A second transport ATPase gene in Saccharomyces cerevisiae

A second transport ATPase gene from Saccharomyces cerevisiae has been identified by hybridization to a PMA1 probe and sequenced. The gene called PMA2 encodes a polypeptide of Mr = 102,157, which, with the exception of the 144 amino-terminal residues, is highly homologous to the structural gene PMA1 for the H+-ATPase. It is localized on the chromosome XVI at 16.7 centimorgan from gal4 and is not essential for haploid growth. Comparison between the upstream, noncoding DNA regions of PMA1 and PMA2 indicates that the two genes are controlled differently. The extensive amino acid sequence homology with the fungal H+-ATPases described so far indicates that the PMA2-encoded protein is also able to function as a H+ pump. This is supported by the observation that in pma1 mutants with reduced plasma membrane ATPase activity, disruption of the PMA2 gene confers the ability to grow under alkaline pH conditions. Slower development of diploids is also observed on normal minimal medium after bilateral disruption of PMA2 in the two parents.     

Mutations in a p-type ATPase gene cause axonal degeneration

Neuronal loss and axonal degeneration are important pathological features of many neurodegenerative diseases. The molecular mechanisms underlying the majority of axonal degeneration conditions remain unknown. To better understand axonal degeneration, we studied a mouse mutant wabbler-lethal (wl). Wabbler-lethal (wl) mutant mice develop progressive ataxia with pronounced neurodegeneration in the central and peripheral nervous system. Previous studies have led to a debate as to whether myelinopathy or axonopathy is the primary cause of neurodegeneration observed in wl mice. Here we provide clear evidence that wabbler-lethal mutants develop an axonopathy, and that this axonopathy is modulated by Wld(s) and Bax mutations. In addition, we have identified the gene harboring the disease-causing mutations as Atp8a2. We studied three wl alleles and found that all result from mutations in the Atp8a2 gene. Our analysis shows that ATP8A2 possesses phosphatidylserine translocase activity and is involved in localization of phosphatidylserine to the inner leaflet of the plasma membrane. Atp8a2 is widely expressed in the brain, spinal cord, and retina. We assessed two of the mutant alleles of Atp8a2 and found they are both nonfunctional for the phosphatidylserine translocase activity. Thus, our data demonstrate for the first time that mutation of a mammalian phosphatidylserine translocase causes axon degeneration and neurodegenerative disease.     

Nucleotide sequence of ATPase subunit 6 gene of maize mitochondria

The ATPase subunit 6, located in the inner mitochondrial membrane, is encoded by mitochondrial genomes in animals and fungi. We have isolated and characterized a mitochondrial gene, designated atp 6, that encodes the subunit 6 polypeptide of Zea mays. Nucleotide and predicted amino acid sequence comparisons have revealed a homology of 44.6 and 33.2% with the yeast ATPase subunit 6 gene and polypeptide, respectively. The predicted protein in maize contains 291 amino acids with a molecular weight of 31,721. Hydropathy profiles generated for the maize and yeast polypeptides are very similar and contain large hydrophobic domains, characteristic of membrane bound proteins. RNA transfer blot analysis indicates that atp 6 is actively transcribed. Interestingly, 122 base pairs of nucleotide sequence interior to atp 6 have extensive homology with the 5′ end of the cytochrome oxidase subunit II gene of maize mitochondria, suggesting recombination between the two genes.     

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).     

DNA-dependent ATPase activity associated with phage P22 gene 12 protein

The product of bacteriophage P22 gene 12 is known from genetic experiments to be essential for phage DNA replication. The P22 12 protein has been purified to near homogeneity from Escherichia coli lysogenic for lambda-P22 hybrid phage containing the replication genes of P22. The protein has a subunit molecular weight of 46,000. The purified protein contains ATPase activity that is stimulated by single-stranded DNA. The ATPase is poorly stimulated by double-stranded DNA. All four ribonucleoside triphosphates are hydrolyzed; none of the deoxynucleoside triphosphates are hydrolyzed. In addition, the P22 12 protein binds to single-stranded DNA in the presence of ATP. Studies of oligonucleotide synthesis by P22 12 protein in conjunction with E. coli dnaG primase are presented in the succeeding paper (Wickner, S. (1984) J. Biol. Chem. 259, 14044-14047).     

The plasma membrane proton pump ATPase: the significance of gene subfamilies

The plasma membrane proton pump ATPase (H(+)-ATPase) plays a central role in transport across the plasma membrane. As a primary transporter, it mediates ATP-dependent H(+) extrusion to the extracellular space, thus creating pH and potential differences across the plasma membrane that activate a large set of secondary transporters. In several species, the H(+)-ATPase is encoded by a family of approximately 10 genes, classified into 5 gene subfamilies and we might ask what can this tell us about the concept, and the evolution, of gene families in plants. All the highly expressed H(+)-ATPase genes are classified into only two gene subfamilies, which diverged before the emergence of present plant species, raising the questions of the significance of the existence of these two well-conserved subfamilies and whether this is related to different kinetic or regulatory properties. Finally, what can we learn from experimental approaches that silence specific genes? In this review, we would like to discuss these questions in the light of recent data.     

A serine-to-proline mutation in the copper-transporting P-type ATPase gene of the macular mouse

We have investigated the cDNA sequence of the copper-transporting P-type ATPase (Atp7a) gene of the macular mouse, a model for human Menkes disease. A point mutation (T to C) that results in substitution of proline for serine in a putative eighth transmembrane domain of the ATP7A was identified. This contrasts with abnormalities identified in the Atp7a of other mottled mouse strains: lack of expression of Atp7a mRNA in the dappled mouse, and a splicing mutation in the blotchy mouse. Received: 1 December 1996 / Accepted: 20 January 1997     

Targeting of the yeast plasma membrane [H+]ATPase: a novel gene AST1 prevents mislocalization of mutant ATPase to the vacuole

We have characterized a class of mutations in PMA1, (encoding plasma membrane ATPase) that is ideal for the analysis of membrane targeting in Saccharomyces cerevisiae. This class of pma1 mutants undergoes growth arrest at the restrictive temperature because newly synthesized ATPase fails to be targeted to the cell surface. Instead, mutant ATPase is delivered to the vacuole, where it is degraded. Delivery to the vacuole occurs without previous arrival at the plasma membrane because degradation of mutant ATPase is not prevented when internalization from the cell surface is blocked. Disruption of PEP4, encoding vacuolar proteinase A, blocks ATPase degradation, but fails to restore growth because the ATPase is still improperly targeted. One of these pma1 mutants was used to select multicopy suppressors that would permit growth at the nonpermissive temperature. A novel gene, AST1, identified by this selection, suppresses several pma1 alleles defective for targeting. The basis for suppression is that multicopy AST1 causes rerouting of mutant ATPase from the vacuole to the cell surface. pma1 mutants deleted for AST1 have a synthetic growth defect at the permissive temperature, providing genetic evidence for interaction between AST1 and PMA1. Ast1 is a cytoplasmic protein that associates with membranes, and is localized to multiple compartments, including the plasma membrane. The identification of AST1 homologues suggests that Ast1 belongs to a novel family of proteins that participates in membrane traffic.     

Structure of the VPATPD gene encoding subunit D of the human vacuolar proton ATPase

The HSD11B2 and VPATPD genes encoding the human kidney isozyme of 11beta-hydroxysteroid dehydrogenase (11-HSD2) and subunit D of the vacuolar proton ATPase, respectively, are located on chromosome 16q22. They are transcribed from complementary DNA strands and their 3' ends are only 0.5 kilobase apart. Because polymorphisms in HSD11B2 have been associated with hypertension and salt sensitivity, we characterized the human VPATPD gene. It spans 19 kb and consists of 8 exons. The encoded protein is 99.5% identical to mouse subunit D at the amino acid level. An alternating purine-pyrimidine tract is located in the 3'-untranslated region of VPATPD. On genotyping 17 hypertensive subjects, no length polymorphism was found. Although VPATPD and HSD11B2 are both expressed in kidney and placenta, they are regulated differently; forskolin upregulates HSD11B2 but not VPATPD in human choriocarcinoma JEG3 cells. The functional significance of the proximity of these two genes remains to be established.     

Cellular and functional analysis of four mutations located in the mitochondrial ATPase6 gene

The smallest rotary motor of living cells, F0F1‐ATP synthase, couples proton flow—generated by the OXPHOS system—from the intermembrane space back to the matrix with the conversion of ADP to ATP. While all mutations affecting the multisubunit complexes of the OXPHOS system probably impact on the cell's output of ATP, only mutations in complex V can be considered to affect this output directly. So far, most of the F0F1‐ATP synthase variations have been detected in the mitochondrial ATPase6 gene. In this study, the four most frequent mutations in the ATPase6 gene, namely L156R, L217R, L156P, and L217P, are studied for the first time together, both in primary cells and in cybrid clones. Arginine (“R”) mutations were associated with a much more severe phenotype than Proline (“P”) mutations, in terms of both biochemical activity and growth capacity. Also, a threshold effect in both “R” mutations appeared at 50% mutation load. Different mechanisms seemed to emerge for the two “R” mutations: the F1 seemed loosely bound to the membrane in the L156R mutant, whereas the L217R mutant induced low activity of complex V, possibly the result of a reduced rate of proton flow through the A6 channel. J. Cell. Biochem. 106: 878–886, 2009. © 2009 Wiley‐Liss, Inc.     

Stable gene amplification and overexpression of sodium- and potassium-activated ATPase in HeLa cells.

Cell lines stably resistant to ouabain were isolated from an unstably resistant HeLa line after growth in nonselective medium. Stable resistant lines bound ouabain at levels 10-fold higher than did HeLa cells and at similar levels to those bound by the unstable C+ line previously described (J. F. Ash, R. M. Fineman, T. Kalka, M. Morgan, and B. Wire, J. Cell Biol. 99: 971-983). Expression and synthesis of the Na+, K+ -ATPase alpha chain showed a similar amplification over that for HeLa cells by Western blots and [35S]methionine pulse-labeling. In addition, a glycoprotein labeled with [3H]fucose and comigrating with the Na+, K+ -ATPase beta chain was eight- to ninefold amplified in stably resistant lines. Dot blots with a cDNA clone specific for Na+, K+ -ATPase alpha chain gene sequences confirmed the amplification of this gene. Karyotyping suggested that the amplification is associated with an expanded, abnormal banded region on the long (q) arm of one chromosome 17.     

Mitochondrial ATPase subunit 6 and cytochrome B gene polymorphisms in young obese adults.

We hypothesized that the mutational strand asymmetry is more strongly exerted upon the mitochondrial cytochrome b (Cytb) gene, which is distant from the origin of the light-strand replication (Ori(L)), than upon the ATPase subunit 6 (ATP6) gene, which is close to the Ori(L). To test this hypothesis, we determined the sequences of these two genes in 96 Japanese young obese adults. The frequency of G-->A transitions was significantly higher than that of C-->T transitions in the Cytb gene, whereas the frequencies of G-->A and C-->T transitions were not significantly different in the ATP6 gene. The marked mutational strand asymmetry in the Cytb gene can be explained by the deamination of C to uracil in the long single-stranded state of the heavy strand during replication. The ratio of the nonsynonymous substitutions at the second codon positions to those at the first codon positions was significantly lower in the Cytb gene than in the ATP6 gene. The physicochemical differences between the standard and the replaced amino acid residues were significantly smaller in the Cytb gene than in ATP6 one. The present study indicates that amino acid sequences are less variable for Cytb than for ATP6 in spite of the strong mutational strand asymmetry for the Cytb gene.     

Nucleotide sequence of tobacco chloroplast gene for the alpha subunit of proton-translocating ATPase.

The tobacco chloroplast gene for the alpha subunit of proton-translocating ATPase has been cloned and sequenced. The coding region contains 1521 bp (507 codons). The nucleotide sequence and the deduced amino acid sequence show 55% and 54% homologies with those of the E. coli alpha subunit, respectively. The deduced amino acid composition is quite similar to that estimated for the spinach alpha subunit.