Molecular cloning of the plant plasma membrane H+-ATPase

Summary Mineral transport across the plasma membrane of plant cells is controlled by an electrochemical gradient of protons. This gradient is generated by an ATP-consuming enzyme in the membrane known as a proton pump, or H⁺-ATPase. The protein has a catalytic subunit of Mr=100,000 and is a prominent band when plasma membrane proteins are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We generated specific rabbit polyclonal antibody against the Mr=100,000 H⁺-ATPase and used the antibody to screen λgtll expression vector libraries of plant DNA. Several phage clones producing immunoreactive protein, and presumably containing DNA sequences for the ATPase structural gene, were isolated and purified from a carrot cDNA library and a Arabidopsis genomic DNA library. These studies represent our first efforts at cloning the structural gene for a plant plasma membrane transport protein. Applicability of the technique to other transport protein genes and the potential for use of recombinant DNA technology in plant mineral transport research are discussed.     

Isolation and characterisation of monoclonal antibodies against hydrophobic membrane subunit 9 of the yeast mitochondrial H+-ATPase

Five stable lines of myeloma-spleen cell hybrids, producing antibodies against the proteolipid subunit 9 of the yeast mitochondrial H+-ATPase F0-sector, have been isolated by immunizing mice with a proteolipid preparation in the presence of sodium dodecyl sulphate. One of these monoclonal antibodies also reacted with subunit 8 of the enzyme complex indicating a shared epitope. The antibodies did not react with the holo-H+-ATPase, suggesting that their epitopes are shielded by other subunits of the enzyme complex.     

Molecular cloning of the rat stomach (H+ + K+)-ATPase

We have isolated cDNA clones for the rat stomach (H+ + K+)-ATPase by employing a novel procedure involving the use of oligonucleotides corresponding to conserved amino acid sequences of related cation transport ATPase and a cross-hybridization with the sheep kidney (Na+ + K+)-ATPase alpha-subunit cDNA. The complete nucleotide sequence of the cDNA has been determined and the amino acid sequence of the protein deduced. The ATPase consists of 1,033 amino acids and has an Mr of 114,012. Amino acid homology and hydropathy plot comparisons between the gastric ATPase and the (Na+ + K+)-ATPase catalytic subunit demonstrate a striking similarity which suggests that their higher order structure and mechanism of action are virtually identical. The greatest homology occurs in the phosphorylation site region and in domains which may be involved in nucleotide binding and energy transduction. The most substantial differences occur in the N-terminal region and in the transmembrane domains. In addition, we report the presence of an open reading frame 5' to the translation initiation site of the gastric ATPase, which raises the possibility that the mRNA is polycistronic.     

Molecular cloning of cDNA encoding the C subunit of H(+)-ATPase from bovine chromaffin granules

A cDNA encoding subunit C of the V-ATPase from bovine chromaffin granules was cloned and sequenced. The gene encodes a hydrophilic protein of 382 amino acids with a calculated molecular weight of 43,989. Hydropathy plots revealed no apparent transmembrane segments and a rather high helix content was detected. A cDNA encoding most of the C subunit of the V-ATPase of human brain was also cloned and sequenced. The deduced amino acid sequence of this gene is almost identical to the bovine polypeptide with only one change of tyrosine 336 that was replaced by histidine in the human gene. Two polypeptide fragments derived from subunit E of V-ATPase from chromaffin granules were sequenced and found to be identical to the predicted amino acid sequence of this subunit from bovine kidney. These observations support the idea that the amino acid sequences of corresponding subunits from different V-ATPases are highly conserved. Unlike the A and B subunits of V-ATPases, that are homologous to the beta and alpha subunits of F-ATPases, subunits C and E showed no homology with analogous subunits of the F-ATPase family. It is proposed that the addition of the C and gamma subunits to the respective V- and F-ATPases during evolution defined them as two separate families of H(+)-ATPases.     

Molecular cloning and sequence analysis of the (Na+ + K+)-ATPase beta subunit from dog kidney

cDNA complementary to mRNA coding for the beta subunit of dog renal (Na+ + K+)-ATPase has been cloned into lambda gt11 and the nucleotide sequence of the DNA has been determined. The amino acid sequence of the beta subunit polypeptide has also been deduced from the DNA. The mature form of the dog kidney beta subunit contains 302 amino acids with three potential asparagine-linked attachment sites for carbohydrate. The initiation methionine is removed during processing of the polypeptide to its mature form. Although the beta subunit is an integral membrane protein there is no signal sequence for the polypeptide, and hydropathy analysis predicts that the beta subunit polypeptide spans the cell membrane only once. Secondary structure predictions and a model for the structure of the beta subunit are proposed. DNA sequencing of the 5' non-coding region of the mRNA revealed a 200 bp inverted repeat from the coding region. Blot hybridization of a fragment of the beta subunit cDNA identified a single mRNA species of 2.7 kb in dog kidney and several rat tissues. RNA from rat liver was deficient in mRNA that hybridized to the dog kidney beta subunit cDNA, although mRNA that hybridized to an alpha subunit cDNA was detected. RNA from a human hepatoma cell line, HepG2, however, contained comparable levels of mRNA for both the alpha and the beta subunits.     

Molecular cloning of cDNA encoding the 16 KDa subunit of vacuolar H(+)-ATPase from mouse cerebellum.

cDNA for the 16 kDa subunit of vacuolar H(+)-ATPase was cloned from mouse cerebellum and sequenced. The deduced polypeptide (155 amino acid residues; molecular weight, 15,808) was highly hydrophobic and homologous to the subunits of bovine adrenal medulla, Torpedo marmorata electric lobe, Drosophila and yeast. Glu-139 (supposed to be essential for proton transport) was also conserved as the potential dicyclohexylcarbodiimide binding site. The subunit had four transmembrane segments: Segment II and IV were highly homologous and Glu-139 was located in Segment IV. The roles of the non-conserved regions are discussed.     

Genetics of antagonistic action and drug resistance inLactobacillus acidophilus

Lactobacillus acidophilus has been recommended as a dietary adjunct because of its antagonistic action toward intestinal pathogens and anti-carcinogenic and hypocholesterolemic activities. ManyL. acidophilus strains harbour plasmids and such strains generally produce bacteriocin(s). Resistance to antibiotics has also been shown to be linked with plasmids. Gene transfer and cloning systems are being developed forL. acidophilus which should permit the rapid genetic characterization of desired species and their modification to obtain predetermined traits. Drug resistance determinants and production of antibiotic-like substances may serve as suitable markers for the study and development of these genetic systems. Recent developments in gene transfer systems have been reviewed here.     

Revised nomenclature for mammalian vacuolar-type H+ -ATPase subunit genes

To date, the nomenclature of mammalian genes encoding the numerous subunits and their many isoforms that comprise the family of vacuolar H(+)-ATPases has not been systematic, resulting in confusion both in the literature and among investigators. We present the official new system for these genes, approved by both Human and Mouse Gene Nomenclature Committees.     

Molecular cloning and chromosomal assignment of the porcine 54 and 56 kDa vacuolar H(+)-ATPase subunit gene (V-ATPase)

Vacuolar proton-translocating ATPases (V-ATPase) are multisubunit enzyme complexes located in the membranes of eukaryotic cells regulating cytoplasmic pH. So far, nothing is known about the genomic organization and chromosomal location of the various subunit genes in higher eukaryotes. Here we describe the isolation and analysis of a cDNA coding for the 54- and 56-kDa porcine V-ATPase subunit alpha and beta isoforms. We have determined the genomic structure of the V-ATPase subunit gene spanning at least 62 kb on Chromosome (Chr) 4q14-q16. It consists of 14 exons with sizes ranging from 54 bp to 346 bp, with a non-coding first exon and an alternatively spliced seventh exon leading to two isoforms. The 5′ end of the V-ATPase cDNA was isolated by RACE-PCR. The V-ATPase alpha isoform mRNA, lacking the seventh exon, has an open reading frame of 1395 nucleotides encoding a hydrophilic protein of 465 amino acids with a calculated molecular mass of 54.2 kDa and a pI of 7.8, whereas the beta isoform has a length of 1449 nucleotides encoding a protein of 483 amino acids with a calculated molecular mass of 55.8 kDa. Amino acid and DNA sequence comparison revealed that the porcine V-ATPase subunit exhibits a significant homology to the VMA13 subunit of Saccharomyces cerevisiae V-ATPase complex and V-ATPase subunit of Caenorhabditis elegans. Received: 14 May 1998 / Accepted: 20 October 1998     

Molecular cloning and sequencing of cDNAs encoding the proteolipid subunit of the vacuolar H(+)-ATPase from a higher plant.

To understand the molecular structure of the vacuolar H(+)-translocating ATPase from plants, cDNAs encoding the N,N'-dicyclohexylcarbodiimide-binding 16-kDa proteolipid from oat (Avena sativa L. var. Lang) have been obtained. A synthetic oligonucleotide corresponding to a region of the bovine proteolipid cDNA (Mandel, M., Moriyama, Y., Hulmes, J.D., Pan, Y.-C.E., Nelson, H., and Nelson, N. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5521-5524) was used to screen an oat cDNA library constructed in lambda gt11. The nucleotide sequences of several positive clones (VATP-P1, clones 12, 54, 93) demonstrated the presence of a small multigene family. The four clones showed extensive divergence in their codon usage and their 3'-untranslated regions; however, the deduced amino acid sequences of the proteins were 97-99% identical. These clones encoded the proteolipid subunit as one of them (clone 12) expressed a fusion protein that reacted with an antibody to the 16-kDa proteolipid. The open reading frame of one cDNA clone (VATP-P1) predicted a polypeptide of 165 amino acids with a molecular mass of 16,641. Based on hydropathy plots, a molecule with four membrane-spanning domains was predicted, in which domain IV was especially conserved among different species. This domain showed 80% identity in nucleotide or amino acid sequences between the oat and the bovine proteolipids and contained a glutamate residue that is the putative N,N'-dicyclohexylcarbodiimide-binding residue. The presence of a small multigene family of the 16-kDa proteolipid was confirmed by Southern blot analysis showing that several distinct restriction fragments of oat nuclear DNA hybridized with the VATP-P1 cDNA.     

A novel cellular survival factor--the B2 subunit of vacuolar H+-ATPase inhibits apoptosis

The ubiquitous vacuolar H(+)-ATPase, a multisubunit proton pump, is essential for intraorganellar acidification. Disruption of its function leads to disturbances of organelle function and cell death. Here, we report that overexpression of the B2 subunit of the H(+)-ATPase inhibits apoptosis. This antiapoptotic effect is not mediated by an increase in H(+)-ATPase activity but through activation of the Ras-mitogen-activated protein kinase (MAPK)-signaling pathway that results in the serine phosphorylation of Bad at residues 112 and 155. Increased Bad phosphorylation reduces its translocation to mitochondria, limits the release of mitochondrial cytochrome c and apoptosis-inducing factor and increases the resistance of the B2 overexpressing cells to apoptosis. Screening experiments of kinase inhibitors, including inhibitors of cAMP-activated protein kinase, protein kinase C, protein kinase B, (MAPK/extracellular signal-regulated (ERK) kinase) MEK and Ste-MEK1(13), a cell permeable ERK activation inhibitor peptide, revealed that the B2 subunit of H(+)-ATPase acts upstream of MEK activation in the MEK/ERK pathway to ameliorate apoptosis.     

cDNA cloning and sequence determination of pig gastric (H+ + K+)-ATPase

Complementary DNA to pig gastric mRNA encoding (H+ + K+)-ATPase was cloned, and its amino acid sequence was deduced from the nucleotide sequence. The enzyme contained 1034 amino acid residues (Mr. 114,285) including the initiation methionine. The sequence of pig (H+ + K+)-ATPase was highly homologous with that of the corresponding enzyme from rat, but had high degree of synonymous codon changes. Potential sites of phosphorylation by cAMP-dependent protein kinase and N-linked glycosylation sites were identified. The amino terminal region contained a lysine-rich sequence similar to that of the alpha subunit of (Na+ + K+)-ATPase, although a cluster of glycine residues was inserted into the sequence of the (H+ + K+)-ATPase. As the pig enzyme is advantageous for biochemical studies, the information of the primary structure is useful for further detailed studies.     

Primary structure of the dicyclohexylcarbodiimide-binding subunit of Streptococcus faecalis H+-ATPase

The complete amino acid sequence of dicyclohexylcarbodiimide (DCC)-binding subunit of proton adenosine triphosphatase from glycolysing bacteria Streptococcus faecalis was established. Isolation of the protein from subbacterial particles was carried out by using extraction with a chloroform/methanol mixture and following gel-filtration on Sephadex LH-60 and Bio-gel P-30. To establish the primary structure, use was made of cyanogen bromide and hydroxylamine cleavages, trypsin and partial acid hydrolyses. Separation of the peptide fragments obtained from cyanogen bromide and hydroxylamine cleavages and partial acid hydrolysis was performed by gel-filtration on Bio-gel P-10 and reversed-phase HPLC. Peptide structures were determined mainly with the aid of 4-N,N-dimethylaminoazobenzene-4'-isothiocyanate. The polypeptide chain of the protein consists of 71 amino acid residues (mol. wt. 7291). The primary structure of the protein from S. faecalis shares all common features of the structural organization of other H+-ATPase DCC-binding subunits and shows a high degree of homology with the corresponding subunit of thermophilic bacterium PS-3. Inactivation of H+-ATPase with DCC was due to modification of Glu54 of the polypeptide chain.     

Properties and subunit composition of the H+-ATPase complex from pea chloroplasts

The H+-ATPase complex from pea chloroplasts was isolated and partially purified. The complex incorporated into the phospholipid membrane can catalyze the 32Pi-ATP exchange reaction. The complex contains eight types of the subunits, five of which belong to the catalytic and three--to the hydrophobic moieties. The molecular weights of the subunits were determined.     

H+-translocating ATPase in Golgi apparatus. Characterization as vacuolar H+-ATPase and its subunit structures

Golgi apparatus was prepared from rat liver, and enzymatic properties and the subunit structure of the H+-ATPase were characterized. GTP (and also ITP) was found to drive H+-transport with about 20% of the initial velocity as that of ATP. Bafilomycin, a specific inhibitor for vacuolar H+-ATPase, inhibited the activity at 2.5 nM. The H+-ATPase was completely inhibited in the cold in the presence of MgATP (5 mM) and NaNO3 (0.1 M). The cold inactivation of the H+-ATPase resulted in release of a set of polypeptides from Golgi membrane, with molecular masses almost identical to that of the hydrophilic sector of chromaffin granule H+-ATPase (72, 57, 41, 34, and 33 kDa). Three of these polypeptides (72, 57, and 34 kDa), cross-reacted with antibodies against the corresponding subunits of the chromaffin granule H+-ATPase. A counterpart of the 39-kDa hydrophobic component of chromaffin granule H+-ATPase was identified in the membrane, but no 115-kDa component was found. Hence, the Golgi H+-ATPase shows typical features of vacuolar H+-ATPase, in relatively low substrate specificity, its response to inhibitors, inactivation by cold treatment in the presence of MgATP, and subunit composition judged by antibody cross-reactivity.     

Binding of Mg2+ to the beta subunit or F1 of H+-ATPase from Escherichia coli

The bindings of Mg2+ to the F1 portion of Escherichia coli H+-ATPase and its isolated alpha and beta subunits were studied with 8-anilinonaphthalene-1-sulfonate (ANS). The fluorescence of ANS increased upon addition of F1 or its alpha subunit or beta subunit, as reported previously (M. Hirano, K. Takeda, H. Kanazawa, and M. Futai (1984) Biochemistry 23, 1652-1656). The fluorescence of ANS bound to F1 or its beta subunit increased significantly with further addition of Mg2+, whereas that of the alpha subunit increased only slightly. Ca2+ and Mn2+ had similar effects on the fluorescence of ANS with F1 and its beta subunit. The Mg2+-induced fluorescence enhancement (delta F) was high at an alkaline pH and was lowered by addition of ethylenediaminetetraacetic acid. Dicyclohexylcarbodiimide and azide had no effect on the delta F. Binding analysis showed that the concentration dependence of Mg2+ on the fluorescence enhancement of the beta subunit is similar to that of F1. These results suggest that both the beta subunit and F1 have binding sites for Mg2+ and that the delta F observed with F1 may be due to the binding of Mg2+ to the beta subunit.     

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.