RESUMEN
A series of naturally occurring compounds reported recently by multiple laboratories defines a new small-molecule class sharing a unique benzolactone enamide core structure and diverse biological actions, including inhibition of growth of tumor cells and oncogene-transformed cell lines. Here we show that representative members of this class, including salicylihalamide A, lobatamides A-F, and oximidines I and II inhibit mammalian vacuolar-type (H+)-ATPases (V-ATPases) with unprecedented selectivity. Data derived from the NCI 60-cell antitumor screen critically predicted the V-ATPase molecular target, while specific biochemical assays provided confirmation and further illumination. The compounds potently blocked representative V-ATPases from human kidney, liver, and osteoclastic giant-cell tumor of bone but were essentially inactive against V-ATPases of Neurospora crassa and Saccharomyces cerevisiae and other membrane ATPases. Essential regulation of pH in cytoplasmic, intraorganellar, and local extracellular spaces is provided by V-ATPases, which are ubiquitously distributed among eukaryotic cells and tissues. The most potent and selective V-ATPase inhibitors heretofore known were the bafilomycins and concanamycins, which do not discriminate between mammalian and nonmammalian V-ATPases. Numerous physiological processes are mediated by V-ATPases, and aberrant V-ATPase functions are implicated in many different human diseases. Previous efforts to develop therapeutic pharmacological modulators of V-ATPases have been frustrated by a lack of synthetically tractable and biologically selective leads. Therefore, availability of the unique benzolactone enamide inhibitor class may enable further elucidation of functional and architectural features of mammalian versus nonmammalian V-ATPase isoforms and provide new opportunities for targeting V-ATPase-mediated processes implicated in diverse pathophysiological phenomena, including cancer.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , ATPasas de Translocación de Protón/antagonistas & inhibidores , Vacuolas/enzimología , Animales , Bovinos , Perros , Relación Dosis-Respuesta a Droga , Neurospora crassa/enzimología , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
By using the process of Repeat-induced Point mutation (Selker, E. U., and Garrett, P. W. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6870-6874), we inactivated vma-1, the gene encoding subunit A of the V-ATPase of Neurospora crassa. Two vma-1 mutant strains were characterized. One was mutated at multiple sites, did not make a protein product, and produced spores that only rarely germinated. The other had four point mutations, made a protein product, and produced viable spores. Neither strain had detectable V-ATPase activity. The vma-1 mutant strains did not grow in medium buffered to pH 7.0 or above or in medium supplemented with the cation Zn(2+). They were completely resistant to inhibition by concanamycin C, supporting our hypothesis that the V-ATPase is the in vivo target of this antibiotic. Inactivation of the vma-1 gene had a pronounced effect on morphology and development of the organism. In the mutants tip growth was inhibited, and multiple branching was induced. The vma-1 mutant strains could not differentiate conidia or perithecia. They could grow slowly as mycelia and could donate nuclei in a sexual cross. A mutation in the plasma membrane ATPase, which suppressed the sensitivity of wild type N. crassa to concanamycin, also proved effective in suppressing the sensitivity of a vma-1 null mutant to basic pH but did not correct the morphological defects.
Asunto(s)
Genes Fúngicos , Neurospora crassa/citología , Neurospora crassa/genética , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón Vacuolares , Vacuolas/enzimología , Cruzamientos Genéticos , Genotipo , Mutación , Neurospora crassa/crecimiento & desarrollo , Fenotipo , Mutación Puntual , Sorbitol/farmacología , Esporas Fúngicas , Supresión Genética , Vacuolas/ultraestructuraRESUMEN
We have identified a gene, named hex-1, that encodes the major protein in the hexagonal crystals, or Woronin bodies, of Neurospora crassa. Analysis of a strain with a null mutation in the hex-1 gene showed that the septal pores in this organism were not plugged when hyphae were damaged, leading to extensive loss of cytoplasm. When grown on agar plates containing sorbose, the hex-1(-) strain showed extensive lysis of hyphal tips. The HEX-1 protein was predicted to be 19,125 Da. Analysis of the N-terminus of the purified protein indicated that 16 residues are cleaved, yielding a protein of 17,377 Da. A polyclonal antibody raised to the HEX-1 protein recognized multiple forms of the protein, apparently dimers and tetramers that were resistant to solubilization by sodium dodecyl sulfate and reducing reagents. Treatment of the protein with phosphatase caused dissociation of these oligomers. Preparations enriched in Woronin bodies contained catalase activity, which was not detected in comparable fractions from the hex-1(-) mutant strain. These results support the hypothesis that the Woronin body is a specialized peroxisome that functions as a plug for septal pores.
Asunto(s)
Proteínas Fúngicas/genética , Genes Fúngicos , Neurospora crassa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Catalasa/química , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/química , Datos de Secuencia Molecular , Peso Molecular , Mutación , Orgánulos/química , Orgánulos/genética , Alineación de SecuenciaRESUMEN
Vacuolar ATPases (V-ATPases) are large complex enzymes that are structural and mechanistic relatives of F(1)F(o)-ATPases. They hydrolyze ATP and pump protons across membranes to hyperpolarize membranes and, often, to acidify cellular compartments. The proton gradients generated are used to drive the movement of various compounds across membranes. V-ATPases are found in membranes of archaebacteria and some eubacteria, in various components of the endomembrane system of all eukaryotes and in the plasma membranes of many specialized eukaryotic cells. They have been implicated in a wide variety of cellular processes and are associated with several diseases. Bafilomycin and concanamycin, specific inhibitors of V-ATPases, have been instrumental in implicating the V-ATPase in many of these roles. To understand further the mechanism of inhibition by these antibiotics and the physiological role of the enzyme in the cell, we have isolated mutants of the filamentous fungus Neurospora crassa that are resistant to concanamycin. Concanamycin has a dramatic effect on hyphal morphology at acid pH and is lethal at basic pH. In the resistant mutants, the cells can germinate and grow, although abnormally, in basic medium. Thus far, none of the mutants we have characterized is mutated in a gene encoding a subunit of the V-ATPase. Instead, the largest class of mutants is mutated in the gene encoding the plasma-membrane H(+)-ATPase. Mutations in at least four uncharacterized genes can also confer resistance. Inactivation of the V-ATPase by disruption of vma-1, which encodes the catalytic subunit (A) of the enzyme, causes a much more severe phenotype than inhibition by concanamycin. A strain lacking vma-1 is seriously impaired in rate of growth, differentiation and capacity to produce viable spores. It is also completely resistant to concanamycin, indicating that the inhibitory effects of concanamycin in vivo are due to inhibition of the V-ATPase. How the multiplicity of ATPases within a cell is regulated and how their activity is integrated with other metabolic reactions is poorly understood. Mutant analysis should help unravel this puzzle.
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Microbiana/genética , Macrólidos , Mutación , Neurospora crassa/enzimología , ATPasas de Translocación de Protón/fisiología , ATPasas de Translocación de Protón Vacuolares , Neurospora crassa/efectos de los fármacos , Neurospora crassa/genética , ATPasas de Translocación de Protón/genéticaRESUMEN
The filamentous fungus Neurospora crassa contains many small vacuoles. These organelles contain high concentrations of polyphosphates and basic amino acids, such as arginine and ornithine. Because of their size and density, the vacuoles can be separated from other organelles in the cell. The ATP-driven proton pump in the vacuolar membrane is a typical V-type ATPase. We examined the size and structure of this enzyme using radiation inactivation and electron microscopy. The vacuolar ATPase is a large and complex enzyme, which appears to contain at least thirteen different types of subunits. We have characterized the genes that encode eleven of these subunits. In this review, we discuss the possible function and structure of these subunits.
Asunto(s)
Neurospora crassa/enzimología , Bombas de Protones/química , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón Vacuolares , Secuencia de Aminoácidos , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Neurospora crassa/genética , Conformación Proteica , Bombas de Protones/genética , ATPasas de Translocación de Protón/genéticaRESUMEN
Concanamycin A (CCA), a specific inhibitor of vacuolar ATPases, inhibited growth of Neurospora crassa in medium adjusted to pH 7 or above. Mutant strains were selected for growth on medium containing 1.0 microM CCA. Sixty-four (of 66) mutations mapped in the region of the pma1 locus, which encodes the plasma membrane H+-ATPase. Analysis of V-ATPase activity in isolated vacuolar membranes from the mutant strains showed wild-type activity and sensitivity to CCA. In contrast, plasma membrane H+-ATPase activity in isolated plasma membranes from the mutants was reduced as compared with wild-type, and in four strains the activity showed increased resistance to vanadate. The most interesting change in the plasma membrane H+-ATPase was in kinetic behavior. The wild-type enzyme showed sigmoid dependence on MgATP concentration with a Hill number of 2.0, while the seven mutants tested exhibited hyperbolic kinetics with a Hill number of 1.0. One interpretation of these data was that the enzyme had changed from a functional dimer to a functional monomer. Mutation of the plasma membrane H+-ATPase did not confer resistance by preventing uptake of CCA. In the presence of CCA both wild-type and mutant strains were unable to accumulate arginine, failed to concentrate chloroquine in acidic vesicles, and exhibited gross alterations in hyphal morphology, indicating that the CCA had entered the cells and inactivated the V-ATPase. Instead, we hypothesize that the mutations conferred resistance because the altered plasma membrane H+-ATPase could more efficiently rid the cell of toxic levels of Ca2+ or protons or other ions accumulated in the cytoplasm following inactivation of the V-ATPase by CCA.
Asunto(s)
Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Genes Fúngicos/efectos de los fármacos , Macrólidos , Neurospora crassa/enzimología , ATPasas de Translocación de Protón/antagonistas & inhibidores , ATPasas de Translocación de Protón/biosíntesis , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón Vacuolares , Membrana Celular/enzimología , Mapeo Cromosómico , Cromosomas Fúngicos , Farmacorresistencia Microbiana , Higromicina B/farmacología , Membranas Intracelulares/enzimología , Cinética , Mitocondrias/enzimología , Mutagénesis , Neurospora crassa/genética , Neurospora crassa/crecimiento & desarrollo , Especificidad de la Especie , Vacuolas/enzimologíaRESUMEN
We have isolated the vma-4 gene, which encodes a 25,746 Dalton subunit of the vacuolar ATPase, from Neurospora crassa. The gene contains two introns and was mapped to the left arm of linkage group I. Comparison of the predicted amino acid sequence with homologous proteins from Saccharomyces cerevisiae, Manduca sexta, and Bos taurus showed only 25% sequence identity. However, computer-assisted predictions of secondary structures gave similar results for all four proteins. Analysis of the sequence and the available biochemical data indicated that the vma-4 gene product may play the same structural role in the vacuolar ATPase as does the gamma-subunit in F-type ATPases.
Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas Fúngicas/genética , Neurospora crassa/genética , Adenosina Trifosfatasas/química , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Fúngicas/química , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , ATPasas de Translocación de Protón/química , Homología de Secuencia de AminoácidoRESUMEN
We have isolated the cDNA and corresponding genomic DNA encoding citrate synthase in Neurospora crassa. Analysis of the protein coding region of this gene, named cit-1, indicates that it specifies the mitochondrial form of citrate synthase. The predicted protein has 469 amino acids and a molecular mass of 52,002 Da. The gene is interrupted by four introns. Hybridization experiments show that a cit-1 probe binds to two different fragments of genomic DNA, which are located on different chromosomes. Neurospora crassa may have two isoforms of citrate synthase, one in the mitochondria and the other in microbodies.
Asunto(s)
Citrato (si)-Sintasa/genética , Genes Fúngicos/genética , Mitocondrias/enzimología , Neurospora crassa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Citrato (si)-Sintasa/química , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Intrones , Datos de Secuencia Molecular , Familia de Multigenes , Neurospora crassa/enzimología , Polimorfismo de Longitud del Fragmento de Restricción , ARN de Hongos/genética , ARN Mensajero/genética , Mapeo RestrictivoRESUMEN
Various ATPases have been tested for their sensitivity to naturally occurring unusual macrolides and their chemically modified derivatives, which are structurally related to bafilomycin A1 (1), the first specific inhibitor of vacuolar ATPases. The structure-activity study showed that in general the concanamycins, 18-membered macrolides, are better and more specific inhibitors than the bafilomycins of this class of membrane-bound ATPases. The additional carbohydrate residue is not responsible for the improved activity. The importance of an intact hemiketal ring, which is part of an intramolecular hydrogen-bonding network, and the effects of the size of the macrolactone ring are discussed. The structurally related elaiophylin (13), a C2-symmetric macrodiolide antibiotic, proved to be inactive on vacuolar ATPases but still retained its inhibitory effect on P-type ATPases.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Antibacterianos/farmacología , Proteínas de Transporte de Catión , Proteínas de Escherichia coli , Macrólidos , Complejos de ATP Sintetasa , Escherichia coli/enzimología , Complejos Multienzimáticos/antagonistas & inhibidores , Neurospora crassa/enzimología , Fosfotransferasas/antagonistas & inhibidores , ATPasas de Translocación de Protón/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
We are using three approaches to investigate the vacuolar ATPase, V-ATPase, from Neurospora crassa. (1) Examination in the electron microscope shows the enzyme has a 'ball and stalk' structure like the F-type ATPases. However, the vacuolar ATPase is significantly larger, has a prominent cleft in the head sector, and has extra components associated with the stalk and membrane sectors. (2) Genes encoding three of the major subunits of the vacuolar ATPase and the homologous subunits of the mitochondrial F-ATPase have been isolated. The exon/intron structures of the genes have been analyzed and the chromosomal locations have been determined. Two of the vacuolar ATPase genes map very close to each other, suggesting the possibility of a cluster of ATPase genes. (3) The function of the ATPase is being investigated by isolating strains with altered or inactivated ATPase. We are characterizing strains that are resistant to bafilomycin A1, a potent and specific inhibitor of the vacuolar ATPase. Initial attempts to inactivate a vacuolar ATPase gene indicate that the enzyme may be essential for growth.
Asunto(s)
Adenosina Trifosfatasas/ultraestructura , Neurospora crassa/enzimología , ATPasas de Translocación de Protón Vacuolares , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/fisiología , Genes Fúngicos , Microscopía Electrónica , Estructura Molecular , Mutación , Neurospora crassa/genética , Vacuolas/enzimologíaRESUMEN
The genes encoding subunits A (vma1) and B (vma2) of the vacuolar H(+)-ATPase from Schizosaccharomyces pombe were cloned by hybridization to cDNAs of the homologous genes in Neurospora crassa. Both genes are interrupted by introns, two in vma1 and four in vma2. Positions of introns do not appear to be conserved when compared to those of N. crassa. The subunit A gene encodes a single product of 619 amino acids and is not interrupted by the coding sequence for a second product as found for Saccharomyces cerevisiae (Kane, P. K., Yamashiro, C. T., Wolczyk, D. F., Neff, N., Goebl, M., and Stevens, T. H. (1990). Science 250, 651-657).
Asunto(s)
Genes Fúngicos , ATPasas de Translocación de Protón/genética , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN de Hongos/genética , Datos de Secuencia Molecular , Neurospora crassa/genética , Conformación Proteica , ATPasas de Translocación de Protón/química , Homología de Secuencia de AminoácidoRESUMEN
The filamentous fungus Neurospora crassa has many small vacuoles which, like mammalian lysosomes, contain hydrolytic enzymes. They also store large amounts of phosphate and basic amino acids. To generate an acidic interior and to drive the transport of small molecules, the vacuolar membranes are densely studded with a proton-pumping ATPase. The vacuolar ATPase is a large enzyme, composed of 8-10 subunits. These subunits are arranged into two sectors, a complex of peripheral subunits called V1 and an integral membrane complex called V0. Genes encoding three of the subunits have been isolated. vma-1 and vma-2 encode polypeptides homologous to the alpha and beta subunits of F-type ATPases. These subunits appear to contain the sites of ATP binding and hydrolysis. vma-3 encodes a highly hydrophobic polypeptide homologous to the proteolipid subunit of vacuolar ATPases from other organisms. This subunit may form part of the proton-containing pathway through the membrane. We have examined the structures of the genes and attempted to inactivate them.
Asunto(s)
Neurospora crassa/enzimología , ATPasas de Translocación de Protón/fisiología , Secuencia de Aminoácidos , Datos de Secuencia Molecular , ATPasas de Translocación de Protón/ultraestructura , Vacuolas/enzimologíaRESUMEN
We have isolated and sequenced cDNA and genomic clones encoding the alpha and beta subunits of the Neurospora crassa ATP synthase. The genes are not linked to each other: atp-1(alpha) maps to either linkage group I or V, and atp-2(beta) lies on linkage group II. The two genes resemble each other in having a large number of introns, five in atp-1 and seven in atp-2, mostly positioned near their 5' ends and varying in length from 60-332 bp. The coding regions of both genes have a high G+C content (59%) and use a low number of codons, 46 (atp-1) and 44 (atp-2), a feature associated with highly expressed genes. Northern-blot analysis shows both genes are expressed at high levels during mycelial growth. Comparison of the exon-intron structures of the beta-subunit-encoding gene with those from human and tobacco showed a similar number of introns, several closely positioned, but no exact conservation in position, size or sequence of introns.
Asunto(s)
Genes Fúngicos , Neurospora crassa/genética , ATPasas de Translocación de Protón/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , ADN de Hongos , Expresión Génica , Humanos , Intrones , Datos de Secuencia Molecular , Neurospora crassa/enzimología , Polimorfismo de Longitud del Fragmento de Restricción , Homología de Secuencia de Ácido NucleicoRESUMEN
Polyclonal antiserum against subunit A (67 kDa) of the vacuolar ATPase from Neurospora crassa reacted with subunit I (87 kDa) from a membrane ATPase of the extremely halophilic archaebacterium Halobacterium saccharovorum. The halobacterial ATPase was inhibited by nitrate and N-ethylmaleimide; the extent of the latter inhibition was diminished in the presence of adenosine di- or triphosphates. 4-Chloro-7-nitrobenzofurazan inhibited the halobacterial ATPase also in a nucleotide-protectable manner; the bulk of inhibitor was associated with subunit II (60 kDa). The data suggested that this halobacterial ATPase may have conserved structural features from both the vacuolar and the F-type ATPases.
Asunto(s)
Adenosina Trifosfatasas/inmunología , Halobacterium/enzimología , ATPasas de Translocación de Protón/inmunología , Vacuolas/enzimología , Adenosina Trifosfatasas/antagonistas & inhibidores , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/inmunología , Western Blotting , Reacciones Cruzadas , Etilmaleimida/farmacología , Proteínas Fúngicas/inmunología , Peso Molecular , Neurospora crassa/enzimología , Nitratos/farmacologíaRESUMEN
We have explored the structure and subunit composition of the vacuolar ATPase of Neurospora crassa by investigating the effects of nitrate. Inhibition of enzyme activity by nitrate was correlated with dissociation of a complex of peripheral polypeptides from the integral membrane part of the enzyme. Surprisingly, this nitrate-induced release of subunits occurred only when nucleotides such as ADP, ATP, or ITP were present. ATPase inhibitors that have been proposed to act at the active site prevented release of subunits. Six polypeptides, 67, 57, 51, 48, 30, and 16 kDa, were coordinately released from the vacuolar membrane. When analyzed by size exclusion chromatography or by centrifugation through glycerol gradients, the six polypeptides behaved as an aggregate of about 440,000 kDa. We also examined vacuolar membranes by electron microscopy, using negative staining. We observed a high density of "ball and stalk" structures on the membranes, similar in size but different in shape from the F0F1-ATPase of mitochondrial membranes. Treatment with nitrate removed the ball and stalk structures from vacuolar membranes but had no visible effect on mitochondrial membranes. We concluded that the overall structure of the vacuolar ATPase is similar to that of F0F1-ATPases; however, the sizes of the component polypeptides and the factors that can cause dissociation are different.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Neurospora crassa/enzimología , Neurospora/enzimología , Compuestos de Potasio , ATPasas de Translocación de Protón/metabolismo , Vacuolas/enzimología , Adenosina Trifosfatasas/aislamiento & purificación , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Membranas Intracelulares/enzimología , Membranas Intracelulares/ultraestructura , Cinética , Sustancias Macromoleculares , Microscopía Electrónica , Mitocondrias/enzimología , Mitocondrias/ultraestructura , Neurospora crassa/ultraestructura , Nitratos/farmacología , Tiocianatos/farmacología , Vacuolas/ultraestructuraRESUMEN
Active transport across the vacuolar components of the eukaryotic endomembrane system is energized by a specific vacuolar H+-ATPase. The amino acid sequences of the 70- and 60-kDa subunits of the vacuolar H+-ATPase are approximately equal to 25% identical to the beta and alpha subunits, respectively, of the eubacterial-type F0F1-ATPases. We now report that the same vacuolar H+-ATPase subunits are approximately equal to 50% identical to the alpha and beta subunits, respectively, of the sulfur-metabolizing Sulfolobus acidocaldarius, an archaebacterium (Archaeobacterium). Moreover, the homologue of an 88-amino acid stretch near the amino-terminal end of the 70-kDa subunit is absent from the F0F1-ATPase beta subunit but is present in the alpha subunit of Sulfolobus. Since the two types of subunits (alpha and beta subunits; 60- and 70-kDa subunits) are homologous to each other, they must have arisen by a gene duplication that occurred prior to the last common ancestor of the eubacteria, eukaryotes, and Sulfolobus. Thus, the phylogenetic tree of the subunits can be rooted at the site where the gene duplication occurred. The inferred evolutionary tree contains two main branches: a eubacterial branch and an eocyte branch that gave rise to Sulfolobus and the eukaryotic host cell. The implication is that the vacuolar H+-ATPase of eukaryotes arose by the internalization of the plasma membrane H+-ATPase of an archaebacterial-like ancestral cell.
Asunto(s)
Archaea/genética , Bacterias/genética , Evolución Biológica , Bacterias Gramnegativas Quimiolitotróficas/genética , ATPasas de Translocación de Protón/genética , Vacuolas/enzimología , Secuencia de Aminoácidos , Archaea/enzimología , Células Eucariotas/enzimología , Bacterias Gramnegativas Quimiolitotróficas/enzimología , Sustancias Macromoleculares , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de Ácido NucleicoRESUMEN
Various membrane ATPases have been tested for their sensitivity to bafilomycin A1, a macrolide antibiotic. F1F0 ATPases from bacteria and mitochondria are not affected by this antibiotic. In contrast, E1E2 ATPases--e.g., the K+-dependent (Kdp) ATPase from Escherichia coli, the Na+,K+-ATPase from ox brain, and the Ca2+-ATPase from sarcoplasmic reticulum--are moderately sensitive to this inhibitor. Finally, membrane ATPases from Neurospora vacuoles, chromaffin granules, and plant vacuoles are extremely sensitive. From this we conclude that bafilomycin A1 is a valuable tool for distinguishing among the three different types of ATPases and represents the first relatively specific potent inhibitor of vacuolar ATPases.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Antibacterianos/farmacología , Macrólidos , Escherichia coli/enzimología , Lactonas/farmacología , Membranas/enzimología , Neurospora/enzimología , Vacuolas/enzimologíaRESUMEN
In partially purified preparations of the vacuolar ATPase from Neurospora crassa, the two most prominent components are polypeptides of Mr = 70,000 and 60,000. We previously reported the isolation of the gene vma-1, which encodes the Mr = 70,000 polypeptide, and presented evidence that the polypeptide contains the site of ATP hydrolysis (Bowman, E. J., Tenney, K., and Bowman, B. J. (1988) J. Biol. Chem. 263, 13994-14001). We now report the isolation of a gene (designated vma-2), that encodes the Mr = 60,000 polypeptide. Analysis of the DNA sequence shows that the polypeptide has 513 amino acids and a molecular mass of 56,808 daltons (and will thus be referred to as the 57-kDa polypeptide). It is fairly rich in polar amino acids and has no apparent membrane-spanning domains. The vma-2 gene contains five short introns (55-71 bases), all clustered in the 5' end of the coding region. The gene maps to the right arm of linkage group II, near 5 S RNA gene 3. Thus, it is unlinked to vma-1 and to other known ATPase genes in N. crassa. The 57-kDa polypeptide shows 25% amino acid sequence identity with the vma-1 gene product. It shows essentially the same degree of similarity (25-28%) to both the alpha and beta subunits of F0F1 ATPases. Analysis of specific regions of the 57-kDa polypeptide, however, suggests it may have a function like that of the alpha subunit in F0F1 ATPases. The data indicate that all four types of ATPase polypeptides have evolved from a common ancestor and that the vacuolar-type ATPases have a structure surprisingly similar to that of the F0F1 ATPases.
Asunto(s)
Adenosina Trifosfatasas/genética , ADN de Hongos/aislamiento & purificación , Neurospora crassa/genética , Neurospora/genética , Vacuolas/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Enzimas de Restricción del ADN , ADN de Hongos/genética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , Neurospora crassa/enzimologíaRESUMEN
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.