Evidence for intersubunit communication during acetyl-CoA cleavage by the multienzyme CO dehydrogenase/acetyl-CoA synthase complex from Methanosarcina thermophila. Evidence that the beta subunit catalyzes C-C and C-S bond cleavage

The carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) from Methanosarcina thermophila is part of a five-subunit complex consisting of alpha, beta, gamma, delta, and epsilon subunits. The multienzyme complex catalyzes the reversible oxidation of CO to CO(2), transfer of the methyl group of acetyl-CoA to tetrahydromethanopterin (H(4)MPT), and acetyl-CoA synthesis from CO, CoA, and methyl-H(4)MPT. The alpha and epsilon subunits are required for CO oxidation. The gamma and delta subunits constitute a corrinoid iron-sulfur protein that is involved in the transmethylation reaction. This work focuses on the beta subunit. The isolated beta subunit contains significant amounts of nickel. When proteases truncate the beta subunit, causing the CODH/ACS complex to dissociate, the amount of intact beta subunit correlates directly with the EPR signal intensity of Cluster A and the activity of the CO/acetyl-CoA exchange reaction. Our results strongly indicate that the beta subunit harbors Cluster A, a NiFeS cluster, that is the active site of acetyl-CoA cleavage and assembly. Although the beta subunit is necessary, it is not sufficient for acetyl-CoA synthesis; interactions between the CODH and the ACS subunits are required for cleavage or synthesis of the C-C bond of acetyl-CoA. We propose that these interactions include intramolecular electron transfer reactions between the CODH and ACS subunits.     

The epsilon subunit of Escherichia coli coupling factor 1 is required for its binding to the cytoplasmic membrane.

The coupling factor, F1-ATPase of Escherichia coli (ECF1) contains five different subunits, alpha, beta, gamma, delta, and epsilon. Properties of delta-deficient ECF1 have previously been described. F1-ATPase containing only the alpha, beta, and gamma subunits was prepared from E. coli by passage of delta-deficient ECF1 through an affinity column containing immobilized antibodies to the epsilon subunit. The delta, epsilon-deficient enzyme has normal ATPase activity but cannot bind to ECF1-depleted membrane vesicles. Both the delta and epsilon subunits are required for the binding of delta, epsilon-deficient ECF1 to membranes and the restoration of oxidative phosphorylation. Either delta or epsilon will bind to the deficient enzyme to form a four-subunit complex. Neither four-subunit enzyme binds to depleted membranes. The epsilon subunit, does, however, slightly improve the binding affinity between delta and delta-deficient enzyme suggesting a possible interaction between the two subunits. Neither subunit binds to trypsin-treated ECF1, which contains only the alpha and beta subunits. A role for gamma in the binding of epsilon to F1 is suggested. epsilon does not bind to ECF1-depleted membranes. Therefore, the in vitro reconstitution of depleted membranes requires an initial complex formation between epsilon and the rest of ECF1 prior to membrane attachment. Reconstitution experiments indicate that only one epsilon is required per functional ECF1 molecule.     

Post-translational modification of actins synthesized in vitro.

It has been shown in two different ways that beta and gamma actins synthesized in vitro are acetylated and that the minor species of actin, delta and epsilon, are nonacetylated forms of beta and gamma actin, respectively. Firstly, additon of acetyl-CoA to the wheat germ system translating poly(A)-containing RNA from unfused rat L6 myoblasts, resulted in an increase in the synthesis of beta and gamma actins at the expense of delta and epsilon actins. Secondly, beta and gamma actins were labeled when synthesized in vitro in the presence of [3H]acetyl-CoA. No label was detectable in delta and epsilon actins. By extrapolation this indicates that beta and gamma actin are acetylated in vivo, probably at the N-terminus. beta and gamma actins synthesized in vivo contain a N tau-methylhistidine residue, but no methylation of beta and gamma actins synthesized in vitro was detectable, using S-[3H]adenosylmethionine as a methyl donor.     

The Evolution of Acetyl-CoA Synthase

Acetyl-coenzyme A synthases (ACS) are Ni-Fe-S containing enzymes found in archaea and bacteria. They are divisible into 4 classes. Class I ACS's catalyze the synthesis of acetyl-CoA from CO2 + 2e-, CoA, and a methyl group, and contain 5 types of subunits (alpha, beta, gamma, delta, and epsilon). Class II enzymes catalyze essentially the reverse reaction and have similar subunit composition. Class III ACS's catalyze the same reaction as Class I enzymes, but use pyruvate as a source of CO2 and 2e-, and are composed of 2 autonomous proteins, an alpha 2 beta 2 tetramer and a gamma delta heterodimer. Class IV enzymes catabolize CO to CO2 and are alpha-subunit monomers. Phylogenetic analyses were performed on all five subunits. ACS alpha sequences divided into 2 major groups, including Class I/II sequences and Class III/IV-like sequences. Conserved residues that may function as ligands to the B- and C-clusters were identified. Other residues exclusively conserved in Class I/II sequences may be ligands to additional metal centers in Class I and II enzymes. ACS beta sequences also separated into two groups, but they were less divergent than the alpha's, and the separation was not as distinct. Class III-like beta sequences contained approximately 300 residues at their N-termini absent in Class I/II sequences. Conserved residues identified in beta sequences may function as ligands to active site residues used for acetyl-CoA synthesis. ACS gamma-sequences separated into 3 groups (Classes I, II, and III), while delta-sequences separated into 2 groups (Class I/II and III). These groups are less divergent than those of alpha sequences. ACS epsilon-sequence topology showed greater divergence and less consistency vis-à-vis the other subunits, possibly reflecting reduced evolutionary constraints due to the absence of metal centers. The alpha subunit phylogeny may best reflect the functional diversity of ACS enzymes. Scenarios of how ACS and ACS-containing organisms may have evolved are discussed.     

Purification of membrane attachment and inhibitory subunits of the proton translocating adenosine triphosphatase from Escherichia coli.

The portion of Escherichia coli adenosine triphosphatase (ATPase) which is peripheral to the membrane (ECFl) is composed of five separate polypeptides referred to as alpha, beta, gamma, delta, and epsilon. Treating purified ECFl with pyridine precipitated the three larger polypeptides (alpha, beta, and gamma), but the two smaller ones (delta and epsilon), which represent only about 10% of ECFl, remained in solution. After removing the pyridine, both delta and epsilon were active and both were obtained in essentially pure form after chromatography on a single molecular-seive column. epsilon strongly inhibited the ATPase activity of ECFl, indicating that epsilon has a regulatory role in the enzyme. epsilon inhibited ECFl missing delta, indicating that delta is not required for inhibition by epsilon. However, enzyme containing just the alpha and beta subunits, which was prepared by treating ECFl with a protease, was fully active hydrolytically but not at all sensitive to inhibition by epsilon. This result suggests that the gamma polypeptide is required for the inhibition of the ATPase by epsilon. delta restored the capacity of ECFl missing delta to recombine with ECFl-depleted membrane vesicles. The ECFl, which became attached to the vesicles by the added delta, was functional in energy transduction, as evidenced by the coupling of ATP hydrolysis to the transhydrogenase reaction in the vesicles. The rebinding of ECFl missing delta was directly proportional to the amount of delta added until all the ECFl receptors in the membranes were occupied. delta may be a stalk which connects the Fl headpiece to the membrane, since the attachment of ECFl to the membrane exhibited an absolute dependence on delta. Although delta is known to have an apparent molecular weight of about 20,000 by gel electrophoresis in the presence of sodium dodecyl sulfate, the active delta eluted from a molecular-seive column with an apparent molecular weight of about 35,000, suggesting that in the active form delta is a dimer or rather elongated in shape. The active epsilon subunit eluted from the same column with an apparent molecular weight of about 16,000.     

Evidence for the existence of protomers in the assembly of encephalomyocarditis virus.

Two capsid precursor subunits, which sediment on glycerol gradients at 13S and 14S, respectively, have been identified in cytoplasmic extracts of encephalomyocarditis virus-infected HeLa cells. The 13S subunit, which was detected after a 10-min pulse label with -3H-labeled amino acids, contained only capsid precursor chain A (mol wt 100,000). When the 10-min pulse label in such cells was chased for 20 min, the A-containing 13S subunit in the cytoplasmic extracts was replaced by a 14S subunit containing equimolar proportions of three chains: epsilon, gamma, and alpha. This (epsilon, gamma, alpha)-containing 14S subunit could be dissociated into 6S subunits with the same polypeptide composition. The sedimentation properties and the polypeptide stoichiometry of these three precursor subunits, when compared with those of the 13S, (beta, gamma, alpha)(5), and 5S, (beta, gamma, alpha), subunits derived by acid dissociation of purified virions, suggest the following structural assignments: 13S, (A)(5); 14S, (epsilon, gamma, alpha)(5), 6S, (epsilon, gamma, alpha). The molecular weights of the individually isolated capsid chains were determined by gel filtration in 6 M guanidine hydrochloride to be: epsilon, 36,000; alpha, 32,000; beta, 29,500; gamma, 26,500; and delta, 7,800. With the exception of the delta-chain, these values are in reasonable agreement with the values previously determined by electrophoresis on sodium dodecyl sulfatepolyacrylamide gels. These data support the hypothesis that picornavirus capsids are assembled from identical protomers according to the following scheme: (A) leads to (A)(5) leads to (epsilon, gamma, alpha)(5) leads to (delta, beta, gamma, alpha)60-n(epsilon, gamma, alpha)n where n is the number of immature protomers per virion.     

Catalytic properties of the F1-adenosine triphosphatase from Escherichia coli K-12 and its genetic variants as revealed by 18O exchanges

We have examined intermediate Pi-water oxygen exchange during [gamma-18O]ATP hydrolysis by the F1 adenosine triphosphatase from Escherichia coli K-12. Water oxygen incorporation into each Pi released was increased as ATP concentration was lowered as observed previously for the same reaction catalyzed by the enzyme from eukaryotic sources. Heterogeneous distributions of 18O in product Pi were produced by coexisting epsilon subunit-replete and epsilon subunit-depleted enzyme molecules. The epsilon-replete enzyme showed a much higher probability for oxygen exchange. These data imply that the epsilon subunit inhibits net ATP hydrolysis by imposing conformational constraints which reduce the cooperative conformational interactions that promote ADP and Pi release. Four enzyme variants altered in alpha or beta subunit structure with reduced net hydrolytic activity showed sharply increased oxygen exchange during ATP hydrolysis. Heterogeneity was apparent in the 18O distribution of the product Pi, however. That behavior could reflect hindered conformational interactions and/or increased affinity of the alpha 3 beta 3 gamma delta complex for the epsilon subunit. In contrast, enzyme from mutant uncA401 showed very little oxygen exchange accompanying hydrolysis of 20 microM ATP. This is the only enzyme so far reported with this unusual property. Its rate limitation appears to be in the hydrolytic rather than the product release step of the catalytic sequence.     

ATPase of Escherichia coli: purification, dissociation, and reconstitution of the active complex from the isolated subunits.

A simple procedure for the purification of Mg2+-stimulated ATPase of Escherichia coli by fractionation with poly(ethylene glycols) and gel filtration is described. The enzyme restores ATPase-linked reactions to membrane preparations lacking these activities. Five different polypeptides (alpha, beta, gamma, delta, epsilon) are observed in sodium dodecyl sulfate electrophoresis. Freezing in salt solutions splits the enzyme complex into subunits which do not possess any catalytic activity. The presence of different subunits is confirmed by electrophoretic and immunological methods. The active enzyme complex can be reconstituted by decreasing the ionic strength in the dissociated sample. Temperature, pH, protein concentration, and the presence of substrate are each important determinants of the rate and extent of reconstitution. The dissociated enzyme has been separated by ion-exchange chromatography into two major fragments. Fragment IA has a molecular weight of about 100000 and contains the alpha, gamma, and epsilon polypeptides. The minor fragment, IB, has about the same molecular weight but contains, besides alpha, gamma, and epsilon, the delta polypeptide. Fragment II, with a molecular weight of about 52000, appears to be identical with the beta polypeptide. ATPase activity can be reconstituted from fragments IA and II, whereas the capacity of the ATPase to drive energy-dependent processes in depleted membrane vesicles is only restored after incubation of these two fractions with fraction IB, which contains the delta subunit.     

F1-ATPase from Micrococcus sp. ATCC 398. Purification by ion-exchange chromatography and further characterization. (Auto)proteolysis and dissociative effects.

The preparation of highly purified F1-ATPase from Micrococcus sp. ATCC 398 by application of DEAE-Sepharose CL-6B chromatography as final step is described. This enzyme consists of five subunits of different molecular weight: alpha (65000), beta (55000),gamma (35000), delta (20000), and epsilon (17000). Disc electrophoresis on 5% polyacrylamide gels removes the epsilon-polypeptide yielding an active ATPase complex with four different subunits: alpha, beta, gamma, delta. Additionally, by variation of the ionic strength delta can (partly) removed allowing the isolation by disc electrophoresis of an active ATPase complex which consists only of three different subunits alpha, beta, and gamma. If the DEAE-Sepharose chromatography is carried out in the absence of diisopropyl phosphofluoridate (auto)proteolysis yields both an active ATPase with the subunits alpha+ (mol. wt 61000), beta, gamma, and delta and an inactive protein complex with the subunits alpha+, beta, gamma, delta, and two additional polypeptides a (mol. wt 38000) and b (mol. wt 23000). The latter two polypeptides are supposedly fragments of alpha+-chains which have become partially cleaved by (auto)proteolysis.     

Hexagonal structure of two-dimensional crystals of the alpha 3 beta 3 complex thermophilic ATP synthase

The highly dissociable alpha 3 beta 3 subunit complex (Mr = 319,582) of thermophilic ATP synthase was crystallized on a mercury surface under oxygen. The two-dimensional crystal was compared with that of TF1 (Mr = 385,351, alpha 3 beta 3 gamma delta epsilon subunit complex) by means of computer image processing. The crystals showed the same hexagonal lattice (a = b = 10 nm), despite the difference in their molecular weights. The color images of the two protein molecules were also hexagonal. However, there was an open hole in the image of the alpha 3 beta 3 complex, where small subunits (gamma, delta, and epsilon) of TF1 may have been located. The structure of this heterohexamer is consistent with that deduced from other physical parameters.     

Structure of the Ni/Fe-S protein subcomponent of the acetyl-CoA decarbonylase/synthase complex from Methanosarcina thermophila at 26-A resolution

The acetyl-CoA decarbonylase/synthase (ACDS) complex is responsible for synthesis and cleavage of acetyl-CoA in methanogens. The complex is composed of five different subunits, with a probable stoichiometry of alpha(8)beta(8)gamma(8)delta(8)epsilon(8). The native molecular mass of a subcomponent of the ACDS complex from Methanosarcina thermophila, the Ni/Fe-S protein containing the 90-kDa alpha and 19-kDa epsilon subunits, was determined by scanning transmission electron microscopy. A value of 218.6 +/- 19.6 kDa (n = 566) was obtained, thus establishing that the oligomeric structure of this subcomponent is alpha(2)epsilon(2). The three-dimensional structure of alpha(2)epsilon(2) was determined at 26-A resolution by analysis of a large number of electron microscopic images of negatively stained, randomly oriented particles. The alpha(2)epsilon(2) subcomponent has a globular appearance, 110 A in diameter, and consists of two large, hemisphere-like masses that surround a hollow internal cavity. The two large masses are connected along one face by a bridge-like structure and have relatively less protein density joining them at other positions. The internal cavity has four main openings to the outside, one of which is directly adjacent to the bridge. The results are consistent with a structure in which the large hemispheric masses are assigned to the two alpha subunits, with epsilon(2) as the bridge forming a structural link between them. The structure of the alpha(2)epsilon(2) subcomponent is discussed in connection with biochemical data from gel filtration, crosslinking, and dissociation experiments and in the context of its function as a major component of the ACDS complex.     

Multiple forms of human glutathione S-transferase and their affinity for bilirubin.

The initial enzymic step in mercapturic acid formation is catalyzed by glutathione S-transferase. Several species of this enzyme, designated as transferases alpha, beta, gamma, delta and epsilon on the basis of increasing isoelectric points, were isolated from human liver. Evidence is presented that each of the purified species is homogeneous with respect to sodium dodecylsulfate-gel electrophoresis. Transferases alpha, beta and epsilon each appear as a single band on gel electrofocusing; transferases gamma and delta are present as two and three bands, respectively, with each band catalytically active. Amino acid analysis indicated the five transferases to be either very closely related or identical in this respect. All enzyme species have a molecular weight of about 48500 and consist of two apparently identical subunits. The spectrum of substrates is the same for each although the enzymes differ slightly in specific activity. As is the case for the rat liver enzymes, each of the human transferases binds bilirubin although this compound is not a substrate.     

Epistatic interactions of deletion mutants in the genes encoding the F1-ATPase in yeast Saccharomyces cerevisiae.

The F1-ATPase is a multimeric enzyme (alpha3 beta3 gamma delta epsilon) primarily responsible for the synthesis of ATP under aerobic conditions. The entire coding region of each of the genes was deleted separately in yeast, providing five null mutant strains. Strains with a deletion in the genes encoding alpha-, beta-, gamma- or delta-subunits were unable to grow, while the strain with a null mutation in epsilon was able to grow slowly on medium containing glycerol as the carbon source. In addition, strains with a null mutation in gamma or delta became 100% rho0/rho- and the strain with the null mutation in gamma grew much more slowly on medium containing glucose. These additional phenotypes were not observed in strains with the double mutations: Delta alpha Delta gamma, Delta beta Delta gamma, Deltaatp11 Delta gamma, Delta alpha Delta delta, Delta beta Delta delta or Deltaatp11 Delta delta. These results indicate that epsilon is not an essential component of the ATP synthase and that mutations in the genes encoding the alpha- and beta-subunits and in ATP11 are epistatic to null mutations in the genes encoding the gamma- and delta-subunits. These data suggest that the propensity to form rho0/rho- mutations in the gamma and delta null deletion mutant stains and the slow growing phenotypes of the null gamma mutant strain are due to the assembly of F1 deficient in the corresponding subunit. These results have profound implications for the physiology of normal cells.     

Biochemical and structural characterization of an essential acyl coenzyme A carboxylase from Mycobacterium tuberculosis

Pathogenic mycobacteria contain a variety of unique fatty acids that have methyl branches at an even-numbered position at the carboxyl end and a long n-aliphatic chain. One such group of acids, called mycocerosic acids, is found uniquely in the cell wall of pathogenic mycobacteria, and their biosynthesis is essential for growth and pathogenesis. Therefore, the biosynthetic pathway of the unique precursor of such lipids, methylmalonyl coenzyme A (CoA), represents an attractive target for developing new antituberculous drugs. Heterologous protein expression and purification of the individual subunits allowed the successful reconstitution of an essential acyl-CoA carboxylase from Mycobacterium tuberculosis, whose main role appears to be the synthesis of methylmalonyl-CoA. The enzyme complex was reconstituted from the alpha biotinylated subunit AccA3, the carboxyltransferase beta subunit AccD5, and the epsilon subunit AccE5 (Rv3281). The kinetic properties of this enzyme showed a clear substrate preference for propionyl-CoA compared with acetyl-CoA (specificity constant fivefold higher), indicating that the main physiological role of this enzyme complex is to generate methylmalonyl-CoA for the biosynthesis of branched-chain fatty acids. The alpha and beta subunits are capable of forming a stable alpha6-beta6 subcomplex but with very low specific activity. The addition of the epsilon subunit, which binds tightly to the alpha-beta subcomplex, is essential for gaining maximal enzyme activity.     

Monoclonal antibody modification of the ATPase activity of Escherichia coli F1 ATPase

Monoclonal antibodies (mAbs) have been made against each of the five subunits of ECF1 (alpha, beta, gamma, delta, and epsilon), and these have been used in topology studies and for examination of the role of individual subunits in the functioning of the enzyme. All of the mAbs obtained reacted with ECF1, while several failed to react with ECF1F0, including three mAbs against the gamma subunit (gamma II, gamma III, and gamma IV), one mAb against delta, and two mAbs against epsilon (epsilon I and epsilon II). These topology data are consistent with the gamma, delta, and epsilon subunits being located at the interface between the F1 and F0 parts of the complex. Two forms of ECF1 were used to study the effects of mAbs on the ATPase activity of the enzyme: ECF1 with the epsilon subunit tightly bound and acting to inhibit activity and ECF1* in which the delta and epsilon subunits had been removed by organic solvent treatment. ECF1* had an ATPase activity under standard conditions of 93 mumol of ATP hydrolyzed min-1 mg-1, cf. an activity of 7.5 units mg-1 for our standard ECF1 preparation and 64 units mg-1 for enzyme in which the epsilon subunit had been removed by trypsin treatment. The protease digestion of ECF1* reduced activity to 64 units mg-1 in a complicated process involving an inhibition of activity by cleavage of the alpha subunit, activation by cleavage of gamma, and inhibition with cleavage of the beta subunit. mAbs to the gamma subunit, gamma II and gamma III, activated ECF1 by 4.4- and 2.4-fold, respectively, by changing the affinity of the enzyme for the epsilon subunit, as evidenced by density gradient centrifugation experiments. The gamma-subunit mAbs did not alter the ATPase activity of ECF1*- or trypsin-treated enzyme. The alpha-subunit mAb (alpha I) activated ECF1 by a factor of 2.5-fold and ECF1F0 by 1.3-fold, but inhibited the ATPase activity of ECF1* by 30%.     

Coupling factor 1 from Escherichia coli lacking subunits delta and epsilon: preparation and specific binding to depleted membranes, mediated by subunits delta or epsilon

A procedure for the preparation of coupling factor 1 (F1) from Escherichia coli lacking subunits delta and epsilon is described. Using chloroform and dimethyl sulfoxide, we can isolate F1 containing only subunits alpha, beta, and gamma [F1(alpha beta gamma)] directly from membrane vesicles in 10-mg quantities. Pure and active subunits delta and epsilon were prepared from five-subunit F1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After addition of these subunits, F1(alpha beta gamma) is as active in reconstituting ATP-dependent transhydrogenase as five-subunit F1. The ATPase activity of F1 (alpha beta gamma) is inhibited by subunit epsilon in a 1:1 stoichiometry to the same extent (approximately equal to 90%) and with the same affinity (Ki = 0.2-0.8 nM) as reported earlier [Dunn, S.D. (1982) J. Biol. Chem. 257, 7354-7359]. In the presence of either delta or epsilon, F1(alpha beta gamma) binds to F1-depleted membrane vesicles and to liposomes containing the membrane sector (F0) of the ATP synthase to an extent commensurate with the F0 content. The binding ratios epsilon/F1 (alpha beta gamma) and probably also delta/F1 (alpha beta gamma) are close to unity. The specific, delta- or epsilon-deficient F1.F0 complexes presumably formed show ATPase activities sensitive to subunit epsilon but not to dicyclohexylcarbodiimide, and no energy-transfer capabilities. Binding studies at different pH values suggest that F1-F0 interactions in the presence of both subunits delta and epsilon are similar to a combination of those mediated by delta or epsilon alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure and subunit stoichiometry of F1-ATPase from bovine mitochondria

The enzyme complex F1-ATPase has been isolated from bovine heart mitochondria by gel filtration of the enzyme released by chloroform from sub-mitochondrial particles. The five individual subunits alpha, beta, gamma, delta and epsilon that comprise the complex have been purified from it, and their amino acid sequences determined almost entirely by direct protein sequence analysis. A single overlap in the gamma-subunit was obtained by DNA sequence analysis of a complementary DNA clone isolated from a bovine cDNA library using a mixture of 32 oligonucleotides as the hybridization probe. The alpha, beta, gamma, delta and epsilon subunits contain 509, 480, 272, 146 and 50 amino acids, respectively. Two half cystine residues are present in the alpha-subunit and one in each of the gamma- and epsilon-chains; they are absent from the beta- and delta-subunits. The stoichiometry of subunits in the complex is estimated to be alpha 3 beta 3 gamma 1 delta 1 epsilon 1 and the molecular weight of the complex is 371,135. Mild trypsinolysis of the F1-ATPase complex, which has little effect on the hydrolytic activity of the enzyme, releases peptides from the N-terminal regions of the alpha- and beta-chains only; the C-terminal regions are unaffected. Sequence analysis of the released peptides demonstrates that the N terminals of the alpha- and beta-chains are ragged. In 65% of alpha-chains, the terminus is pyrrolidone carboxylic acid; in the remainder this residue is absent and the chains commence at residue 2, i.e. lysine. In the beta-subunit a minority of chains (16%) have N-terminal glutamine, or its deamidation product, glutamic acid (6%), or the cyclized derivative, pyrrolidone carboxylic acid (5%). A further 28% commence at residue 2, alanine, and 45% at residue 3, serine. The delta-chains also are heterogeneous; in 50% of chains the N-terminal alanine residue is absent. The sequences of the alpha- and beta-chains show that they are weakly homologous, as they are in bacterial F1-ATPases. The sequence of the bovine delta-subunit of F1-ATPase shows that it is the counterpart of the bacterial epsilon-subunit. The bovine epsilon-subunit is not related to any known bacterial or chloroplast H+-ATPase subunit, nor to any other known sequence. The counterpart of the bacterial delta-subunit is bovine oligomycin sensitivity conferral protein, which helps to bind F1 to the inner mitochondrial membrane.(ABSTRACT TRUNCATED AT 400 WORDS)     

Total reconstitution of DNA polymerase III holoenzyme reveals dual accessory protein clamps

DNA polymerase III holoenzyme (holoenzyme) is the 10-subunit replicase of the Escherichia coli chromosome. In this report, pure preparations of delta, delta', and a gamma chi psi complex are resolved from the five protein gamma complex subassembly. Using these subunits and other holoenzyme subunits isolated from overproducing plasmid strains of E. coli, the rapid and highly processive holoenzyme has been reconstituted from only five pure single subunits: alpha, epsilon, gamma, delta, and beta. The preceding report showed that of the three subunits in the core polymerase, only a complex of alpha (DNA polymerase) and epsilon (3'-5' exonuclease) are required to assemble a processive holoenzyme on a template containing a preinitiation complex (Studwell, P.S., and O'Donnell, M. (1990) J. Biol. Chem. 265, 1171-1178). This report shows that of the five proteins in the gamma complex only a heterodimer of gamma and delta is required with the beta subunit to form the ATP-activated preinitiation complex with a primed template. Surprisingly, the delta' subunit does not form an active complex with gamma but forms a fully active heterodimer complex with the tau subunit (as does delta). Hence, the tau delta' and gamma delta heterodimers are fully active in the preinitiation complex reaction with beta and primed DNA. Holoenzymes reconstituted using the alpha epsilon complex, beta subunit, and either gamma delta or tau delta' are fully processive in DNA synthesis, and upon completing the template they rapidly cycle to a new primed template endowed with a preinitiation complex clamp. Since the holoenzyme molecule contains all of these accessory subunits (gamma, delta, tau, delta', and beta) in all likelihood it has the capacity to form two preinitiation complex clamps simultaneously at two primer termini. Two primer binding components within one holoenzyme may mediate its rapid cycling to multiple primers on the lagging strand and also provides functional evidence for the hypothesis of holoenzyme as a dimeric polymerase capable of simultaneous replication of both leading and lagging strands of a replication fork.     

CD3.TCR1, a human CD3 epitope expressed on viable gamma/delta lymphocytes exclusively.

T lymphocytes express either the alpha/beta or the gamma/delta receptor (TCR) in a mutually exclusive fashion. Both structures are associated on the cell membrane with the CD3 proteins which are thought to transduce signals resulting from antigen recognition. The CD3 complex is present in both alpha/beta and gamma/delta cells and includes at least five proteins (designated gamma, delta, epsilon, zeta and eta). We have developed here a novel mAb, anti-CD3.TCR1, which immunoprecipitates the CD3 molecules from both alpha/beta and gamma/delta cells lysates following solubilization with Triton X-100. While the SDS-PAGE migration profile of the material recognized by either anti-CD3.TCR1 or anti-OKT3 are superimposable in both cell types, this mAb recognizes viable untreated gamma/delta T lymphocytes exclusively. These findings further support the view that molecular interactions within the TCR/CD3 protein complex are distinct in the two T lymphocyte populations.     

Added subunit beta of CF1 as well as gamma/delta/epsilon restore photophosphorylation in partially CF1-depleted thylakoids.

We investigated the ability of subunits beta, gamma, delta, and epsilon of CF1, the F1-ATPase of chloroplasts, to interact with exposed CF0 in EDTA-treated, partially CF1-depleted thylakoid membranes. We measured the ability of subunits beta, gamma, delta, and epsilon to stimulate the rate of photophosphorylation under continuous light and, for subunit beta, also the ability to diminish the proton leakage through exposed CF0 by deceleration of the decay of electrochromic absorption transients under flashing light. The greatest effect was caused by subunit beta, followed by gamma/delta/epsilon. Pairwise combinations of gamma, delta, and epsilon or each of these subunits alone were only marginally effective. Subunit gamma from the thermophilic bacterium PS 3 in combination with chloroplast delta and epsilon was as effective as chloroplast gamma. The finding that the small CF1 subunits in concert and the beta subunit by itself specifically interacted with the exposed proton channel CF0, qualifies the previous concept of subunit delta acting particularly as a plug to the open CF0 channel. The interactions between the channel and the catalytic portion of the enzyme seem to involve most of the small, and at least beta of the large subunits.