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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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Leaky energetics in Bcl-xL–deficientmitochondria. (A and B) Total ATP levels corrected for totalprotein in paired DIV3–6 cortical cultures after addition of 5µg/ml oligomycin (control [n = 8] and cKO[n = 7] at each of six time points in threeindependent experiments) or 20 µg/ml aurovertin B (control[n = 5] and cKO [n =4] at each of six time points from two independent experiments).Vertical dashed lines mark the time frame evaluated for TMRMfluorescence in C and D. Data are presented as the mean ± SEM. (Cand D) Time course of TMRM fluorescence intensities (mean ± SEM)after addition of 5 µg/ml oligomycin. Analyses of 15–35neurons from three to five fields per genotype per time point arepresented, and results are representative of five similar independentexperiments. Parallel experiments were performed with 20 µg/mlF1 inhibitor aurovertin B (n =13–35 cells from two to three fields at each time point; total of136 control and 193 cKO cells) from two independent experiments. (E)Two-photon microscopy images of cortical neurons (DIV4) stained with 100nM TMRM to assess ΔΨm as described for Fig. 1 C after addition of 6 nMoligomycin (Oligo.). A representative of five independent experiments isshown. Bar, 10 µm. (F and G) Relative NAD(P)H levels weredetermined in the same cellular subregions analyzed in C and D, asdescribed for but not included in Fig. 1B. Horizontal dashed lines mark starting NAD(P)H levels. Dataare presented as relative ratios for direct comparisons. Mean ±SEM is represented, and values are the same as in C and D.
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fig6: Leaky energetics in Bcl-xL–deficientmitochondria. (A and B) Total ATP levels corrected for totalprotein in paired DIV3–6 cortical cultures after addition of 5µg/ml oligomycin (control [n = 8] and cKO[n = 7] at each of six time points in threeindependent experiments) or 20 µg/ml aurovertin B (control[n = 5] and cKO [n =4] at each of six time points from two independent experiments).Vertical dashed lines mark the time frame evaluated for TMRMfluorescence in C and D. Data are presented as the mean ± SEM. (Cand D) Time course of TMRM fluorescence intensities (mean ± SEM)after addition of 5 µg/ml oligomycin. Analyses of 15–35neurons from three to five fields per genotype per time point arepresented, and results are representative of five similar independentexperiments. Parallel experiments were performed with 20 µg/mlF1 inhibitor aurovertin B (n =13–35 cells from two to three fields at each time point; total of136 control and 193 cKO cells) from two independent experiments. (E)Two-photon microscopy images of cortical neurons (DIV4) stained with 100nM TMRM to assess ΔΨm as described for Fig. 1 C after addition of 6 nMoligomycin (Oligo.). A representative of five independent experiments isshown. Bar, 10 µm. (F and G) Relative NAD(P)H levels weredetermined in the same cellular subregions analyzed in C and D, asdescribed for but not included in Fig. 1B. Horizontal dashed lines mark starting NAD(P)H levels. Dataare presented as relative ratios for direct comparisons. Mean ±SEM is represented, and values are the same as in C and D.

Mentions: Our vesicle models predict that the increased membrane leakiness (productive andnonproductive ion flux) across the inner mitochondrial membrane inbcl-x–deficient neurons will result in decreasedenergetic performance. To test this prediction, cultured bcl-xcKO and control cortical neurons were energetically stressed by the addition ofmitochondrial ATP synthase inhibitors and analyzed for ATP levels and formitochondrial parameters by two-photon microscopy. Extensive genetic andbiochemical evidence indicates that oligomycin inhibits mitochondrial ATPsynthesis by acting on FO to disrupt the proton path (Walker and Dickson, 2006), and a crystalstructure reveals that aurovertin B inhibits the enzymatic F1 subunitby binding near the ATP-binding site on β subunit (van Raaij et al., 1996). Treatment with oligomycin orwith aurovertin B caused cellular ATP levels to decline similarly in control andknockout neurons (Fig. 6, A and B).Therefore, the F1FO ATP synthase was an importantcontributor to ATP production and concomitant dissipation of membrane potentialin both genotypes before treatment.


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)

Leaky energetics in Bcl-xL–deficientmitochondria. (A and B) Total ATP levels corrected for totalprotein in paired DIV3–6 cortical cultures after addition of 5µg/ml oligomycin (control [n = 8] and cKO[n = 7] at each of six time points in threeindependent experiments) or 20 µg/ml aurovertin B (control[n = 5] and cKO [n =4] at each of six time points from two independent experiments).Vertical dashed lines mark the time frame evaluated for TMRMfluorescence in C and D. Data are presented as the mean ± SEM. (Cand D) Time course of TMRM fluorescence intensities (mean ± SEM)after addition of 5 µg/ml oligomycin. Analyses of 15–35neurons from three to five fields per genotype per time point arepresented, and results are representative of five similar independentexperiments. Parallel experiments were performed with 20 µg/mlF1 inhibitor aurovertin B (n =13–35 cells from two to three fields at each time point; total of136 control and 193 cKO cells) from two independent experiments. (E)Two-photon microscopy images of cortical neurons (DIV4) stained with 100nM TMRM to assess ΔΨm as described for Fig. 1 C after addition of 6 nMoligomycin (Oligo.). A representative of five independent experiments isshown. Bar, 10 µm. (F and G) Relative NAD(P)H levels weredetermined in the same cellular subregions analyzed in C and D, asdescribed for but not included in Fig. 1B. Horizontal dashed lines mark starting NAD(P)H levels. Dataare presented as relative ratios for direct comparisons. Mean ±SEM is represented, and values are the same as in C and D.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3198165&req=5

fig6: Leaky energetics in Bcl-xL–deficientmitochondria. (A and B) Total ATP levels corrected for totalprotein in paired DIV3–6 cortical cultures after addition of 5µg/ml oligomycin (control [n = 8] and cKO[n = 7] at each of six time points in threeindependent experiments) or 20 µg/ml aurovertin B (control[n = 5] and cKO [n =4] at each of six time points from two independent experiments).Vertical dashed lines mark the time frame evaluated for TMRMfluorescence in C and D. Data are presented as the mean ± SEM. (Cand D) Time course of TMRM fluorescence intensities (mean ± SEM)after addition of 5 µg/ml oligomycin. Analyses of 15–35neurons from three to five fields per genotype per time point arepresented, and results are representative of five similar independentexperiments. Parallel experiments were performed with 20 µg/mlF1 inhibitor aurovertin B (n =13–35 cells from two to three fields at each time point; total of136 control and 193 cKO cells) from two independent experiments. (E)Two-photon microscopy images of cortical neurons (DIV4) stained with 100nM TMRM to assess ΔΨm as described for Fig. 1 C after addition of 6 nMoligomycin (Oligo.). A representative of five independent experiments isshown. Bar, 10 µm. (F and G) Relative NAD(P)H levels weredetermined in the same cellular subregions analyzed in C and D, asdescribed for but not included in Fig. 1B. Horizontal dashed lines mark starting NAD(P)H levels. Dataare presented as relative ratios for direct comparisons. Mean ±SEM is represented, and values are the same as in C and D.
Mentions: Our vesicle models predict that the increased membrane leakiness (productive andnonproductive ion flux) across the inner mitochondrial membrane inbcl-x–deficient neurons will result in decreasedenergetic performance. To test this prediction, cultured bcl-xcKO and control cortical neurons were energetically stressed by the addition ofmitochondrial ATP synthase inhibitors and analyzed for ATP levels and formitochondrial parameters by two-photon microscopy. Extensive genetic andbiochemical evidence indicates that oligomycin inhibits mitochondrial ATPsynthesis by acting on FO to disrupt the proton path (Walker and Dickson, 2006), and a crystalstructure reveals that aurovertin B inhibits the enzymatic F1 subunitby binding near the ATP-binding site on β subunit (van Raaij et al., 1996). Treatment with oligomycin orwith aurovertin B caused cellular ATP levels to decline similarly in control andknockout neurons (Fig. 6, A and B).Therefore, the F1FO ATP synthase was an importantcontributor to ATP production and concomitant dissipation of membrane potentialin both genotypes before treatment.

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