The ATP synthase of Halobacterium salinarium (halobium) is an archaebacterial type as revealed from the amino acid sequences of its two major subunits.

The head piece of the A-type ATP synthase in an extremely halophilic archaebacterium, namely Halobacterium salinarium (halobium), is composed of two kinds of subunit, alpha and beta, and is associated with ATP-hydrolyzing activity. The genes encoding these subunits with hydrolytic activity have been cloned and sequenced. The putative amino acid sequences of the alpha and beta subunits deduced from the nucleotide sequences of the genomic DNA consist of 585 and 471 residues, respectively. The amino acid sequence of the alpha subunit of the halobacterial ATPase is 63 and 49% identical to the alpha subunits of ATPases from two other archaebacteria, Methanosarcina barkeri and Sulfolobus acidocaldarius, respectively. The sequence of the beta subunit is 66 and 55% identical to the beta subunits from these respective organisms. The homology between the alpha and beta subunits is around 30%. In contrast, the sequences of the halobacterial ATPase is less than 30% identical to F1 ATPase when any combination of subunits is considered. However, they are greater than 50% identical to a eukaryotic vacuolar ATPase when alpha and a, beta and b combinations are considered. These data fully confirm the first demonstration of this kind of relationship which was achieved by immunoblotting with an antibody raised against the halobacterial ATPase. We concluded that the archaebacterial ATP synthase is an A-type and not an F-type ATPase. This classification is also demonstrated by a "rooted" phylogenetic tree where halobacteria locate close to other archaebacteria and eukaryotes and distant from eubacteria.     

The genes encoding the two carboxyltransferase subunits of Escherichia coli acetyl-CoA carboxylase.

We report characterization of the component proteins and molecular cloning of the genes encoding the two subunits of the carboxyltransferase component of the Escherichia coli acetyl-CoA carboxylase. Peptide mapping of the purified enzyme component indicates that the carboxyltransferase component is a complex of two nonidentical subunits, a 35-kDa alpha subunit and a 33-kDa beta subunit. The alpha subunit gene encodes a protein of 319 residues and is located immediately downstream of the polC gene (min 4.3 of the E. coli genetic map). The deduced amino acid composition, molecular mass, and amino acid sequence match those determined for the purified alpha subunit. Six sequenced internal peptides also match the deduced sequence. The amino-terminal sequence of the beta subunit was found within a previously identified open reading frame of unknown function called dedB and usg (min 50 of the E. coli genetic map) which encodes a protein of 304 residues. Comparative peptide mapping also indicates that the dedB/usg gene encodes the beta subunit. Moreover, the deduced molecular mass and amino acid composition of the dedB/usg-encoded protein closely match those determined for the beta subunit. The deduced amino acid sequences of alpha and beta subunits show marked sequence similarities to the COOH-terminal half and the NH2-terminal halves, respectively, of the rat propionyl-CoA carboxylase, a biotin-dependent carboxylase that catalyzes a similar carboxyltransferase reaction reaction. Several conserved regions which may function as CoA-binding sites are noted.     

Isolation of genes encoding the Neurospora vacuolar ATPase. Analysis of vma-1 encoding the 67-kDa subunit reveals homology to other ATPases

The vacuolar membrane of Neurospora crassa contains a H+-translocating ATPase composed of at least three subunits with approximate molecular weights of 70,000, 60,000, and 15,000. Both genomic and cDNA clones encoding the largest subunit, which appears to contain the active site of the enzyme, have been isolated and sequenced. The gene for this subunit, designated vma-1, contains six small introns (60-131 base pairs) and encodes a hydrophilic protein of 607 amino acids, Mr 67,121. Within the sequence is a putative nucleotide-binding region, consistent with the proposal that this subunit contains the site of ATP hydrolysis. This 67-kDa polypeptide shows high homology (62% identical residues overall and 84% in the middle of the protein) to the analogous polypeptide of a higher plant vacuolar ATPase. The hypothesis that the vacuolar ATPase is related to F0F1 ATPases is strongly supported by the finding of considerable homology between the 67-kDa subunit of the Neurospora vacuolar ATPase and both the alpha and beta subunits of F0F1 ATPases.     

Cloning and sequencing of the genes for A and B subunits of the V-type Na(+)-ATPase of a facultatively anaerobic alkaliphile

The structural genes for A and B subunits of the V-type Na(+)-ATPase from a facultatively anaerobic alkaliphile (Amphibacillus sp.), strain M-12, were cloned and sequenced. Transformation of Escherichia coli with the genes overexpressed two proteins, which crossreacted with an antiserum against A and B subunits of the V-type Na(+)-ATPase from Enterococcus hirae. The deduced amino acid sequence (594 amino acids; Mr, 66,144) of A subunit of the M-12 enzyme exhibited 73%, 51%, 49% and 53% identities with those of V-type ATPases from E. hirae, Thermus thermophilus, Neurospora crassa and Drosophila melanogaster, respectively. The amino acid sequence (458 amino acids; Mr, 51,308) of B subunit of the M-12 enzyme was 74%, 53%, 52% and 54% identical with those of the ATPases from E. hirae, T. thermophilus, N. crassa and D. melanogaster, respectively. The fact indicates that the amino acid sequences of A and B subunits of the M-12 enzyme exhibit significantly higher homologies with those of the E. hirae Na(+)-ATPase as compared with those of the H(+)-ATPases from T. thermophilus, N. crassa and D. melanogaster.     

High performance liquid chromatography purification and amino terminal sequence analysis of the subunits of bovine heart mitochondrial F1-ATPase

All five subunits of bovine heart mitochondrial F1-ATPase have been isolated by reverse-phase HPLC and NH2-terminal sequences determined by gas phase Edman degradations. Bovine gamma exhibits 16 identities in the first 30 residues compared with the NH2-terminus of gamma from E.coli F1. Bovine delta exhibit about 27% identity with residues 28-59 of precursor delta from N.crassa and in the first six residues is identical with delta from S.cerevisiae. Approximately half of bovine epsilon has been sequenced. Possibly significant sequence similarities exist between bovine gamma and epsilon and kinase-related gene and oncogene products. The bovine alpha subunit has a blocked NH2-terminus.     

Topological studies suggest that the pathway of the protons through F0 is provided by amino acid residues accessible from the lipid phase

The structure of the F0 part of ATP synthases from E. coli and Neurospora crassa was analyzed by hydrophobic surface labeling with [125I]TID. In the E. coli F0 all three subunits were freely accessible to the reagent, suggesting that these subunits are independently integrated in the membrane. Labeled amino acid residues were identified by Edman degradation of the dicyclohexylcarbodiimide binding (DCCD) proteins from E. coli and Neurospora crassa. The very similar patterns obtained with the two homologous proteins suggested the existence of tightly packed alpha-helices. The oligomeric structure of the DCCD binding protein appeared to be very rigid since little, if any, change in the labeling pattern was observed upon addition of oligomycin or DCCD to membranes from Neurospora crassa. When membranes were pretreated with DCCD prior to the reaction with [125I]TID an additionally labeled amino acid appeared at the position of Glu-65 which binds DCCD covalently, indicating the location of this inhibitor on the outside of the oligomer. It is suggested that proton conduction occurs at the surface of the oligomer of the DCCD binding protein. Possibly this oligomer rotates against the subunit alpha or beta and thus enables proton translocation. Conserved residues in subunit alpha, probably located in the lipid bilayer, might participate in the proton translocation mechanism.     

Characterization of the subunits of beta-conglycinin

Four subunits of beta-conglycinin were purified from soybean cultivar CX 635-1-1-1, and were designated alpha, alpha', beta, and beta' in accordance with nomenclature proposed by Thanh and Shibasaki [(1977) Biochim. Biophys. Acta 490, 370-384]. Of these subunits, beta' has not previously been reported or characterized. Consistent with the low levels of methionine in these proteins, cyanogen bromide cleavage of alpha', alpha, and beta' subunits produced only a few fragments. The beta subunit contains no methionine and was not cleaved by cyanogen bromide. The NH2-terminal amino acid sequences of the alpha and alpha' subunits are homologous, and each has valine at its amino terminus. The beta subunit has a very different NH2-terminal sequence from those of the alpha and alpha' subunits, and has leucine at its amino terminus. The NH2-terminal sequence of the beta' subunit could not be determined, as it appeared to be blocked to Edman degradation. Although alpha and alpha' subunits have similar NH2-terminal sequences, they differ in the number of methionine residues and so yielded different numbers of cyanogen bromide fragments. Two cyanogen bromide fragments (CB-1 and CB-2) were purified from the alpha subunit. CB-1 originated from the NH2-terminal end of the subunit. The amino acid sequence of CB-2 was identical to that predicted from the nucleotide sequence of cDNA clone pB36. The insert in pB36 encoded 216 amino acids from the COOH-terminal end of the alpha subunit and contained a 138-bp trailer sequence which was followed by a poly-(A) tail. Maps showing the relative positions of methionine residues and carbohydrate moieties in the alpha and alpha' subunits were drawn, based on primary sequence data, and the size and carbohydrate content of the CNBr fragments derived from the subunits.     

Molecular cloning of genes encoding major two subunits of a eubacterial V-type ATPase from Thermus thermophilus.

The atpAB genes which encode the alpha and beta subunits of membrane ATPase from a thermophilic eubacterium, Thermus thermophilus HB8, were cloned. The deduced amino-acid sequences of the alpha subunit (583 amino acids) and the beta subunit (478 amino acids) are only moderately similar to the alpha beta subunits of the F0F1-ATPases, while they are highly similar to the major two subunits of the V-type ATPases, a family of ATPases which have been so far found in eukaryotic endomembrane vacuolar vesicles and archaebacterial plasma membranes. Thus, T. thermophilus ATPase belongs to the V-type ATPase family, even though this bacterium is a eubacterium. The hypothesis that the differentiation of an ancestral ATPase into V-type and F0F1-ATPase occurred after the evolution of a primordial cell into archaebacteria and eubacteria should be modified accordingly.     

Isolation and preliminary characterization of the subunits of the terminal component of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4.

The terminal oxygenase component (ISPNAP) of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4 was purified to homogeneity. The protein contained approximately 4 g-atoms each of iron and acid-labile sulfide per mol of ISPNAP, and enzyme activity was stimulated significantly by addition of exogenous iron. The large (alpha) and small (beta) subunits of ISPNAP were isolated by two different procedures. The NH2-terminal amino acid sequences of the alpha and beta subunits were identical to the deduced amino acid sequences reported for the ndoB and ndoC genes from P. putida NCIB 9816 and almost identical to the NH2-terminal amino acid sequences determined for the large and small subunits of ISPNAP from P. putida G7. Gel filtration in the presence of 6 M urea gave an alpha subunit with an absorption maximum at 325 nm and broad absorption between 420 and 450 nm. The alpha subunit contained approximately 2 g-atoms each of iron and acid-labile sulfide per mol of the subunit. The beta subunit did not contain iron or acid-labile sulfide. These results, taken in conjunction with the deduced amino acid sequences of the large subunits from several iron-sulfur oxygenases, indicate that each alpha subunit of ISPNAP contains a Rieske [2Fe-2S] center.     

Conformational interactions between alpha and beta subunits in the F1 ATPase of Escherichia coli as shown by chemical modification of uncA401 and uncD412 mutant enzymes

In contrast to wild-type F1 adenosine triphosphatase, the beta subunits of soluble ATPase from Escherichia coli mutant strains AN120 (uncA401) and AN939 (uncD412) were not labeled by the fluorescent thiol-specific reagents 5-iodoacetamidofluorescein, 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid or 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenzo-2-oxa-1,3-diazole. The mutation in the alpha subunit (uncA401) of F1 ATPase thus influences the accessibility of the single cysteinyl residue in the beta subunit. Following reaction of ATPase with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole or N,N'-dicyclohexylcarbodiimide, the alpha and beta subunits of the uncA401, but not of the uncD412 mutant F1 ATPase were intensely labeled by a fluorescent thiol reagent. The mutation in the beta subunit (uncD412) thus influences the accessibility of the cysteinyl residues in the alpha subunit. In other work [Stan-Lotter, H. and Bragg, P.D. (1986) Arch. Biochem. Biophys. 248] we have shown that 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid react with a different beta subunit from that labeled by N,N'-dicyclohexylcarbodiimide. This asymmetry with respect to modification by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and N,N'-dicyclohexylcarbodiimide was seen in both mutant enzymes. In addition, the modification of one beta subunit of the uncA401 F1 ATPase induced the previously unreactive sulfhydryl group of another beta subunit to react with 2-(4'-iodoacetamidoanilino-naphthalene-6-sulfonic acid. These results provide evidence for at least three types of conformational interactions of the major subunits of F1 ATPase: from alpha to beta, from beta to alpha, and from beta to beta. As in wild-type ATPase, labeling of membrane-bound unc mutant ATPase by a fluorescent thiol reagent modified the alpha subunits. This suggests that a conformational change of yet a different type occurs when the enzyme binds to the membrane.     

Amino acid and nucleotide sequence homologies among E. coli RNA polymerase core enzyme subunits, DNA primase, elongation factor Tu, F1-ATPase alpha, ribosomal protein L3, DNA polymerase I, T7 phage DNA polymerase, and MS2 phage RNA replicase beta subunit

The 330 residue-long N-terminal domains (NTDs) of beta and beta' subunits of the Escherichia coli RNA polymerase (RPase) core enzyme were found to be significantly homologous to the entire length of its alpha subunit. The C-terminal domains (CTDs) of the RPase beta subunit and DNA primase (dnaG protein) were not only strongly homologous to each other but also considerably homologous to the RPase alpha, suggesting that an alpha subunit-like enzyme must have been commonly ancestral to core enzyme subunits and primase. The N-terminal region (NTR) of RPase alpha was also found to show significant homologies with NTRs of the E. coli EF-Tu and F1-ATPase alpha subunit, and a possible weak homology with ribosomal protein L3. A most important finding was that the C-terminal regions (CTRs) of DNA polymerase (DPase) I, T7 phage DPase and MS2 phage RNA replicase beta subunit are closely homologous with one another. These CTRs showed considerable homologies to RPase alpha NTD and RPase beta CTD. These conclusions are based on statistical evaluations of homologies in base and/or amino acid sequence alignments.     

Sulfhydryl groups of the F1 adenosine triphosphatase of Escherichia coli and the stoichiometry of the subunits

The distribution and total number of sulfhydryl groups present in the F1 adenosine triphosphatase of Escherichia coli were used to calculate the stoichiometry of the alpha-delta subunits. Titration with 5,5'-dithiobis (2-nitrobenzoate) gave 19.1 +/- 2.2 sulfhydryl groups/mol ATPase. Labeling with [14C]iodoacetamide and [14C]N-ethylmaleimide showed that 11.9, 3.1, 1.9, and 1.8 sulfhydryl groups per molecule of ATPase were associated with the alpha, beta, gamma, and delta subunits, respectively. The epsilon subunit was not labeled. Application of the method of Creighton [Nature (London) (1980) 284, 487-489] showed that 4, 1, and 2 sulfhydryl groups were present in the alpha, beta, and gamma subunits, respectively. This, together with published data for the delta subunit, allowed a subunit stoichiometry of alpha 3 beta 3 gamma delta to be calculated. The presence of four cysteinyl residues in the alpha subunit, as shown by several different methods, does not agree with the results of DNA sequencing of the ATPase genes [H. Kanazawa, T. Kayano, K. Mabuchi, and M. Futai (1981) Biochem. Biophys. Res. Commun. 103, 604-612; N. J. Gay and J. E. Walker (1981) Nucl. Acids Res. 9, 2187-2194] where three cysteinyl residues/alpha subunit have been found. It is suggested that post-translational modification of the alpha subunit to add a fourth cysteinyl residue might occur.     

Molecular cloning of the beta-subunit of a possible non-F0F1 type ATP synthase from the acidothermophilic archaebacterium, Sulfolobus acidocaldarius

The gene which encodes the beta subunit of the novel membrane-associated ATPase has been identified and characterized. The beta subunit, which is most likely the soluble part of the non-F0F1 type H+-ATPase, was obtained from the archaebacterium, Sulfolobus acidocaldarius. In terms of its location, it follows just after the gene for its alpha subunit. It is comprised of 1398 nucleotides, corresponding to a protein of 465 amino acids, and the consensus sequence in the nucleotide binding proteins is poorly conserved. Together with previously described results, the distant homology of the S. acidocaldarius ATPase alpha and beta subunits when compared to those of F0F1-ATPases indicates that this archaebacterial ATPase belongs to an ion-translocating ATPase family uniquely different than F0F1-ATPases even if S. acidocaldarius ATPase and F0F1-ATPases have been derived from a common ancestral ATPase.     

The membrane-associated ATPase from Sulfolobus acidocaldarius is distantly related to F1-ATPase as assessed from the primary structure of its alpha-subunit

Isolation of novel membrane-associated ATPases, presumably soluble parts of the H+-ATPases, from archaebacteria has been recently reported, and their properties were found to be significantly different from the usual F1-ATPase. In order to assess the relationship of the archaebacterial ATPases to the F1-ATPases and other known ATPases, the amino acid sequence of the alpha subunit of the ATPase from Sulfolobus acidocaldarius, an acidothermophilic archaebacterium, was compared with the sequences of other ATPases. The gene encoding its alpha subunit was cloned from the genomic library of S. acidocaldarius, and the nucleotide sequence was determined. The 591-amino acid sequence deduced from the nucleotide sequence contains a small number of short stretches that shows sequence similarity to the alpha and beta subunits of F1-ATPase. However, the overall similarity is too weak to consider it to be a typical member of the F1-ATPase family when the highly conserved sequences of the F1-ATPase subunits among various organisms are taken into account. Moreover, most of these stretches overlap the consensus sequences that are commonly found in some nucleotide-binding proteins. There is no significant sequence similarity to the ion-translocating ATPases, which form phosphorylated intermediates, such as animal Na+,K+-ATPases. Thus, the S. acidocaldarius ATPase and probably other archaebacterial ATPases also appear to belong to a new group of ion-translocating ATPases that has only a distant relationship to F1-ATPase.     

Thiol modification as a probe of conformational forms of the F1 ATPase of Escherichia coli and of the structural asymmetry of its beta subunits

The sulfhydryl groups of soluble and membrane-bound F1 adenosine triphosphatase of Escherichia coli were modified by reaction with the fluorescent thiol reagents 5-iodoacetamidofluorescein, 2-[(4'-iodoacetamido)anilino]naphthalene-6-sulfonic acid 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenzo-2-oxa-1,3-d iaz ole and 2-[(4'-maleimidyl)anilino]naphthalene-6-sulfonic acid. Whereas gamma and delta subunits were always labeled by these reagents, the beta subunit reacted preferentially in the soluble enzyme, and the alpha subunit in the membrane-bound enzyme. This suggests that the soluble enzyme undergoes a conformational change on binding to the membrane. The three beta subunits of the soluble ATPase did not react with chemical reagents in a similar manner. One beta subunit was cross-linked to the epsilon subunit on treatment of the ATPase with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide, as observed previously by L?tscher et al. [Biochemistry (1984) 23, 4134-4140]. A second beta subunit, which did not cross-link to the epsilon subunit, was modified preferentially by the fluorescent thiol reagents and by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. The third beta subunit was less chemically reactive than the others. Both alpha and beta subunits of the soluble 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole-modified enzyme were labeled by the fluorescent thiol reagents. Thus, the modified enzyme, which is inactive, probably has a different conformation from the native soluble ATPase.     

Pike eel (Muraenesox cinereus) gonadotropin. Amino acid sequences of both alpha and beta subunits

The amino acid sequences of pike eel gonadotropin alpha and beta subunits have been determined by standard sequencing analytical methods. The alpha subunit is composed of 93 amino acid residues while the beta subunit comprises 113 amino acid residues. All the invariant half-cystine residues are in the same positions as those found in other gonadotropins. It is noteworthy that the first, putative glycosylation site (Asn56) found in the alpha subunit of other gonadotropins was replaced by Asp56 in the alpha subunit of pike eel gonadotropin. Similarity analyses indicate that both subunits are structurally more similar to other known fish gonadotropin subunits than to those of the mammalian gonadotropins.     

Subunit location and sequences of the cysteinyl peptides of pig heart NAD-dependent isocitrate dehydrogenase

Pig heart NAD-dependent isocitrate dehydrogenase has a subunit structure consisting of alpha 2 beta gamma, with the alpha subunit exhibiting a molecular weight of 39,000 and the beta and gamma each having molecular weights of 41,000. The amino-terminal sequences (33-35 residues) and the cysteinyl peptide sequences have now been determined by using subunits separated by chromatofocusing or isoelectric focusing and electroblotting. Displacement of the N-terminal sequence of the alpha subunit by 11-12 amino acids relative to that of the larger beta and gamma subunits reveals a 17 amino acid region of great similarity in which 10 residues are identical in all three subunits. The complete enzyme has 6.0 free SH groups per average subunit of 40,000 daltons, but yields 15 distinguishable cysteines in isolated tryptic peptides. Six distinct cysteines in sequenced peptides have been located in the alpha subunit. The beta and gamma subunits contain seven and five cysteines, respectively, with tryptic peptides containing three cysteines being common to the beta and gamma subunits. The three subunits appear to be closely related, but beta and gamma are more similar to each other than either is to the alpha subunit. The NAD-specific isocitrate dehydrogenase from pig heart has been shown to have 2 binding sites/enzyme tetramer for isocitrate, manganous ion, NAD+, and the allosteric activator ADP [Colman, R. F. (1983) Pept. Protein Rev. 1, 41-69]. It is proposed that the catalytically active tetrameric enzyme is organized as a dimer of dimers in which the alpha beta and alpha gamma dimers are nonidentical but functionally similar.     

Isolation and sequencing of cDNA clones encoding alpha and beta subunits of Drosophila melanogaster casein kinase II.

Cloned cDNAs encoding both subunits of Drosophila melanogaster casein kinase II have been isolated by immunological screening of lambda gt11 expression libraries, and the complete amino acid sequence of both polypeptides has been deduced by DNA sequencing. The alpha cDNA contained an open reading frame of 336 amino acid residues, yielding a predicted molecular weight for the alpha polypeptide of 39,833. The alpha sequence contained the expected semi-invariant residues present in the catalytic domain of previously sequenced protein kinases, confirming that it is the catalytic subunit of the enzyme. Pairwise homology comparisons between the alpha sequence and the sequences of a variety of vertebrate protein kinase suggested that casein kinase II is a distantly related member of the protein kinase family. The beta subunit was derived from an open reading frame of 215 amino acid residues and was predicted to have a molecular weight of 24,700. The beta subunit exhibited no extensive homology to other proteins whose sequences are currently known.     

A novel site on gamma 3 subunits important for assembly of GABA(A) receptors

Gamma-aminobutyric acid, type A (GABA(A)) receptors are ligand-gated chloride channels and are the major inhibitory transmitter receptors in the central nervous system. The majority of these receptors is composed of two alpha, two beta, and one gamma subunits. To identify sequences important for subunit assembly, we generated C-terminally truncated and chimeric gamma(3) constructs. From their ability to associate with full-length alpha(1) and beta(3) subunits, we concluded that amino acid sequence gamma(3)(70-84) either directly interacts with alpha(1) or beta(3) subunits or stabilizes a contact site elsewhere in the protein. The observation that this sequence contains amino acid residues homologous to gamma(2) residues contributing to the benzodiazepine-binding site at the alpha(1)/gamma(2) interface suggested that in alpha(1)beta(3)gamma(3) receptors the sequence gamma(3)(70-84) is located at the alpha(1)/gamma(3) interface. In the absence of alpha(1) subunits this sequence might allow assembly of beta(3) with gamma(3) subunits. Other experiments indicated that sequences gamma(3)(86-95) and gamma(3)(94-107), which are homologous to previously identified sequences important for assembly of gamma(2) subunits, are also important for assembly of gamma(3) subunits. This indicates that during assembly of the GABA(A) receptor, more than one N-terminal sequence is important for binding to the same neighboring subunit. Whether the three sequences investigated are involved in direct interaction or stabilize other regions involved in intersubunit contacts has to be further studied.     

Stable structure of thermophilic proton ATPase beta subunit

F1-ATPase is the major enzyme for ATP synthesis in mitochondria, chloroplasts, and bacterial plasma membranes. F1-ATPase obtained from thermophilic bacterium PS3 (TF1) is the only ATPase which can be reconstituted from its primary structure. Its beta subunit constitutes the catalytic site, and is capable of forming hybrid F1's with E. coli alpha and gamma subunits. Since the stability of TF1 resides in its primary structure, we cloned a gene coding for TF1, and the primary structure of the beta subunit was deduced from the nucleotide sequence of the gene to compare the sequence with those of beta's of three major categories of F1's; prokaryotic membranes, chloroplasts, and mitochondria. The following results were obtained. Homology: The primary structure of the TF1 beta subunit (473 residues, Mr = 51,995.6) showed 89.3% homology with 270 residues which are identical in the beta subunits from human mitochondria, spinach chloroplasts, and E. coli. It contained regions homologous to several nucleotide-binding proteins. Secondary structure: The deduced alpha-helical (30.1%) and beta-sheet (22.3%) contents were consistent with those determined from the circular dichroism spectra. Residues forming reverse turns (Gly and Pro) were highly conserved among the F1 beta subunits. Substituted residues and stability of TF1: We compared the amino acid sequence of the TF1 beta subunit with those of the other F1 beta subunits mentioned above. The observed substitutions in the thermophilic subunit increased its propensities to form secondary structures, and its external polarity to form tertiary structure. Codon usage: The codon usage of the TF1 beta gene was found to be unique. The changes in codons that achieved these amino acid substitutions were much larger than those caused by minimal mutations, and the third letters of the optimal codons were either guanine or cytosine, except in codons for Gln, Lys, and Glu.