al.  The mitochondrial permeability transition , 2022, BioFactors.

[44]  J. Martinou,et al.  The Release of Cytochrome c from Mitochondria during Apoptosis of NGF-deprived Sympathetic Neurons Is a Reversible Event , 1999, The Journal of cell biology.

[45]  Francesco Cecconi,et al.  Apaf1 (CED-4 Homolog) Regulates Programmed Cell Death in Mammalian Development , 1998, Cell.

[46]  A. Ito,et al.  Transport of the precursor for sulfite oxidase into intermembrane space of liver mitochondria: characterization of import and processing activities. , 1984, Journal of biochemistry.

[47]  S. Novgorodov,et al.  The Peptide Mastoparan Is a Potent Facilitator of the Mitochondrial Permeability Transition (*) , 1995, The Journal of Biological Chemistry.

[48]  G A Perkins,et al.  Electron tomography of large, multicomponent biological structures. , 1997, Journal of structural biology.

[49]  D. Newmeyer,et al.  Cell-free apoptosis in Xenopus laevis egg extracts. , 2000, Methods in enzymology.

[50]  P. Gruss,et al.  Interdigital cell death can occur through a necrotic and caspase-independent pathway , 1999, Current Biology.