The effect of oral melatonin supplementation on MT-ATP6 gene expression and IVF outcomes in Iranian infertile couples: a nonrandomized controlled trial.

This study aims to evaluate the effect of melatonin supplementation on the outcomes of in vitro fertilization (IVF) and mitochondrial adenosine triphosphate production (MT-ATP6) gene expression in Iranian infertile couples. A single-blind nonrandomized controlled trial was conducted, recruiting 90 infertile couples who underwent IVF at an infertility center in Tehran, Iran. Patients who were assigned to the intervention group received melatonin as a supplementation to the standard controlled ovarian stimulation (COS). The control group received a COS protocol only. Primary outcome was the mRNA level of the MT-ATP6 gene in cumulus cells of ovarian follicles. Secondary outcomes were the mean number of mature oocytes retrieved, the embryo quality, and biochemical and clinical pregnancy rates. The mRNA level of the MT-ATP6 gene in cumulus cells between intervention and control groups was not statistically different (0.931 vs.1; P Ëƒ 0.05). The mean number of poor-quality embryos was significantly lower in the intervention group than that in the control group (0.27 vs. 0.80; P = 0.028). The biochemical and clinical pregnancy rates were higher in the intervention group (24% vs. 14%, P = 0.089, and 14% vs. 7%, P = 0.302, respectively); however, the difference was not significant. Melatonin supplementation did not increase the odds of clinical pregnancy and the number of mature oocytes retrieved, but significantly reduced the number of low-quality embryos. More extensive studies focusing on the level of MT-ATP6 gene expression in the oocyte or blastomere cells may further elucidate the effect of supplementation with melatonin in infertile couples who have poor clinical outcomes. Trial registration: Current Controlled Trials: IRCT2015042912307N4.

Targeted inhibition of ATP5B gene prevents bone erosion in collagen-induced arthritis by inhibiting osteoclastogenesis.

Bone resorption by osteoclasts is an energy consuming activity, which depends on mitochondrial ATP. ATP5B, a mitochondrial ATP synthase beta subunit, is a catalytic core involved in producing ATP. Here, we investigated the contribution of ATP5B in osteoclast differentiation and joint destruction. ATP5B (LV-ATP5B) targeting or non-targeting (LV-NC) siRNA containing lentivirus particles were transduced into bone marrow macrophage derived osteoclasts or locally administered to arthritic mouse joints. Inhibition of ATP5B reduced the expression of osteoclast related genes and proteins, suppressed bone resorption by significantly impairing F-actin formation and decreased the levels of adhesion-associated proteins. In addition, ATP5B deficiency caused osteoclast mitochondrial dysfunction and, impaired the secretion of vacuole protons and MMP9. Importantly, inhibition of ATP5B expression, protected arthritis mice from joint destructions although serum levels of inflammatory mediators (TNF-α, IL-1ß) and IgG2α antibodies were unaffected. These results demonstrate an essential function of ATP5B in osteoclast differentiation and bone resorption, and suggest it as a potential therapeutic target for protecting bones in RA.

Current understanding of structure, function and biogenesis of yeast mitochondrial ATP synthase.

The yeast mitochondrial ATP synthase is a rotary molecular machine primarily responsible for the production of energy used to drive cellular processes. The enzyme complex is composed of 17 different subunits grouped into a soluble F1 sector and a membrane-embedded F0 sector. The catalytic head of the F1 sector and the membrane integrated motor module in the F0 sector are connected by two stalks, the F1 central stalk and the F0 peripheral stalk. Proton translocation through the F0 motor module drives the rotation of the subunit 910-ring that generates torque which is transmitted to the calaytic head through the γ subunit of the central stalk. The rotation of the γ subunit causes changes in conformation of the catalytic head which leads to the synthesis of ATP. Biogenesis of the enzyme involves modular assembly of polypeptides of dual genetic origin, the nuclear and the mitochondrial genomes. Most of the yeast ATP synthase subunits are encoded by the genome of the nucleus, translated on cytosolic ribosomes and imported into mitochondria. In the mitochondria, the enzyme forms a dimer which contributes to the formation of cristae, a characteristic of mitochondrial morphology. Substantial progress has recently been made on the elucidation of detailed stucture, function and biogenesis of yeast mitochondrial ATP synthase. The recent availability of high-resolution structure of the complete monomeric form, as well as the atomic model for the dimeric F0 sector, has advanced the understanding of the enzyme complex. This review is intended to provide an overview of current understanding of the molecular structure, catalytic mechanism, subunit import into mitochondria, and the subunit assembly into the enzyme complex. This is important as the yeast mitochondrial ATP synthase may be used as a model for understanding the corresponding enzyme complexes from human and other eukaryotic cells in physiological and diseased states.

All trans retinoic acid depresses the content and activity of the mitochondrial ATP synthase in human keratinocytes.

Proteomic analysis shows that treatment of keratinocytes cultures with all trans retinoic acid (ATRA), under condition in which it inhibits cell growth, results in marked decrease of the level of the F1-ß subunit of the catalytic sector of the mitochondrial FoF1 ATP synthase complex. Enzymatic analysis shows in ATRA-treated keratinocytes a consistent depression of the ATPase activity, with decreased olygomycin sensitivity, indicating an overall alteration of the ATP synthase complex. These findings, together with the previously reported inhibition of respiratory complex I, show that depression of the activity of oxidative phosphorylation enzymes is involved in the cell growth inhibitory action of ATRA.

Developmental defect of cytochrome oxidase mutants of Streptomyces coelicolor A3(2).

To study the link between energy metabolism and secondary metabolism/morphological development in Streptomyces, knockout mutants were generated with regard to the subunits of the cytochrome oxidase supercomplex (CcO) in Streptomyces coelicolor A3(2). All mutants exhibited an identical phenotype: viable but defective in antibiotic production and cell differentiation when grown in both complex and minimal media. The growth yield of the CcO mutant was about half of that of the WT strain on glucose medium while both strains grew similarly on maltose medium. Intracellular ATP measurement demonstrated that the CcO mutant exhibited high intracellular ATP level. A similar elevation of intracellular ATP level was observed with regard to the WT strain cultured in the presence of BCDA, a copper-chelating agent. Reverse transcriptase PCR analysis demonstrated that the transcription of ATP synthase operon is upregulated in the CcO mutant. Addition of carbonylcyanide m-chlorophenylhydrazone, an inhibitor of ATP synthesis, promoted antibiotic production and aerial mycelia formation in the CcO mutant and BCDA-treated WT cells. We hypothesize that the deficiency of CcO causes accumulation of intracellular ATP, and that the high ATP level inhibits the onset of development in S. coelicolor.

Knockout of Tmem70 alters biogenesis of ATP synthase and leads to embryonal lethality in mice.

TMEM70, a 21-kDa protein localized in the inner mitochondrial membrane, has been shown to facilitate the biogenesis of mammalian F1Fo ATP synthase. Mutations of the TMEM70 gene represent the most frequent cause of isolated ATP synthase deficiency resulting in a severe mitochondrial disease presenting as neonatal encephalo-cardiomyopathy (OMIM 604273). To better understand the biological role of this factor, we generated Tmem70-deficient mice and found that the homozygous Tmem70-/- knockouts exhibited profound growth retardation and embryonic lethality at ∼9.5 days post coitum. Blue-Native electrophoresis demonstrated an isolated deficiency in fully assembled ATP synthase in the Tmem70-/- embryos (80% decrease) and a marked accumulation of F1 complexes indicative of impairment in ATP synthase biogenesis that was stalled at the early stage, following the formation of F1 oligomer. Consequently, a decrease in ADP-stimulated State 3 respiration, respiratory control ratio and ATP/ADP ratios, indicated compromised mitochondrial ATP production. Tmem70-/- embryos exhibited delayed development of the cardiovascular system and a disturbed heart mitochondrial ultrastructure, with concentric or irregular cristae structures. Tmem70+/- heterozygous mice were fully viable and displayed normal postnatal growth and development of the mitochondrial oxidative phosphorylation system. Nevertheless, they presented with mild deterioration of heart function. Our results demonstrated that Tmem70 knockout in the mouse results in embryonic lethality due to the lack of ATP synthase and impairment of mitochondrial energy provision. This is analogous to TMEM70 dysfunction in humans and verifies the crucial role of this factor in the biosynthesis and assembly of mammalian ATP synthase.

Deletion of capn4 Protects the Heart Against Endotoxemic Injury by Preventing ATP Synthase Disruption and Inhibiting Mitochondrial Superoxide Generation.

BACKGROUND: Our recent study has demonstrated that inhibition of calpain by transgenic overexpression of calpastatin reduces myocardial proinflammatory response and dysfunction in endotoxemia. However, the underlying mechanisms remain to be determined. In this study, we used cardiomyocyte-specific capn4 knockout mice to investigate whether and how calpain disrupts ATP synthase and induces mitochondrial superoxide generation during endotoxemia. METHODS AND RESULTS: Cardiomyocyte-specific capn4 knockout mice and their wild-type littermates were injected with lipopolysaccharides. Four hours later, calpain-1 protein and activity were increased in mitochondria of endotoxemic mouse hearts. Mitochondrial calpain-1 colocalized with and cleaved ATP synthase-α (ATP5A1), leading to ATP synthase disruption and a concomitant increase in mitochondrial reactive oxygen species generation during lipopolysaccharide stimulation. Deletion of capn4 or upregulation of ATP5A1 increased ATP synthase activity, prevented mitochondrial reactive oxygen species generation, and reduced proinflammatory response and myocardial dysfunction in endotoxemic mice. In cultured cardiomyocytes, lipopolysaccharide induced mitochondrial superoxide generation that was prevented by overexpression of mitochondria-targeted calpastatin or ATP5A1. Upregulation of calpain-1 specifically in mitochondria sufficiently induced superoxide generation and proinflammatory response, both of which were attenuated by ATP5A1 overexpression or mitochondria-targeted superoxide dismutase mimetics. CONCLUSIONS: Cardiomyocyte-specific capn4 knockout protects the heart against lipopolysaccharide-induced injury in endotoxemic mice. Lipopolysaccharides induce calpain-1 accumulation in mitochondria. Mitochondrial calpain-1 disrupts ATP synthase, leading to mitochondrial reactive oxygen species generation, which promotes proinflammatory response and myocardial dysfunction during endotoxemia. These findings uncover a novel mechanism by which calpain mediates myocardial dysfunction in sepsis.

Molecular Analysis by Gene Expression of Mitochondrial ATPase Subunits in Papillary Thyroid Cancer: Is ATP5E Transcript a Possible Early Tumor Marker?

BACKGROUND: Cancer development involves an "injury" to the respiratory machinery (Warburg effect) due to decreased or impaired mitochondrial function. This circumstance results in a down regulation of some of the ATPase subunits of the malignant tissue. The objective of this work was to assess and compare the relative expression of mRNA of mitochondrial ATPase subunits between samples of thyroid cancer and benign nodules. MATERIAL AND METHODS: Samples from 31 patients who had an operation for PTC at the General Hospital of Mexico were snap-frozen and stored at -70°C. Thirty-five patients who had an operation for benign tumors were also included in the study. mRNA expression levels of alpha, beta, gamma, and epsilon subunits of F1 and "c12" of subunit Fo were determined by real-time RT-PCR (by duplicate), in order to determine if abnormal expression of these genes could partially explain the Warburg effect in papillary thyroid cancer (PTC). RESULTS: ATP5E transcript alteration (down-expression) was highly associated to PTC diagnosis OR=11.76 (95% confidence interval, 1.245-237.98; p=0.04). CONCLUSIONS: Relative down-expression of ATP5E transcript was highly associated with PTC diagnosis. This transcript alteration may be used as a tumoral marker in papillary thyroid cancer.

Humanin Derivatives Inhibit Necrotic Cell Death in Neurons.

Humanin and its derivatives are peptides known for their protective antiapoptotic effects against Alzheimer's disease. Herein, we identify a novel function of the humanin-derivative AGA(C8R)-HNG17 (namely, protection against cellular necrosis). Necrosis is one of the main modes of cell death, which was until recently considered an unmoderated process. However, recent findings suggest the opposite. We have found that AGA(C8R)-HNG17 confers protection against necrosis in the neuronal cell lines PC-12 and NSC-34, where necrosis is induced in a glucose-free medium by either chemohypoxia or by a shift from apoptosis to necrosis. Our studies in traumatic brain injury models in mice, where necrosis is the main mode of neuronal cell death, have shown that AGA(C8R)-HNG17 has a protective effect. This result is demonstrated by a decrease in a neuronal severity score and by a reduction in brain edema, as measured by magnetic resonance imaging (MRI). An insight into the peptide's antinecrotic mechanism was attained through measurements of cellular ATP levels in PC-12 cells under necrotic conditions, showing that the peptide mitigates a necrosis-associated decrease in ATP levels. Further, we demonstrate the peptide's direct enhancement of the activity of ATP synthase activity, isolated from rat-liver mitochondria, suggesting that AGA(C8R)-HNG17 targets the mitochondria and regulates cellular ATP levels. Thus, AGA(C8R)-HNG17 has potential use for the development of drug therapies for necrosis-related diseases, for example, traumatic brain injury, stroke, myocardial infarction, and other conditions for which no efficient drug-based treatment is currently available. Finally, this study provides new insight into the mechanisms underlying the antinecrotic mode of action of AGA(C8R)-HNG17.

Glucocorticoid receptor is involved in the breed-dependent transcriptional regulation of mtDNA- and nuclear-encoded mitochondria genes in the liver of newborn piglets.

BACKGROUND: Mitochondria, which are essential for the functionality of eukaryotic cells, are particularly important in metabolically active tissues such as liver. Different breeds of pigs demonstrate distinct metabolic profiles in the liver, yet little is known whether the expression and transcriptional regulation of mitochondrial genes differ between breeds. RESULTS: Here we used male newborn Large White (LW) and Erhualian (EHL) piglets to delineate the difference in hepatic mitochondrial gene regulation between breeds. The hepatic content of ATP was significantly higher (p < 0.01) in EHL piglets, which was associated with lower mtDNA copy number (p < 0.05). Most of the mtDNA-encoded genes (10 of 13), however, were more abundantly expressed in EHL compared to LW piglets. We also detected 3 differentially expressed nuclear-encoded mitochondrial genes, among which isocitrate dehydrogenase 2 (IDH2) and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit d (ATP5H) were expressed significantly lower, while adenylate kinase 1 (AK1) was significantly over expressed in EHL piglets. Compared to LW, the over expression of mtDNA-encoded genes in EHL was associated with significantly higher (p < 0.01) glucocorticoid receptor (GR) binding to the control region of mtDNA with no alterations in the methylation status. For nuclear-encoded genes, however, a negative correlation was observed between GR binding and mRNA expression of AK1 and ATP5H. Moreover, higher expression of AK1 in EHL piglets was also associated with lower cytosine methylation (p < 0.05) and hydroxymethylation (p < 0.05). In the promoter region. CONCLUSIONS: These results indicate a role of the GR in the breed-dependent regulation of mitochondrial genes in the liver of newborn piglets.

Deletion of the transcriptional regulator opi1p decreases cardiolipin content and disrupts mitochondrial metabolism in Saccharomyces cerevisiae.

Cardiolipin, the main anionic phospholipid in the inner mitochondrial membrane, provides shape, charge and osmotic support to this membrane due to its biophysical properties. In addition, it helps form respiratory supercomplexes and provides functionality to mitochondrial proteins. Defects in the biosynthesis or remodeling of cardiolipin have been related to severe diseases, such as Barth syndrome. Opi1p, a transcriptional repressor for most enzymes in phospholipid biosynthesis found in Saccharomyces cerevisiae, has been demonstrated not to affect the biosynthesis of this mitochondrial phospholipid. However, we found that opi1 deletion compromises mitochondrial metabolism producing severe respiratory defects. The mechanism producing this phenotype was explored and found to be a mitochondrial cardiolipin depletion of almost 50%, resulting in low cytochrome content and high mitochondrial DNA instability. The origin of this low cardiolipin content strongly correlated with the overproduction of inositol, an intrinsic phenotype of this mutation. Overall, our results show that adequate regulation of phospholipid synthesis is essential for the maintenance of mitochondrial function.

Nucleo-cytoplasmic interactions affect RNA editing of cox2, atp6 and atp9 in alloplasmic male-sterile rice (Oryza sativa L.) lines.

RNA editing plays an important role in the regulation of mitochondrial gene expression in flowering plants. In this study, we examined RNA editing of the mitochondrial genes cox2, atp6 and atp9 in five isonuclear alloplasmic male-sterile lines (IAMSLs) of rice to investigate whether different cytoplasmic types affect RNA editing. Although many editing sites were conserved among the three genes, we found that the editing efficiency of certain sites was significantly different between different IAMSLs or between IAMSLs and their corresponding cytoplasmic donor CMS lines. Furthermore, several editing sites were found to be either present or absent in certain IAMSLs and their corresponding CMS lines. These results indicate that nuclear loci, as well as unknown editing factors within the mitochondria of different cytoplasmic types, may be involved in RNA editing, and they suggest that RNA editing in plant mitochondria is affected by nucleo-cytoplasmic interactions.

Low-intensity laser irradiation at 660 nm stimulates transcription of genes involved in the electron transport chain.

BACKGROUND DATA: Low-intensity laser irradiation (LILI) has been shown to stimulate cellular functions leading to increased adenosine triphosphate (ATP) synthesis. This study was undertaken to evaluate the effect of LILI on genes involved in the mitochondrial electron transport chain (ETC, complexes I-IV) and oxidative phosphorylation (ATP synthase). METHODS: Four human skin fibroblast cell models were used in this study: normal non-irradiated cells were used as controls while wounded, diabetic wounded, and ischemic cells were irradiated. Cells were irradiated with a 660 nm diode laser with a fluence of 5 J/cm(2) and gene expression determined by quantitative real-time reverse transcription (RT) polymerase chain reaction (PCR). RESULTS: LILI upregulated cytochrome c oxidase subunit VIb polypeptide 2 (COX6B2), cytochrome c oxidase subunit VIc (COX6C), and pyrophosphatase (inorganic) 1 (PPA1) in diabetic wounded cells; COX6C, ATP synthase, H+transporting, mitochondrial Fo complex, subunit B1 (ATP5F1), nicotinamide adenine dinucleotide (NADH) dehydrogenase (ubiquinone) 1 alpha subcomplex, 11 (NDUFA11), and NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) in wounded cells; and ATPase, H+/K+ exchanging, beta polypeptide (ATP4B), and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C2 (subunit 9) (ATP5G2) in ischemic cells. CONCLUSIONS: LILI at 660 nm stimulates the upregulation of genes coding for subunits of enzymes involved in complexes I and IV and ATP synthase.

Nuclear expression of a mitochondrial DNA gene: mitochondrial targeting of allotopically expressed mutant ATP6 in transgenic mice.

Nuclear encoding of mitochondrial DNA transgenes followed by mitochondrial targeting of the expressed proteins (allotopic expression; AE) represents a potentially powerful strategy for creating animal models of mtDNA disease. Mice were created that allotopically express either a mutant (A6M) or wildtype (A6W) mt-Atp6 transgene. Compared to non-transgenic controls, A6M mice displayed neuromuscular and motor deficiencies (wire hang, pole, and balance beam analyses; P < 0.05), no locomotor differences (gait analysis; P < 0.05) and enhanced endurance in Rota-Rod evaluations (P < 0.05). A6W mice exhibited inferior muscle strength (wire hang test; P < 0.05), no difference in balance beam footsteps, accelerating Rota-Rod, pole test and gait analyses; (P < 0.05) and superior performance in balance beam time-to-cross and constant velocity Rota-Rod analyses (P < 0.05) in comparison to non-transgenic control mice. Mice of both transgenic lines did not differ from non-transgenic controls in a number of bioenergetic and biochemical tests including measurements of serum lactate and mitochondrial MnSOD protein levels, ATP synthesis rate, and oxygen consumption (P > 0.05). This study illustrates a mouse model capable of circumventing in vivo mitochondrial mutations. Moreover, it provides evidence supporting AE as a tool for mtDNA disease research with implications in development of DNA-based therapeutics.

EF-1α and mitochondrial ATP synthase ß chain: alteration of their expression in encystment-induced Colpoda cucullus.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the total proteins contained in encystment-induced Colpoda cucullus showed that a 50-kDa protein (p50) disappeared, whereas the expression of a 49-kDa protein (p49) was enhanced in early phase of morphogenetic transformation into the resting cyst (i.e. 2-5 h after the onset of encystment induction). Puromycin or actinomycin D inhibited the alteration in the expression of p50 and p49 by the induction of encystment. These results suggest that the encystment-specific alteration in expression of these proteins is performed by a transcriptional regulation. Liquid chromatography tandem mass spectrometry analysis revealed that p50 is mitochondrial ATP synthase ß chains, and that p49 is elongation factor 1α.

A deubiquitylating complex required for neosynthesis of a yeast mitochondrial ATP synthase subunit.

The ubiquitin system is known to be involved in maintaining the integrity of mitochondria, but little is known about the role of deubiquitylating (DUB) enzymes in such functions. Budding yeast cells deleted for UBP13 and its close homolog UBP9 displayed a high incidence of petite colonies and slow respiratory growth at 37°C. Both Ubp9 and Ubp13 interacted directly with Duf1 (DUB-associated factor 1), a WD40 motif-containing protein. Duf1 activates the DUB activity of recombinant Ubp9 and Ubp13 in vitro and deletion of DUF1 resulted in the same respiratory phenotype as the deletion of both UBP9 and UBP13. We show that the mitochondrial defects of these mutants resulted from a strong decrease at 37°C in the de novo biosynthesis of Atp9, a membrane-bound component of ATP synthase encoded by mitochondrial DNA. The defect appears at the level of ATP9 mRNA translation, while its maturation remained unchanged in the mutants. This study describes a new role of the ubiquitin system in mitochondrial biogenesis.

Shrimp ATP synthase genes complement yeast null mutants for ATP hydrolysis but not synthesis activity.

The aim of this study was to examine the feasibility of employing a yeast functional complementation assay for shrimp genes by using the shrimp mitochondrial F(1)F(0)-ATP synthase enzyme complex as a model. Yeast mutants defective in this complex are typically respiratory-deficient and cannot grow on non-fermentable carbon sources such as glycerol, allowing easy verification of functional complementation by yeast growth on media with them as the only carbon source. We cloned the previous published sequence of ATP2 (coding for ATP synthase ß subunit) from the Pacific white shrimp Penaeus vannamei (Pv) and also successfully amplified a novel PvATP3 (coding for the ATP synthase γ subunit). Analysis of the putative amino acid sequence of PvATP3 revealed a significant homology with the ATP synthase γ subunit of crustaceans and insects. Complementation assays were performed using full-length ATP2 and ATP3 as well as a chimeric form of ATP2 containing a leader peptide sequence from yeast and a mature sequence from shrimp. However, the shrimp genes were unable to complement the growth of respective yeast mutants on glycerol medium, even though transcriptional expression of the shrimp genes from plasmid-borne constructs in the transformed yeast cells was confirmed by RT-PCR. Interestingly, both PvATP2 and PvATP3 suppressed the lethality of the yeast F(1) mutants after the elimination of mitochondrial DNA, which suggests the assembly of a functional F(1) complex necessary for the maintenance of membrane potential in the ρ(0) state. These data suggest an incompatibility of the shrimp/yeast chimeric F(1)-ATPase with the stalk and probably also the F(0) sectors of the ATP synthase, which is essential for coupled energy transduction and ATP synthesis.

Purification, characterization and reconstitution into membranes of the oligomeric c-subunit ring of thermophilic F(o)F(1)-ATP synthase expressed in Escherichia coli.

F(o)F(1)-ATP synthase catalyzes ATP synthesis coupled with proton-translocation across the membrane. The membrane-embedded F(o) portion is responsible for the H(+) translocation coupled with rotation of the oligomeric c-subunit ring, which induces rotation of the γ subunit of F(1). For solid-state NMR measurements, F(o)F(1) of thermophilic Bacillus PS3 (TF(o)F(1)) was overexpressed in Escherichia coli and the intact c-subunit ring (TF(o)c-ring) was isolated by new procedures. One of the key improvement in this purification was the introduction of a His residue to each c-subunit that acts as a virtual His(10)-tag of the c-ring. After solubilization from membranes by sodium deoxycholate, the c-ring was purified by Ni-NTA affinity chromatography, followed by anion-exchange chromatography. The intactness of the isolated c-ring was confirmed by high-resolution clear native PAGE, sedimentation analysis, and H(+)-translocation activity. The isotope-labeled intact TF(o)c-ring was successfully purified in such an amount as enough for solid-state NMR measurements. The isolated TF(o)c-rings were reconstituted into lipid membranes. A solid-state NMR spectrum at a high quality was obtained with this membrane sample, revealing that this purification procedure was suitable for the investigation by solid-state NMR. The purification method developed here can also be used for other physicochemical investigations.

Turnover of ATP synthase subunits in F1-depleted HeLa and yeast cells.

Mitochondrial translation of the Saccharomyces cerevisiae Atp6p subunit of F(1)-F(0) ATP synthase is regulated by the F(1) ATPase. Here we show normal expression of Atp6p in HeLa cells depleted of the F(1) ß subunit. Instead of being translationally down-regulated, HeLa cells lacking F(1) degrade Atp6p, thereby preventing proton leakage across the inner membrane. Mammalian mitochondria also differ in the way they minimize the harmful effect of unassembled F(1) α subunit. While yeast mutants lacking ß subunit have stable aggregated F(1) α subunit in the mitochondrial matrix, the human α subunit is completely degraded in cells deficient in F(1) ß subunit. These results are discussed in light of the different properties of the proteins and environments in which yeast and human mitochondria exist.