Coupling factor activity of the purified pea mitochondrial F1-ATPase

The pea cotyledon mitochondrial F₁-ATPase was released from the submitochondrial particles by a washing procedure using 300 mM sucrose /2 mM Tricine (pH 7.4). The enzyme was purified by DEAE-cellulose chromatography and subsequent sucrose density gradient centrifugation. Using polyacrylamide gel electrophoresis under non-denaturing conditions, the purified protein exhibited a single sharp band with slightly lower mobility than the purified pea chloroplast CF₁-ATPase. The molecular weights of pea mitochondrial F₁-ATPase and pea chloroplast CF₁-ATPase were found to be 409 000 and 378 000, respectively. The purified pea mitochondrial F₁-ATPase dissociated into six types of subunits on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Most of these subunits had mobilities different from the subunits of the pea chloroplast CF₁-ATPase. The purified mitochondrial F₁-ATPase exhibited coupling factor activity. In spite of the observed differences between CF₁ and F₁, the mitochondrial enzyme stimulated ATP formation in CF₁-depleted pea chloroplast membranes. Thus, the mitochondrial F₁ was able to substitute functionally for the chloroplast CF₁ in reconstituting photophosphorylation.     

N-Ethylmaleimide inhibition of the catalytic activities of the Dunaliella salina coupling factor 1 (CF1) and the restoration of the inhibition of the CF1 ATPase activity by N-ethylmaleimide

The sensitivity of the catalytic activities of the D. salina chloroplast coupling factor 1 (CF₁) to chemical modification by N-ethylmaleimide has been investigated. (i) When D. salina thylakoid membranes are treated with N-ethylmaleimide, both photophosphorylation and the inducible CF₁ ATPase activity are partially (approx. 60%) inhibited. The inhibition of both activities does not require the presence of a proton-motive force, and the inhibition of photophosphorylation is directly related to the N-ethylmaleimide-covalent modification of CF₁ as shown by (a) the time-course for the inhibition and (b) the maximal extent of inhibition. (ii) Treatment of the purified, latent, D. salina CF₁ with low concentrations of N-ethylmaleimide also results in the partial (approx. 60%) inhibition of the inducible ATPase activity (I₅₀ ≈ 50 μM). The inhibition does not require the presence of the chemical modifier during the activation of the enzyme. (iii) N-ethylmaleimide-induced inhibition of the ATPase activity of either membrane-bound or solubilized CF₁ is partially reversed by either (a) prolonged incubation at low concentrations of N-ethylmaleimide or (b) short incubation times at high concentrations of N-ethylmaleimide. The results are interpreted as indicating multiple binding sites on the D. salina CF₁ that have different rates of reactivity with N-ethylmaleimide. Those sites (or site) that react rapidly with N-ethylmaleimide cause(s) an inhibition of both ATP synthase and ATPase activities, whereas those sites (or site) that react more slowly partially restore(s) the original-ATPase activity. The effects of N-ethylmaleimide on the catalytic activity of D. salina CF₁ are probably mediated by N-ethylmaleimide-induced conformational changes of the enzyme.     

The products of the mitochondrial orf25 and orfB genes are FO components in the plant F1FO ATP synthase

The F_O portion of the mitochondrial ATP synthase contains a range of different subunits in bacteria, yeast and mammals. A search of the Arabidopsis genome identified sequence orthologs for only some of these subunits. Blue native polyacrylamide gel electrophoresis separation of Arabidopsis mitochondrial respiratory chain complexes revealed intact F₁F_O, and separated F₁ and F_O components. The subunits of each complex were analysed by mass spectrometry and matched to Arabidopsis genes. In the F₁F_O complex a series of nine known subunits were identified along with two additional proteins matching the predicted products of the mitochondrial encoded orfB and orf25 genes. The F₁ complex contained the five well-characterised F₁ subunits, while four subunits in the F_O complex were identified: subunit 9, d subunit, and the orfB and orf25 products. Previously, orfB has been suggested as the plant equivalent of subunit 8 based on structural and sequence similarity. We propose that orf25 is the plant b subunit based on structural similarity and its presence in the F_O complex. Chimerics of orf25, orfB, subunit 9 and subunit 6 have been associated with cytoplasmic male sterility in a variety of plant species, our additional findings now place all these proteins in the same protein complex.     

Genes encoding the alpha, gamma, delta, and four F0 subunits of ATP synthase constitute an operon in the cyanobacterium Anabaena sp. strain PCC 7120.

A cluster of genes encoding subunits of ATP synthase of Anabaena sp. strain PCC 7120 was cloned, and the nucleotide sequences of the genes were determined. This cluster, denoted atp1, consists of four F0 genes and three F1 genes encoding the subunits a (atpI), c (atpH), b' (atpG), b (atpF), delta (atpD), alpha (aptA), and gamma (atpC) in that order. Closely linked upstream of the ATP synthase subunit genes is an open reading frame denoted gene 1, which is equivalent to the uncI gene of Escherichia coli. The atp1 gene cluster is at least 10 kilobase pairs distant in the genome from apt2, a cluster of genes encoding the beta (atpB) and epsilon (atpE) subunits of the ATP synthase. This two-clustered ATP synthase gene arrangement is intermediate between those found in chloroplasts and E. coli. A unique feature of the Anabaena atp1 cluster is overlap between the coding regions for atpF and atpD. The atp1 cluster is transcribed as a single 7-kilobase polycistronic mRNA that initiates 140 base pairs upstream of gene 1. The deduced translation products for the Anabaena sp. strain PCC 7120 subunit genes are more similar to chloroplast ATP synthase subunits than to those of E. coli.     

The binding of eosin-labeled subunit δ to the isolated chloroplast ATPase, CF1, as revealed by rotational diffusion in solution

We investigated the binding of subunit δ to solubilized chloroplast ATPase. Purified δ was covalently labeled with eosin 5-isothiocyanate and its rotational correlation time was determined by a photoselection technique as a function of added CF₁ (containing δ) and of CF₁(−δ) (lacking δ). In aqueous buffer the rotational correlation time of labeled δ was 33 ns. This is compatible with a rather elongated shape with the dimensions 2b = 100 Å/2a = 28 Å. Binding of δ to CF₁ decreased the rotational correlation time about 10-fold. The result was a biphasic decay of the laser flash-induced absorption anisotropy which was analyzed to yield the proportion of δ (bound to CF₁) relative to δ (free). CF₁(−δ), which completely lacked the δ-subunit, bound one δ (mol/mol) with high affinity (K_d ≈ 100 nM) and at least another δ with about 20-fold lower affinity. The δ-containing CF₁, revealed only the low-affinity site(s) for δ. This was compatible with a 1:1 stoichiometry of δ in isolated CF₁.     

Aspects of Subunit Interactions in the Chloroplast ATP Synthase (I. Isolation of a Chloroplast Coupling Factor 1-Subunit III Complex from Spinach Thylakoids)

A chloroplast ATP synthase complex (CF1 [chloroplast-coupling factor 1]-CF0 [membrane-spanning portion of chloroplast ATP synthase]) depleted of all CF0 subunits except subunit III (also known as the proteolipid subunit) was purified to study the interaction between CF1 and subunit III. Subunit III has a putative role in proton translocation across the thylakoid membrane during photophosphorylation; therefore, an accurate model of subunit inter-actions involving subunit III will be valuable for elucidating the mechanism and regulation of energy coupling. Purification of the complex from a crude CF1-CF0 preparation from spinach (Spinacia oleracea) thylakoids was accomplished by detergent treatment during anion-exchange chromatography. Subunit III in the complex was positively identified by amino acid analysis and N-terminal sequencing. The association of subunit III with CF1 was verified by linear sucrose gradient centrifugation, immunoprecipitation, and incorporation of the complex into asolectin liposomes. After incorporation into liposomes, CF1 was removed from the CF1-III complex by ethylenediaminetetracetate treatment. The subunit III-proteoliposomes were competent to rebind purified CF1. These results indicate that subunit III directly interacts with CF1 in spinach thylakoids.     

Light-induced proton slip and proton leak at the thylakoid membrane

A treatment of leaves of Spinacia oleracea L. with light or with the thiol reagent dithiothreitol in the dark led to partly uncoupled thylakoids. After induction in intact leaves, the partial uncoupling was irreversible at the level of isolated thylakoids. We distinguish between uncoupling by proton slip, which means a decrease of the H⁺/e⁻-ratio due to less efficient proton pumping, and proton leak as defined by enhanced kinetics of proton efflux. Proton slip and proton leak made about equal contributions to the total uncoupling. The enhanced proton efflux kinetics corresponded to reduction of subunit CF₁-γ of the ATP synthase as shown by fluorescence labeling of thylakoid proteins with the sulfhydryl probe 5-iodoacetamido fluorescein. The maximum value of the fraction of reduced CF₁-γ was only 36%, which indicates that in vivo the reduction of CF₁-γ could be limited by fast reoxidation and/or restricted accessibility of CF₁-γ to thioredoxin. Measurements of the ratio ATP/2e indicated that only the uncoupling related to less efficient proton pumping led to a decrease in the ATP yield.     

Subunit interactions within the chloroplast ATP synthase (CF0-CF1) as deduced by specific depletion of CF0 polypeptides

The proton-linked ATP synthase (CF1-CF0) of chloroplasts consists of a catalytic component (CF1) and a membrane-embedded part (CF0) that interacts with CF1 and contains a proton channel. The subunits of CF0 which are involved in binding of CF1 were studied by examining the effect of selective depletion of subunits I, II, and IV of CF0 from the chloroplast ATP synthase on the association of the remaining CF0 subunits with CF1. Dissociated CF0 subunits were identified by sucrose density gradient centrifugation. Removal of subunit IV alone from CF0-CF1 did not cause dissociation of the other CF0 subunits from CF1. Upon removal of both subunits I and IV from CF0-CF1, subunit II also dissociated, but subunit III was still bound to CF1. Thus, at least two subunits of CF0, I and III, directly associate with CF1. Subunit II is unlikely to bind CF1 directly and may associate with subunit I. Although depletion of subunit IV does not cause dissociation of CF0 from CF1, its interaction with CF1 subunits is uncertain.     

Insights into ATP synthase assembly and function through the molecular genetic manipulation of subunits of the yeast mitochondrial enzyme complex

Development of an increasingly detailed understanding of the eucaryotic mitochondrial ATP synthase requires a detailed knowledge of the stoichiometry, structure and function of F(0) sector subunits in the contexts of the proton channel and the stator stalk. Still to be resolved are the precise locations and roles of other supernumerary subunits present in mitochondrial ATP synthase complexes, but not found in the bacterial or chloroplast enzymes. The highly developed system of molecular genetic manipulation available in the yeast Saccharomyces cerevisiae, a unicellular eucaryote, permits testing for gene function based on the effects of gene disruption or deletion. In addition, the genes encoding ATP synthase subunits can be manipulated to introduce specific amino acids at desired positions within a subunit, or to add epitope or affinity tags at the C-terminus, enabling questions of stoichiometry, structure and function to be addressed. Newly emerging technologies, such as fusions of subunits with GFP are being applied to probe the dynamic interactions within mitochondrial ATP synthase, between ATP synthase complexes, and between ATP synthase and other mitochondrial enzyme complexes.     

The partial purification of a factor from Dunaliella salina that causes the rapid in situ inactivation of light-activated chloroplast coupling factor 1 (CF1)

A factor having the expected properties of the in vivo oxidant responsible for inactivating the in vivo light-activated chloroplast coupling factor 1 (CF₁) has been partially purified from cell-free extracts of Dunaliella salina. This factor is highly polar, weakly acidic, and relatively temperature stable. The ability of this factor to inactivate light-activated CF₁ is prevented if it is pretreated with reductants such as dithiothreitol. The factor has virtually no effect on the ethanol-induced, Mg²⁺ -dependent ATPase activity of the isolated CF₁.     

Positioning of protein-coding genes on the soybean chloroplast genome

Seven major plastid protein encoding genes were positioned on the soybean chloroplast DNA by heterologous hybridization. These include the genes for the alpha, beta and epsilon subunits of the CF₁ component of ATP synthase (atpA, atpB and atpE respectively), for subunit III of the CF₀ component of ATP synthase (atpH), for the cytochrome f (cytF), for the ‘32 Kd’ thylakoid protein (psbA), and for the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcL), all of which map in the large single copy region. The atpB, atpE and rbcL genes are located in the region adjacent to one of the segments of the inverted repeat. The genetic organization of the soybean chloroplast DNA is compared to that of other plastid genomes.     

Guanosine and formycin triphosphates bind at non-catalytic nucleotide binding sites of CF1 ATPase and inhibit ATP hydrolysis

Guanosine triphosphate and formycin triphosphate (FTP) in the presence of excess Mg²⁺ can bind to empty non-catalytic sites of spinach chloroplast ATPase (CF₁). This results in a greatly reduced capacity for ATP hydrolysis compared to the enzyme with non-catalytic sites filled with ATP. With two GTP bound at non-catalytic sites the inhibition is about 90%; with two FTP bound about 80% inhibition is obtained. Binding and release of the nucleotides from the non-catalytic sites are relatively slow processes. Exposure of CF₁ with one or two empty non-catalytic sites to 5–10 μM FTP or GTP for 15 min suffices for about 50% of the maximum inhibition. Reactivation of CF₁ after exposure to higher FTP or GTP concentrations requires long exposure to 2 μM EDTA. The findings show that, contrary to previous assumptions, GTP can bind tightly to non-catalytic sites of CF₁. They suggest that the presence of adenine nucleotides at non-catalytic sites might be essential for high catalytic capacity of the F₁ ATPases.     

[I]Iodonaphtylazide labeling selectively a cysteine residue in the F0 of the ATP-synthase from E. coli is unsuitable for topographic studies of membrane proteins

The ATP synthase from E. coli was reacted with the hydrophobic photolabel [¹²⁵I]iodonaphtylazide. Subunit b in the F₀-part was selectively labelled. Label was traced back to the single cysteine₂₁ in subunit b. Thus the reactive intermediate of INA generated by photolysis had a high preference for nucleophiles. Due to this high selectivity the detection of membrane spanning peptide segments by labelling with INA is not reliable.     

Molecular characterization of two point mutants in the chloroplast atpB gene of the green alga Chlamydomonas reinhardtii defective in assembly of the ATP synthase complex

Two point mutants of Chlamydomonas reinhardtii, previously found by recombination and complementation analysis to map in the chloroplast atpB gene encoding the beta subunit of the CF1/CF0 ATP synthase, are here shown to be missense alterations near the 5' end of that gene. One mutant (ac-u-c-2-9) has a change at amino acid position 47 of the beta subunit from leucine (CTA) to arginine (CGA). In the second mutant (ac-u-c-2-29), the codon AAA (lysine) is changed to AAC (asparagine) at position 154. Spontaneous revertants of each mutant were isolated that restore the original wild type base pair. Northern analysis of total RNA and in vivo pulse labeling followed by immunoprecipitation reveals that both mutant atpB genes are transcribed and translated normally. However, immunoblots show that the amount of beta subunit associated with mutant thylakoids is only approximately 3% of that seen in wild type and that the CF1 alpha and gamma subunits are missing entirely. The disruption of ATP synthase complex assembly in these mutants is much more severe than in Escherichia coli beta subunit gene point mutants, which retain significant amounts of alpha and beta subunits on their membranes (Noumi, T., Oka, N., Kanazawa, H., and Futai, M. (1986) J. Biol. Chem. 261, 7070-7075). These results support the hypothesis that there are differences in assembly of the ATP synthase between E. coli and chloroplasts. In particular they indicate that beta must be present for assembly of the alpha and gamma subunits of CF1 onto chloroplast membranes.     

The Drosophila PROS-28.1 gene is a member of the proteasome gene family

In the present communication, we report the identification of a new gene family which encodes the protein subunits of the proteasome. The proteasome is a high-M_r complex possessing proteolytic activity. Screening a Drosophila λgt11 cDNA expression library with the proteasome-specific antibody N19-28 we isolated a clone encoding the 28-kDa No. 1 proteasome protein subunit. In accordance with the nomenclature of proteasome subunits in Drosophila, the corresponding gene is designated PROS-28.1, and it encodes an mRNA of 1.1 kb with an open reading frame of 249 amino acids (aa). Genomic Southern-blot hybridization shows PROS-28.1 to be a member of a family of related genes. Analysis of the predicted aa sequence reveals a potential nuclear targeting signal, a potential site for tyrosine kinase and a potential cAMP/cGMP-dependent phosphorylation site. The aa sequence comparison of the products of PROS-28.1 and PROS-35 with the C2 proteasome subunit of rat shows a strong sequence similarity between the different proteasome subunits. The data suggest that at least a subset of the proteasome-encoding genes belongs to a family of related genes (PROS gene family) which may have evolved from a common ancestral PROS gene.