Quantitation and origin of the mitochondrial membrane potential in human cells lacking mitochondrial DNA

Appleby, Ruth D.; Porteous, William K.; Hughes, Gillian; James, Andrew M.; Shannon, Daniel; Wei, Yau‐Huei; Murphy, Michael P.

doi:10.1046/j.1432-1327.1999.00350.x

Cited by 150 publications

(149 citation statements)

References 47 publications

(63 reference statements)

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“…The mitochondrial pellet was washed in STE, and the supernatant was acetone-precipitated. Assays of the mitochondrial enzyme citrate synthase and of the cytosolic enzyme lactate dehydrogenase confirmed that this fractionation procedure was effective, with less than 5% lactate dehydrogenase activity in the mitochondrial fraction that contained about 40% of the total citrate synthase activity (36).…”

mentioning

confidence: 80%

Specific Modification of Mitochondrial Protein Thiols in Response to Oxidative Stress

Lin¹,

Hughes²,

Muratovska³

et al. 2002

Journal of Biological Chemistry

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182

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Mitochondria play a central role in redox-linked processes in the cell through mechanisms that are thought to involve modification of specific protein thiols, but this has proved difficult to assess. In particular, specific labeling and quantitation of mitochondrial protein cysteine residues have not been achieved due to the lack of reagents available that can be applied to the intact organelle or cell. To overcome these problems we have used a combination of mitochondrial proteomics and targeted labeling of mitochondrial thiols using a novel compound, (4-iodobutyl)triphenylphosphonium (IBTP). This lipophilic cation is accumulated by mitochondria and yields stable thioether adducts in a thiol-specific reaction. The selective uptake into mitochondria, due to the large membrane potential across the inner membrane, and the high pH of the matrix results in specific labeling of mitochondrial protein thiols by IBTP. Individual mitochondrial proteins that changed thiol redox state following oxidative stress could then be identified by their decreased reaction with IBTP and isolated by two-dimensional electrophoresis. We demonstrate the selectivity of IBTP labeling and use it to show that glutathione oxidation and exposure to an S-nitrosothiol or to peroxynitrite cause extensive redox changes to mitochondrial thiol proteins. In conjunction with blue native gel electrophoresis, we used IBTP labeling to demonstrate that thiols are exposed on the matrix faces of respiratory Complexes I, II, and IV. This novel approach enables measurement of the thiol redox state of individual mitochondrial proteins during oxidative stress and cell death. In addition the methodology has the potential to identify novel redox-dependent modulation of mitochondrial proteins.Changes in the thiol redox state of mitochondrial proteins are significant in a number of cellular processes including the permeability transition, cell death due to calcium loading and oxidative stress, the response of cells to nitric oxide, tumor necrosis factor signaling, commitment to apoptosis, and in regulating respiratory chain function (1-9). However the detailed mechanisms and the proteins involved are uncertain. This is partly because of the technical challenges presented by determining thiol modifications of proteins in general and the difficulties inherent in mitochondrial proteomics. Potential protein thiol alterations include formation of mixed disulfides or internal disulfides from vicinal dithiols, S-nitrosation, and the formation of higher oxidation states (10 -15). The differential reactivity of individual protein thiols and the range of lifetimes of altered redox states can act as signal sensors or transducers to influence mitochondrial function (13-17). Nitric oxide may be a particularly important regulator of mitochondrial protein thiols because it diffuses easily into mitochondria and partitions selectively into the lipid bilayer where it can modify otherwise inaccessible thiols (18 -20). Modification of protein thiols by nitric oxide most likely occurs...

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mentioning

confidence: 80%

Specific Modification of Mitochondrial Protein Thiols in Response to Oxidative Stress

Lin¹,

Hughes²,

Muratovska³

et al. 2002

Journal of Biological Chemistry

Self Cite

182

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“…Mitochondrial dysfunction resulting from PNPase deficiency led to secondary effects including reduced ATP production. In addition to reduced ATP production from defective OX-PHOS, a complementary mechanism for further reducing ATP might include increased ATP catabolism as an adaptive response, through the ATP synthase acting as an ATP hydrolase, which would help stabilize ⌬ and inhibit the onset of apoptosis (2). Reduced cell energy charge, in turn, would enhance the phosphorylation of AMPK, which could slow cell growth through a potential link to p53-mediated inhibition of the cyclin E cell cycle checkpoint protein (21,31).…”

Section: Discussionmentioning

confidence: 99%

“…Oligomycin-sensitive complex V ATP hydrolysis activity was determined by incubating 30 g mitochondria in 1 ml 100 mM Tris buffer, pH 8.0, containing 50 mM KCl, 2 mM MgCl 2 , 0.2 mM EDTA, 200 M NADH, 1 mM phosphoenolpyruvate, 2 U/ml lactate dehydrogenase, 4 U/ml pyruvate kinase, 2.5 mM Mg-ATP, 4 g/ml rotenone, 300 nM antimycin A, and 2 mM KCN in the presence or absence of 5 g/ml oligomycin. The oxidation of NADH was followed at 340 minus 400 nm for 7 min at 30°C (2). Enzyme activities are shown as nmol/min/mg protein.…”

Section: Methodsmentioning

confidence: 99%

Mammalian Polynucleotide Phosphorylase Is an Intermembrane Space RNase That Maintains Mitochondrial Homeostasis

Chen¹,

Rainey²,

Balatoni³

et al. 2006

Molecular and Cellular Biology

133

150

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We recently identified polynucleotide phosphorylase (PNPase) as a potential binding partner for the TCL1 oncoprotein. Mammalian PNPase exhibits exoribonuclease and poly(A) polymerase activities, and PNPase overexpression inhibits cell growth, induces apoptosis, and stimulates proinflammatory cytokine production. A physiologic connection for these anticancer effects and overexpression is difficult to reconcile with the presumed mitochondrial matrix localization for endogenous PNPase, prompting this study. Here we show that basal and interferon-␤-induced PNPase was efficiently imported into energized mitochondria with coupled processing of the N-terminal targeting sequence. Once imported, PNPase localized to the intermembrane space (IMS) as a peripheral membrane protein in a multimeric complex. Apoptotic stimuli caused PNPase mobilization following cytochrome c release, which supported an IMS localization and provided a potential route for interactions with cytosolic TCL1. Consistent with its IMS localization, PNPase knockdown with RNA interference did not affect mitochondrial RNA levels. However, PNPase reduction impaired mitochondrial electrochemical membrane potential, decreased respiratory chain activity, and was correlated with altered mitochondrial morphology. This resulted in F o F 1 -ATP synthase instability, impaired ATP generation, lactate accumulation, and AMP kinase phosphorylation with reduced cell proliferation. Combined, the data demonstrate an unexpected IMS localization and a key role for PNPase in maintaining mitochondrial homeostasis.

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“…Ϫ by ANT (Buchet and Godinot, 1998;Appleby et al, 1999;Chandel and Schumacker, 1999;Arnould et al, 2003). Oligomycin (which binds and inhibits only the F 0 portion of F 0 F 1 -ATPase) does not eliminate ATPase activity and ⌬⌿m in rho zero cells, because they are devoid of F 0 (Buchet andGodinot, 1998 Appleby et al, 1999).…”

Section: Discussionmentioning

confidence: 99%

Apoptosome-deficient Cells Lose Cytochromecthrough Proteasomal Degradation but Survive by Autophagy-dependent Glycolysis

Ferraro

Pulicati

Cencioni

et al. 2008

MBoC

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Cytochrome c release from mitochondria promotes apoptosome formation and caspase activation. The question as to whether mitochondrial permeabilization kills cells via a caspase-independent pathway when caspase activation is prevented is still open. Here we report that proneural cells of embryonic origin, when induced to die but rescued by apoptosome inactivation are deprived of cytosolic cytochrome c through proteasomal degradation. We also show that, in this context, those cells keep generating ATP by glycolysis for a long period of time and that they keep their mitochondria in a depolarized state that can be reverted. Moreover, under these conditions, such apoptosome-deficient cells activate a Beclin 1-dependent autophagy pathway to sustain glycolytic-dependent ATP production. Our findings contribute to elucidating what the point-of-no-return in apoptosis is. They also help in clarifying the issue of survival of apoptosomedeficient proneural cells under stress conditions. Unraveling this issue could be highly relevant for pharmacological intervention and for therapies based on neural stem cell transfer in the treatment of neurological disorders.

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Quantitation and origin of the mitochondrial membrane potential in human cells lacking mitochondrial DNA

Cited by 150 publications

References 47 publications

Specific Modification of Mitochondrial Protein Thiols in Response to Oxidative Stress

Specific Modification of Mitochondrial Protein Thiols in Response to Oxidative Stress

Mammalian Polynucleotide Phosphorylase Is an Intermembrane Space RNase That Maintains Mitochondrial Homeostasis

Apoptosome-deficient Cells Lose Cytochromecthrough Proteasomal Degradation but Survive by Autophagy-dependent Glycolysis

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