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A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast.

Pavlov EV, Priault M, Pietkiewicz D, Cheng EH, Antonsson B, Manon S, Korsmeyer SJ, Mannella CA, Kinnally KW - J. Cell Biol. (2001)

Bottom Line: Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2.Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax.These findings implicate this channel, named mitochondrial apoptosis-induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, New York University College of Dentistry, New York, NY 10010, USA.

ABSTRACT
During apoptosis, proapoptotic factors are released from mitochondria by as yet undefined mechanisms. Patch-clamping of mitochondria and proteoliposomes formed from mitochondrial outer membranes of mammalian (FL5.12) cells has uncovered a novel ion channel whose activity correlates with onset of apoptosis. The pore diameter inferred from the largest conductance state of this channel is approximately 4 nm, sufficient to allow diffusion of cytochrome c and even larger proteins. The activity of the channel is affected by Bcl-2 family proteins in a manner consistent with their pro- or antiapoptotic properties. Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2. Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax. These findings implicate this channel, named mitochondrial apoptosis-induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.

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Proteoliposomes enriched with Bax fail to retain cytochrome c. (A) Immunoblots show Bax (but not VDAC) levels are higher (5–10-fold) in outer membranes purified from mitochondria isolated from FL5.12 cells 12 h after IL-3 withdrawal (i.e., apoptotic cells) than from normal cells (i.e., control). Silver-stained gels and Western blots with antibodies against cytochrome oxidase subunit IV indicate equivalent content of overall protein and low contamination by inner membranes for both preparations (data not shown). (B) Immunoblots show that less cytochrome c is present in salt-washed proteoliposomes made from mitochondrial outer membranes of apoptotic cells than of normal cells (control).
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fig1: Proteoliposomes enriched with Bax fail to retain cytochrome c. (A) Immunoblots show Bax (but not VDAC) levels are higher (5–10-fold) in outer membranes purified from mitochondria isolated from FL5.12 cells 12 h after IL-3 withdrawal (i.e., apoptotic cells) than from normal cells (i.e., control). Silver-stained gels and Western blots with antibodies against cytochrome oxidase subunit IV indicate equivalent content of overall protein and low contamination by inner membranes for both preparations (data not shown). (B) Immunoblots show that less cytochrome c is present in salt-washed proteoliposomes made from mitochondrial outer membranes of apoptotic cells than of normal cells (control).

Mentions: Hematopoietic FL5.12 cells enter apoptosis upon withdrawal of IL-3 by a process that has been well characterized (Bojes et al., 1997; Gross et al., 1998). The translocation of Bax from the cytoplasm to the mitochondrial outer membrane is an early event in this cascade. As shown in Fig. 1 A, outer membranes of mitochondria isolated 12 h after IL-3 withdrawal contained significantly more Bax (5–10-fold increase when normalized to voltage-dependent anion-selective channel [VDAC] levels) than the same membranes before IL-3 withdrawal, as expected (Gross et al., 1998).


A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast.

Pavlov EV, Priault M, Pietkiewicz D, Cheng EH, Antonsson B, Manon S, Korsmeyer SJ, Mannella CA, Kinnally KW - J. Cell Biol. (2001)

Proteoliposomes enriched with Bax fail to retain cytochrome c. (A) Immunoblots show Bax (but not VDAC) levels are higher (5–10-fold) in outer membranes purified from mitochondria isolated from FL5.12 cells 12 h after IL-3 withdrawal (i.e., apoptotic cells) than from normal cells (i.e., control). Silver-stained gels and Western blots with antibodies against cytochrome oxidase subunit IV indicate equivalent content of overall protein and low contamination by inner membranes for both preparations (data not shown). (B) Immunoblots show that less cytochrome c is present in salt-washed proteoliposomes made from mitochondrial outer membranes of apoptotic cells than of normal cells (control).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2150879&req=5

fig1: Proteoliposomes enriched with Bax fail to retain cytochrome c. (A) Immunoblots show Bax (but not VDAC) levels are higher (5–10-fold) in outer membranes purified from mitochondria isolated from FL5.12 cells 12 h after IL-3 withdrawal (i.e., apoptotic cells) than from normal cells (i.e., control). Silver-stained gels and Western blots with antibodies against cytochrome oxidase subunit IV indicate equivalent content of overall protein and low contamination by inner membranes for both preparations (data not shown). (B) Immunoblots show that less cytochrome c is present in salt-washed proteoliposomes made from mitochondrial outer membranes of apoptotic cells than of normal cells (control).
Mentions: Hematopoietic FL5.12 cells enter apoptosis upon withdrawal of IL-3 by a process that has been well characterized (Bojes et al., 1997; Gross et al., 1998). The translocation of Bax from the cytoplasm to the mitochondrial outer membrane is an early event in this cascade. As shown in Fig. 1 A, outer membranes of mitochondria isolated 12 h after IL-3 withdrawal contained significantly more Bax (5–10-fold increase when normalized to voltage-dependent anion-selective channel [VDAC] levels) than the same membranes before IL-3 withdrawal, as expected (Gross et al., 1998).

Bottom Line: Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2.Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax.These findings implicate this channel, named mitochondrial apoptosis-induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, New York University College of Dentistry, New York, NY 10010, USA.

ABSTRACT
During apoptosis, proapoptotic factors are released from mitochondria by as yet undefined mechanisms. Patch-clamping of mitochondria and proteoliposomes formed from mitochondrial outer membranes of mammalian (FL5.12) cells has uncovered a novel ion channel whose activity correlates with onset of apoptosis. The pore diameter inferred from the largest conductance state of this channel is approximately 4 nm, sufficient to allow diffusion of cytochrome c and even larger proteins. The activity of the channel is affected by Bcl-2 family proteins in a manner consistent with their pro- or antiapoptotic properties. Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2. Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax. These findings implicate this channel, named mitochondrial apoptosis-induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.

Show MeSH