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Bcl-xL regulates mitochondrial energetics by stabilizing the inner membrane potential.

Chen YB, Aon MA, Hsu YT, Soane L, Teng X, McCaffery JM, Cheng WC, Qi B, Li H, Alavian KN, Dayhoff-Brannigan M, Zou S, Pineda FJ, O'Rourke B, Ko YH, Pedersen PL, Kaczmarek LK, Jonas EA, Hardwick JM - J. Cell Biol. (2011)

Bottom Line: Computational, biochemical, and genetic evidence indicated that Bcl-x(L) reduces futile ion flux across the inner mitochondrial membrane to prevent a wasteful drain on cellular resources, thereby preventing an energetic crisis during stress.Given that F(1)F(O)-ATP synthase directly affects mitochondrial membrane potential and having identified the mitochondrial ATP synthase β subunit in a screen for Bcl-x(L)-binding partners, we tested and found that Bcl-x(L) failed to protect β subunit-deficient yeast.Thus, by bolstering mitochondrial energetic capacity, Bcl-x(L) may contribute importantly to cell survival independently of other Bcl-2 family proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.

ABSTRACT
Mammalian Bcl-x(L) protein localizes to the outer mitochondrial membrane, where it inhibits apoptosis by binding Bax and inhibiting Bax-induced outer membrane permeabilization. Contrary to expectation, we found by electron microscopy and biochemical approaches that endogenous Bcl-x(L) also localized to inner mitochondrial cristae. Two-photon microscopy of cultured neurons revealed large fluctuations in inner mitochondrial membrane potential when Bcl-x(L) was genetically deleted or pharmacologically inhibited, indicating increased total ion flux into and out of mitochondria. Computational, biochemical, and genetic evidence indicated that Bcl-x(L) reduces futile ion flux across the inner mitochondrial membrane to prevent a wasteful drain on cellular resources, thereby preventing an energetic crisis during stress. Given that F(1)F(O)-ATP synthase directly affects mitochondrial membrane potential and having identified the mitochondrial ATP synthase β subunit in a screen for Bcl-x(L)-binding partners, we tested and found that Bcl-x(L) failed to protect β subunit-deficient yeast. Thus, by bolstering mitochondrial energetic capacity, Bcl-x(L) may contribute importantly to cell survival independently of other Bcl-2 family proteins.

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Submitochondrial localization of endogenousBcl-xL. (A) Immunoblots of total cell lysates(clarified), cytosolic fractions, and heavy membranes prepared fromdissected cortexes pooled from three 3-d-old mice per sample. Blots wereprobed with antibodies to Bcl-xL (1:1,000; provided by L.Boise), cytochrome c (Cyt c; 7H8.2C12[1:1,000]), cytochrome oxidase subunit IV (COX IV; 1:1,000; Invitrogen),SMAC (1:1,000; Invitrogen), voltage-dependent anion channel (VDAC;1:1,000; EMD), actin (1:1,000; MP Biomedicals), and UCP-2 (6525 [1:100;Santa Cruz Biotechnology, Inc.]). A representative of three independentexperiments is shown. (B) Summary of immunogold EM staining forBcl-xL and ATP synthase β proteins in control andcKO mouse brain representing three independent experiments. (C) EM ofmicrodissected CA1 hippocampus (where CA1 synapses onto CA3) from mousebrains stained with gold-labeled Bcl-xL antibody detectsBcl-xL on mitochondrial inner membranes/cristae (blackarrows), outer membrane polar clusters (arrowheads), and adjacentmembranes (line arrows). Bars, 0.1 µm. (D) Immunogold staining ofbcl-x cKO CA1 mouse brain prepared in parallel asin C. Bar, 0.1 µm. (E) Immunoblot analysis of Percoll-purifiednonsynaptic adult rat brain mitochondria. Mitochondria were incubatedwith the indicated proteases (for 30 min) with or without 0.01%digitonin to permeabilize the outer membrane and were blotted forBcl-xL (1:1,000; Abcam), Tom20 (11415 [1:2,000; SantaCruz Biotechnology, Inc.]), and ATP synthase β subunit (A21351[1:1,000; Invitrogen]).
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fig2: Submitochondrial localization of endogenousBcl-xL. (A) Immunoblots of total cell lysates(clarified), cytosolic fractions, and heavy membranes prepared fromdissected cortexes pooled from three 3-d-old mice per sample. Blots wereprobed with antibodies to Bcl-xL (1:1,000; provided by L.Boise), cytochrome c (Cyt c; 7H8.2C12[1:1,000]), cytochrome oxidase subunit IV (COX IV; 1:1,000; Invitrogen),SMAC (1:1,000; Invitrogen), voltage-dependent anion channel (VDAC;1:1,000; EMD), actin (1:1,000; MP Biomedicals), and UCP-2 (6525 [1:100;Santa Cruz Biotechnology, Inc.]). A representative of three independentexperiments is shown. (B) Summary of immunogold EM staining forBcl-xL and ATP synthase β proteins in control andcKO mouse brain representing three independent experiments. (C) EM ofmicrodissected CA1 hippocampus (where CA1 synapses onto CA3) from mousebrains stained with gold-labeled Bcl-xL antibody detectsBcl-xL on mitochondrial inner membranes/cristae (blackarrows), outer membrane polar clusters (arrowheads), and adjacentmembranes (line arrows). Bars, 0.1 µm. (D) Immunogold staining ofbcl-x cKO CA1 mouse brain prepared in parallel asin C. Bar, 0.1 µm. (E) Immunoblot analysis of Percoll-purifiednonsynaptic adult rat brain mitochondria. Mitochondria were incubatedwith the indicated proteases (for 30 min) with or without 0.01%digitonin to permeabilize the outer membrane and were blotted forBcl-xL (1:1,000; Abcam), Tom20 (11415 [1:2,000; SantaCruz Biotechnology, Inc.]), and ATP synthase β subunit (A21351[1:1,000; Invitrogen]).

Mentions: To pursue the role of Bcl-xL in regulating mitochondrial parameters,we determined the subcellular localization of endogenous Bcl-xLprotein in neurons of the brain. Endogenous Bcl-xL in HeLa cellsresides predominantly in the cytosol as a homodimer and translocates tomitochondria via its C-terminal transmembrane domain after a death stimulus(Jeong et al., 2004). However,crude fractionation of mouse cortex suggests that a significant proportion ofendogenous Bcl-xL localizes to mitochondria in the brain (Fig. 2 A), which is consistent with anearlier finding (Soane et al., 2008).Deletion of bcl-x (except in interneurons and glial cells whereNEX-Cre is not expressed; Berman et al.,2009) did not significantly alter other mitochondrial markers (Fig. 2 A).


Bcl-xL regulates mitochondrial energetics by stabilizing the inner membrane potential.

Chen YB, Aon MA, Hsu YT, Soane L, Teng X, McCaffery JM, Cheng WC, Qi B, Li H, Alavian KN, Dayhoff-Brannigan M, Zou S, Pineda FJ, O'Rourke B, Ko YH, Pedersen PL, Kaczmarek LK, Jonas EA, Hardwick JM - J. Cell Biol. (2011)

Submitochondrial localization of endogenousBcl-xL. (A) Immunoblots of total cell lysates(clarified), cytosolic fractions, and heavy membranes prepared fromdissected cortexes pooled from three 3-d-old mice per sample. Blots wereprobed with antibodies to Bcl-xL (1:1,000; provided by L.Boise), cytochrome c (Cyt c; 7H8.2C12[1:1,000]), cytochrome oxidase subunit IV (COX IV; 1:1,000; Invitrogen),SMAC (1:1,000; Invitrogen), voltage-dependent anion channel (VDAC;1:1,000; EMD), actin (1:1,000; MP Biomedicals), and UCP-2 (6525 [1:100;Santa Cruz Biotechnology, Inc.]). A representative of three independentexperiments is shown. (B) Summary of immunogold EM staining forBcl-xL and ATP synthase β proteins in control andcKO mouse brain representing three independent experiments. (C) EM ofmicrodissected CA1 hippocampus (where CA1 synapses onto CA3) from mousebrains stained with gold-labeled Bcl-xL antibody detectsBcl-xL on mitochondrial inner membranes/cristae (blackarrows), outer membrane polar clusters (arrowheads), and adjacentmembranes (line arrows). Bars, 0.1 µm. (D) Immunogold staining ofbcl-x cKO CA1 mouse brain prepared in parallel asin C. Bar, 0.1 µm. (E) Immunoblot analysis of Percoll-purifiednonsynaptic adult rat brain mitochondria. Mitochondria were incubatedwith the indicated proteases (for 30 min) with or without 0.01%digitonin to permeabilize the outer membrane and were blotted forBcl-xL (1:1,000; Abcam), Tom20 (11415 [1:2,000; SantaCruz Biotechnology, Inc.]), and ATP synthase β subunit (A21351[1:1,000; Invitrogen]).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3198165&req=5

fig2: Submitochondrial localization of endogenousBcl-xL. (A) Immunoblots of total cell lysates(clarified), cytosolic fractions, and heavy membranes prepared fromdissected cortexes pooled from three 3-d-old mice per sample. Blots wereprobed with antibodies to Bcl-xL (1:1,000; provided by L.Boise), cytochrome c (Cyt c; 7H8.2C12[1:1,000]), cytochrome oxidase subunit IV (COX IV; 1:1,000; Invitrogen),SMAC (1:1,000; Invitrogen), voltage-dependent anion channel (VDAC;1:1,000; EMD), actin (1:1,000; MP Biomedicals), and UCP-2 (6525 [1:100;Santa Cruz Biotechnology, Inc.]). A representative of three independentexperiments is shown. (B) Summary of immunogold EM staining forBcl-xL and ATP synthase β proteins in control andcKO mouse brain representing three independent experiments. (C) EM ofmicrodissected CA1 hippocampus (where CA1 synapses onto CA3) from mousebrains stained with gold-labeled Bcl-xL antibody detectsBcl-xL on mitochondrial inner membranes/cristae (blackarrows), outer membrane polar clusters (arrowheads), and adjacentmembranes (line arrows). Bars, 0.1 µm. (D) Immunogold staining ofbcl-x cKO CA1 mouse brain prepared in parallel asin C. Bar, 0.1 µm. (E) Immunoblot analysis of Percoll-purifiednonsynaptic adult rat brain mitochondria. Mitochondria were incubatedwith the indicated proteases (for 30 min) with or without 0.01%digitonin to permeabilize the outer membrane and were blotted forBcl-xL (1:1,000; Abcam), Tom20 (11415 [1:2,000; SantaCruz Biotechnology, Inc.]), and ATP synthase β subunit (A21351[1:1,000; Invitrogen]).
Mentions: To pursue the role of Bcl-xL in regulating mitochondrial parameters,we determined the subcellular localization of endogenous Bcl-xLprotein in neurons of the brain. Endogenous Bcl-xL in HeLa cellsresides predominantly in the cytosol as a homodimer and translocates tomitochondria via its C-terminal transmembrane domain after a death stimulus(Jeong et al., 2004). However,crude fractionation of mouse cortex suggests that a significant proportion ofendogenous Bcl-xL localizes to mitochondria in the brain (Fig. 2 A), which is consistent with anearlier finding (Soane et al., 2008).Deletion of bcl-x (except in interneurons and glial cells whereNEX-Cre is not expressed; Berman et al.,2009) did not significantly alter other mitochondrial markers (Fig. 2 A).

Bottom Line: Computational, biochemical, and genetic evidence indicated that Bcl-x(L) reduces futile ion flux across the inner mitochondrial membrane to prevent a wasteful drain on cellular resources, thereby preventing an energetic crisis during stress.Given that F(1)F(O)-ATP synthase directly affects mitochondrial membrane potential and having identified the mitochondrial ATP synthase β subunit in a screen for Bcl-x(L)-binding partners, we tested and found that Bcl-x(L) failed to protect β subunit-deficient yeast.Thus, by bolstering mitochondrial energetic capacity, Bcl-x(L) may contribute importantly to cell survival independently of other Bcl-2 family proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.

ABSTRACT
Mammalian Bcl-x(L) protein localizes to the outer mitochondrial membrane, where it inhibits apoptosis by binding Bax and inhibiting Bax-induced outer membrane permeabilization. Contrary to expectation, we found by electron microscopy and biochemical approaches that endogenous Bcl-x(L) also localized to inner mitochondrial cristae. Two-photon microscopy of cultured neurons revealed large fluctuations in inner mitochondrial membrane potential when Bcl-x(L) was genetically deleted or pharmacologically inhibited, indicating increased total ion flux into and out of mitochondria. Computational, biochemical, and genetic evidence indicated that Bcl-x(L) reduces futile ion flux across the inner mitochondrial membrane to prevent a wasteful drain on cellular resources, thereby preventing an energetic crisis during stress. Given that F(1)F(O)-ATP synthase directly affects mitochondrial membrane potential and having identified the mitochondrial ATP synthase β subunit in a screen for Bcl-x(L)-binding partners, we tested and found that Bcl-x(L) failed to protect β subunit-deficient yeast. Thus, by bolstering mitochondrial energetic capacity, Bcl-x(L) may contribute importantly to cell survival independently of other Bcl-2 family proteins.

Show MeSH
Related in: MedlinePlus