F<Subscript>1</Subscript>F<Subscript>0</Subscript>-ATP Synthase Complex Interactions <Emphasis Type="Italic">In Vivo</Emphasis> Can Occur in the Absence of the Dimer Specific Subunit e

Evidence suggests membrane bound F₁F₀-ATPase complexes form stable associations such that dimers can be retrieved from detergent lysates of mitochondria isolated from a range of sources including algae, higher plants, yeast and bovine heart, and plant chloroplasts. The physiological relevance of these interactions is not clear but may be connected with the formation and structure of mitochondrial cristae. We sought to demonstrate, in vivo, the association of F₁F₀-ATPases in yeast cells co-expressing two b subunits each fused at its C-terminus to a GFP variant appropriate for fluorescence resonance energy transfer (FRET; BFP as the donor and GFP as the acceptor fluorophore). Both subunit b-GFP and b-BFP fusions were assembled into functional complexes. FRET was observed from enzyme complexes in molecular proximity in respiring cells providing the first demonstration of the association, in vivo, of F₁F₀-ATPase complexes. Moreover, FRET was observed within cells lacking the dimer specific subunit e, indicating structured associations can occur within the inner membrane in the absence of subunit e.     

Intramolecular ex vivo Fluorescence Resonance Energy Transfer (FRET) of Dihydropyridine Receptor (DHPR) β1a Subunit Reveals Conformational Change Induced by RYR1 in Mouse Skeletal Myotubes

The dihydropyridine receptor (DHPR) β_1a subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of β_1a as part of the macromolecular DHPR-ryanodine receptor type I (RyR1) complex is still debatable. We used fluorescence resonance energy transfer (FRET) to probe proximity relationships within the β_1a subunit in cultured skeletal myotubes lacking or expressing RyR1. The fluorescein biarsenical reagent FlAsH was used as the FRET acceptor, which exhibits fluorescence upon binding to specific tetracysteine motifs, and enhanced cyan fluorescent protein (CFP) was used as the FRET donor. Ten β_1a reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of β_1a with either carboxyl or amino terminal fused CFP. FRET efficiency was largest when CCPGCC was positioned next to CFP, and significant intramolecular FRET was observed for all constructs suggesting that in situ the β_1a subunit has a relatively compact conformation in which the carboxyl and amino termini are not extended. Comparison of the FRET efficiency in wild type to that in dyspedic (lacking RyR1) myotubes revealed that in only one construct (H458 CCPGCC β_1a -CFP) FRET efficiency was specifically altered by the presence of RyR1. The present study reveals that the C-terminal of the β_1a subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of β_1a and RyR1 in resting myotubes.     

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-ε improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-α and PKC-ε are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-ε mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.Here, we present methods for assessing the role of PKC-ε in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-ε is activated by overexpressing the constitutively active PKC-ε mutant. PKC-ε is inhibited by overexpressing the inactive mutant of PKC-ε. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F₀F₁-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F₀F₁-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.     

Subunit Movements in Single Membrane-bound H+-ATP Synthases from Chloroplasts during ATP Synthesis

Subunit movements within the H⁺-ATP synthase from chloroplasts (CF₀F₁) are investigated during ATP synthesis. The γ-subunit (γCys-322) is covalently labeled with a fluorescence donor (ATTO532). A fluorescence acceptor (adenosine 5′-(β,γ-imino)triphosphate (AMPPNP)-ATTO665) is noncovalently bound to a noncatalytic site at one α-subunit. The labeled CF₀F₁ is integrated into liposomes, and a transmembrane pH difference is generated by an acid base transition. Single-pair fluorescence resonance energy transfer is measured in freely diffusing proteoliposomes with a confocal two-channel microscope. The fluorescence time traces reveal a repetitive three-step rotation of the γ-subunit relative to the α-subunit during ATP synthesis. Some traces show splitting into sublevels with fluctuations between the sublevels. During catalysis the central stalk interacts, with equal probability, with each αβ-pair. Without catalysis the central stalk interacts with only one specific αβ-pair, and no stepping between FRET levels is observed. Two inactive states of the enzyme are identified: one in the presence of AMPPNP and one in the presence of ADP.     

A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells

G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gα_i1, Gα_i2 and Gα_i3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gα_i subunit, and cp173Venus fused to the Gγ₂ subunit as acceptor. The Gα_i FRET biosensors constructs are expressed together with Gβ₁ from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gα_i FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gα_i FRET sensor in single cells upon stimulation of several GPCRs, including the LPA₂, M₃ and BK₂ receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gα_i1, Gα_i2 and Gα_i3 activation will be valuable for live-cell measurements that probe Gα_i activation.     

The depletion of F1 subunit ε in yeast leads to an uncoupled respiratory phenotype that is rescued by mutations in the proton-translocating subunits of F0

The central stalk of the ATP synthase is an elongated hetero-oligomeric structure providing a physical connection between the catalytic sites in F₁ and the proton translocation channel in F₀ for energy transduction between the two subdomains. The shape of the central stalk and relevance to energy coupling are essentially the same in ATP synthases from all forms of life, yet the protein composition of this domain changed during evolution of the mitochondrial enzyme from a two- to a three-subunit structure (γ, δ, ε). Whereas the mitochondrial γ- and δ-subunits are homologues of the bacterial central stalk proteins, the deliberate addition of subunit ε is poorly understood. Here we report that down-regulation of the gene (ATP15) encoding the ε-subunit rapidly leads to lethal F₀-mediated proton leaks through the membrane because of the loss of stability of the ATP synthase. The ε-subunit is thus essential for oxidative phosphorylation. Moreover, mutations in F₀ subunits a and c, which slow the proton translocation rate, are identified that prevent ε-deficient ATP synthases from dissipating the electrochemical potential. Cumulatively our data lead us to propose that the ε-subunit evolved to permit operation of the central stalk under the torque imposed at the normal speed of proton movement through mitochondrial F₀.     

F1-ATPase of Escherichia coli: THE ?-INHIBITED STATE FORMS AFTER ATP HYDROLYSIS,IS DISTINCT FROM THE ADP-INHIBITED STATE,AND RESPONDS DYNAMICALLY TO CATALYTIC SITE LIGANDS*

F₁-ATPase is the catalytic complex of rotary nanomotor ATP synthases. Bacterial ATP synthases can be autoinhibited by the C-terminal domain of subunit ϵ, which partially inserts into the enzyme''s central rotor cavity to block functional subunit rotation. Using a kinetic, optical assay of F₁·ϵ binding and dissociation, we show that formation of the extended, inhibitory conformation of ϵ (ϵ_X) initiates after ATP hydrolysis at the catalytic dwell step. Prehydrolysis conditions prevent formation of the ϵ_X state, and post-hydrolysis conditions stabilize it. We also show that ϵ inhibition and ADP inhibition are distinct, competing processes that can follow the catalytic dwell. We show that the N-terminal domain of ϵ is responsible for initial binding to F₁ and provides most of the binding energy. Without the C-terminal domain, partial inhibition by the ϵ N-terminal domain is due to enhanced ADP inhibition. The rapid effects of catalytic site ligands on conformational changes of F₁-bound ϵ suggest dynamic conformational and rotational mobility in F₁ that is paused near the catalytic dwell position.     

Fluorescent Fusion Proteins of Soluble Guanylyl Cyclase Indicate Proximity of the Heme Nitric Oxide Domain and Catalytic Domain

Background

To examine the structural organisation of heterodimeric soluble guanylyl cyclase (sGC) Förster resonance energy transfer (FRET) was measured between fluorescent proteins fused to the amino- and carboxy-terminal ends of the sGC β₁ and α subunits.

Methodology/Principal Findings

Cyan fluorescent protein (CFP) was used as FRET donor and yellow fluorescent protein (YFP) as FRET acceptor. After generation of recombinant baculovirus, fluorescent-tagged sGC subunits were co-expressed in Sf9 cells. Fluorescent variants of sGC were analyzed in vitro in cytosolic fractions by sensitized emission FRET. Co-expression of the amino-terminally tagged α subunits with the carboxy-terminally tagged β₁ subunit resulted in an enzyme complex that showed a FRET efficiency of 10% similar to fluorescent proteins separated by a helix of only 48 amino acids. Because these findings indicated that the amino-terminus of the α subunits is close to the carboxy-terminus of the β₁ subunit we constructed fusion proteins where both subunits are connected by a fluorescent protein. The resulting constructs were not only fluorescent, they also showed preserved enzyme activity and regulation by NO.

Conclusions/Significance

Based on the ability of an amino-terminal fragment of the β₁ subunit to inhibit activity of an heterodimer consisting only of the catalytic domains (α_catβ_cat), Winger and Marletta (Biochemistry 2005, 44:4083–90) have proposed a direct interaction of the amino-terminal region of β₁ with the catalytic domains. In support of such a concept of “trans” regulation of sGC activity by the H-NOX domains our results indicate that the domains within sGC are organized in a way that allows for direct interaction of the amino-terminal regulatory domains with the carboxy-terminal catalytic region. In addition, we constructed “fluorescent-conjoined” sGC''s by fusion of the α amino-terminus to the β₁ carboxy-terminus leading to a monomeric, fluorescent and functional enzyme complex. To our knowledge this represents the first example where a fluorescent protein links two different subunits of a higher ordered complex to yield a stoichometrically fixed functionally active monomer.     

Mutations on the N-Terminal Edge of the DELSEED Loop in either the α or β Subunit of the Mitochondrial F1-ATPase Enhance ATP Hydrolysis in the Absence of the Central γ Rotor

F₁-ATPase is a rotary molecular machine with a subunit stoichiometry of α₃β₃γ₁δ₁ε₁. It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central γ rotor dictates the sequential conformational changes to the catalytic sites in the α₃β₃ core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F₁-ATPase have suggested that the α₃β₃ core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this γ-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the α and β subunits that stimulate ATP hydrolysis by the mitochondrial F₁-ATPase in the absence of γ. We found that the F446I mutation in the α subunit and G419D mutation in the β subunit suppress cell death by the loss of mitochondrial DNA (ρ^o) in a Kluyveromyces lactis mutant lacking γ. In organello ATPase assays showed that the mutant but not the wild-type γ-less F₁ complexes retained 21.7 to 44.6% of the native F₁-ATPase activity. The γ-less F₁ subcomplex was assembled but was structurally and functionally labile in vitro. Phe446 in the α subunit and Gly419 in the β subunit are located on the N-terminal edge of the DELSEED loops in both subunits. Mutations in these two sites likely enhance the transmission of catalytically required conformational changes to an adjacent α or β subunit, thereby allowing robust ATP hydrolysis and cell survival under ρ^o conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the α₃β₃ subcomplex.     

Fluorescence resonance energy transfer study of subunit exchange in human lens crystallins and congenital cataract crystallin mutants

Lens α-crystallin is an oligomeric protein with a molecular mass of 500–1000 kDa and a polydispersed assembly. It consists of two types of subunits, αA and αB, each with a molecular mass of 20 kDa. The subunits also form homo-oligomers in some other tissues and in vitro. Their quaternary structures, which are dynamic and characterized by subunit exchange, have been studied by many techniques, including fluorescence resonance energy transfer (FRET) and mass spectrometry analysis. The proposed mechanism of subunit exchange has been either by dissociation/association of monomeric subunits or by rapid equilibrium between oligomers and suboligomers. To explore the nature of subunit exchange further, we performed additional FRET measurements and analyses using a fluorescent dye-labeled W9F αA-crystallin as the acceptor probe and Trp in other crystallins (wild-type and R116C αA, wild-type and R120G αB, wild-type and Q155* βB2) as the donor probe and calculated the transfer efficiency, Förster distance, and average distance between two probes. The results indicate only slight decreased efficiency and increased distance between two probes for the R116C αA and R120G αB mutations despite conformational changes.     

Conformational Transitions of Subunit ? in ATP Synthase from Thermophilic Bacillus PS3

Subunit ɛ of bacterial and chloroplast F_OF₁-ATP synthase is responsible for inhibition of ATPase activity. In Bacillus PS3 enzyme, subunit ɛ can adopt two conformations. In the “extended”, inhibitory conformation, its two C-terminal α-helices are stretched along subunit γ. In the “contracted”, noninhibitory conformation, these helices form a hairpin. The transition of subunit ɛ from an extended to a contracted state was studied in ATP synthase incorporated in Bacillus PS3 membranes at 59°C. Fluorescence energy resonance transfer between fluorophores introduced in the C-terminus of subunit ɛ and in the N-terminus of subunit γ was used to follow the conformational transition in real time. It was found that ATP induced the conformational transition from the extended to the contracted state (half-maximum transition extent at 140 μM ATP). ADP could neither prevent nor reverse the ATP-induced conformational change, but it did slow it down. Acid residues in the DELSEED region of subunit β were found to stabilize the extended conformation of ɛ. Binding of ATP directly to ɛ was not essential for the ATP-induced conformational change. The ATP concentration necessary for the half-maximal transition (140 μM) suggests that subunit ɛ probably adopts the extended state and strongly inhibits ATP hydrolysis only when the intracellular ATP level drops significantly below the normal value.     

Protein Kinase C-α Interaction with F0F1-ATPase Promotes F0F1-ATPase Activity and Reduces Energy Deficits in Injured Renal Cells

We showed previously that active PKC-α maintains F₀F₁-ATPase activity, whereas inactive PKC-α mutant (dnPKC-α) blocks recovery of F₀F₁-ATPase activity after injury in renal proximal tubules (RPTC). This study tested whether mitochondrial PKC-α interacts with and phosphorylates F₀F₁-ATPase. Wild-type PKC-α (wtPKC-α) and dnPKC-α were overexpressed in RPTC to increase their mitochondrial levels, and RPTC were exposed to oxidant or hypoxia. Mitochondrial levels of the γ-subunit, but not the α- and β-subunits, were decreased by injury, an event associated with 54% inhibition of F₀F₁-ATPase activity. Overexpressing wtPKC-α blocked decreases in γ-subunit levels, maintained F₀F₁-ATPase activity, and improved ATP levels after injury. Deletion of PKC-α decreased levels of α-, β-, and γ-subunits, decreased F₀F₁-ATPase activity, and hindered the recovery of ATP content after RPTC injury. Mitochondrial PKC-α co-immunoprecipitated with α-, β-, and γ-subunits of F₀F₁-ATPase. The association of PKC-α with these subunits decreased in injured RPTC overexpressing dnPKC-α. Immunocapture of F₀F₁-ATPase and immunoblotting with phospho(Ser) PKC substrate antibody identified phosphorylation of serine in the PKC consensus site on the α- or β- and γ-subunits. Overexpressing wtPKC-α increased phosphorylation and protein levels, whereas deletion of PKC-α decreased protein levels of α-, β-, and γ-subunits of F₀F₁-ATPase in RPTC. Phosphoproteomics revealed phosphorylation of Ser¹⁴⁶ on the γ subunit in response to wtPKC-α overexpression. We concluded that active PKC-α 1) prevents injury-induced decreases in levels of γ subunit of F₀F₁-ATPase, 2) interacts with α-, β-, and γ-subunits leading to increases in their phosphorylation, and 3) promotes the recovery of F₀F₁-ATPase activity and ATP content after injury in RPTC.     

Anomalous Surplus Energy Transfer Observed with Multiple FRET Acceptors

Background

Förster resonance energy transfer (FRET) is a mechanism where energy is transferred from an excited donor fluorophore to adjacent chromophores via non-radiative dipole-dipole interactions. FRET theory primarily considers the interactions of a single donor-acceptor pair. Unfortunately, it is rarely known if only a single acceptor is present in a molecular complex. Thus, the use of FRET as a tool for measuring protein-protein interactions inside living cells requires an understanding of how FRET changes with multiple acceptors. When multiple FRET acceptors are present it is assumed that a quantum of energy is either released from the donor, or transferred in toto to only one of the acceptors present. The rate of energy transfer between the donor and a specific acceptor (k_D→A) can be measured in the absence of other acceptors, and these individual FRET transfer rates can be used to predict the ensemble FRET efficiency using a simple kinetic model where the sum of all FRET transfer rates is divided by the sum of all radiative and non-radiative transfer rates.

Methodology/Principal Findings

The generality of this approach was tested by measuring the ensemble FRET efficiency in two constructs, each containing a single fluorescent-protein donor (Cerulean) and either two or three FRET acceptors (Venus). FRET transfer rates between individual donor-acceptor pairs within these constructs were calculated from FRET efficiencies measured after systematically introducing point mutations to eliminate all other acceptors. We find that the amount of energy transfer observed in constructs having multiple acceptors is significantly greater than the FRET efficiency predicted from the sum of the individual donor to acceptor transfer rates.

Conclusions/Significance

We conclude that either an additional energy transfer pathway exists when multiple acceptors are present, or that a theoretical assumption on which the kinetic model prediction is based is incorrect.     

Quantitative Intensity-Based FRET Approaches—A Comparative Snapshot

Förster resonance energy transfer (FRET) has become an important tool for analyzing different aspects of interactions among biological macromolecules in their native environments. FRET analysis has also been successfully applied to study the spatiotemporal regulation of various cellular processes using genetically encoded FRET-based biosensors. A variety of procedures have been described for measuring FRET efficiency or the relative abundance of donor-acceptor complexes, based on analysis of the donor fluorescence lifetime or the spectrally resolved fluorescence intensity. The latter methods are preferable if one wants to not only quantify the apparent FRET efficiencies but also calculate donor-acceptor stoichiometry and observe fast dynamic changes in the interactions among donor and acceptor molecules in live cells. This review focuses on a comparison of the available intensity-based approaches used to measure FRET. We discuss their strengths and weaknesses in terms of FRET quantification, and provide several examples of biological applications.     

Intensity Correlation-Based Calibration of FRET

Dual-laser flow cytometric resonance energy transfer (FCET) is a statistically efficient and accurate way of determining proximity relationships for molecules of cells even under living conditions. In the framework of this algorithm, absolute fluorescence resonance energy transfer (FRET) efficiency is determined by the simultaneous measurement of donor-quenching and sensitized emission. A crucial point is the determination of the scaling factor α responsible for balancing the different sensitivities of the donor and acceptor signal channels. The determination of α is not simple, requiring preparation of special samples that are generally different from a double-labeled FRET sample, or by the use of sophisticated statistical estimation (least-squares) procedures. We present an alternative, free-from-spectral-constants approach for the determination of α and the absolute FRET efficiency, by an extension of the presented framework of the FCET algorithm with an analysis of the second moments (variances and covariances) of the detected intensity distributions. A quadratic equation for α is formulated with the intensity fluctuations, which is proved sufficiently robust to give accurate α-values on a cell-by-cell basis in a wide system of conditions using the same double-labeled sample from which the FRET efficiency itself is determined. This seemingly new approach is illustrated by FRET measurements between epitopes of the MHCI receptor on the cell surface of two cell lines, FT and LS174T. The figures show that whereas the common way of α determination fails at large dye-per-protein labeling ratios of mAbs, this presented-as-new approach has sufficient ability to give accurate results. Although introduced in a flow cytometer, the new approach can also be straightforwardly used with fluorescence microscopes.     

Resonance energy transfer between green fluorescent protein variants: complexities revealed with myosin fusion proteins

Green fluorescent protein and its variants are frequently used as F?rster (fluorescence) resonance energy transfer (FRET) pairs to determine the proximity of protein domains. We prepared fusion proteins comprising yellow fluorescent protein-Dictyostelium myosin II motor domain-cyan fluorescent protein (YFP-myosin-CFP) and compared their FRET properties with an existing construct (GFP-myosin-BFP), containing a green fluorescent protein acceptor and blue fluorescent protein donor [Suzuki, Y., Yasunaga, T., Ohkura, R., Wakabayashi, T. and Sutoh, K. (1998) Nature 396, 380-383]. The latter construct showed an apparent 40% reduction in acceptor fluorescence on ATP addition, when excited via the donor, compared with the YFP-myosin-CFP constructs which showed a small increase (相似文献   

Isolation and Antigenic Characterization of Corn Mitochondrial F(1)-ATPase

Corn mitochondrial F₁-ATPase was purified from submitochondrial particles by chloroform extraction. Enzyme stored in ammonium sulfate at 4°C was substantially activated by ATP, while enzyme stored at −70°C in 25% glycerol was not. Enzyme in glycerol remained fully active (8-9 micromoles P_i released per minute per milligram), while the ammonium sulfate preparations steadily lost activity over a 2-month storage period. The enzyme was cold labile, and inactived by 4 minutes at 60°C. Treatment with octylglucoside resulted in complete loss of activity, while vanadate had no effect on activity. The apparent subunit molecular weights of corn mitochondrial F₁-ATPase were determined by SDS-polyacrylamide gel electrophoresis to be 58,000 (α), 55,000 (β), 35,000 (γ), 22,000 (δ), and 12,000 (ε). Monoclonal and polyclonal antibodies used in competitive binding assays demonstrated that corn mitochondrial F₁-ATPase was antigenically distinct from the chloroplastic CF₁-ATPases of corn and spinach. Monoclonal antibodies against antigenic sites on spinach CF₁-ATPase β and γ subunits were used to demonstrate that those sites were either changed substantially or totally absent from the mitochondrial F₁-ATPase.     

The phototroph-specific β-hairpin structure of the γ subunit of FoF1-ATP synthase is important for efficient ATP synthesis of cyanobacteria

The F_oF₁ synthase produces ATP from ADP and inorganic phosphate. The γ subunit of F_oF₁ ATP synthase in photosynthetic organisms, which is the rotor subunit of this enzyme, contains a characteristic β-hairpin structure. This structure is formed from an insertion sequence that has been conserved only in phototrophs. Using recombinant subcomplexes, we previously demonstrated that this region plays an essential role in the regulation of ATP hydrolysis activity, thereby functioning in controlling intracellular ATP levels in response to changes in the light environment. However, the role of this region in ATP synthesis has long remained an open question because its analysis requires the preparation of the whole F_oF₁ complex and a transmembrane proton-motive force. In this study, we successfully prepared proteoliposomes containing the entire F_oF₁ ATP synthase from a cyanobacterium, Synechocystis sp. PCC 6803, and measured ATP synthesis/hydrolysis and proton-translocating activities. The relatively simple genetic manipulation of Synechocystis enabled the biochemical investigation of the role of the β-hairpin structure of F_oF₁ ATP synthase and its activities. We further performed physiological analyses of Synechocystis mutant strains lacking the β-hairpin structure, which provided novel insights into the regulatory mechanisms of F_oF₁ ATP synthase in cyanobacteria via the phototroph-specific region of the γ subunit. Our results indicated that this structure critically contributes to ATP synthesis and suppresses ATP hydrolysis.     

satFRET: estimation of Förster resonance energy transfer by acceptor saturation

We demonstrate theoretically and experimentally the quantification of Förster resonance energy transfer (FRET) by direct and systematic saturation of the excited state of acceptor molecules. This version of acceptor depletion methods for FRET estimation, denoted as “satFRET” is reversible and suitable for time-resolved measurements. The technique was investigated theoretically using the steady-state solution of the differential equation system of donor and acceptor molecular states. The influence of acceptor photobleaching during measurement was included in the model. Experimental verification was achieved with the FRET-pair Alexa 546- Alexa 633 loaded on particles in different stoichiometries and measured in a confocal microscope. Estimates of energy transfer efficiency by excited state saturation were compared to those obtained by measurements of sensitised emission and acceptor photobleaching. The results lead to a protocol that allows time-resolved FRET measurements of fixed and living cells on a conventional confocal microscope. This procedure was applied to fixed Chinese hamster ovary cells containing a cyan fluorescent protein and yellow fluorescent protein pair. The time resolution of the technique was demonstrated in a live T cell activation assay comparing the FRET efficiencies measured using a genetically encoded green and red fluorescent protein biosensor for GTP/GDP turnover to those measured by acceptor photobleaching of fixed cells.     

Torque Transmission Mechanism via DELSEED Loop of F1-ATPase

F₁-ATPase (F₁) is an ATP-driven rotary motor in which the three catalytic β subunits in the stator ring sequentially induce the unidirectional rotation of the rotary γ subunit. Many lines of evidence have revealed open-to-closed conformational transitions in the β subunit that swing the C-terminal domain inward. This conformational transition causes a C-terminal protruding loop with conserved sequence DELSEED to push the γ subunit. Previous work, where all residues of DELSEED were substituted with glycine to disrupt the specific interaction with γ and introduce conformational flexibility, showed that F₁ still rotated, but that the torque was halved, indicating a remarkable impact on torque transmission. In this study, we conducted a stall-and-release experiment on F₁ with a glycine-substituted DELSEED loop to investigate the impact of the glycine substitution on torque transmission upon ATP binding and ATP hydrolysis. The mutant F₁ showed a significantly reduced angle-dependent change in ATP affinity, whereas there was no change in the equilibrium for ATP hydrolysis. These findings indicate that the DELSEED loop is predominantly responsible for torque transmission upon ATP binding but not for that upon ATP hydrolysis.