Mitochondrial ATPase Inhibitor Research Articles

A new linear peptide, higapeptin, isolated from the mud flat-derived fungus Acremonium persicinum inhibits mitochondrial energy metabolism.

A combination of LC-MS/MS and feature-based molecular networking analyses led to the isolation of a new adenopeptin analog, higapeptin (1), and four known peptides, adenopeptin (2), adenopeptins B and C (3 and 4), and acremopeptin (5), from the rice culture of the fungus Acremonium persicinum (18F04103) isolated from a mud flat of the Ariake Sea in Kyushu, Japan. The structure of 1 was determined by NMR and MS/MS fragmentation analyses. The absolute configuration of the constituent amino acids was determined by Marfey's analysis after acid hydrolysis. The C-terminal residue was synthesized, and its absolute configuration was established by Marfey's analysis. Compounds 1 and 2 were found to inhibit mitochondrial energy metabolism, similar to efrapeptin D (6), a known mitochondrial ATPase inhibitor.

Inhibition of ATP synthase reverse activity restores energy homeostasis in mitochondrial pathologies

The maintenance of cellular function relies on the close regulation of adenosine triphosphate (ATP) synthesis and hydrolysis. ATP hydrolysis by mitochondrial ATP Synthase (CV) is induced by loss of proton motive force and inhibited by the mitochondrial protein ATPase inhibitor (ATPIF1). The extent of CV hydrolytic activity and its impact on cellular energetics remains unknown due to the lack of selective hydrolysis inhibitors of CV. We find that CV hydrolytic activity takes place in coupled intact mitochondria and is increased by respiratory chain defects. We identified (+)‐Epicatechin as a selective inhibitor of ATP hydrolysis that binds CV while preventing the binding of ATPIF1. In cells with Complex‐III deficiency, we show that inhibition of CV hydrolytic activity by (+)‐Epichatechin is sufficient to restore ATP content without restoring respiratory function. Inhibition of CV–ATP hydrolysis in a mouse model of Duchenne Muscular Dystrophy is sufficient to improve muscle force without any increase in mitochondrial content. We conclude that the impact of compromised mitochondrial respiration can be lessened using hydrolysis‐selective inhibitors of CV.

Expression and function of mitochondrial inhibitor factor-1 and TASK channels in adrenal cells

Adrenal medullary chromaffin (AMC) cells in the perinatal period and carotid body glomus cells after birth respond to hypoxia with catecholamine secretion. The hypoxia detection mechanism in such O2-sensitive cells is still not well defined. One hypothesis is that a decrease in cellular ATP may be involved in the hypoxia detection. This idea is based on ATP dependence of TASK channel activity that regulates the resting membrane potential and is suppressed by hypoxia in glomus cells. Mitochondrial ATPase inhibitor factor-1 (IF1), a physiological regulator of ATP synthase, helps prevent ATP hydrolysis under hypoxic conditions. In cells where IF1 expression is high, exposure to hypoxia is expected to have no effect on TASK channel activity. This possibility was electrophysiologically and immunocytochemically explored. Single channel recordings revealed that 36-pS TASK3-like channels contribute to the resting membrane potential in young rat adrenal cortical (AC) cells. TASK3-like channel activity in a cell-attached patch was not affected by bath application of mitochondrial inhibitors. Consistent with this finding, IF1-like immunoreactive material was well expressed in rat AC cells. In further support of our hypothesis, IF1-like immunoreactive material was well expressed in adult rat AMC cells that are known to be hypoxia-insensitive and minimally expressed in newborn AMC cells that are hypoxia-sensitive. These results provide evidence for the functional relevance of IF1 expression in excitability in O2-sensitive cells in response to mitochondrial inhibition.

ITRAQ Proteomic Analysis of Wheat (Triticum aestivum L.) Genotypes Differing in Waterlogging Tolerance.

Transient and chronic waterlogging constrains crop production in many regions of the world. Here, we invoke a novel iTRAQ-based proteomic strategy to elicit protein synthesis and regulation responses to waterlogging in tolerant (XM 55) and sensitive genotypes (YM 158). Of the 7,710 proteins identified, 16 were distinct between the two genotypes under waterlogging, partially defining a proteomic basis for waterlogging tolerance (and sensitivity). We found that 11 proteins were up-regulated and 5 proteins were down-regulated; the former included an Fe-S cluster assembly factor, heat shock cognate 70, GTP-binding protein SAR1A-like and CBS domain-containing protein. Down-regulated proteins contained photosystem II reaction center protein H, carotenoid 9, 10 (9′, 10′)-cleavage dioxygenase-like, psbP-like protein 1 and mitochondrial ATPase inhibitor. We showed that nine proteins responded to waterlogging with non-cultivar specificity: these included 3-isopropylmalate dehydratase large subunit, solanesyl-diphosphate synthase 2, DEAD-box ATP-dependent RNA helicase 3, and 3 predicted or uncharacterized proteins. Sixteen of the 28 selected proteins showed consistent expression patterns between mRNA and protein levels. We conclude that waterlogging stress may redirect protein synthesis, reduce chlorophyll synthesis and enzyme abundance involved in photorespiration, thus influencing synthesis of other metabolic enzymes. Collectively, these factors accelerate the accumulation of harmful metabolites in leaves in waterlogging-susceptible genotypes. The differentially expressed proteins enumerated here could be used as biological markers for enhancing waterlogging tolerance as part of future crop breeding programs.

Mitochondrial Fission Complex Is Required for Long-Term Heat Tolerance of Arabidopsis.

Plants are often exposed not only to short-term (S) heat stress but also to long-term (L) heat stress over several consecutive days. A few Arabidopsis mutants defective in L-heat tolerance have been identified, but the molecular mechanisms involved are less well understood than those involved in S-heat tolerance. To elucidate the mechanisms, we isolated the new sensitive to long-term heat5 (sloh5) mutant from EMS-mutagenized seeds of L-heat-tolerant Col-0. The sloh5 mutant was hypersensitive to L-heat but not to S-heat, osmo-shock, salt-shock or oxidative stress. The causal gene, SLOH5, is identical to elongatedmitochondria1 (ELM1), which plays an important role in mitochondrial fission in conjunction with dynamin-related proteins DRP3A and DRP3B. Transcript levels of ELM1, DRP3A and DRP3B were time-dependently increased by L-heat stress, and drp3a drp3b double mutants were hypersensitive to L-heat stress. The sloh5 mutant contained massively elongated mitochondria. L-heat stress caused mitochondrial dysfunction and cell death in sloh5. Furthermore, WT plants treated with a mitochondrial myosin ATPase inhibitor were hypersensitive to L-heat stress. These findings suggest that mitochondrial fission and function are important in L-heat tolerance of Arabidopsis.

Crystal and solution structures of a novel antimicrobial peptide from Chrysomya megacephala.

Antimicrobial peptides (AMPs) are small amphipathic peptides that exhibit bactericidal activity against a wide range of pathogenic microorganisms and are considered to be potential substitutes for antibiotics effective against microbial infection. PSK, an 84-amino-acid AMP recently isolated from Chrysomya megacephala larvae, probably belongs to the mitochondrial ATPase inhibitor family according to its sequence. No member of this family from an insect has been structurally characterized to date. In this study, the crystal structure of full-length PSK determined by molecular replacement using an ab initio modeled ensemble as a search model and a solution structure obtained from small-angle X-ray scattering (SAXS) measurements are reported. The crystal structure reveals a distinct fold compared with those of homologous peptides, in that PSK comprises two antiparallel α-helices rather than a single long helix, which is in good agreement with the SAXS-based ab initio model. However, the peptide exists as a monomer in solution, even though a stable dimer was observed in the crystal structure. This apparent contradiction may reflect different oligomerization states that may be implicated in its bioactivity. The data presented here have established a solid basis for further mechanistic studies of this novel insect AMP.

Fructose substitution enhances derivation and self-renewal expansion of induced pluripotent stem cells

Background & Aim Induced pluripotent stem cells (iPSCs), which can be generated from patients’ somatic cells and grown in cultures maintaining their pluripotency and possessing the unlimited self-renewal capability, have been utilized for developing cell therapeutic strategies, dissecting disease mechanisms and validating drug response. Recent studies imply pluripotent stem cells (PSCs) possess a unique metabolic features and require specific metabolites in maintaining their cell fate. In the current work, we have deciphered the specific metabolites required for maintaining self-renewal expansion of iPSCs. Methods, Results & Conclusion Utilizing RNA-Seq analysis, we showed that, in comparing with fibroblast, ESCs and iPSCs were found to retain the expression of critical enzymes involved in Krebs cycle. We then determined if the specific bioenergetics adaptation in cultures can affect pluripotency acquirement or maintenance. Oct4-GFP(+) iPSCs derived mouse fibroblast were seeded in medium containing either 4500mM Glucose (high glucose) with or without Oligomycin (mitochondrial ATPase inhibitor), 500mM Glucose (low glucose) and 0mM Glucose (no glucose) with or without pyruvate (to be utilized in TCA cycles). We found that depletion of glucose resulted in reduced growth of Oct4-GFP(+) iPSC colonies. In contrast, addition of pyruvate could rescue the growth of Oct4-GFP(+) iPSC colonies suggesting the retaining mitochondrial respiration may aid self-renewal expansion of pluripotent stem cells. The further analysis revealed that mouse and human ESCs/iPSCs had evaluated level of GLUT5 & KHK suggesting pluripotent stem cells can utilize fructose efficiently. We further validated that fructose substitution was sufficient to maintain the self-renewal growth of ESCs/iPSCs. Importantly, fructose substitution was found to enhance generation of iPSCs from Yamanaka factor mediated fibroblast reprogramming possibly via selectively suppressing the growth of partial reprogrammed cells and upregulating alpha 2,6-sialylation as sialyltransferase ST6Gal1 is known to play an crucial role in regulating pluripotency. In summary, the current work elucidated that the specific bioenergetics adaptation in cultures linking to pluripotency maintenance and identified ESCs/iPSCs can utilize fructose efficiently. Fructose substitution can therefore be used to enhance derivation of iPSCs.

Mitochondrial ATPase inhibitor factor 1, MjATPIF1, is beneficial for WSSV replication in kuruma shrimp (Marsupenaeus japonicus)

ATPase Inhibitory Factor 1 (IF1) is a mitochondrial protein that functions as a physiological inhibitor of F1F0-ATP synthase. In the present study, a mitochondrial ATPase inhibitor factor 1 (MjATPIF1) was identified from kuruma shrimp (Marsupenaeus japonicus), which was demonstrated to participate in the viral immune reaction of white spot syndrome virus (WSSV). MjATPIF1 contained a mitochondrial ATPase inhibitor (IATP) domain, and was widely distributed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine of shrimp. MjATPIF1 transcription was upregulated in hemocytes and intestines by WSSV. WSSV replication decreased after MjATPIF1 knockdown by RNA interference and increased following recombinant MjATPIF1 protein injection. Further study found that MjATPIF1 promoted the production of superoxide and activated the transcription factor nuclear factor kappa B (NF-κB, Dorsal) to induce the transcription of WSSV RNAs. These results demonstrate that MjATPIF1 benefits WSSV replication in kuruma shrimp by inducing superoxide production and NF-κB activation.

Development and Characterization of Monoclonal Antibodies for the Mycotoxin Citreoviridin.

Citreoviridin (CTV) in an inhibitor of mitochondrial ATPase that has been isolated from molded yellow rice and linked to the human disease Shoshin-kakke (acute cardiac beriberi). The disease results from a deficiency of thiamine, however, purified CTV can reproduce the symptoms in experimental animals. The link between CTV and Shoshin-kakke has been difficult to resolve, in part because cases of the disease are rare. In addition to rice, CTV has been found in maize, pecan nuts, and wheat products. A method to screen for CTV and its geometric isomer, iso-CTV, in commodities was developed, based upon the isolation of two novel monoclonal antibodies (mAb). In an antigen-immobilized competitive enzyme-linked immunosorbent assay format (CI-ELISA), the observed IC50s for CTV were 11 ng/mL and 18 ng/mL (mAbs 2-2 and 2-4, respectively). The assays were relatively tolerant to methanol and acetonitrile, which allowed their application to the detection of CTV in spiked polished white rice. For quantification, a standard mixture of CTV and iso-CTV was used, along with matrix matched calibration. The dynamic range of the ELISA using mAb 2-4 was equivalent to 0.23 to 2.22 mg/kg in rice. Recoveries over the range of 0.36 to 7.23 mg/kg averaged 97 ± 10%. The results suggest that the mAb 2-4-based immunoassay can be applied to the screening of white rice for CTV. Both mAbs were also observed to significantly enhance the fluorescence of the toxin.

Phosphorylation Level of Complex V Proteins of Higher Plant Mitochondria Correlates with Their Redox State

The effect of synthetic cytokinin BAP (6-benzylaminopurine) on protein phosphorylation in isolated maize mitochondria was investigated. The effect of BAP in comparison with classical inhibitors of the mitochondria respiratory complexes, such as rotenone, malonate, SHAM, and KCN on the level of mitochondrial protein phosphorylation was studied. Assessment of the activity of respiratory complexes I and II by the BN-PAGE (blue native polyacrylamide gel electrophoresis) in mitochondria untreated and treated with BAP has not revealed any difference in the activity of these complexes. Investigation of the mitochondrial complex V activity under BAP treatment has shown a significant decrease in the mitochondrial ATPase activity. The effect of BAP was similar to the effect of such a specific inhibitor of the membrane-bound mitochondrial ATPase as oligomycin and an inhibitor of protein tyrosine kinases genistein.

307. Metabolic heterogeneity among Glioblastoma stem-like cells reflects differences in response to drug treatments

Purpose Glioblastoma multiforme (GBM) is the most aggressive brain tumor. The cancer stem cell hypothesis postulates the existence within GBM of a small fraction of self-renewing cells with stem like properties (GSCs), resistant to conventional treatments. Metabolic profiling may contribute to discover new diagnostic or prognostic biomarkers to be used for personalized therapies. Methods Forty-four GSC lines, derived from GBM patients were analyzed via MR spectroscopy as described in [1] . Unsupervised Cluster analysis of MR data was performed to identify GSC subgroups with different metabolic profiles. Responses to treatment with oligomycin and etomoxir were examined by MRS and confocal microscopy. Results Clustering of GSCs evidenced three subgroups: Clusters 1a and 1b, with high intergroup similarity and neural fingerprints, and Cluster 2, with a metabolism typical of commercial tumor lines (Fig. 1) according to [2] . Subclones generated by the same GSC line showed different metabolic phenotypes. Aerobic glycolysis prevailed in Cluster 2 cells as demonstrated by higher lactate production compared to Cluster 1 cells. Oligomycin, a mitochondrial ATPase inhibitor, induced high lactate extrusion only in Cluster 1 cells, where it produced neutral lipid accumulation detected as mobile lipid signals (MRS) and as lipid droplets (confocal microscopy). Conclusions These results indicate a relevant role of mitochondrial fatty acids oxidation for energy production in GSCs. On the other hand, further metabolic differences, likely accounting for different therapy responsiveness observed after etomoxir treatment, suggest that caution must be used in considering patient treatment with mitochondria FAO blockers.

Proteome modifications of Pacific white shrimp (Litopenaeus vannamei) muscle under biofloc system

The objective of the study was to examine the effects of biofloc technology on the muscle proteome of Litopenaeus vannamei. Two biofloc treatments and one control were compared: biofloc‐based tanks under zero‐water exchange fed with 150 g/kg crude protein (BF15), or with 250 g/kg crude protein (BF25) diets, and clear water tanks with 50% of daily water exchange stocked with shrimp fed with similar amount of a 250 g/kg crude protein diet, referred to as control. The shrimp (5.28 ± 0.42 g) were divided into the 300‐L fibreglass tanks (water volume of 200 L) at a density of 35 shrimp per tank and were cultured for 35 days. The biofloc groups displayed better growth and survival compared to the control. The muscle tissue from the control and BF25 groups was subjected to proteomic analysis. Lactate dehydrogenase, enolase, arginine kinase, mitochondrial ATP synthase subunit alpha, mitochondrial ATPase inhibitor factor 1 precursor, serpin 3 and myeloid differentiation factor 88 had an increased abundance in the BF25 group, while myosin heavy chain type 1 and myosin heavy chain type 2 showed a decreased abundance. The results indicate that biofloc technology could alter the expression of proteins involved in structure, metabolism and immune status of cultured shrimp.

Particular Effects of Genistein on Tyrosine Phosphorylation of Maize Mitochondrial Proteins

The activity of specific tyrosine protein kinases is known to be associated with the production of oncogenes. A specific inhibitor of tyrosine protein kinases can be effective not only as an antitumor agent but also as a tool for studying the physiological role of tyrosine phosphorylation. Among similar inhibitors, the flavonoid genistein was shown to have the most specific effect. This agent almost completely inhibited phosphorylation of tyrosine residues, while the activity of serine and threonine protein kinases was slightly suppressed. At the same time, another flavonoid quercetin inhibited activities of tyrosine kinase and other protein kinases with equal efficacy. The aim of the work was to study the effect of genistein on phosphorylation of maize mitochondrial proteins at the tyrosine residues for the assessment of signal cascades associated with tyrosine protein kinases. It was first revealed that tyrosine phosphorylation occurred in the maize mitochondrial heat shock protein 60 (HSP60) and the protein of 90 kDa. Phosphorylation of the 90-kDa protein is observed only during the treatment of isolated mitochondria with genistein. The study of the activity of mitochondrial respiratory complex V showed a significant decrease in the hydrolytic activity of the mitochondrial ATPase after exposure to genistein. The activating effect of this agent on mitochondrial protein kinases can be explained by changes in the redox state of mitochondria due to inhibition of mitochondrial ATPase by genistein.

Influence of phosphatidylserine and phosphatidylethanolamine on farnesol tolerance in Candida albicans.

Candida albicans is among the most common human fungal pathogens. The ability to undergo the morphological transition from yeast to hyphal growth is critical for its pathogenesis. Farnesol, a precursor in the isoprenoid/sterol pathway, is a quorum-sensing molecule produced by C. albicans that inhibits hyphal growth in this polymorphic fungus. Interestingly, C. albicans can tolerate farnesol concentrations that are toxic to other fungi. We hypothesized that changes in phospholipid composition are one of the factors contributing to farnesol tolerance in C. albicans. In this study, we found that loss of enzymes that synthesize the phospholipids phosphatidylserine (PS) and/or phosphatidylethanolamine (PE) compromise the tolerance of C. albicans to farnesol. Compared with wild type, the phospholipid mutant cho1∆/∆ (loss of PS and decreased PE synthesis) shows greater inhibition of growth, loss of ATP production, increased consumption of oxygen, and increased formation of reactive oxygen species in the presence of farnesol. The cho1∆/∆ mutant also exhibits decreased sensitivity to mitochondrial ATPase inhibition, suggesting that cells lacking PS and/or downstream PE rely less on mitochondrial function for ATP synthesis. These data reveal that PS and PE play roles in farnesol tolerance and maintaining mitochondrial respiratory function.

Metabolic Heterogeneity Evidenced by MRS among Patient-Derived Glioblastoma Multiforme Stem-Like Cells Accounts for Cell Clustering and Different Responses to Drugs.

Clustering of patient-derived glioma stem-like cells (GSCs) through unsupervised analysis of metabolites detected by magnetic resonance spectroscopy (MRS) evidenced three subgroups, namely clusters 1a and 1b, with high intergroup similarity and neural fingerprints, and cluster 2, with a metabolism typical of commercial tumor lines. In addition, subclones generated by the same GSC line showed different metabolic phenotypes. Aerobic glycolysis prevailed in cluster 2 cells as demonstrated by higher lactate production compared to cluster 1 cells. Oligomycin, a mitochondrial ATPase inhibitor, induced high lactate extrusion only in cluster 1 cells, where it produced neutral lipid accumulation detected as mobile lipid signals by MRS and lipid droplets by confocal microscopy. These results indicate a relevant role of mitochondrial fatty acid oxidation for energy production in GSCs. On the other hand, further metabolic differences, likely accounting for different therapy responsiveness observed after etomoxir treatment, suggest that caution must be used in considering patient treatment with mitochondria FAO blockers. Metabolomics and metabolic profiling may contribute to discover new diagnostic or prognostic biomarkers to be used for personalized therapies.

Abstract 286: ATPase Inhibitory Factor 1 Regulates Glycolysis in Cardiomyocytes

It is known that pathological cardiac hypertrophy is associated with decreased fatty acid oxidation (FAO) and increased reliance on glycolysis. This metabolic remodeling is generally recognized and considered ultimately maladaptive for sustaining myocardial energetics and function. The mechanisms responsible for the switch are poorly understood but appear to be coupled with impaired mitochondrial function. We found that mitochondrial ATPase inhibitor factor 1 (ATPIF1) was up-regulated (by 4-fold, p=0.01) in the failing heart induced by TAC surgery as well as in hypertrophied adult rat cardiomyocytes (CMs) induced by 10 uM phenylephrine (PE, by 3-fold, p=0.01), but not in the physiological hypertrophied heart (p=0.9). ATPIF1 is an inhibitor of ATPase in the mitochondrial ATP synthase, or Complex V. It was shown that upregulation of ATPIF1 increased glycolysis in non-cardiomyocytes. Here we wish to test the hypothesis that ATPIF1 stimulates glycolysis in the heart undergoing pathological hypertrophy. Overexpress ATPIF1 (OE) by adenovirus vector in CMs, which mimics the up-regulation of ATPIF1 induced by PE, increased glycolysis in CMs (control: 8.54±0.61 mpH/min, OE: 11.46±0.48 mpH/min; p=0.006), while ATPIF1 knockdown (KD) by shRNA in PE treated CMs abolished the upregulation of glycolytic capacity (control: 29.27±1.31 mpH/min, KD: 28.61±1.81 mpH/min, control-PE: 40.12±1.33 mpH/min, KD-PE: 32.51±2.09 mpH/min; p=0.003). ATPIF1OE or pathological hypertrophy induced by PE increased the expression of glycolytic enzymes, e.g. GAPDH, GLUT1, LDHA and PKM2, which were abolished by ATPIF1KD. We also found that elevation of ATPIF1 decreased mitochondrial oxygen consumption rate (OCR) and promoted mitochondrial reactive oxygen species (mtROS) production, while the generation of mtROS caused by PE was abrogated in the ATPIF1 deficient CMs. Blockade of mtROS production by expressing mitochondria localized catalase suppressed the increased glycolysis in ATPIF1-OE CMs. In conclusion, our results identify ATPIF1 as a regulator of glycolysis in pathological cardiac hypertrophy, which may provide a mechanism for the regulation of glycolysis through mitochondrial bioenergetics.

Pyruvate kinase M2 and the mitochondrial ATPase Inhibitory Factor 1 provide novel biomarkers of dermatomyositis: a metabolic link to oncogenesis

BackgroundMetabolic alterations play a role in the development of inflammatory myopathies (IMs). Herein, we have investigated through a multiplex assay whether proteins of energy metabolism could provide biomarkers of IMs.MethodsA cohort of thirty-two muscle biopsies and forty plasma samples comprising polymyositis (PM), dermatomyositis (DM) and sporadic inclusion body myositis (sIBM) and control donors was interrogated with monoclonal antibodies against proteins of energy metabolism using reverse phase protein microarrays (RPPA).ResultsWhen compared to controls the expression of the proteins is not significantly affected in the muscle of PM patients. However, the expression of β-actin is significantly increased in DM and sIBM in consistence with muscle and fiber regeneration. Concurrently, the expression of some proteins involved in glucose metabolism displayed a significant reduction in muscle of sIBM suggesting a repression of glycolytic metabolism in these patients. In contrasts to these findings, the expression of the glycolytic pyruvate kinase isoform M2 (PKM2) and of the mitochondrial ATPase Inhibitor Factor 1 (IF1) and Hsp60 were significantly augmented in DM when compared to other IMs in accordance with a metabolic shift prone to cancer development. PKM2 alone or in combination with other biomarkers allowed the discrimination of control and IMs with very high (>95%) sensitivity and specificity. Unfortunately, plasma levels of PKM2 were not significantly altered in DM patients to recommend its use as a non-invasive biomarker of the disease.ConclusionsExpression of proteins of energy metabolism in muscle enabled discrimination of patients with IMs. RPPA identified the glycolysis promoting PKM2 and IF1 proteins as specific biomarkers of dermatomyositis, providing a biochemical link of this IM with oncogenesis.

Measurement of Energy-dependent Rhodamine 6G Efflux in Yeast Species.

Rhodamine 6G is a highly fluorescent dye often used to determine the transport activity of yeast membrane efflux pumps. The ATP-binding cassette transporter KlPdr5p confers resistance to several unrelated drugs in Kluyveromyces lactis. KlPdr5p also extrudes rhodamine 6G (R6G) from intact yeast cells in an energy-dependent manner. Incubation of yeast cells in the presence of 2-deoxy-D-glucose (inhibitor of glycolysis) and R6G (mitochondrial ATPase inhibitor) leads to marked depletion of intracellular ATP pool ( Kolaczkowski et al., 1996 ). An active KlPdr5p mediated extrusion of R6G from intact yeast cells can be followed by direct measurement of the fluorescence of extruded R6G in the assay buffer.

Dimerization interface and dynamic properties of yeast IF1 revealed by Site-Directed Spin Labeling EPR spectroscopy

The mitochondrial ATPase inhibitor, IF1, regulates the activity of the mitochondrial ATP synthase. The oligomeric state of IF1 related to pH is crucial for its inhibitory activity. Although extensive structural studies have been performed to characterize the oligomeric states of bovine IF1, only little is known concerning those of yeast IF1. While bovine IF1 can be found as an inhibitory dimer at low pH and a non-inhibitory tetramer at high pH, a monomer/dimer equilibrium has been described for yeast IF1, high pH values favoring the monomeric state. Combining different strategies involving the grafting of nitroxide spin labels combined with Electron Paramagnetic Resonance (EPR) spectroscopy, the present study brings the first structural characterization, at the residue level, of yeast IF1 in its dimeric form. The results show that the dimerization interface involves the central region of the peptide revealing that the dimer corresponds to a non-inhibitory state. Moreover, we demonstrate that the C-terminal region of the peptide is highly dynamic and that this segment is probably folded back onto the central region. Finally, the pH-dependence of the inter-label distance distribution has been observed indicating a conformational change between two structural states in the dimer.

The region from phenylalanine-28 to lysine-50 of a yeast mitochondrial ATPase inhibitor (IF1) forms an α-helix in solution.

A mitochondrial ATPase inhibitor, IF1, is a 63 amino acid residue protein that regulates the activity of ATP synthase (F(1)F(o)-ATPase). In the present study, we constructed mutant IF1 proteins with proline residues inserted into a wide range of their primary structures to determine the location and function of α-helix in the protein. A total of 11 yeast IF1 protein mutants were expressed and purified. Proline insertions in the region 28-50 reduced α-helical contents, indicating that the region formed a helix in solution. Oligomer formation of proline mutants at the C-terminal 38-60 region was markedly reduced, indicating that the region is required for oligomerization of the protein. Proline mutants at the N-terminal 18-39 region did not inhibit F(1)F(o)-ATPase, indicating that the region is required for ATPase inhibitory activity. Inhibition of a proline insertion mutant between residues 44 and 45 that lost a large portion of the α-helix was slower, although the maximal inhibition level of the mutant protein was comparable to that of wild-type IF1. The results suggest that the helix of yeast IF1 facilitates binding to F(1) by promoting initial interaction of the proteins.