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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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Bcl-xL association with inner mitochondrial membranecomponents. (A) Summary of yeast two-hybrid interactionsbetween BCL-2 family proteins and the ATP synthase β subunit,including wild-type (WT) and Bcl-xL mutants mt1(F131V/D133A), mt7 (V135A/N136I/W137L), and mt8 (G138E/R139L/I140N). (B)Yeast (BY4741) transformed with the indicated plasmids were heat ramptreated and presented as colony counts from four determinations in twoindependent experiments. Data are presented as the mean ± SEM.Student’s t test was used; *, P =0.012 compared with control; **, P = 0.001. (C)Immunoblots for Bcl-xL (provided by L. Boise) or βsubunit in total rat liver mitochondria (T), inner membrane vesicles(F1), and highly purified ATP synthasomes (F2 and F3) of increasingpurity (Ko et al., 2003). Thereplicate blot in the middle was probed with Bcl-xL antibodypreincubated with recombinant Bcl-xL (rBcl-xL)protein purified from Escherichia coli (lanesF1–F3 only). (D) Coomassie blue–stained preparative SDSgel of final purification step for Bcl-xL–bindingpartners from WEHI 7.1 cells. (E) Immunoblots for Bcl-xL andβ subunit in size column chromatography fractions (F) and totallysates (T). Eluted size markers are indicated.
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fig3: Bcl-xL association with inner mitochondrial membranecomponents. (A) Summary of yeast two-hybrid interactionsbetween BCL-2 family proteins and the ATP synthase β subunit,including wild-type (WT) and Bcl-xL mutants mt1(F131V/D133A), mt7 (V135A/N136I/W137L), and mt8 (G138E/R139L/I140N). (B)Yeast (BY4741) transformed with the indicated plasmids were heat ramptreated and presented as colony counts from four determinations in twoindependent experiments. Data are presented as the mean ± SEM.Student’s t test was used; *, P =0.012 compared with control; **, P = 0.001. (C)Immunoblots for Bcl-xL (provided by L. Boise) or βsubunit in total rat liver mitochondria (T), inner membrane vesicles(F1), and highly purified ATP synthasomes (F2 and F3) of increasingpurity (Ko et al., 2003). Thereplicate blot in the middle was probed with Bcl-xL antibodypreincubated with recombinant Bcl-xL (rBcl-xL)protein purified from Escherichia coli (lanesF1–F3 only). (D) Coomassie blue–stained preparative SDSgel of final purification step for Bcl-xL–bindingpartners from WEHI 7.1 cells. (E) Immunoblots for Bcl-xL andβ subunit in size column chromatography fractions (F) and totallysates (T). Eluted size markers are indicated.

Mentions: The possibility that Bcl-xL regulates mitochondrial membrane potentialby acting at the inner mitochondrial membrane led us to revisit our earlieryeast two-hybrid screen (Chau et al.,2000). Seeking to identify prosurvival functions distinct fromantiapoptotic functions of Bcl-xL in an unbiased screen, the BH1domain mutant of Bcl-xL (mt1; F131V/D133A), which inhibits cell deathwithout binding prodeath family members Bax or Bak (Cheng et al., 1996), was used to screen a human B celllibrary (Chau et al., 2000). Among thesix hits, we identified an unexpected Bcl-xL–binding partner,the β subunit of the mitochondrial F1FO ATPsynthase. This interaction was confirmed in a secondary yeast two-hybrid screenin which the β subunit interacted with wild-type Bcl-xL andBcl-2 but did not interact with mutants lacking antideath activity(Bcl-xL mt7 and mt8) and did not interact with Bax or Bak (Fig. 3 A). Because Bcl-xL mt1could potentially inhibit mammalian cell death by binding BH3-only proteins(Billen et al., 2008), we assayedthe function of mt1 and mt8 in yeast, which lack Bcl-2 and BH3-only proteins.Bcl-xL mt1 but not mt8 protected yeast from dose-dependent celldeath (Fig. 3 B).


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)

Bcl-xL association with inner mitochondrial membranecomponents. (A) Summary of yeast two-hybrid interactionsbetween BCL-2 family proteins and the ATP synthase β subunit,including wild-type (WT) and Bcl-xL mutants mt1(F131V/D133A), mt7 (V135A/N136I/W137L), and mt8 (G138E/R139L/I140N). (B)Yeast (BY4741) transformed with the indicated plasmids were heat ramptreated and presented as colony counts from four determinations in twoindependent experiments. Data are presented as the mean ± SEM.Student’s t test was used; *, P =0.012 compared with control; **, P = 0.001. (C)Immunoblots for Bcl-xL (provided by L. Boise) or βsubunit in total rat liver mitochondria (T), inner membrane vesicles(F1), and highly purified ATP synthasomes (F2 and F3) of increasingpurity (Ko et al., 2003). Thereplicate blot in the middle was probed with Bcl-xL antibodypreincubated with recombinant Bcl-xL (rBcl-xL)protein purified from Escherichia coli (lanesF1–F3 only). (D) Coomassie blue–stained preparative SDSgel of final purification step for Bcl-xL–bindingpartners from WEHI 7.1 cells. (E) Immunoblots for Bcl-xL andβ subunit in size column chromatography fractions (F) and totallysates (T). Eluted size markers are indicated.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig3: Bcl-xL association with inner mitochondrial membranecomponents. (A) Summary of yeast two-hybrid interactionsbetween BCL-2 family proteins and the ATP synthase β subunit,including wild-type (WT) and Bcl-xL mutants mt1(F131V/D133A), mt7 (V135A/N136I/W137L), and mt8 (G138E/R139L/I140N). (B)Yeast (BY4741) transformed with the indicated plasmids were heat ramptreated and presented as colony counts from four determinations in twoindependent experiments. Data are presented as the mean ± SEM.Student’s t test was used; *, P =0.012 compared with control; **, P = 0.001. (C)Immunoblots for Bcl-xL (provided by L. Boise) or βsubunit in total rat liver mitochondria (T), inner membrane vesicles(F1), and highly purified ATP synthasomes (F2 and F3) of increasingpurity (Ko et al., 2003). Thereplicate blot in the middle was probed with Bcl-xL antibodypreincubated with recombinant Bcl-xL (rBcl-xL)protein purified from Escherichia coli (lanesF1–F3 only). (D) Coomassie blue–stained preparative SDSgel of final purification step for Bcl-xL–bindingpartners from WEHI 7.1 cells. (E) Immunoblots for Bcl-xL andβ subunit in size column chromatography fractions (F) and totallysates (T). Eluted size markers are indicated.
Mentions: The possibility that Bcl-xL regulates mitochondrial membrane potentialby acting at the inner mitochondrial membrane led us to revisit our earlieryeast two-hybrid screen (Chau et al.,2000). Seeking to identify prosurvival functions distinct fromantiapoptotic functions of Bcl-xL in an unbiased screen, the BH1domain mutant of Bcl-xL (mt1; F131V/D133A), which inhibits cell deathwithout binding prodeath family members Bax or Bak (Cheng et al., 1996), was used to screen a human B celllibrary (Chau et al., 2000). Among thesix hits, we identified an unexpected Bcl-xL–binding partner,the β subunit of the mitochondrial F1FO ATPsynthase. This interaction was confirmed in a secondary yeast two-hybrid screenin which the β subunit interacted with wild-type Bcl-xL andBcl-2 but did not interact with mutants lacking antideath activity(Bcl-xL mt7 and mt8) and did not interact with Bax or Bak (Fig. 3 A). Because Bcl-xL mt1could potentially inhibit mammalian cell death by binding BH3-only proteins(Billen et al., 2008), we assayedthe function of mt1 and mt8 in yeast, which lack Bcl-2 and BH3-only proteins.Bcl-xL mt1 but not mt8 protected yeast from dose-dependent celldeath (Fig. 3 B).

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