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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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Nonapoptotic function of Bcl-xL. (A) Two-photonmicroscopy of bcl-x–deficient cultured neuronswas performed as described for Fig. 1B, except using a flow chamber. The measurements shown beginwith the flow of oligomycin-free medium after 40 min of 5 µg/mloligomycin (n = 4–11 per time point pergenotype). Data are presented as the mean ± SD. (B) A modeldepicting an increase in membrane leak in the absence ofBcl-xL. M, matrix. (C) Heat ramp cell death assay ofyeast strains with mutant ATP2 orFIS1/whi2-1 (Cheng et al., 2008; Tenget al., 2011). Representative images of yeast growth areshown; arrows indicate fivefold dilutions. Data are presented as mean± SD for four independent strains per plasmid, each tested induplicate in each of two independent experiments and plotted as theratio of colony numbers for Bcl-xL/empty vector(FIS1/whi2, n = 10;ATP2, n = 16).Student’s t test was used; *, P =5.7 × 10−10.
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fig7: Nonapoptotic function of Bcl-xL. (A) Two-photonmicroscopy of bcl-x–deficient cultured neuronswas performed as described for Fig. 1B, except using a flow chamber. The measurements shown beginwith the flow of oligomycin-free medium after 40 min of 5 µg/mloligomycin (n = 4–11 per time point pergenotype). Data are presented as the mean ± SD. (B) A modeldepicting an increase in membrane leak in the absence ofBcl-xL. M, matrix. (C) Heat ramp cell death assay ofyeast strains with mutant ATP2 orFIS1/whi2-1 (Cheng et al., 2008; Tenget al., 2011). Representative images of yeast growth areshown; arrows indicate fivefold dilutions. Data are presented as mean± SD for four independent strains per plasmid, each tested induplicate in each of two independent experiments and plotted as theratio of colony numbers for Bcl-xL/empty vector(FIS1/whi2, n = 10;ATP2, n = 16).Student’s t test was used; *, P =5.7 × 10−10.

Mentions: In contrast to controls, bcl-x–deficient cortical neuronsconsistently underwent delayed mitochondrial depolarization 30–45 minafter the addition of oligomycin (Fig. 6, C andE). Consistent with an energy-wasting crisis unique tobcl-x–deficient neurons, oligomycin also causesmitochondrial NAD(P)H levels to decline to ∼50% of pretreatment levels in<1 h, whereas NAD(P)H levels rebound and stabilize after oligomycintreatment in controls (Fig. 6 F). Theseresults suggest that bcl-x–deficient mitochondriacontinue to deplete the substrate of complex I, as would be expected for a leakymitochondrial membrane that allows the respiratory chain to continue running.Consistent with this conclusion, rates of oxygen uptake by cells decrease withoverexpression of Bcl-xL and increase with shRNA knockdown ofBcl-xL (Alavian et al.,2011). NAD(P)H depletion and membrane depolarization were not simplya result of inhibition of mitochondrial ATP synthesis because NAD(P)H levels andmembrane potential were sustained for at least 1 h after aurovertin B treatment,although at lower steady-state levels relative to controls (see Discussion;Fig. 6 G). To verify that depletionof NAD(P)H and mitochondrial depolarization is not simply a marker of celldeath, oligomycin was washed away from depolarizedbcl-x–deficient neurons in a flow chamber. Upon washout,we observed simultaneous increases in NAD(P)H levels and TMRM intensity,indicating cell recovery (Fig. 7 A). Theevidence presented suggests that Bcl-xL increases the efficiency ofmitochondrial energetics by decreasing inner membrane leakiness, therebypreventing membrane potential fluctuations and the resulting energy deficits(Fig. 7 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)

Nonapoptotic function of Bcl-xL. (A) Two-photonmicroscopy of bcl-x–deficient cultured neuronswas performed as described for Fig. 1B, except using a flow chamber. The measurements shown beginwith the flow of oligomycin-free medium after 40 min of 5 µg/mloligomycin (n = 4–11 per time point pergenotype). Data are presented as the mean ± SD. (B) A modeldepicting an increase in membrane leak in the absence ofBcl-xL. M, matrix. (C) Heat ramp cell death assay ofyeast strains with mutant ATP2 orFIS1/whi2-1 (Cheng et al., 2008; Tenget al., 2011). Representative images of yeast growth areshown; arrows indicate fivefold dilutions. Data are presented as mean± SD for four independent strains per plasmid, each tested induplicate in each of two independent experiments and plotted as theratio of colony numbers for Bcl-xL/empty vector(FIS1/whi2, n = 10;ATP2, n = 16).Student’s t test was used; *, P =5.7 × 10−10.
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fig7: Nonapoptotic function of Bcl-xL. (A) Two-photonmicroscopy of bcl-x–deficient cultured neuronswas performed as described for Fig. 1B, except using a flow chamber. The measurements shown beginwith the flow of oligomycin-free medium after 40 min of 5 µg/mloligomycin (n = 4–11 per time point pergenotype). Data are presented as the mean ± SD. (B) A modeldepicting an increase in membrane leak in the absence ofBcl-xL. M, matrix. (C) Heat ramp cell death assay ofyeast strains with mutant ATP2 orFIS1/whi2-1 (Cheng et al., 2008; Tenget al., 2011). Representative images of yeast growth areshown; arrows indicate fivefold dilutions. Data are presented as mean± SD for four independent strains per plasmid, each tested induplicate in each of two independent experiments and plotted as theratio of colony numbers for Bcl-xL/empty vector(FIS1/whi2, n = 10;ATP2, n = 16).Student’s t test was used; *, P =5.7 × 10−10.
Mentions: In contrast to controls, bcl-x–deficient cortical neuronsconsistently underwent delayed mitochondrial depolarization 30–45 minafter the addition of oligomycin (Fig. 6, C andE). Consistent with an energy-wasting crisis unique tobcl-x–deficient neurons, oligomycin also causesmitochondrial NAD(P)H levels to decline to ∼50% of pretreatment levels in<1 h, whereas NAD(P)H levels rebound and stabilize after oligomycintreatment in controls (Fig. 6 F). Theseresults suggest that bcl-x–deficient mitochondriacontinue to deplete the substrate of complex I, as would be expected for a leakymitochondrial membrane that allows the respiratory chain to continue running.Consistent with this conclusion, rates of oxygen uptake by cells decrease withoverexpression of Bcl-xL and increase with shRNA knockdown ofBcl-xL (Alavian et al.,2011). NAD(P)H depletion and membrane depolarization were not simplya result of inhibition of mitochondrial ATP synthesis because NAD(P)H levels andmembrane potential were sustained for at least 1 h after aurovertin B treatment,although at lower steady-state levels relative to controls (see Discussion;Fig. 6 G). To verify that depletionof NAD(P)H and mitochondrial depolarization is not simply a marker of celldeath, oligomycin was washed away from depolarizedbcl-x–deficient neurons in a flow chamber. Upon washout,we observed simultaneous increases in NAD(P)H levels and TMRM intensity,indicating cell recovery (Fig. 7 A). Theevidence presented suggests that Bcl-xL increases the efficiency ofmitochondrial energetics by decreasing inner membrane leakiness, therebypreventing membrane potential fluctuations and the resulting energy deficits(Fig. 7 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