Membrane-bound ATP Synthase Research Articles

Plasticity and structural alterations of mitochondria and sarcoplasmic organelles in muscles of mice deficient in α-dystrobrevin, a component of the dystrophin-glycoprotein complex.

The dystrophin-glycoprotein complex (DGC) plays a crucial role in maintaining the structural integrity of the plasma membrane and the neuromuscular junction. In this study, we investigated the impact of the deficiency of α-dystrobrevin (αdbn), a component of the DGC, on the homeostasis of intracellular organelles, specifically mitochondria and the sarcoplasmic reticulum (SR). In αdbn deficient muscles, we observed a significant increase in the membrane-bound ATP synthase complex levels, a marker for mitochondria in oxidative muscle fiber types compared to wild-type. Furthermore, examination of muscle fibers deficient in αdbn using electron microscopy revealed profound alterations in the organization of mitochondria and the SR within certain myofibrils of muscle fibers. This included the formation of hyper-branched intermyofibrillar mitochondria with extended connections, an extensive network spanning several myofibrils, and a substantial increase in the number/density of subsarcolemmal mitochondria. Concurrently, in some cases, we observed significant structural alterations in mitochondria, such as cristae loss, fragmentation, swelling, and the formation of vacuoles and inclusions within the mitochondrial matrix cristae. Muscles deficient in αdbn also displayed notable alterations in the morphology of the SR, along with the formation of distinct anomalous concentric SR structures known as whorls. These whorls were prevalent in αdbn-deficient mice but were absent in wild-type muscles. These results suggest a crucial role of the DGC αdbn in regulating intracellular organelles, particularly mitochondria and the SR, within muscle cells. The remodeling of the SR and the formation of whorls may represent a novel mechanism of the unfolded protein response (UPR) in muscle cells.

Inhibition of Escherichia coliATP synthase by dietary pomegranate phenolics

BackgroundA wide range of natural and synthetic compounds bind and inhibit ATP synthase. Dietary polyphenols found in fruits, vegetables, and spices such as tea, olives, grapes, cantaloupes, garlic, and pomegranate possess antimicrobial activities. Dietary pomegranate polyphenols (DPPs) have been shown to exhibit anticancer, antioxidant, and antimicrobial properties. In the current study we are exploring the connection between antimicrobial properties of DPPs and the inhibition of microbial ATP synthase. ATP synthase is the main source of ATP in almost all organisms from bacteria to man. Selective inhibition of bacterial ATP synthase could help in combating microbial infections.MethodsWild type E. coli pBWU13.4 is grown on minimal media to late log phase. Cells are harvested, French Pressed and centrifuged to isolate and purify membrane bound F1FO ATP synthase. Our null control is E. coli pUC118 with deleted ATPase gene. DPPs and its analogs induced inhibition of wild‐type and mutant membrane‐bound ATP synthase is performed at room temperature. Wild‐type, mutant, and null cell growth assays on limiting glucose are evaluated at OD595 using AccuSkan Plate reader over a period of 24 hours.ResultsVariable oxidative phosphorylation and ATPase activity pattern was observed between wild type phytochemical binding site mutant strains. Western blot showed purity and integrity of wild type and mutant enzymes. DPPs and its analogs caused variable inhibition of ATP synthase. The wild‐type enzyme was fully inhibited while mutant enzyme showed variable resistance in presence of DPPs. Variation in the growth pattern was in agreement with the inhibitory profiles of isolated enzymes.ConclusionsThe inhibitory profiles of isolated E. coli membrane‐bound ATP synthase shows that DPPs are potent inhibitors of ATP synthase. Inhibition of E. coli cell growth was also related to DPPs caused ATP synthase inhibition suggesting that ATP synthase is a valuable molecular drug target to combat microbial infection.

Inhibition of Escherichia coli ATP synthase by dietary ginger phenolics

For centuries, dietary ginger has been known for its antioxidant, anticancer, and antibacterial properties. In the current study, we examined the link between antibacterial properties of 7 dietary ginger phenolics (DGPs)—gingerenone A, 6-gingerol, 8-gingerol, 10-gingerol, paradol, 6-shogaol, and zingerone—and inhibition of bacterial ATP synthase. DGPs caused complete (100%) inhibition of wild-type Escherichia coli membrane-bound F1Fo ATP synthase, but partial and variable (0%–87%) inhibition of phytochemical binding site mutant enzymes αR283D, αE284R, βV265Q, and γT273A. The mutant enzyme ATPase activity was 16-fold to 100-fold lower than that of the wild-type enzyme. The growth of wild-type, null, and mutant strains in the presence of the 7 DGPs were abrogated to variable degrees on limiting glucose and succinate media. DGPs-caused variable inhibitory profiles of wild-type and mutant ATP synthase confirm that residues of α-, β-, and γ-subunits are involved in the formation of phytochemical binding site. The variable degree of growth in the presence of DGPs also indicates the possibility of molecular targets other than ATP synthase. Our results establish that antibacterial properties of DGPs can be linked to the binding and inhibition of bacterial ATP synthase. Therefore, bacterial ATP synthase is a valuable molecular target for DGPs.

Venom peptides as selective inhibitors of bacterial ATP synthase

BackgroundIt is predicted that by the year 2050 world will face 10 million additional deaths per year due to antibiotic resistance. Misuse and overuse of antibiotics, is making microbes resistant to normally effective antibiotics. Therefore, it is of necessary to find different ways to combat microbial infections. ATP synthase is the main source of cellular energy production in almost all organisms from bacteria to men. Selective inhibition of bacterial ATP synthase to kill bacteria provides an effective way to eradicate antibiotic resistant bacteria. Venom peptides from many sources bind and inhibit ATP synthase. Venom peptides bind at the βDELSEED‐motif of ATP synthase. Although βDELSEED‐motif is highly conserved throughout the evolution but is skirted by more than twenty variable amino acids. We are identifying and characterizing the selective venom peptide inhibitors of ATP synthase using Escherichia coli as a model organism.MethodWild type, null, and mutant E. coli strains are used to select the strong venom peptide inhibitors of ATP synthase. Growth of E. coli strains is measured on fermentable and non‐fermentable carbon sources in presence and absence of venom peptides. F1Fo ATP synthase is isolated by harvesting cells in the minimal media, lysing cells by French Press, and isolating membrane bound ATP synthase by multiple high‐speed ultracentrifugation. A variety of venom peptides are used to inhibit ATP synthase. Inhibitory potency of venom peptides is augmented by incremental addition of charge and α helicity.ResultsVenom peptides cause variable degree of ATP synthase inhibition. Modifications of venom peptides cause increased inhibition. Addition of a c‐terminal NH2 group in venom peptides augment the inhibitory effects by about 100‐fold. Venom peptides also show synergistic inhibitory effects on ATP synthase.ConclusionsVenom peptides potently kill E. coli cells by binding at βDELSEED‐motif of ATP synthase. ATP synthase can be used as a potent molecular drug target to combat antibiotic resistant bacterial infections.Support or Funding InformationWarner/Fermaturo and ATSU Board of Trustees Research Grant

Whole-cell biocatalysis for hydrogen storage and syngas conversion to formate using a thermophilic acetogen

BackgroundIn times of global climate change, the conversion and capturing of inorganic CO2 have gained increased attention because of its great potential as sustainable feedstock in the production of biofuels and biochemicals. CO2 is not only the substrate for the production of value-added chemicals in CO2-based bioprocesses, it can also be directly hydrated to formic acid, a so-called liquid organic hydrogen carrier (LOHC), by chemical and biological catalysts. Recently, a new group of enzymes were discovered in the two acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui which catalyze the direct hydrogenation of CO2 to formic acid with exceptional high rates, the hydrogen-dependent CO2 reductases (HDCRs). Since these enzymes are promising biocatalysts for the capturing of CO2 and the storage of molecular hydrogen in form of formic acid, we designed a whole-cell approach for T. kivui to take advantage of using whole cells from a thermophilic organism as H2/CO2 storage platform. Additionally, T. kivui cells were used as microbial cell factories for the production of formic acid from syngas.ResultsThis study demonstrates the efficient whole-cell biocatalysis for the conversion of H2 + CO2 to formic acid in the presence of bicarbonate by T. kivui. Interestingly, the addition of KHCO3 not only stimulated formate formation dramatically but it also completely abolished unwanted side product formation (acetate) under these conditions and bicarbonate was shown to inhibit the membrane-bound ATP synthase. Cell suspensions reached specific formate production rates of 234 mmol gprotein−1 h−1 (152 mmol gCDW−1 h−1), the highest rates ever reported in closed-batch conditions. The volumetric formate production rate was 270 mmol L−1 h−1 at 4 mg mL−1. Additionally, this study is the first demonstration that syngas can be converted exclusively to formate using an acetogenic bacterium and high titers up to 130 mM of formate were reached.ConclusionsThe thermophilic acetogenic bacterium T. kivui is an efficient biocatalyst which makes this organism a promising candidate for future biotechnological applications in hydrogen storage, CO2 capturing and syngas conversion to formate.

Insect venom peptides as potent inhibitors of Escherichia coli ATP synthase

Insect venom peptides (IVPs) eumenitin, lasiocepsin, lycosin1, mastoparanB, panurgine1, and protonectin possess antibacterial properties, and the ubiquitous enzyme ATP synthase has a peptide-binding site. In the present study, we studied the effect of IVPs on binding and inhibition of three Escherichia coli strains (wild type, mutant, and null) and isolated E. coli ATP synthase. IVPs and their C-terminal amide (–NH2) analogs caused variable inhibition of membrane-bound F1Fo ATP synthase. While wild type E. coli growth was substantially hampered, null E. coli growth was near normal in the presence of IVPs and their C-terminal–NH2 analogs. The presence of C-terminal–NH2 groups on IVPs resulted in increased inhibition of ATP synthase and reduced growth of E. coli strains. Insignificant inhibition of the βDELSEED-motif mutant enzyme with the βAAAAAAA-motif confirmed that IVPs interact with the βDELSEED-motif, also known as the peptide-binding site. The higher level of growth loss in E. coli strains by eumenitin, lasiocepsin, lycosin1, mastoparanB, panurgine1, and protonectin and their C-terminal–NH2 analogs suggested the likelihood of additional cellular or molecular targets. IVPs caused inhibition of E. coli strains, which demonstrates an association between antimicrobial traits of IVPs and bacterial ATP synthase.

Growth Kinetics, Carbon Isotope Fractionation, and Gene Expression in the Hyperthermophile Methanocaldococcus jannaschii during Hydrogen-Limited Growth and Interspecies Hydrogen Transfer.

Hyperthermophilic methanogens are often H2 limited in hot subseafloor environments, and their survival may be due in part to physiological adaptations to low H2 conditions and interspecies H2 transfer. The hyperthermophilic methanogen Methanocaldococcus jannaschii was grown in monoculture at high (80 to 83 μM) and low (15 to 27 μM) aqueous H2 concentrations and in coculture with the hyperthermophilic H2 producer Thermococcus paralvinellae The purpose was to measure changes in growth and CH4 production kinetics, CH4 fractionation, and gene expression in M. jannaschii with changes in H2 flux. Growth and cell-specific CH4 production rates of M. jannaschii decreased with decreasing H2 availability and decreased further in coculture. However, cell yield (cells produced per mole of CH4 produced) increased 6-fold when M. jannaschii was grown in coculture rather than monoculture. Relative to high H2 concentrations, isotopic fractionation of CO2 to CH4 (εCO2-CH4) was 16‰ larger for cultures grown at low H2 concentrations and 45‰ and 56‰ larger for M. jannaschii growth in coculture on maltose and formate, respectively. Gene expression analyses showed H2-dependent methylene-tetrahydromethanopterin (H4MPT) dehydrogenase expression decreased and coenzyme F420-dependent methylene-H4MPT dehydrogenase expression increased with decreasing H2 availability and in coculture growth. In coculture, gene expression decreased for membrane-bound ATP synthase and hydrogenase. The results suggest that H2 availability significantly affects the CH4 and biomass production and CH4 fractionation by hyperthermophilic methanogens in their native habitats.IMPORTANCE Hyperthermophilic methanogens and H2-producing heterotrophs are collocated in high-temperature subseafloor environments, such as petroleum reservoirs, mid-ocean ridge flanks, and hydrothermal vents. Abiotic flux of H2 can be very low in these environments, and there is a gap in our knowledge about the origin of CH4 in these habitats. In the hyperthermophile Methanocaldococcus jannaschii, growth yields increased as H2 flux, growth rates, and CH4 production rates decreased. The same trend was observed increasingly with interspecies H2 transfer between M. jannaschii and the hyperthermophilic H2 producer Thermococcus paralvinellae With decreasing H2 availability, isotopic fractionation of carbon during methanogenesis increased, resulting in isotopically more negative CH4 with a concomitant decrease in H2-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression and increase in F420-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression. The significance of our research is in understanding the nature of hyperthermophilic interspecies H2 transfer and identifying biogeochemical and molecular markers for assessing the physiological state of methanogens and possible source of CH4 in natural environments.

Identification of the salusin-\u03b2 receptor using proteoliposomes embedded with endogenous membrane proteins

Although orphan G protein-coupled receptors (GPCRs) have been used as targets to discover unidentified natural ligands, increasing numbers of non-GPCRs have been found to mediate important biological functions. Bioinformatics of genome and cDNA resources predict putative bioactive peptides, demanding an alternative approach to efficiently unravel cell surface targets. In silico analysis of a full-length cDNA library previously allowed us to identify salusin-β, a parasympathomimetic/pro-atherosclerotic peptide with unique physicochemical properties. Here, we show that the β-chain of ATP synthase is a cell surface receptor for salusin-β by utilizing artificial liposomes embedded with endogenous membrane proteins directly transferred from animal tissues while retaining the ligand-binding capability. Conventional techniques using detergents identified a β-actin-profilin complex as membrane-associated salusin-β-binding proteins, but failed to identify the cell surface receptor. Since the α-chain of ATP synthase is a principal cell surface target for angiostatin, a potent endogenous angiogenesis inhibitor, we investigated whether salusin-β modulates angiogenesis. Salusin-β inhibited cell surface ATP synthase activity and prevented sarcoma cell-induced angiogenesis in an in vivo mouse air sac model. Therefore, salusin-β binds to membrane-bound ATP synthase and acts as an angiogenesis inhibitor. The current methodology allows the identification of novel cell surface targets, irrespective of the receptor structure.

A connection between antimicrobial properties of venom peptides and microbial ATP synthase

Venom peptides anoplin, cupiennin 1a, latarcin 1, latarcin 3a, latarcin 5, melittin, and pandinin 2 are known to have antibacterial properties. In the current study, we examined whether the antimicrobial properties of these venom peptides have any connection to the binding and inhibition of bacterial ATP synthase. Venom peptides inhibited Escherichia coli wild type and mutant membrane-bound F1Fo ATP synthase to varying degrees. Although significant loss of oxidative phosphorylation was observed for wild type, very little loss occurred for null and mutant E. coli strains in the presence of venom peptides. This study also reaffirms that βDELSEED-motif residues of ATP synthase are required for peptide binding. Modified venom peptides with C-terminal amide (NH2) groups caused augmented inhibition of ATP synthase and E. coli cell death. Growth patterns of wild type, null, and mutant strains in the presence of melittin, anoplin, cupiennin 1a, latarcin 1, latarcin 3a, latarcin 5, pandinin 2, and their modified variants suggested the possibility of additional molecular targets. Our results demonstrate that the antimicrobial properties of venom peptides are connected to the binding and inhibition of bacterial ATP synthase. Moreover, selective inhibition of ATP synthase by venom peptides suggests a viable alternative to combat antibiotic-resistant microbial infections.

Understanding the link between antimicrobial properties of dietary olive phenolics and bacterial ATP synthase

The naturally occurring olive phenolics tyrosol, hydroxytyrosol, dihydroxyphenylglycol (DHPG), and oleuropein are known to have antioxidant, antitumor, and antibacterial properties. In the current study, we examined whether the antimicrobial properties of tyrosol, hydroxytyrosol, DHPG, and oleuropein were linked to the inhibition of bacterial ATP synthase. Tyrosol, hydroxytyrosol, DHPG, and oleuropein inhibited Escherichia coli wild-type and mutant membrane-bound F1Fo ATP synthase to variable degrees. The growth properties of wild-type, null, and mutant strains in presence of above olive phenolics were also abrogated to variable degrees on limiting glucose and succinate. Tyrosol and oleuropein synergistically inhibited the wild-type enzyme. Comparative wild-type and mutant F1Fo ATP synthase inhibitory profiles suggested that αArg-283 is an important residue and olive phenolics bind at the polyphenol binding pocket of ATP synthase. Growth patterns of wild-type, null, and mutant strains in the presence of tyrosol, hydroxytyrosol, DHPG, and oleuropein also hint at the possibility of additional molecular targets. Our results demonstrated that ATP synthase can be used as a molecular target and the antimicrobial properties of olive phenolics in general and tyrosol in particular can be linked to the binding and inhibition of bacterial ATP synthase.

Patterns of protein expression in water-stressed wheat chloroplasts

The performance of control and water-stressed 10-d-old wheat seedlings was compared. During short-term water stress (irrigation was withheld for 9 d), rates of photosynthesis and transpiration, stomatal conductance, and relative water content decreased whereas the proline content increased. Chloroplast proteins were extracted from the leaves, separated by iso-electric focusing through two-dimensional electrophoresis, and stained with CBB R-250. Differentially expressed proteins were detected and analyzed with MALDI-TOF/TOF mass spectrometry. Under water stress, 9 proteins were up-regulated whereas 11 proteins were not affected. The ribulose-1,5-bisphospate carboxylase/oxygenase (Rubisco) small and large subunits, chloride carrier/channel family, and H+-ATPase were up-regulated by water stress whereas membrane-bound ATP synthase subunit b and cytochrome b6-f complex were down-regulated.

Targeting bacterial membrane function: an underexploited mechanism for treating persistent infections

Persistent infections involving slow-growing or non-growing bacteria are hard to treat with antibiotics that target biosynthetic processes in growing cells. Consequently, there is a need for antimicrobials that can treat infections containing dormant bacteria. In this Review, we discuss the emerging concept that disrupting the bacterial membrane bilayer or proteins that are integral to membrane function (including membrane potential and energy metabolism) in dormant bacteria is a strategy for treating persistent infections. The clinical applicability of these approaches is exemplified by the efficacy of lipoglycopeptides that damage bacterial membranes and of the diarylquinoline TMC207, which inhibits membrane-bound ATP synthase. Despite some drawbacks, membrane-active agents form an important new means of eradicating recalcitrant, non-growing bacteria.

Identification of key proteins involved in the anammox reaction

Bacteria performing anaerobic ammonium oxidation (anammox) are key players in the global nitrogen cycle due to their inherent ability to convert biologically available nitrogen to N(2). Anammox is increasingly being exploited during wastewater treatment worldwide, and about 50% of the total N(2) production in marine environments is estimated to proceed by the anammox pathway. To fully understand the microbial functionality and mechanisms that control environmental feedbacks of the anammox reaction, key proteins involved in the reaction must be identified. In this study we have utilized an analytical protocol that facilitates detection of proteins associated with the anammoxosome, an intracellular membrane compartment within the anammox bacterium. The protocol enabled us to identify several key proteins of the anammox reaction including a hydrazine hydrolase producing hydrazine, a hydrazine-oxidizing enzyme converting hydrazine to N(2) and a membrane-bound ATP synthase generating ATP from the gradients of protons formed in the anammox reaction. We also performed immunogold labelling electron microscopy to determine the subcellular location of the hydrazine hydrolase. The results from our study support the hypothesis that proteins associated with the anammoxosome host the complete suite of reactions during anammox.

Coupling factor 6 as a novel vasoactive and proatherogenic peptide in vascular endothelial cells

Coupling factor 6 (CF6) is composed of 76 amino acids and is present in the peripheral stalk of mitochondrial ATP synthase. The generation of CF6 is positively regulated by tumor necrosis factor alpha and shear stress via nuclear factor kappaB, and by high glucose via protein kinase C and p38 mitogen-activated protein kinase. CF6 is released outside of the cells from vascular endothelial cells, and binds to the beta-subunit of the plasma membrane-bound ATP synthase in vascular endothelial cells and leads to intracellular acidosis. CF6 produces vasoconstriction, and the biological active site resides at the C-terminal portion. CF6 suppresses prostacyclin generation via inhibition of cytosolic phospholipase A(2). CF6 also suppresses nitric oxide synthase activity via an increase in asymmetric dimethylarginine and a decrease in platelet/endothelial cell adhesion molecule-1. CF6 induces the gene and protein expression of proatherogenic molecules such as endothelin 2, urokinase type plasminogen activator receptor, estrogen receptor beta, a soluble short form of vascular endothelial growth factor receptor-1, and lectin-like oxidized low-density lipoprotein receptor-1. The plasma level of CF6 is elevated in patients with essential hypertension, diabetes mellitus, end-stage renal disease, acute myocardial infarction, and coronary heart disease. It is likely that CF6 contributes to the pathogenesis of cardiovascular diseases, but further intensive investigation is needed.

F0F1 ATP synthase of Streptomycetes: Modulation of activity and oligomycin resistance by protein Ser/Thr kinases

Inverted membrane vesicles of Gram-positive actinobacteria Streptomyces fradiae, S. lividans, and S. avermitilis have been prepared and membrane-bound F0F1 ATP synthase has been biochemically characterized. It has been shown that the ATPase activity of membrane-bound F0F1 complex is Mg2+-dependent and moderately stimulated by high concentrations of Ca2+ ions (10–20 mM). The ATPase activity is inhibited by N,N′-dicyclohexylcarbodiimide and oligomycin A, typical F0F1 ATPase inhibitors that react with the membrane-bound F0 complex. The assay of biochemical properties of the F0F1 ATPases of Streptomycetes in all cases showed the presence of ATPase populations highly susceptible and insensitive to oligomycin A. The in vitro labeling and inhibitory assay showed that the inverted phospholipid vesicles of S. fradiae contained active membrane-bound Ser/Thr protein kinase(s) phosphorylating the proteins of the F0F1 complex. Inhibition of phosphorylation leads to decrease of the ATPase activity and increase of its susceptibility to oligomycin. The in vivo assay confirmed the enhancement of actinobacteria cell sensitivity to oligomycin after inhibition of endogenous phosphorylation. The sequencing of the S. fradiae genes encoding oligomycin-binding A and C subunits of F0F1 ATP synthase revealed their close phylogenetic relation to the genes of S. lividans and S. avermitilis.

Tethering polypeptides through bifunctional PEG cross‐linking agents to probe protein function: Application to ATP synthase

Chemical crosslinking mediated by short bifunctional reagents has been widely used for determining physical relationships among polypeptides in multisubunit proteins, but less often for functional studies. Here we introduce the approach of tethering polypeptides by using bifunctional reagents containing a lengthy, flexible PEG linker as a form of crosslinking especially suited to functional analyses. The rotary molecular motor ATP synthase was used as a model subject. Single cysteine residues were introduced into selected positions of ATP synthase epsilon subunit, a component of the rotor subcomplex of the enzyme, and the unrelated maltose binding protein (MBP), then the two purified recombinant proteins were crosslinked by means of a dimaleimido-PEG cross-linking agent. Following purification, the epsilon-PEG-MBP was incorporated into membrane-bound ATP synthase by reconstitution with epsilon-depleted F(1)-ATPase and membrane vesicles that had been stripped of endogenous F(1). ATP synthase reconstituted using epsilon-PEG-MBP had reduced ATP hydrolytic activity that was uncoupled from the pumping of H(+), indicating the physical blockage of rotation of the gammaepsilonc(10) rotor by the conjugated MBP, whereas enzyme reconstituted with epsilon-PEG was normal. These results directly demonstrate the feasibility of studying mechanistic features of molecular motors through PEG-based conjugation of unrelated proteins. Since tethering polypeptides provides a means of maintaining proximity without directly specifying or modifying interactions, application of the general method to other types of protein functional studies is envisioned.

Chemical modification of mono-cysteine mutants allows a more global look at conformations of the ε subunit of the ATP synthase from Escherichia coli

The epsilon subunit of the ATP synthase from E. coli undergoes conformational changes while rotating through 360 degrees during catalysis. The conformation of epsilon was probed in the membrane-bound ATP synthase by reaction of mono-cysteine mutants with 3-N-maleimidyl-propionyl biocytin (MPB) under resting conditions, during ATP hydrolysis, and after inhibition by ADP-AlF(3). The relative extents of labeling were quantified after electrophoresis and blotting of the partially purified epsilon subunit. Residues from the N-terminal beta-sandwich domain showed a position-specific pattern of labeling, consistent with prior structural studies. Some residues near the epsilon-gamma interface showed changes up to two-fold if labeling occurred during ATP hydrolysis or after inhibition by ADP-AlF(3). In contrast, residues found in the C-terminal alpha-helices were all labeled to a moderate or high level with a pattern that was consistent with a partially opened helical hairpin. The results indicate that the two C-terminal alpha-helices do not adopt a fixed conformation under resting conditions, but rather exhibit intrinsic flexibility.

Protein Complexes in the Archaeon Methanothermobacter thermautotrophicus Analyzed by Blue Native/SDS-PAGE and Mass Spectrometry

Methanothermobacter thermautotrophicus is a thermophilic archaeon that produces methane as the end product of its primary metabolism. The biochemistry of methane formation has been extensively studied and is catalyzed by individual enzymes and proteins that are organized in protein complexes. Although much is known of the protein complexes involved in methanogenesis, only limited information is available on the associations of proteins involved in other cell processes of M. thermautotrophicus. To visualize and identify interacting and individual proteins of M. thermautotrophicus on a proteome-wide scale, protein preparations were separated using blue native electrophoresis followed by SDS-PAGE. A total of 361 proteins, corresponding to almost 20% of the predicted proteome, was identified using peptide mass fingerprinting after MALDI-TOF MS. All previously characterized complexes involved in energy generation could be visualized. Furthermore the expression and association of the heterodisulfide reductase and methylviologen-reducing hydrogenase complexes depended on culture conditions. Also homomeric supercomplexes of the ATP synthase stalk subcomplex and the N5-methyl-5,6,7,8-tetrahydromethanopterin:coenzyme M methyltransferase complex were separated. Chemical cross-linking experiments confirmed that the multimerization of both complexes was not experimentally induced. A considerable number of previously uncharacterized protein complexes were reproducibly visualized. These included an exosome-like complex consisting of four exosome core subunits, which associated with a tRNA-intron endonuclease, thereby expanding the constituency of archaeal exosomes. The results presented show the presence of novel complexes and demonstrate the added value of including blue native gel electrophoresis followed by SDS-PAGE in discovering protein complexes that are involved in catabolic, anabolic, and general cell processes.

Ectopic beta-chain of ATP synthase is an apolipoprotein A-I receptor in hepatic HDL endocytosis.

The effect of high-density lipoprotein (HDL) in protecting against atherosclerosis is usually attributed to its role in 'reverse cholesterol transport'. In this process, HDL particles mediate the efflux and the transport of cholesterol from peripheral cells to the liver for further metabolism and bile excretion. Thus, cell-surface receptors for HDL on hepatocytes are chief partners in the regulation of cholesterol homeostasis. A high-affinity HDL receptor for apolipoprotein A-I (apoA-I) was previously identified on the surface of hepatocytes. Here we show that this receptor is identical to the beta-chain of ATP synthase, a principal protein complex of the mitochondrial inner membrane. Different experimental approaches confirm this ectopic localization of components of the ATP synthase complex and the presence of ATP hydrolase activity at the hepatocyte cell surface. Receptor stimulation by apoA-I triggers the endocytosis of holo-HDL particles (protein plus lipid) by a mechanism that depends strictly on the generation of ADP. We confirm this effect on endocytosis in perfused rat liver ex vivo by using a specific inhibitor of ATP synthase. Thus, membrane-bound ATP synthase has a previously unsuspected role in modulating the concentrations of extracellular ADP and is regulated by a principal plasma apolipoprotein.

Surprises and revelations in biochemistry: 1950–2000

Surprises and revelations in biochemistry: 1950–2000