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.     

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.     

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.     

Biochemical characterization of the yeast vacuolar H(+)-ATPase

The yeast vacuolar proton-translocating ATPase was isolated by two different methods. A previously reported purification of the enzyme (Uchida, E., Ohsumi, Y., and Anraku, Y. (1985) J. Biol. Chem. 260, 1090-1095) was repeated. This procedure consisted of isolation of vacuoles, solubilization with the zwitterionic detergent ZW3-14, and glycerol gradient centrifugation of the solubilized vacuoles. The fraction with the highest specific activity (11 mumol of ATP hydrolyzed mg-1 min-1) included eight polypeptides of apparent molecular masses of 100, 69, 60, 42, 36, 32, 27, and 17 kDa, suggesting that the enzyme may be more complex than the three-subunit composition proposed from the original purification. The 69-kDa polypeptide was recognized by antisera against the catalytic subunits of two other vacuolar ATPases and labeled with the ATP analog 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, indicating that it contains all or part of the catalytic site. A monoclonal antibody was prepared against this subunit. Under nondenaturing conditions, the antibody immunoprecipitated eight polypeptides, of the same molecular masses as those seen in the glycerol gradient fraction, from solubilized vacuolar vesicles. All eight of these polypeptides are therefore good candidates for being genuine subunits of the enzyme. The structure and function of the yeast vacuolar H+-ATPase were further characterized by examining the inhibition of ATPase activity by KNO3. In the presence of 5 mM MgATP, 100 mM KNO3 inhibited 71% of the ATPase activity of vacuolar vesicles, and the 69- and 60-kDa subunits (and possibly the 42-kDa subunit) were removed from the vacuolar membrane to a similar extent. At concentrations of less than 200 mM KNO3, the stripping of the ATPase subunits and the inhibition of ATPase activity were dependent on the presence of MgATP, suggesting that this is a conformation-specific disassembly of the enzyme. The yeast vacuolar H+-ATPase is a multisubunit enzyme, consisting of a combination of peripheral and integral membrane subunits. Its structure and subunit composition are very similar to other vacuolar ATPase, and it shares some characteristics with the F1F0-ATPases.     

Is the Paracoccus halodenitrificans ATPase a chimeric enzyme?

Membranes from Paracoccus halodenitrificans contain an ATPase that is most active in the absence of NaCl. The most unusual characteristic of the enzyme is its pattern of sensitivity to various inhibitors. Azide and rhodamine 6G, inhibitors of F1F0-ATPases, inhibit ATP hydrolysis as do bafilomycin A1, concanamycin A (folimycin), N-ethylmaleimide, and p-chloromercuriphenylsulfonate which are inhibitors of vacuolar ATPases. This indiscriminate sensitivity suggests that this ATPase may be a hybrid and that caution should be exercised when using inhibition as a diagnostic for distinguishing between F1F0-ATPases and vacuolar ATPases.     

The halobacterial H+-translocating ATP synthase relates to the eukaryotic anion-sensitive H+-ATPase

The H+-translocating ATP synthase of Halobacterium halobium (Y. Mukohata and M. Yoshida (1987) J. Biochem. 102, 797-802) includes a catalytic moiety of 320 kDa which is isolated as an azide-insensitive ATPase (T. Nanba and Y. Mukohata (1987) J. Biochem. 102, 591-598). The polyclonal antibody against this archaebacterial ATPase cross-reacts with the anion-sensitive H+-ATPase of red beet, Beta vulgaris, tonoplast as well as with another archaebacterial ATPase from Sulfolobus acidocaldarius. The affinity is much higher than to F1-ATPase from spinach chloroplasts or to Ca2+-ATPase from sarcoplasmic reticulum of rabbit skeletal muscle.     

F-type or V-type? The chimeric nature of the archaebacterial ATP synthase.

Archaebacterial plasma membranes contain an ATPase acting in vivo as a delta mu H(+)-driven ATP synthase. While functional features and their general structural design are resembling F-type ATPases, primary sequences of the two large polypeptides from the catalytic part are closely related to V-type ATPases from eucaryotic vacuolar membranes. The chimeric nature of archaebacterial ATPase from Sulfolobus was investigated in terms of nucleotide interactions and related to specific sequence parameters in a comparison to well known F- and V-type ATPases. The study disclosed a general difference of F- and V-type ATPases at one class of the nucleotide binding sites.     

Amino acid sequence of the alpha and beta subunits of Methanosarcina barkeri ATPase deduced from cloned genes. Similarity to subunits of eukaryotic vacuolar and F0F1-ATPases

The atpA and atpB genes coding for the alpha and beta subunits, respectively, of membrane ATPase were cloned from a methanogen Methanosarcina barkeri, and the amino acid sequences of the two subunits were deduced from the nucleotide sequences. The methanogenic alpha (578 amino acid residues) and beta (459 amino acid residues) subunits were highly homologous to the large and small subunits, respectively, of vacuolar H+-ATPases; 52% of the residues of the methanogenic alpha subunit were identical with those of the large subunit of vacuolar enzyme of carrot or Neurospora crassa, respectively, and 59, 60, and 59% of the residues of the methanogenic beta subunit were identical with those of the small subunits of N. crassa, Arabidopsis thaliana, and Sacharomyces cerevisiae, respectively. The methanogenic subunits were also highly homologous to the corresponding subunits of Sulfolobus acidocaldarius ATPase. The methanogenic alpha and beta subunits showed 22 and 24% identities with the beta and the alpha subunits of Escherichia coli F1, respectively. Furthermore, important amino acid residues identified genetically in the E. coli enzyme were conserved in the methanogenic enzyme. This sequence conservation suggests that vacuolar, F1, methanogenic, and S. acidocaldarius ATPases were derived from a common ancestral enzyme.     

Localization of the plasma membrane and mitochondrial H(+)-ATPases in Leishmania donovani promastigotes.

Immunochemical methods were used to characterize the proton-translocating ATPases (H(+)-ATPases) of the plasma membrane and mitochrondrion of Leishmania donovani promastigotes. Antisera directed against the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae reacted with a 66 kDa membrane protein of L. donovani promastigotes. By immunocytochemistry, the antiserum was shown to label the cell and flagellar surface of promastigotes as well as the Golgi apparatus and the membrane of intracellular organelles. The target antigen was shown to possess ATPase activity resembling the leishmanial H(+)-ATPase activity. Antisera raised against the beta-subunit of the F0F1-ATPase of Escherichia coli reacted with a 56 kDa protein in L. donovani promastigotes. Ultrastructurally, the anti-beta-subunit antibody was exclusively associated with the mitochondrion in these cells. This antiserum immunoprecipitates ATP hydrolytic activity typical of the F1 beta-subunit activity of the mitochondria of higher eukaryotes.     

Thermus thermophilus membrane-associated ATPase. Indication of a eubacterial V-type ATPase

An ATPase with Mr of 360,000 was purified from plasma membranes of a thermophilic eubacterium Thermus thermophilus, and was characterized. ATP hydrolytic activity of the purified enzyme was extremely low, 0.07 mumol of Pi released mg-1 min-1, and it was stimulated up to 30-fold by bisulfite. The following properties of the enzyme indicate that it is not a usual F1-ATPase but that it belongs to the V-type ATPase family, another class of ATPases found in membranes of archaebacteria and eukaryotic endomembranes. Among its four kinds of subunits with approximate Mr values of 66,000 (alpha), 55,000 (beta), 30,000 (gamma), and 12,000 (delta), the alpha subunit had a similar molecular size to the catalytic subunits of the V-type ATPases but was significantly larger than the alpha subunit of F1-ATPases. ATP hydrolytic activity was not affected by azide, an inhibitor of F1-ATPases, but was inhibited by nitrate, an inhibitor of the V-type ATPase. N-terminal amino acid sequences determined for the purified alpha and beta subunits showed much higher similarity to those of the V-type ATPases than those of F1-ATPases. Thus the distribution of the V-type ATPase in the prokaryotic kingdom may not be restricted to archaebacteria.     

cDNA sequence and homologies of the "57-kDa" nucleotide-binding subunit of the vacuolar ATPase from Arabidopsis

Functional and structural similarities among a wide variety of endomembrane H+-ATPases suggest that they form a distinct class with a common origin. Immunological studies (Manolson, M. F., Percy, J. M., Apps, D. K., Xie, X. S., Stone, D. K., and Poole, R. J. (1987) in Proceedings of the Membrane Protein Symposium (Goheen, S. C., ed) pp. 427-434, Bio-Rad, Richmond, CA, and M. F. Manolson, J. M. Percy, D. K. Apps, X. S. Xie, D. K. Stone, M. Harrison, D. J. Clarke, R. J. Poole, unpublished data) support this idea and suggest an evolutionary relationship between the endomembrane and F0F1 ATPases. Further examination of relationships necessitates comparison of protein/nucleic acid sequence data. To this end, we have cloned and sequenced the cDNA encoding the 57-kDa polypeptide of the Arabidopsis vacuolar membrane H+-ATPase. To our knowledge, this is the first report of the sequence of a "57-kDa" subunit for plant or animal endomembrane H+-ATPase. This cDNA encodes a hydrophilic polypeptide containing a putative ATP binding site. Lack of a secretion signal sequence suggests it is not processed through the endoplasmic reticulum but translated on cytosolic ribosomes. Comparison of protein sequences shows the 57-kDa subunit from Arabidopsis to be nearly identical with the corresponding subunit in Neurospora vacuolar membrane H+-ATPase, very similar to the beta subunit of the archaebacterium Sulfolobus, and slightly, but nevertheless significantly, homologous to the alpha and beta subunits of the F0F1-ATPases. These results suggest that these different classes of ATPases have evolved from a common ancestor.     

Structure of an ATPase operon of an acidothermophilic archaebacterium, Sulfolobus acidocaldarius

The nucleotide sequence of the operon of the ATPase complex of an acidothermophilic archaebacterium, Sulfolobus acidocaldarius, has been determined. In addition to the three previously reported genes for the alpha, beta, and c (proteolipid) subunits of the ATPase complex (Denda, K., Konishi, J., Oshima, T., Date, T., and Yoshida, M. (1989) J. Biol. Chem. 264, 7119-7121), the operon contained three other genes encoding hydrophilic proteins with molecular masses 25, 13, and 7 kDa. The 25-kDa protein is the third largest subunit (gamma), the 13-kDa protein is most likely the fourth subunit (delta), and the 7-kDa protein may correspond to an unknown subunit of the ATPase, tentatively named as epsilon subunit. They do not have significant sequence similarity to subunits in F0F1-ATPases and eukaryotic V-type ATPases, whereas the other three subunits, alpha, beta, and c, have homologous counterparts in F0F1- and V-type ATPases. The order of the genes in the operon was delta alpha beta gamma epsilon c. The S. acidocaldarius ATPase operon differed from the eucabacterial F0F1-ATPase operon in that the former contains only one gene for a hydrophobic subunit at the most downstream part of the operon whereas the latter has three hydrophobic F0 genes preceding five hydrophilic F1 genes.     

Identification and partial purification of a cytosolic activator of vacuolar H(+)-ATPases from mammalian kidney.

A factor that activates affinity-purified vacuolar H(+)-ATPase from bovine kidney microsomes was identified and partially purified from bovine kidney cytosol. The activator is a heat-stable, trypsin-sensitive acidic protein with a Mr by gel filtration of approximately 35,000. The activator increased the activity of renal microsomal and brush border H(+)-ATPase by over 60% but stimulated lysosomal H(+)-ATPase activity by only 28%; it had little or no activity against the remaining N-ethylmaleimide-insensitive ATPase in kidney microsomes and other transport ATPases. Stimulation of ATPase activity appeared to result from binding of the activator to the H(+)-ATPase. Activation was saturable, with a Hill coefficient of 1 at low protein concentrations. Both activator binding and stimulation of H(+)-ATPase activity were enhanced at pH values less than or equal to 6.5. The activator has selective effects on different H(+)-ATPases and is poised to activate the enzyme at low physiologic values of cytosolic pH; this newly identified cytosolic proteins may participate in the physiologic regulation of the vacuolar H(+)-ATPase.     

A gene encoding the proteolipid subunit of Sulfolobus acidocaldarius ATPase complex

An analysis of genes for the major two subunits of the membrane-associated ATPase from an acidothermophilic archaebacterium, Sulfolobus acidocaldarius, suggested that it belongs to a different ATPase family from the F1-ATPase (Denda, K., Konishi, J., Oshima, T., Date, T., and Yoshida, M. (1988) J. Biol. Chem. 263, 17251-17254). In the same operon of the above two genes we found a gene encoding a very hydrophobic protein of 101 amino acids (Mr = 10,362). A proteolipid was purified from the membranes of this bacteria in which partial amino acid sequences matched with the sequence deduced from the gene. Significant amino acid sequence homology and a similar hydropathy profile appeared when the sequence was compared with the 8-kDa proteolipid subunit of F0F1-ATPases. It is about 30 amino acids larger than the 8-kDa proteolipid and has a small (11-amino acid) repeat sequence. However, it is distinct from the 16-kDa proteolipid subunit of an eukaryotic vacuolar H+-ATPase (Mandel, M., Moriyama, Y., Hulmes, J.D., Pan, Y.-E., Nelson, H., and Nelson, N. (1988) Proc. Natl. Acad. Sci. U.S.A. 85,5521-5524).     

The cDNA sequence of the 69-kDa subunit of the carrot vacuolar H+-ATPase. Homology to the beta-chain of F0F1-ATPases

Vacuolar ATPases constitute a novel class of N-ethylmaleimide- and nitrate-sensitive proton pumps associated with the endomembrane system of eukaryotic cells. They resemble F0F1-ATPases in that they are large multimeric proteins, 400-500 kDa, composed of three to nine different subunits. Previous studies have indicated that the active site is located on the approximately 70-kDa subunit. Using antibodies to the approximately 70-kDa subunit of corn to screen a carrot root lambda gt11 cDNA library, we have isolated cDNA clones of the carrot 69-kDa subunit. The complete primary structure of the 69-kDa subunit was then determined from the nucleotide sequence of its cDNA. The 69-kDa subunit consists of 623 amino acids (Mr 68,835), with no obvious membrane-spanning regions. The carrot cDNA sequence was over 70% homologous with exons of a Neurospora 69-kDa genomic clone. The protein sequence of the carrot 69-kDa subunit also exhibited 34.3% identity to four representative F0F1-ATPase beta-chains over a 275-amino-acid core stretch of similar sequence. Alignment studies revealed several regions which were highly homologous to beta-chains, including sequences previously implicated in catalytic function. This provides definitive evidence that the vacuolar ATPase is closely related to the F0F1-type ATPases. A major functional difference between the 69-kDa and beta-subunits is the location of 3 critical cysteine residues: two in the putative catalytic region (Cys-248 and Cys-256) and one in the proposed Mg2+-binding site (Cys-279). These cysteines (and two others) probably account for the sensitivity of the vacuolar H+-ATPase to the sulfhydryl reagent, N-ethylmaleimide. It is proposed that the two ATPases may have arisen from a common ancestor by the insertion or deletion of a large stretch of nonhomologous sequence near the amino-terminal end of the subunit.     

A cytosolic inhibitor of vacuolar H(+)-ATPases from mammalian kidney.

Regulation of the vacuolar H(+)-ATPase in organellar and transepithelial acidification has been attributed to the effects of the proton electrochemical gradient across the membrane or to changes in the number of proton pumps. We now report the identification and purification of a protein from bovine kidney cytosol that inhibits both ATPase activity and proton translocating activity of vacuolar H(+)-ATPases. Its relative molecular weight (M(r)) is 6300, similar to that for protein inhibitors of the mitochondrial F0F1-ATPase. The newly identified cytosolic inhibitor protein may participate in the physiologic regulation of the vacuolar H(+)-ATPase by suppressing activity directly.     

Subunit Composition and Organization of the Vacuolar H-ATPase from Oat Roots

The vacuolar H⁺-translocating ATPase (H⁺-ATPase), originally reported to consist of three major subunits, has been further purified from oat roots (Avena sativa var Lang) to determine the complete subunit composition. Triton-solubilized ATPase activity was purified by gel filtration on Sephacryl S400 and ion-exchange chromatography (Q-Sepharose). ATP hydrolysis activity of purified preparations was inhibited by 100 nanomolar bafilomycin A₁, a specific vacuolar-type ATPase inhibitor. The purified oat H⁺-ATPase (relative molecular weight = 650,000) was composed of polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. To analyze the organization of the H⁺-ATPase subunits, native vacuolar membranes were treated with KI and MgATP to dissociate peripheral proteins. Release of 70, 60, 44, 42, 36, and 29 kilodalton polypeptides from the membrane was accompanied by a loss of ATP hydrolysis and ATP-dependent H⁺-pumping activities. Five of the peripheral subunits were released from the membrane as a large complex of 540 kilodaltons. Vesicles that had lost the peripheral sector of the ATPase could hold a pH gradient generated by the proton-translocating pyrophosphatase, suggesting that the integral sector of the ATPase did not form a H⁺-conducting pathway. Negative staining of native vesicles revealed knob-like structures of 10 to 12 nanometers in dense patches on the surface of vacuolar membranes. These structures were removed by MgATP and KI, which suggested that they were the peripheral sectors of the H⁺-ATPase. These results demonstrate that the vacuolar H⁺-ATPase from oat roots has 10 different subunits. The oat vacuolar ATPase is organized as a large peripheral sector and an integral sector with a subunit composition similar, although not identical to, other eukaryotic vacuolar ATPases. Variations in subunit composition observed among several ATPases support the idea that distinct types of vacuolar H⁺-ATPases exist in plants.     

Lysosomal H+-translocating ATPase has a similar subunit structure to chromaffin granule H+-ATPase complex

Subunit structure of the lysosomal H+-ATPase was investigated using cold inactivation, immunological cross-reactivity with antibodies against individual subunits of the H+-ATPase from chromaffin granules and chemical modification with N,N'-dicyclohexyl[14C]carbodiimide. The lysosomal H+-ATPase was irreversibly inhibited when incubated at 0 degrees C in the presence of chloride or nitrate and MgATP. Inactivation in the cold resulted in the release of several polypeptides (72, 57, 41, 34 and 33 kDa) from the membrane, which had the same electrophoretic mobility as the corresponding subunits of chromaffin granule H+-ATPase. Cross-reactivity of antibodies revealed that the 72, 57 and 34 kDa polypeptides were immunologically identical to the corresponding subunits of chromaffin granule H+-ATPase. Dicyclohexylcarbodiimide, which inhibits proton translocation in the vacuolar ATPase, predominantly labeled two polypeptides of 18 and 15 kDa, which compose the membrane sector of the enzyme. These results suggest that the lysosomal H+-ATPase is a multimeric enzyme, whose subunit structure is similar to the chromaffin granule H+-ATPase. The subunit structure of other vacuolar H+-ATPases, revealed by cold inactivation and immunological cross-reactivity, is also presented.     

Purification and properties of the coupling-factor ATPases F1 from Rhodopseudomonas palustris and Rhodopseudomonas sphaeroides

The coupling-factor ATPases from photosynthetically grown Rhodopseudomonas palustris and Rhodopseudomonas sphaeroides were purified by the same procedure to homogeneity. Gel chromatography on Sephacryl S-300 Superfine shortened the process of purification and improved its yield. Solubilization of the ATPase from both bacteria was found to be dependent on a specific sonication treatment of the cell suspensions, indicating a very weakly bound F1-ATPase in R. palustris. Depleted chromatophores could be restored in photophosphorylation and membrane-bound ATPase activities by adding the solubilized ATPase protein. The purified enzymes did not show a markedly trypsin-stimulated or dithiothreitol-stimulated activity. Isoelectric focusing and chromatofocusing revealed isoelectric points of 5.0 for both F1-ATPases. The molecular weights were determined by gel chromatography plus high-performance liquid chromatography. Hence, we calculated a molecular weight of 350000 for both F1-ATPases. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed five subunits for both enzymes. Kinetic parameters, regarding substrate specificity, the effect of divalent cations, Km and Ki values for the membrane-bound and solubilized ATPases were determined.