A systematic assessment of mitochondrial function identified novel signatures for drug-induced mitochondrial disruption in cells.

Mitochondrial perturbation has been recognized as a contributing factor to various drug-induced organ toxicities. To address this issue, we developed a high-throughput flow cytometry-based mitochondrial signaling assay to systematically investigate mitochondrial/cellular parameters known to be directly impacted by mitochondrial dysfunction: mitochondrial membrane potential (MMP), mitochondrial reactive oxygen species (ROS), intracellular reduced glutathione (GSH) level, and cell viability. Modulation of these parameters by a training set of compounds, comprised of established mitochondrial poisons and 60 marketed drugs (30 nM to 1mM), was tested in HL-60 cells (a human pro-myelocytic leukemia cell line) cultured in either glucose-supplemented (GSM) or glucose-free (containing galactose/glutamine; GFM) RPMI-1640 media. Post-hoc bio-informatic analyses of IC50 or EC50 values for all parameters tested revealed that MMP depolarization in HL-60 cells cultured in GSM was the most reliable parameter for determining mitochondrial dysfunction in these cells. Disruptors of mitochondrial function depolarized MMP at concentrations lower than those that caused loss of cell viability, especially in cells cultured in GSM; cellular GSH levels correlated more closely to loss of viability in vitro. Some mitochondrial respiratory chain inhibitors increased mitochondrial ROS generation; however, measuring an increase in ROS alone was not sufficient to identify mitochondrial disruptors. Furthermore, hierarchical cluster analysis of all measured parameters provided confirmation that MMP depletion, without loss of cell viability, was the key signature for identifying mitochondrial disruptors. Subsequent classification of compounds based on ratios of IC50s of cell viability:MMP determined that this parameter is the most critical indicator of mitochondrial health in cells and provides a powerful tool to predict whether novel small molecule entities possess this liability.

Uniform nomenclature for the mitochondrial contact site and cristae organizing system.

The mitochondrial inner membrane contains a large protein complex that functions in inner membrane organization and formation of membrane contact sites. The complex was variably named the mitochondrial contact site complex, mitochondrial inner membrane organizing system, mitochondrial organizing structure, or Mitofilin/Fcj1 complex. To facilitate future studies, we propose to unify the nomenclature and term the complex "mitochondrial contact site and cristae organizing system" and its subunits Mic10 to Mic60.

ALS-linked mutant superoxide dismutase 1 (SOD1) alters mitochondrial protein composition and decreases protein import.

Mutations in superoxide dismutase 1 (SOD1) cause familial ALS. Mutant SOD1 preferentially associates with the cytoplasmic face of mitochondria from spinal cords of rats and mice expressing SOD1 mutations. Two-dimensional gels and multidimensional liquid chromatography, in combination with tandem mass spectrometry, revealed 33 proteins that were increased and 21 proteins that were decreased in SOD1(G93A) rat spinal cord mitochondria compared with SOD1(WT) spinal cord mitochondria. Analysis of this group of proteins revealed a higher-than-expected proportion involved in complex I and protein import pathways. Direct import assays revealed a 30% decrease in protein import only in spinal cord mitochondria, despite an increase in the mitochondrial import components TOM20, TOM22, and TOM40. Recombinant SOD1(G93A) or SOD1(G85R), but not SOD1(WT) or a Parkinson's disease-causing, misfolded α-synuclein(E46K) mutant, decreased protein import by >50% in nontransgenic mitochondria from spinal cord, but not from liver. Thus, altered mitochondrial protein content accompanied by selective decreases in protein import into spinal cord mitochondria comprises part of the mitochondrial damage arising from mutant SOD1.

Novel antibody-based strategies for the rapid diagnosis of mitochondrial disease and dysfunction.

We are developing rapid immunoassays to measure the protein levels, enzymatic activities and post-translational modifications of mitochondrial proteins. These assays can be arrayed in multi-analyte panels for biomarker discovery and they can also be used individually at point of care where the level or activity of a small number proteins or even a single protein is highly informative. For example, we have characterized OXPHOS deficits associated with lipoatrophy, an adverse metabolic side-effect of anti-retroviral therapy, and have shown that OXPHOS deficits observed in vitro are also exhibited not only in clinically affected tissue (peripheral fat) but also in more easily accessible tissue (peripheral blood mononucleated cells). Similarly, we have shown that a small set of assays can be used to identify almost all patients with genetic deficits in OXPHOS complexes I or IV, the most common cause of inherited mitochondrial disease. Finally, we recently reported that Friedreich's Ataxia (FA) patients and carriers can be identified on the basis of a simple dipstick test to measure levels of a single protein, frataxin, an iron regulatory protein whose disrupted expression is the proximal cause of neurodegeneration in FA. Because each of these tests can be performed in an extremely simple, rapid dipstick format using non-invasive samples such as cheek swabs and fingerprick blood, they have potential for use as point of care diagnostics for mitochondrial disease and as front-line screening tools to help guide drug therapies and minimize adverse off-target drug effects.

Lateral-flow immunoassay for detecting drug-induced inhibition of mitochondrial DNA replication and mtDNA-encoded protein synthesis.

Drug-induced mitochondrial toxicity can occur as a result of inhibition of mitochondrial DNA (mtDNA) replication as with certain nucleoside reverse transcriptase inhibitors or inhibition of mtDNA-encoded protein synthesis as with certain antibacterials. Both types of dysfunction have the overall effect of reducing the level of proteins encoded by mtDNA. A lateral-flow immunoassay which measures the levels of both a mtDNA-encoded protein and a nuclear DNA-encoded protein allows simple and rapid determination of the ratio of these 2 proteins and, hence, identifies changes in mtDNA-encoded protein levels. Here, we describe an assay that compares the level of Complex IV (cytochrome c oxidase), a mitochondrial protein which has 3 subunits encoded by mtDNA and made by mitochondrial ribosomes, with that of frataxin, a protein encoded by nuclear DNA and made by cytosolic ribosomes. We tested a selection of antibacterials and antiretrovirals in cells and show that the ratio of Complex IV: frataxin decreases when a drug inhibits either mtDNA replication or mtDNA-encoded protein synthesis. The results obtained with the assay were confirmed by Western blotting and immunocytochemical analysis. The assay has high reproducibility, requires small amounts of sample, is quantitative, and is able to identify drugs which ultimately lead to a decrease in mtDNA-encoded proteins.

Screening for the metabolic basis of neurodegeneration: developing a focused proteomic approach.

Metabolism is controlled by a complex system of transcriptional events and posttranslational modifications stimulated by substrate and metabolite availability. It is becoming clear that neurodegenerative diseases are a symptom of a deficiency in the regulation or execution of metabolic reactions. Mitochondria, as the central organelles in metabolic regulation as well as the chief generators of reactive species, clearly have a role to play in the etiology of neurodegenerative conditions. We are developing antibody-based capture arrays to determine multiple parameters of key mitochondrial proteins. Parameters include enzyme activity, quantity, oxidative modification (including nitrative and oxidative stress), and regulation (phosphorylation and acetylation). At this time the core of this array focuses on the enzymes of oxidative phosphorylation. We continue to expand this array as antibodies for enzyme isolation and modification detection become available. Here we demonstrate the use of this array by analyzing the proteomic differences in oxidative phosphorylation enzymes between human heart and liver tissues, cells grown in media promoting aerobic versus anaerobic metabolism, and the catalytic/proteomic effects of mitochondria exposed to oxidative stress.

Mitochondrial oxidative phosphorylation protein levels in peripheral blood mononuclear cells correlate with levels in subcutaneous adipose tissue within samples differing by HIV and lipoatrophy status.

Depletion of mitochondrial DNA (mtDNA) and mtDNA-encoded respiratory chain proteins in subcutaneous (SC) fat from patients with HIV lipoatrophy have clearly demonstrated the role of mitochondrial dysfunction in this syndrome. Research in HIV lipoatrophy, however, has been severely hampered by the lack of a suitable surrogate marker in blood or other easily obtained clinical specimens as fat biopsies are invasive and mtDNA levels in peripheral blood mononuclear cells (PBMC) do not consistently correlate with the disease process. We used a simple, rapid, quantitative 2-site dipstick immunoassay to measure OXPHOS enzymes Complex I (CI) and Complex IV (CIV), and rtPCR to measure mtDNA in 26 matched SC fat and PBMC specimens previously banked from individuals on potent antiretroviral (ARV) therapy with HIV lipoatrophy, on similar ARV therapy without lipoatrophy, and in HIV seronegative controls. Significant correlations were found between the respective PBMC and fat levels for both CI (r = 0.442, p = 0.024) and for CIV (r = 0.507, p = 0.008). Both CI and CIV protein levels were also significantly reduced in both PBMCs and fat in lipoatrophic subjects compared to HIV seronegative controls (p < or = 0.05), while a comparative reduction in mtDNA levels in lipoatrophic subjects was observed only in fat. We conclude that CI and CIV levels in PBMCs correlate to their respective levels in fat and may have utility as surrogate markers of mitochondrial dysfunction in lipoatrophy.

Monitoring oxidative and nitrative modification of cellular proteins; a paradigm for identifying key disease related markers of oxidative stress.

High levels of free radicals produced by the mitochondrial respiratory chain, with subsequent damage to mitochondria have been implicated in a large and growing number of diseases. The underlying pathology of these diseases is oxidative damage to mitochondrial DNA, lipids and proteins which accumulate over time to produce a metabolic deficiency. We are developing an antibody based immunocapture array for many important mitochondrial proteins involved in free radical production, detoxification and mitochondrial energy production. Our array is capable of a multi-parameter measurement including enzyme activity, quantity, and oxidative protein modifications. Here we demonstrate the use of this array by analyzing the proteomic differences in OXPHOS (oxidative phosphorylation) enzymes between human heart and liver tissues, cells grown in media promoting aerobic versus anaerobic metabolism, and the catalytic/proteomic effects of mitochondria exposed to oxidative stress. Protein oxidation is identified as carbonyl formation arising from reactive oxygen species and 3-nitrotyrosine as a marker of reactive nitrogen species. Several identified modifications are confirmed by electrophoresis and mass spectrometry of immunocaptured material. We continue to expand this array as antibodies for enzyme isolation and detection become available.

Lateral-flow immunoassay for the frataxin protein in Friedreich's ataxia patients and carriers.

Friedreich's Ataxia (FA) is an inherited neurodegenerative disease caused by reduction in levels of the mitochondrial protein frataxin. Currently there are no simple, reliable methods to accurately measure the concentrations of frataxin protein. We designed a lateral-flow immunoassay that quantifies frataxin protein levels in a variety of sample materials. Using recombinant frataxin we evaluated the accuracy and reproducibility of the assay. The assay measured recombinant human frataxin concentrations between 40 and 4000 pg/test or approximately 0.1-10 nM of sample. The intra and inter-assay error was <10% throughout the working range. To evaluate clinical utility of the assay we used genetically defined lymphoblastoid cells derived from FA patients, FA carriers and controls. Mean frataxin concentrations in FA patients and carriers were significantly different from controls and from one another (p=0.0001, p=0.003, p=0.005, respectively) with levels, on average, 29% (patients) and 64% (carriers) of the control group. As predicted, we observed an inverse relationship between GAA repeat number and frataxin protein concentrations within the FA patient cohort. The lateral flow immunoassay provides a simple, accurate and reproducible method to quantify frataxin protein in whole cell and tissue extracts, including primary samples obtained by non-invasive means, such as cheek swabs and whole blood. The assay is a novel tool for FA research that may facilitate improved diagnostic and prognostic evaluation of FA patients and could also be used to evaluate efficacy of therapies designed to cure FA by increasing frataxin protein levels.

Early developmental pathology due to cytochrome c oxidase deficiency is revealed by a new zebrafish model.

Deficiency of cytochrome c oxidase (COX) is associated with significant pathology in humans. However, the consequences for organogenesis and early development are not well understood. We have investigated these issues using a zebrafish model. COX deficiency was induced using morpholinos to reduce expression of CoxVa, a structural subunit, and Surf1, an assembly factor, both of which impaired COX assembly. Reduction of COX activity to 50% resulted in developmental defects in endodermal tissue, cardiac function, and swimming behavior. Cellular investigations revealed different underlying mechanisms. Apoptosis was dramatically increased in the hindbrain and neural tube, and secondary motor neurons were absent or abnormal, explaining the motility defect. In contrast, the heart lacked apoptotic cells but showed increasingly poor performance over time, consistent with energy deficiency. The zebrafish model has revealed tissue-specific responses to COX deficiency and holds promise for discovery of new therapies to treat mitochondrial diseases in humans.

The mitochondrial inner membrane protein mitofilin exists as a complex with SAM50, metaxins 1 and 2, coiled-coil-helix coiled-coil-helix domain-containing protein 3 and 6 and DnaJC11.

A monoclonal antibody (mAb) has been produced which reacts with human mitofilin, a mitochondrial inner membrane protein. This mAb immunocaptures its target protein in association with six other proteins, metaxins 1 and 2, SAM50, CHCHD3, CHCHD6 and DnaJC11, respectively. The first three are outer membrane proteins, CHCHD3 has been assigned to the matrix space, and the other two proteins have not been described in mitochondria previously. The functional role of this new complex is uncertain. However, a role in protein import related to maintenance of mitochondrial structure is suggested as mitofilin helps regulate mitochondrial morphology and at least four of the associated proteins (metaxins 1 and 2, SAM50 and CHCHD3) have been implicated in protein import, while DnaJC11 is a chaperone-like protein that may have a similar role.

Mitochondrial impairment by PPAR agonists and statins identified via immunocaptured OXPHOS complex activities and respiration.

Mitochondrial impairment is increasingly implicated in the etiology of toxicity caused by some thiazolidinediones, fibrates, and statins. We examined the effects of members of these drug classes on respiration of isolated rat liver mitochondria using a phosphorescent oxygen sensitive probe and on the activity of individual oxidative phosphorylation (OXPHOS) complexes using a recently developed immunocapture technique. Of the six thiazolidinediones examined, ciglitazone, troglitazone, and darglitazone potently disrupted mitochondrial respiration. In accord with these data, ciglitazone and troglitazone were also potent inhibitors of Complexes II+III, IV, and V, while darglitazone predominantly inhibited Complex IV. Of the six statins evaluated, lovastatin, simvastatin, and cerivastatin impaired mitochondrial respiration the most, with simvastatin and lovastatin impairing multiple OXPHOS Complexes. Within the class of fibrates, gemfibrozil more potently impaired respiration than fenofibrate, clofibrate, or ciprofibrate. Gemfibrozil only modestly inhibited Complex I, fenofibrate inhibited Complexes I, II+III, and V, and clofibrate inhibited Complex V. Our findings with the two complementary methods indicate that (1) some members of each class impair mitochondrial respiration, whereas others have little or no effect, and (2) the rank order of mitochondrial impairment accords with clinical adverse events observed with these drugs. Since the statins are frequently co-prescribed with the fibrates or thiazolidinediones, various combinations of these three drug classes were also analyzed for their mitochondrial effects. In several cases, the combination additively uncoupled or inhibited respiration, suggesting that some combinations are more likely to yield clinically relevant drug-induced mitochondrial side effects than others.

Angiostatin-like activity of a monoclonal antibody to the catalytic subunit of F1F0 ATP synthase.

The antiangiogenic protein angiostatin inhibits ATP synthase on the endothelial cell surface, blocking cellular proliferation. To examine the specificity of this interaction, we generated monoclonal antibodies (mAb) directed against ATP synthase. mAb directed against the beta-catalytic subunit of ATP synthase (MAb3D5AB1) inhibits the activity of the F(1) domain of ATP synthase and recognizes the catalytic beta-subunit of ATP synthase. We located the antibody recognition site of MAb3D5AB1 in domains containing the active site of the beta-subunit. MAb3D5AB1 also binds to purified Escherichia coli F(1) with an affinity 25-fold higher than the affinity of angiostatin for this protein. MAb3D5AB1 inhibits the hydrolytic activity of F(1) ATP synthase at lower concentrations than angiostatin. Like angiostatin, MAb3D5AB1 inhibits ATP generation by ATP synthase on the endothelial cell surface in acidic conditions, the typical tumor microenvironment where cell surface ATP synthase exhibits greater activity. MAb3D5AB1 disrupts tube formation and decreases intracellular pH in endothelial cells exposed to low extracellular pH. Neither angiostatin nor MAb3D5AB1 showed an antiangiogenic effect in the corneal neovascularization assay; however, both were effective in the low-pH environment of the chicken chorioallantoic membrane assay. Thus, MAb3D5AB1 shows angiostatin-like properties superior to angiostatin and may be exploited in cancer chemotherapy.

Small-scale immunopurification of cytochrome c oxidase for a high-throughput multiplexing analysis of enzyme activity and amount.

COX (cytochrome c oxidase) deficiency is one of the main causes of genetic mitochondrial disease and presents with multiple phenotypes, depending on whether the causative mutation exists in a mitochondrial or nuclear gene and on whether it involves an altered catalytic or structural component or an assembly factor for this membrane-embedded 13-subunit enzyme complex. COX deficiency is routinely observed in AD (Alzheimer's disease), although there is continuing debate about whether this is a causative or a secondary consequence of the condition. Altered levels of COX and reduced oxidative phosphorylation capacity have been reported in other common diseases, including cancer, and are seen as unwanted side effects in a number of drug treatments, particularly with antiretroviral and antibiotic treatments. Here, we introduce a simple, rapid, high-throughput 96-well plate protocol that uses a multiplex approach to determine the amount and activity of COX, which should find widespread use in evaluating the above diseases and in drug safety studies. Importantly, the method uses very small amounts of cell material or tissue and does not require the isolation of mitochondria. We show the utility of this approach by example of the analysis of fibroblasts from patients with COX activity deficiency and the effect of the antiretroviral drug ddC (2',3'-dideoxycytidine) on the biogenesis of the enzyme.

The F(1)F(0) ATP synthase and mitochondrial respiratory chain complexes are present on the plasma membrane of an osteosarcoma cell line: An immunocytochemical study.

F(1)F(0) ATP synthase is ectopically expressed on the surface of several cell types, including endothelium and cancer cells. This study uses immunocytochemical detection methods via highly specific monoclonal antibodies to explore the possibility of plasma membrane localization of other mitochondrial proteins using an osteosarcoma cell line in which the location of the mitochondrial reticulum can be clearly traced by green fluorescent protein tagging of the organelle. We found that subunits of three of the four respiratory chain complexes were present on the surface of these cells. Additionally, we show for the first time that F(0) subunits d and OSCP of the ATP synthase are ectopically expressed. In all cases the OXPHOS proteins show a punctate distribution, consistent with data from proteome analysis of isolated lipid rafts that place the various mitochondrial proteins in plasma membrane microdomains. We also examined the cell surface for marker membrane proteins from several other intracellular organelles including ER, golgi and nuclear envelope. They were not found on the surface of the osteosarcoma cells. We conclude that mitochondrial membrane proteins are ectopically expressed, but not proteins from other cellular organelles. A specific mechanism by which the mitochondrion and plasma membrane fuse to deliver organellar proteins is suggested.

Parkinson's disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled.

Loss of mitochondrial complex I catalytic activity in the electron transport chain (ETC) is found in multiple tissues from individuals with sporadic Parkinson's disease (PD) and is a property of some PD model neurotoxins. Using special ETC subunit-specific and complex I immunocapture antibodies directed against the entire complex I macroassembly, we quantified ETC proteins and protein oxidation of complex I subunits in brain mitochondria from 10 PD and 12 age-matched control (CTL) samples. We measured nicotinamide adenine dinucleotide (NADH)-driven electron transfer rates through complex I and correlated these with complex I subunit oxidation levels and reductions of its 8 kDa subunit. PD brain complex I shows 11% increase in ND6, 34% decrease in its 8 kDa subunit and contains 47% more protein carbonyls localized to catalytic subunits coded for by mitochondrial and nuclear genomes We found no changes in levels of ETC proteins from complexes II-V. Oxidative damage patterns to PD complex I are reproduced by incubation of CTL brain mitochondria with NADH in the presence of rotenone but not by exogenous oxidant. NADH-driven electron transfer rates through complex I inversely correlate with complex I protein oxidation status and positively correlate with reduction in PD 8 kDa subunit. Reduced complex I function in PD brain mitochondria appears to arise from oxidation of its catalytic subunits from internal processes, not from external oxidative stress, and correlates with complex I misassembly. This complex I auto-oxidation may derive from abnormalities in mitochondrial or nuclear encoded subunits, complex I assembly factors, rotenone-like complex I toxins, or some combination.

Proteomic analysis of succinate dehydrogenase and ubiquinol-cytochrome c reductase (Complex II and III) isolated by immunoprecipitation from bovine and mouse heart mitochondria.

The oxidative phosphorylation system (OXPHOS) consists of five multi-enzyme complexes, Complexes I-V, and is a key component of mitochondrial function relating to energy production, oxidative stress, cell signaling and apoptosis. Defects or a reduction in activity in various components that make up the OXPHOS enzymes can cause serious diseases, including neurodegenerative disease and various metabolic disorders. Our goal is to develop techniques that are capable of rapid and in-depth analysis of all five OXPHOS complexes. Here, we describe a mild, micro-scale immunoisolation and mass spectrometric/proteomic method for the characterization of Complex II (succinate dehydrogenase) and Complex III (ubiquinol-cytochrome c reductase) from bovine and rodent heart mitochondria. Extensive protein sequence coverage was obtained after immunocapture, 1D SDS PAGE separation and mass spectrometric analysis for a majority of the 4 and 11 subunits, respectively, that make up Complexes II and III. The identification of several posttranslational modifications, including the covalent FAD modification of flavoprotein subunit 1 from Complex II, was possible due to high mass spectrometric sequence coverage.

Mass spectrometric identification of a novel phosphorylation site in subunit NDUFA10 of bovine mitochondrial complex I.

Mitochondrial Complex I (NADH:ubiquinone oxidoreductase) consists of at least 46 subunits. Phosphorylation of the 42-kDa subunit NDUFA10 was recently reported using a novel phosphoprotein stain [Schulenberg et al. (2003) Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye. J. Biol. Chem. 278, 27251]. Two smaller Complex I phosphoproteins, ESSS and MWFE, and their sites of modification, have since been determined [Chen et al. (2004) The phosphorylation of subunits of complex I from bovine heart mitochondria. J. Biol. Chem. 279, 26036]. Here we identify the site of phosphorylation in NDUFA10 from bovine heart mitochondria by tandem mass spectrometry. A single phosphopeptide spanning residues 47-60 was identified and confirmed by synthesis to be (47)LITVDGNICSGKpSK(60), establishing serine-59 as the site of phosphorylation.

Rapid purification and mass spectrometric characterization of mitochondrial NADH dehydrogenase (Complex I) from rodent brain and a dopaminergic neuronal cell line.

Oxidative stress and mitochondrial dysfunction signify important biochemical events associated with the loss of dopaminergic neurons in Parkinson's disease (PD). Studies using in vitro and in vivo PD models or tissues from diseased patients have demonstrated a selective inhibition of mitochondrial NADH dehydrogenase (Complex I of the OXPHOS electron transport chain) that affects normal mitochondrial physiology leading to neuronal death. In an earlier study, we demonstrated that oxidative stress due to glutathione depletion in dopaminergic cells, a hallmark of PD, leads to Complex I inhibition via cysteine thiol oxidation (Jha et al. (2000) J. Biol. Chem. 275, 26096-26101). Complex I is a approximately 980-kDa multimeric enzyme spanning the inner mitochondrial membrane comprising at least 45 protein subunits. As a prerequisite to investigating modifications to Complex I using a rodent disease model for PD, we developed two independent rapid and mild isolation procedures based on sucrose gradient fractionation and immunoprecipitation to isolate Complex I from mouse brain and a cultured rat mesencephalic dopaminergic neuronal cell line. Both protocols are capable of purifying Complex I from small amounts of rodent tissue and cell cultures. Blue Native gel electrophoresis, one-dimensional and two-dimensional SDS-PAGE were employed to assess the purity and composition of isolated Complex I followed by extensive mass spectrometric characterization. Altogether, 41 of 45 rodent Complex I subunits achieved MS/MS sequence coverage. To our knowledge, this study provides the first detailed mass spectrometric analysis of neuronal Complex I proteins and provides a means to investigate the role of cysteine oxidation and other posttranslational modifications in pathologies associated with mitochondrial dysfunction.

An Inhibitor of the F1 subunit of ATP synthase (IF1) modulates the activity of angiostatin on the endothelial cell surface.

Angiostatin binds to endothelial cell (EC) surface F(1)-F(0) ATP synthase, leading to inhibition of EC migration and proliferation during tumor angiogenesis. This has led to a search for angiostatin mimetics specific for this enzyme. A naturally occurring protein that binds to the F1 subunit of ATP synthase and blocks ATP hydrolysis in mitochondria is inhibitor of F1 (IF1). The present study explores the effect of IF1 on cell surface ATP synthase. IF1 protein bound to purified F(1) ATP synthase and inhibited F(1)-dependent ATP hydrolysis consistent with its reported activity in studies of mitochondria. Although exogenous IF1 did not inhibit ATP production on the surface of EC, it did conserve ATP on the cell surface, particularly at low extracellular pH. IF1 inhibited ATP hydrolysis but not ATP synthesis, in contrast to angiostatin, which inhibited both. In cell-based assays used to model angiogenesis in vitro, IF1 did not inhibit EC differentiation to form tubes and only slightly inhibited cell proliferation compared with angiostatin. From these data, we conclude that inhibition of ATP synthesis is necessary for an anti-angiogenic outcome in cell-based assays. We propose that IF1 is not an angiostatin mimetic, but it can serve a protective role for EC in the tumor microenvironment. This protection may be overridden in a concentration-dependent manner by angiostatin. In support of this hypothesis, we demonstrate that angiostatin blocks IF1 binding to ATP synthase and abolishes its ability to conserve ATP. These data suggest that there is a relationship between the binding sites of IF1 and angiostatin on ATP synthase and that IF1 could be employed to modulate angiogenesis.