The ∈ subunit of the chloroplast ATP synthase of Pisum sativum

In contrast to the well-characterized spinach ( Spinacea oleracea) chloroplast ATP synthase (CF1–CFo), the properties of the chloroplast ATP synthase from pea (Pisum sativum ) have not been as intensively studied. Preliminary data suggested that the regulatory properties of the two enzymes differ. In the absence of activating treatments the ATPase activity of pea thylakoids in the dark was higher than that in spinach thylakoids. When assayed in the presence of sulfite, the MgATPase activity of pea thylakoids was inhibited to a maximum of 67% by tentoxin, indicating that the dark ATPase activity is in part catalyzed by CF1–CFo. The ATPase activity of purified pea CF1 was also higher than that of spinach CF1 in the absence of activating treatments. These differences could result from the different regulatory properties of the pea or subunit or both. The pea subunit was less effective in binding to or inhibiting the ATPase activity of pea o r spinach CF1 deficient in (CF1-). Spinach inhibited the ATPase activity of pea CF1- at lower concentrations than pea . The gene encoding the pea subunit was cloned and over-expressed. Recombinant pea did not restore low proton permeability to spinach thylakoid membranes reconstitituted with spinach CF1-, although pea was effective when tested with pea thylakoids reconstitituted with pea CF1-. These results confirm earlier suggestions that the C-terminal region of is important in -CF1 and -CFo interactions.     

Conformational change of the α subunit of Escherichia coli F1 ATPase: ATP changes the trypsin sensitivity of the subunit

Conformational change in the α subunit of Escherichia coli proton-translocating ATPase was studied using trypsin. The subunit was cleaved with a small amount of trypsin (1 μg/mg subunit) to peptides of less than 8000 daltons. On the other hand, the subunit was cleaved to two main polypeptides (30,000 and 25,000 daltons) in the presence of sufficient ATP (1 mm-0.5 μm) to saturate the high-affinity site of the subunit. Analysis of digests of the subunit combined with fluorescent maleimide suggested that the subunit was digested in the middle of the polypeptide chain in the presence of the nucleotide. ADP and adenylyl imidodiphosphate had the same effect as ATP. These results suggest that the conformation of the subunit changed to form two trypsin-resistant domains upon binding of ATP to the high-affinity site.     

Effects of site-directed mutation on the function of the chloroplast ATP synthase ε subunit

The previous work in our lab showed that the spinach chloroplast ATP synthase ε mutant with 3 amino acid residues deleted from the N-terminus had much lower ability to inhibit ATP hydrolysis and block proton leakage in comparison to a mutant with 1 or 2 residues deleted from the N-terminus. The present study aimed at determining whether there is special importance in the structure and function of the N-terminal third residue of the chloroplast ε subunit. The leucine residue at the N-terminal third site (Leu3) of the spinach chloroplast ε subunit was replaced with Ile, Phe, Thr, Arg, Glu or Pro by site-directed mutagenesis, forming mutants εL3I, εL3F, εL3T, εL3R, εL3E and εL3P, respectively. These ε variants all showed lower abilities to inhibit ATP hydrolysis and to block proton leakage, as compared to the wild type ε subunit (εWT). The abilities of mutants εL3I and εL3F to restore the ATP synthesis activity of reconstituted membranes were higher than those of εWT, but the abilities of the other ε variants were lower than that of εWT. These results indicate that the hydrophobic and neutral characteristics of Leu3 of the chloroplast ε subunit are very important for its ability to inhibit ATP hydrolysis and block proton leakage, and for the ATP synthesis ability of ATP synthase.     

Multiple phosphorylation sites in the β subunit of thylakoid ATP synthase

Proteomic analyses of the β subunit of the plastid ATP synthase of barley (Hordeum vulgare L.) revealed that mature protein was not carboxy terminus processed and suggested the correction of the 274 codon (GAT to AAT) in the data bank that was confirmed by DNA sequencing. Six isoforms of the ATP synthase β subunit with pI ranging from 4.95 to 5.14 were resolved by two-dimensional electrophoresis (2-DE). Mass spectrometry analyses indicated that the six isoforms differ in their phosphorylation degree, which was confirmed by the disappearance of more acidic forms after incubation with the protein phosphatase calcineurin. Six Ser and/or Thr were detected as phosphorylated, among them the conserved Thr-179 that is also phosphorylated in the β subunit of human mitochondria. The results are discussed in relation with the proposed regulation of the ATP synthase by phosphorylation and 14-3-3 proteins.     

The carboxyl-terminal helical domain of the ATP synthase γ subunit is involved in ε subunit conformation and energy coupling

The γ subunit located at the center of ATP synthase (F_OF₁) plays critical roles in catalysis. Escherichia coli mutant with Pro substitution of the γ subunit residue γLeu218, which are located the rotor shaft near the c subunit ring, decreased NADH-driven ATP synthesis activity and ATP hydrolysis-dependent H⁺ transport of membranes to ~60% and ~40% of the wild type, respectively, without affecting F_OF₁ assembly. Consistently, the mutant was defective in growth by oxidative phosphorylation, indicating that energy coupling is impaired by the mutation. The ε subunit conformations in the γLeu218Pro mutant enzyme were investigated by cross-linking between cysteine residues introduced into both the ε subunit (εCys118 and εCys134, in the second helix and the hook segment, respectively) and the γ subunit (γCys99 and γCys260, located in the globular domain and the carboxyl-terminal helix, respectively). In the presence of ADP, the two γ260 and ε134 cysteine residues formed a disulfide bond in both the γLeu218Pro mutant and the wild type, indicating that the hook segment of ε subunit penetrates into the α₃β₃-ring along with the γ subunits in both enzymes. However, γ260/ε134 cross-linking in the γLeu218Pro mutant decreased significantly in the presence of ATP, whereas this effect was small in the wild type. These results suggested that the γ subunit carboxyl-terminal helix containing γLeu218 is involved in the conformation of the ε subunit hook region during ATP hydrolysis and, therefore, is required for energy coupling in F_OF₁.     

Modulation of nucleotide binding to the catalytic sites of thermophilic F1-ATPase by the ε subunit: Implication for the role of the ε subunit in ATP synthesis

Effect of ε subunit on the nucleotide binding to the catalytic sites of F₁-ATPase from the thermophilic Bacillus PS3 (TF₁) has been tested by using α₃β₃γ and α₃β₃γε complexes of TF₁ containing βTyr341 to Trp substitution. The nucleotide binding was assessed with fluorescence quenching of the introduced Trp. The presence of the ε subunit weakened ADP binding to each catalytic site, especially to the highest affinity site. This effect was also observed when GDP or IDP was used. The ratio of the affinity of the lowest to the highest nucleotide binding sites had changed two orders of magnitude by the ε subunit. The differences may relate to the energy required for the binding change in the ATP synthesis reaction and contribute to the efficient ATP synthesis.     

ATP binding to the ϵ subunit of thermophilic ATP synthase is crucial for efficient coupling of ATPase and H+ pump activities

ATP binding to the ? subunit of F1-ATPase, a soluble subcomplex of TFoF1 (FoF1-ATPase synthase from the thermophilic Bacillus strain PS3), affects the regulation of F1-ATPase activity by stabilizing the compact, ATPase-active, form of the ? subunit [Kato, S., Yoshida, M. and Kato-Yamada, Y. (2007) J. Biol. Chem. 282, 37618-37623]. In the present study, we report how ATP binding to the ? subunit affects ATPase and H+ pumping activities in the holoenzyme TFoF1. Wild-type TFoF1 showed significant H+ pumping activity when ATP was used as the substrate. However, GTP, which bound poorly to the ? subunit, did not support efficient H+ pumping. Addition of small amounts of ATP to the GTP substrate restored coupling between GTPase and H+ pumping activities. Similar uncoupling was observed when TFoF1 contained an ATP-binding-deficient ? subunit, even with ATP as a substrate. Further analysis suggested that the compact conformation of the ? subunit induced by ATP binding was required to couple ATPase and H+ pumping activities in TFoF1 unless the ? subunit was in its extended-state conformation. The present study reveals a novel role of the ? subunit as an ATP-sensitive regulator of the coupling of ATPase and H+ pumping activities of TFoF1.     

Subunit F modulates ATP binding and migration in the nucleotide-binding subunit B of the A1AO ATP synthase of Methanosarcina mazei Gö1

The interaction of the nucleotide-binding subunit B with subunit F is essential in coupling of ion pumping and ATP synthesis in A₁A_O ATP synthases. Here we provide structural and thermodynamic insights on the nucleotide binding to the surface of subunits B and F of Methanosarcina mazei Gö1 A₁A_O ATP synthase, which initiated migration to its final binding pocket via two transitional intermediates on the surface of subunit B. NMR- and fluorescence spectroscopy as well as ITC data combined with molecular dynamics simulations of the nucleotide bound subunit B and nucleotide bound B-F complex in explicit solvent, suggests that subunit F is critical for the migration to and eventual occupancy of the final binding site by the nucleotide of subunit B. Rotation of the C-terminus and conformational changes in subunit B are initiated upon binding with subunit F causing a perturbation that leads to the migration of ATP from the transition site 1 through an intermediate transition site 2 to the final binding site 3. This mechanism is elucidated on the basis of change in binding affinity for the nucleotide at the specific sites on subunit B upon complexation with subunit F. The change in enthalpy is further explained based on the fluctuating local environment around the binding sites.     

Is Kir6.1 a subunit of mitoK(ATP)?

The subunit composition of the mitochondrial ATP-sensitive K⁺-channel (mitoK_ATP) is unknown, though some suspect a role for the inward rectifier, Kir6.1, based largely on antibody studies of heart mitochondria. To ascertain the molecular identity of mitoK_ATP we therefore sought to purify this putative mitochondrial Kir6.1, and conclusively identify the subunits by mass spectrometry. Immunoblots, conducted with two commercially available antibodies, revealed two distinct signals in isolated heart mitochondria, of 51 and 48 kDa, respectively. Localization was confirmed by either immuno-gold electron microscopy or by immunofluorescence. Each putative Kir6.1 species was extracted, purified, and identified by LC-MS/MS. The 51 kDa band was identified as NADH-dehydrogenase flavoprotein 1, while the preponderant protein in the 48-kDa band was mitochondrial isocitrate dehydrogenase (NADP form). 1D-, 2D-, and native gel analyses were consistent with these assignments. The data suggest it is premature to assign Kir6.1 a role in mitoK_ATP on the basis of immunoreactivity alone.     

The precursor of the F1β subunit of the ATP synthase is covalently modified upon binding to plant mitochondria

We present evidence for a unique covalent modification of a nuclear-encoded precursor protein targeted to plant mitochondria. We investigated the early events of in vitro import for the mitochondrial precursor of the ATP synthase F₁ subunit from Nicotiana plumbaginifolia (pF₁) into plant mitochondria. When pF₁ of 59 kDa was incubated with mitochondria isolated from different higher-plant species, a band of 61 kDa was generated. The 61 kDa protein was a covalently modified form of the 59 kDa pF₁. The modification was dependent on the 25 amino acid long N-terminal region of the presequence of pF₁. The modification was catalysed by an enzyme located in the outer mitochondrial membrane which was specific for higher plants and could not be washed off from the membrane by urea, KCl or EDTA. The modification was ATP- and Ca²⁺-dependent, but it was not affected by inhibitors of protein kinases. No inhibition of the modification was observed with phosphatase, methylation or acylation inhibitors. The modification occurs prior to translocation through the mitochondrial outer membrane. Inhibition of the modification process does not affect the import of the precursor protein, hence precursor modification was not a prerequisite for import. Both the modified and the unmodified pF₁ proteins were strongly associated with the mitochondrial outer membrane.     

ATP synthase: Subunit–subunit interactions in the stator stalk

In ATP synthase, proton translocation through the F_o subcomplex and ATP synthesis/hydrolysis in the F₁ subcomplex are coupled by subunit rotation. The static, non-rotating portions of F₁ and F_o are attached to each other via the peripheral “stator stalk”, which has to withstand elastic strain during subunit rotation. In Escherichia coli, the stator stalk consists of subunits b₂δ; in other organisms, it has three or four different subunits. Recent advances in this area include affinity measurements between individual components of the stator stalk as well as a detailed analysis of the interaction between subunit δ (or its mitochondrial counterpart, the oligomycin-sensitivity conferring protein, OSCP) and F₁. The current status of our knowledge of the structure of the stator stalk and of the interactions between its subunits will be discussed in this review.     

Isolation and characterization of the mitochondrial ATP synthase fromChlamydomonas reinhardtii. cDNA sequence and deduced protein sequence of the α subunit

We have isolated the F₀F₁-ATP synthase complex from oligomycin-sensitive mitochondria of the green algaChlamydomonas reinhardtii. A pure and active ATP synthase was obtained by eans of sonication, extraction with dodecyl maltoside and ion exchange and gel permeation chromatography in the presence of glycerol, DTT, ATP and-21. The enzyme consists of 14 subunits as judged by SDS-PAGE. A cDNA clone encoding the ATP synthase subunit has been sequenced. The deduced protein sequence contains a presequence of 45 amino acids which is not present in the mature protein. The mature protein is 58–70% identical to corresponding mitochondrial proteins from other organisms. In contrast to the ATP synthase subunit fromC. reinhardtii (Franzen and Falk, Plant Mol Biol 19 (1992) 771–780), the protein does not have a C-terminal extension. However, the N-terminal domain of the mature protein is 15–18 residues longer than in ATP synthase subunits from other organisms. Southern blot analysis indicates that the protein is encoded by a single-copy gene.Abbreviations DM dodecyl--D-maltoside - OSCP oligomycin sensitivity conferring protein - PMSF phenyl-methylsulfonylfluoride - DTT dithiothreitol - EDTA ethylenediaminotetraacetic disodium salt     

Modulation of coupling in the Escherichia coli ATP synthase by ADP and Pi: Role of the ε subunit C-terminal domain

The ε-subunit of ATP-synthase is an endogenous inhibitor of the hydrolysis activity of the complex and its α-helical C-terminal domain (εCTD) undergoes drastic changes among at least two different conformations. Even though this domain is not essential for ATP synthesis activity, there is evidence for its involvement in the coupling mechanism of the pump. Recently, it was proposed that coupling of the ATP synthase can vary as a function of ADP and P_i concentration. In the present work, we have explored the possible role of the εCTD in this ADP- and P_i-dependent coupling, by examining an εCTD-lacking mutant of Escherichia coli. We show that the loss of P_i-dependent coupling can be observed also in the εCTD-less mutant, but the effects of P_i on both proton pumping and ATP hydrolysis were much weaker in the mutant than in the wild-type. We also show that the εCTD strongly influences the binding of ADP to a very tight binding site (half-maximal effect ≈ 1 nM); binding at this site induces higher coupling in EF_OF₁ and increases responses to P_i. It is proposed that one physiological role of the εCTD is to regulate the kinetics and affinity of ADP/P_i binding, promoting ADP/P_i-dependent coupling.     

Singlet oxygen affects the activity of the thylakoid ATP synthase and has a strong impact on its γ subunit

Singlet oxygen is reported to have the most potent damaging effect upon the photosynthetic machinery. Usually this reactive oxygen molecule acts in concert with other ROS types under stressful conditions. To understand the specific role of singlet oxygen we took advantage of the conditional flu mutant of Arabidopsis thaliana. In flu, the negative feedback loop is abolished, which blocks chlorophyll biosynthesis in the dark. Therefore high amounts of free protochlorophyllide accumulate during darkness. If flu gets subsequently illuminated, free protochlorophyllide acts as a photosensitiser leading almost exclusively to high amounts of ¹O₂. Analysing the thylakoid protein pattern by using 2D PAGE and subsequent MALDI-TOF analysis, we could show, in addition to previous described effects on photosystem II, that singlet oxygen has a massive impact on the thylakoid ATP synthase, especially on its γ subunit. Additionally, it could be shown that the activity of the ATP synthase is reduced upon singlet oxygen exposure and that the rate of non-photochemical quenching is affected in flu mutants exposed to ¹O₂.     

Amputation of a C-terminal helix of the γ subunit increases ATP-hydrolysis activity of cyanobacterial F1 ATP synthase

F₁ is a soluble part of F_oF₁-ATP synthase and performs a catalytic process of ATP hydrolysis and synthesis. The γ subunit, which is the rotary shaft of F₁ motor, is composed of N-terminal and C-terminal helices domains, and a protruding Rossman-fold domain located between the two major helices parts. The N-terminal and C-terminal helices domains of γ assemble into an antiparallel coiled-coil structure, and are almost embedded into the stator ring composed of α₃β₃ hexamer of the F₁ molecule. Cyanobacterial and chloroplast γ subunits harbor an inserted sequence of 30 or 39 amino acids length within the Rossman-fold domain in comparison with bacterial or mitochondrial γ. To understand the structure–function relationship of the γ subunit, we prepared a mutant F₁-ATP synthase of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, in which the γ subunit is split into N-terminal α-helix along with the inserted sequence and the remaining C-terminal part. The obtained mutant showed higher ATP-hydrolysis activities than those containing the wild-type γ. Contrary to our expectation, the complexes containing the split γ subunits were mostly devoid of the C-terminal helix. We further investigated the effect of post-assembly cleavage of the γ subunit. We demonstrate that insertion of the nick between two helices of the γ subunit imparts resistance to ADP inhibition, and the C-terminal α-helix is dispensable for ATP-hydrolysis activity and plays a crucial role in the assembly of F₁-ATP synthase.     

NMR structure of the transmembrane domain of the n-acetylcholine receptor β2 subunit

Nicotinic acetylcholine receptors (nAChRs) are involved in fast synaptic transmission in the central and peripheral nervous system. Among the many different types of subunits in nAChRs, the β2 subunit often combines with the α4 subunit to form α4β2 pentameric channels, the most abundant subtype of nAChRs in the brain. Besides computational predictions, there is limited experimental data available on the structure of the β2 subunit. Using high-resolution NMR spectroscopy, we solved the structure of the entire transmembrane domain (TM1234) of the β2 subunit. We found that TM1234 formed a four-helix bundle in the absence of the extracellular and intracellular domains. The structure exhibited many similarities to those previously determined for the Torpedo nAChR and the bacterial ion channel GLIC. We also assessed the influence of the fourth transmembrane helix (TM4) on the rest of the domain. Although secondary structures and tertiary arrangements were similar, the addition of TM4 caused dramatic changes in TM3 dynamics and subtle changes in TM1 and TM2. Taken together, this study suggests that the structures of the transmembrane domains of these proteins are largely shaped by determinants inherent in their sequence, but their dynamics may be sensitive to modulation by tertiary and quaternary contacts.     

Expression profile of the α subunit of the heterotrimeric G protein in rice

Previous studies on the activity of the rice Gα promoter using a β-Glucuronidase (GUS) reporter construct indicated that Gα expression was highest in developing organs and changed in a developmental stage-dependent manner. In this paper, GUS activity derived from the rice Gα promoter was analyzed in seeds and developing leaves. In seeds, GUS activity was detected in the aleurone layer, embryo, endosperm and scutellar epithelium. In developing leaves, the activity was detected in the mesophyll tissues, phloem and xylem of the leaf sheath and in the mesophyll tissue of the leaf blade. The activity in the aleurone layer and scutellar epithelium suggests that the Gα subunit may be involved in gibberellin signaling. The activity in the mesophyll tissues of the leaf blade suggests that the Gα subunit may be related to the intensity of disease resistance. The pattern of the activity in the developing leaf also indicates that the expression of Gα follows a developmental profile at the tissue level.Key words: expression pattern, Gα subunit, GUS staining pattern, heterotrimeric G protein, riceThe rice mutant d1 is deficient in the heterotrimeric G protein α subunit (Gα). Recently it was found that the dwarfism phenotype of d1 is due to a reduction in cell numbers.¹ This discovery has led to new questions regarding how rice Gα regulates cell number, and which other signaling molecules are involved in this process in various tissues and at different development stages. Studies of d1 suggest that rice Gα participates in both gibberellin signaling^2–4 and brassinosteroid signaling.^5–8 Promoter studies using the β-Glucuronidase (GUS) reporter indicate that Gα expression is highest in developing organs.¹ In this paper, we report on the expression pattern of a Gα promoter::GUS construct in seeds and developing leaves of rice.     

The C-terminus of the G protein α subunit controls the affinity of nucleotides

The C-terminus of the G protein α subunit has a well-known role in determining the selective coupling with the cognate G protein-coupled receptor (GPCR). In fact, rhodopsin, a prototypical GPCR, exhibits active state [metarhodopsin II (MII)] stabilization by interacting with G protein [extra formation of MII (eMII)], and the extent of stabilization is affected by the C-terminal sequence of Gα. Here we examine the relationship between the amount of eMII and the activation efficiency of Gi mutants whose Giα forms have different lengths of the C-terminal sequence of Goα. The results show that both the activation efficiencies of Gi and the amounts of eMII were affected by mutations; however, there was no correlation between them. This finding suggested that the C-terminal region of Gα not only stabilizes MII (active state) but also affects the nucleotide-binding site of Gα. Therefore, we measured the activation efficiency of these mutants by MII at several concentrations of GDP and GTP and calculated the rate constants of GDP release, GDP uptake, and GTP uptake. These rate constants of the Gi mutants were substantially different from those of the wild type, indicating that the replacement of the amino acid residues in the C-terminus alters the affinity of nucleotides. The rate constants of GDP uptake and GTP uptake showed a strong correlation, suggesting that the C-terminus of Giα controls the accessibility of the nucleotide-binding site. Therefore, our results strongly suggest that there is a long-range interlink between the C-terminus of Giα and its nucleotide-binding site.