P-type ATPase Genes Research Articles

Identification of the potato (Solanum tuberosum L.) P-type ATPase gene family and investigating the role of PHA2 in response to Pep13.

P-type ATPase family members play important roles in plant growth and development and are involved in plant resistance to various biotic and abiotic factors. Extensive studies have been conducted on the P-type ATPase gene families in Arabidopsis thaliana and rice but our understanding in potato remains relatively limited. Therefore, this study aimed to screen and analyze 48 P-type ATPase genes from the potato (Solanum tuberosum L.) genome database at the genome-wide level. Potato P-type ATPase genes were categorized into five subgroups based on the phylogenetic classification of the reported species. Additionally, several bioinformatic analyses, including gene structure analysis, chromosomal position analysis, and identification of conserved motifs and promoter cis-acting elements, were performed. Interestingly, the plasma membrane H+-ATPase (PM H+-ATPase) genes of one of the P3 subgroups showed differential expression in different tissues of potato. Specifically, PHA2, PHA3, and PHA7 were highly expressed in the roots, whereas PHA8 was expressed in potatoes only under stress. Furthermore, the small peptide Pep13 inhibited the expression of PHA1, PHA2, PHA3, and PHA7 in potato roots. Transgenic plants heterologously overexpressing PHA2 displayed a growth phenotype sensitive to Pep13 compared with wild-type plants. Further analysis revealed that reducing potato PM H+-ATPase enzyme activity enhanced resistance to Pep13, indicating the involvement of PM H+-ATPase in the physiological process of potato late blight and the enhancement of plant disease resistance. This study confirms the critical role of potato PHA2 in resistance to Pep13.

Genome-Wide Identification, Characterization, and Expression Analyses of P-Type ATPase Superfamily Genes in Soybean

P-type ATPases are transmembrane pumps of cations and phospholipids. They are energized by hydrolysis of ATP and play important roles in a wide range of fundamental cellular and physiological processes during plant growth and development. However, the P-type ATPase superfamily genes have not been characterized in soybean. Here, we performed genome-wide bioinformatic and expression analyses of the P-type ATPase superfamily genes in order to explore the potential functions of P-type ATPases in soybean. A total of 105 putative P-type ATPase genes were identified in the soybean genome. Phylogenetic relationship analysis of the P-type ATPase genes indicated that they can be divided into five subfamilies including P1B, P2A/B, P3A, P4 and P5. Proteins belonging to the same subfamily shared conserved domains. Forty-seven gene pairs were related to segmental duplication, which contributed to the expansion of the P-type ATPase genes during the evolution of soybean. Most of the P-type ATPase genes contained hormonal- and/or stress-related cis-elements in their promoter regions. Expression analysis by retrieving RNA-sequencing datasets suggested that almost all of the P-type ATPase genes could be detected in soybean tissues, and some genes showed tissue-specific expression patterns. Nearly half of the P-type ATPase genes were found to be significantly induced or repressed under stresses like salt, drought, cold, flooding, and/or phosphate starvation. Four genes were significantly affected by rhizobia inoculation in root hairs. The induction of two P2B-ATPase genes, GmACA1 and GmACA2, by phosphate starvation was confirmed by quantitative RT-PCR. This study provides information for understanding the evolution and biological functions of the P-type ATPase superfamily genes in soybean.

Low-Frequency Magnetic Field of Appropriate Strengths Changed Secondary Metabolite Production and Na+ Concentration of Intracellular and Extracellular Monascus purpureus.

Monascus purpureus is used to yield edible pigments accompanied by mycotoxin-citrinin. A low-frequency (<300 Hz) magnetic field (LF-MF) affects microbial metabolism. The link of LF-MF with secondary metabolites and intracellular and extracellular Na+ levels in M. purpureus was determined. The fermentation broth was exposed to LF-MF during the first 2 days of fermentation and continuously cultured at 30°C and 200 rpm until the 8th day of fermentation. Results showed that LF-MF treatments didn't affect the growth of M. purpureus in liquid-state fermentation. Compared with the control, citrinin production showed a decrease of 45.0%, while yellow, red, and orange pigment production showed an increase of 72.9, 73.9, and 40.1%, respectively, with LF-MF treatment of 1.6 mT. This was in agreement with downregulation of pksCT and ctnA, and upregulation of pksPT, pigR, veA, and laeA at the transcriptional level. Moreover, 1.6 mT LF-MF exposure caused the transfer of Na+ from extracellular to intracellular, which was validated through the upregulation of transmembrane sensor synthesis genes and the changes in the relative expression levels of the P-type ATPase and protein phosphatase genes. This study established that LF-MF could inhibit citrinin and stimulate pigment production and changeintracellular and extracellular Na+ concentrations. Bioelectromagnetics. 2020;41:289-297 © 2020 Bioelectromagnetics Society.

Comparative analysis of the P-type ATPase gene family in seven Rosaceae species and an expression analysis in pear (Pyrus bretschneideri Rehd.)

P-type ATPases are integral membrane transporters that play important roles in transmembrane transport in plants. However, a comprehensive analysis of the P-type ATPase gene family has not been conducted in Chinese white pear (Pyrus bretschneideri) or other Rosaceae species. Here, we identified 419 P-type ATPase genes from seven Rosaceae species (Pyrus bretschneideri, Malus domestica, Prunus persica, Fragaria vesca, Prunus mume, Pyrus communis and Pyrus betulifolia). Structural and phylogenetic analyses revealed that P-type ATPase genes can be divided into five subfamilies. Different subfamilies have different conserved motifs and cis-acting elements, which may lead to functional divergence within one gene family. Dispersed duplication and whole-genome duplication may play critical roles in the expansion of the P-type ATPase family. Purifying selection was the primary force driving the evolution of P-type ATPase family genes. Based on the dynamic transcriptome analysis and transient transformation of Chinese white pear fruit, Pbr029767.1 in the P3A subfamily were found to be associated with malate accumulation during pear fruit development. Using a co-expression network, we identified several transcription factors that may have regulatory relationships with the P-type ATPase gene family. Overall, this study lays a solid foundation for understanding the evolution and functions of P-type ATPase genes in Chinese white pear and six other Rosaceae species.

Evolution of P2A and P5A ATPases: ancient gene duplications and the red algal connection to green plants revisited.

In a search for slowly evolving nuclear genes that may cast light on the deep evolution of plants, we carried out phylogenetic analyses of two well‐characterized subfamilies of P‐type pumps (P2A and P5A ATPases) from representative branches of the eukaryotic tree of life. Both P‐type ATPase genes were duplicated very early in eukaryotic evolution and before the divergence of the present eukaryotic supergroups. Synapomorphies identified in the sequences provide evidence that green plants and red algae are more distantly related than are green plants and eukaryotic supergroups in which secondary or tertiary plastids are common, such as several groups belonging to the clade that includes Stramenopiles, Alveolata, Rhizaria, Cryptophyta and Haptophyta (SAR). We propose that red algae branched off soon after the first photosynthesizing eukaryote had acquired a primary plastid, while in another lineage that led to SAR, the primary plastid was lost but, in some cases, regained as a secondary or tertiary plastid.

P-type transport ATPases in Leishmania and Trypanosoma.

P-type ATPases are critical to the maintenance and regulation of cellular ion homeostasis and membrane lipid asymmetry due to their ability to move ions and phospholipids against a concentration gradient by utilizing the energy of ATP hydrolysis. P-type ATPases are particularly relevant in human pathogenic trypanosomatids which are exposed to abrupt and dramatic changes in their external environment during their life cycles. This review describes the complete inventory of ion-motive, P-type ATPase genes in the human pathogenic Trypanosomatidae; eight Leishmania species (L. aethiopica, L. braziliensis, L. donovani, L. infantum, L. major, L. mexicana, L. panamensis, L. tropica), Trypanosoma cruzi and three Trypanosoma brucei subspecies (Trypanosoma brucei brucei TREU927, Trypanosoma brucei Lister strain 427, Trypanosoma brucei gambiense DAL972). The P-type ATPase complement in these trypanosomatids includes the P1B (metal pumps), P2A (SERCA, sarcoplasmic-endoplasmic reticulum calcium ATPases), P2B (PMCA, plasma membrane calcium ATPases), P2D (Na+ pumps), P3A (H+ pumps), P4 (aminophospholipid translocators), and P5B (no assigned specificity) subfamilies. These subfamilies represent the P-type ATPase transport functions necessary for survival in the Trypanosomatidae as P-type ATPases for each of these seven subfamilies are found in all Leishmania and Trypanosoma species included in this analysis. These P-type ATPase subfamilies are correlated with current molecular and biochemical knowledge of their function in trypanosomatid growth, adaptation, infectivity, and survival.

Neo1 and phosphatidylethanolamine contribute to vacuole membrane fusion in Saccharomyces cerevisiae.

ABSTRACTNEO1 is an essential gene in budding yeast and belongs to a highly conserved subfamily of P-type ATPase genes that encode phospholipid flippases. Inactivation of temperature sensitive neo1ts alleles produces pleiomorphic defects in the secretory and endocytic pathways, including fragmented vacuoles. A screen for multicopy suppressors of neo1-2ts growth defects yielded YPT7, which encodes a Rab7 homolog involved in SNARE-dependent vacuolar fusion. YPT7 suppressed the vacuole fragmentation phenotype of neo1-2, but did not suppress Golgi-associated protein trafficking defects. Neo1 localizes to Golgi and endosomal membranes and was only observed in the vacuole membrane, where Ypt7 localizes, in retromer mutants or when highly overexpressed in wild-type cells. Phosphatidylethanolamine (PE) has been implicated in Ypt7-dependent vacuolar membrane fusion in vitro and is a potential transport substrate of Neo1. Strains deficient in PE synthesis (psd1Δ psd2Δ) displayed fragmented vacuoles and the neo1-2 fragmented vacuole phenotype was also suppressed by overexpression of PSD2, encoding a phosphatidylserine decarboxylase that produces PE at endosomes. In contrast, neo1-2 was not suppressed by overexpression of VPS39, an effector of Ypt7 that forms a membrane contact site potentially involved in PE transfer between vacuoles and mitochondria. These results support the crucial role of PE in vacuole membrane fusion and implicate Neo1 in concentrating PE in the cytosolic leaflet of Golgi and endosomes, and ultimately the vacuole membrane.

The calcineurin-responsive transcription factor Crz1 is required for conidation, full virulence and DMI resistance in Penicillium digitatum

The Ca2+/calmodulin-dependent pathway is a critical signal transduction pathway that mediates various cellular responses in multiple organisms. Calcineurin acts through zinc finger transcription factor Crz1 in fungi. In the present study, we identified an ortholog of Crz1, PdCrz1, in Penicillium digitatum, the most important pathogen of postharvest citrus. To determine the function(s) of PdCrz1, a gene-disruption mutant was generated using an Agrobacterium tumefaciens-mediated transformation (ATMT) procedure. The PdCrz1 disruption (ΔPdCrz1) mutant was defective in conidiation, cell wall integrity and virulence, and was hypersensitivity to stresses caused by Ca2+, H2O2 or 14α-demethylation inhibitor (DMI) fungicides. Expressions of cell wall synthase genes (CHS2, CHS3 and FKS1) and P-type ATPase genes (PMR1 and PMC1) were defective in the ΔPdCrz1 mutant when compared with the wild type. Calcineurin inhibitors were shown to enhance the effect of imazalil on P. digitatum. In contrast, Ca2+ (≤0.2moll−1) could alleviate the antifungal activity by imazalil. These results indicate that PdCrz1 has important roles in conidiation, virulence and DMI resistance in P. digitatum. In addition, this study also indicates that specific calcium signaling pathway inhibitors could be used as adjuvants in the conventional DMI fungicides.

Functional characterization of the copper-transporting P-type ATPase gene of Penicillium janthinellum strain GXCR

Copper (Cu)-transporting P-type ATPase (CTPA) genes have been documented to play an important role in resistance to heavy metals. However, our understanding of roles of CTPA genes of the filamentous fungi was based only on sequence similarity prediction before. In a previous study, we isolated a Penicillum janthinellum strain GXCR of higher tolerance to Cu (200 mM). In this study, we cloned the partial cDNA of CTPA gene, named PcpA, from the strain GXCR. Sequence alignment indicated that the cloned cDNA sequence has the highest identity (94.4%) with a predictive CTPA gene of Aspergillus clavatus. The PcpA-encoded protein, termed PcpA, has classical functional domains of CTPAs, and shows differences from reported CTPAs in some specific sequence motifs and transmembrane regions. Expression of the PcpA was induced by extracellular Cu, cadmium (Cd), and silver (Ag). PcpA RNA interference (RNAi) mutants with a reduced level of PcpA mRNA were more sensitive to Cu, iron, Cd, and Ag than the wild-type (WT) strain GXCR. When grown in the presence of Cu, iron, and Cd, intracellular Cu and iron contents in the PcpA RNAi mutant were significantly (P<0.05) lower than those in the WT; However, intracellular Cd content in the mutant was significantly (P<0.05) higher than that in the WT. Taken together, it can be concluded that the PcpA functions in Cu uptake and homeostasis, iron uptake, and Cd export from the cytosol to the extracytosol.

Copper bioaccumulation by the actinobacterium Amycolatopsis sp. AB0

Amycolatopsis sp. AB0, a copper resistant actinobacterium isolated from polluted sediments, has shown high copper specific biopsortion ability (25 mg g(-1)). Two approaches were used to confirm metal accumulation in growing cells of Amycolatopsis sp. AB0; we performed subcellular fractioning assays which showed that the retained copper was associated with the extra-cellular fraction (exopolymer, 40%), but mainly within the cells. Intracellular distribution of copper was: 86% in the cytosolic fraction, 11% at the cell wall and 3% associated with the ribosome/membrane fraction. Its copper bioaccumulation ability was corroborated by using silver enhanced staining of copper with the Timm's reagent technique, which has not been used to detect metal deposits in bacteria before. In addition, we constructed specific oligonucleotides for targeting genes coding for copper P-Type ATPases that could be involved in the copper uptake ability of this strain. A 607 bp DNA fragment was amplified and sequenced from Amycolatopsis sp AB0. BLAST search analysis showed 71% protein homology of the deduced sequence with a putative cation-transporting ATPase of Nocardia farcinica and 65% with a copper translocating ATPase of Mycobacterium flavescens. To our knowledge this is the first report of the presence of copper P-type ATPase genes in the Amycolotopsis genus.

An unexpectedly high degree of specialization and a widespread involvement in sterol metabolism among the C. elegans putative aminophospholipid translocases

BackgroundP-type ATPases in subfamily IV are exclusively eukaryotic transmembrane proteins that have been proposed to directly translocate the aminophospholipids phosphatidylserine and phosphatidylethanolamine from the exofacial to the cytofacial monolayer of the plasma membrane. Eukaryotic genomes contain many genes encoding members of this subfamily. At present it is unclear why there are so many genes of this kind per organism or what individual roles these genes perform in organism development.ResultsWe have systematically investigated expression and developmental function of the six, tat-1 through 6, subfamily IV P-type ATPase genes encoded in the Caenorhabditis elegans genome. tat-5 is the only ubiquitously-expressed essential gene in the group. tat-6 is a poorly-transcribed recent duplicate of tat-5. tat-2 through 4 exhibit tissue-specific developmentally-regulated expression patterns. Strong expression of both tat-2 and tat-4 occurs in the intestine and certain other cells of the alimentary system. The two are also expressed in the uterus, during spermatogenesis and in the fully-formed spermatheca. tat-2 alone is expressed in the pharyngeal gland cells, the excretory system and a few cells of the developing vulva. The expression pattern of tat-3 is almost completely different from those of tat-2 and tat-4. tat-3 expression is detectable in the steroidogenic tissues: the hypodermis and the XXX cells, as well as in most cells of the pharynx (except gland), various tissues of the reproductive system (except uterus and spermatheca) and seam cells. Deletion of tat-1 through 4 individually interferes little or not at all with the regular progression of organism growth and development under normal conditions. However, tat-2 through 4 become essential for reproductive growth during sterol starvation.Conclusiontat-5 likely encodes a housekeeping protein that performs the proposed aminophospholipid translocase function routinely. Although individually dispensable, tat-1 through 4 seem to be at most only partly redundant. Expression patterns and the sterol deprivation hypersensitivity deletion phenotype of tat-2 through 4 suggest that these genes carry out subtle metabolic functions, such as fine-tuning sterol metabolism in digestive or steroidogenic tissues. These findings uncover an unexpectedly high degree of specialization and a widespread involvement in sterol metabolism among the genes encoding the putative aminophospholipid translocases.

The type 4 subfamily of P-type ATPases, putative aminophospholipid translocases with a role in human disease

The maintenance of phospholipid asymmetry in membrane bilayers is a paradigm in cell biology. However, the mechanisms and proteins involved in phospholipid translocation are still poorly understood. Members of the type 4 subfamily of P-type ATPases have been implicated in the translocation of phospholipids from the outer to the inner leaflet of membrane bilayers. In humans, several inherited disorders have been identified which are associated with loci harboring type 4 P-type ATPase genes. Up to now, one inherited disorder, Byler disease or progressive familial intrahepatic cholestasis type 1 (PFIC1), has been directly linked to mutations in a type 4 P-type ATPase gene. How the absence of an aminophospholipid translocase activity relates to this severe disease is, however, still unclear. Studies in the yeast Saccharomyces cerevisiae have recently identified important roles for type 4 P-type ATPases in intracellular membrane- and protein-trafficking events. These processes require an (amino)phospholipid translocase activity to initiate budding or fusion of membrane vesicles from or with other membranes. The studies in yeast have greatly contributed to our cell biological insight in membrane dynamics and intracellular-trafficking events; if this knowledge can be translated to mammalian cells and organs, it will help to elucidate the molecular mechanisms which underlie severe inherited human diseases such as Byler disease.

Genomic Comparison of P-Type ATPase Ion Pumps in Arabidopsis and Rice

Members of the P-type ATPase ion pump superfamily are found in all three branches of life. Forty-six P-type ATPase genes were identified in Arabidopsis, the largest number yet identified in any organism. The recent completion of two draft sequences of the rice (Oryza sativa) genome allows for comparison of the full complement of P-type ATPases in two different plant species. Here, we identify a similar number (43) in rice, despite the rice genome being more than three times the size of Arabidopsis. The similarly large families suggest that both dicots and monocots have evolved with a large preexisting repertoire of P-type ATPases. Both Arabidopsis and rice have representative members in all five major subfamilies of P-type ATPases: heavy-metal ATPases (P1B), Ca2+-ATPases (endoplasmic reticulum-type Ca2+-ATPase and autoinhibited Ca2+-ATPase, P2A and P2B), H+-ATPases (autoinhibited H+-ATPase, P3A), putative aminophospholipid ATPases (ALA, P4), and a branch with unknown specificity (P5). The close pairing of similar isoforms in rice and Arabidopsis suggests potential orthologous relationships for all 43 rice P-type ATPases. A phylogenetic comparison of protein sequences and intron positions indicates that the common angiosperm ancestor had at least 23 P-type ATPases. Although little is known about unique and common features of related pumps, clear differences between some members of the calcium pumps indicate that evolutionarily conserved clusters may distinguish pumps with either different subcellular locations or biochemical functions.

P-type ATPases in Caenorhabditis and Drosophila: implications for evolution of the P-type ATPase subunit families with special reference to the Na,K-ATPase and H,K-ATPase subgroup.

Here we show a complete list of the P-type ATPase genes in Caenorhabditis elegans and Drosophila melanogaster. A detailed comparison of the deduced amino-acid sequences in combination with phylogenetic and chromosomal analyses has revealed the following: (1) The diversity of this gene family has been achieved by two major evolutionary steps; the establishment of the major P-type ATPase subgroups with distinct substrate (ion) specificities in a common ancestor of vertebrate and invertebrate, followed by the evolution of multiple isoforms occurring independently in vertebrate and invertebrate phyla. (2) Pairs of genes that have intimate phylogenetic relationship are frequently found in proximity on the same chromosome. (3) Some of the Na,K- and H,K-ATPase isoforms in D. melanogaster and C. elegans lack motifs shown to be important for alpha/beta-subunit assembly, suggesting that such alpha- and beta-subunits might exist by themselves (lonely subunits). The mutation rates for these subunits are much faster than those for the subunits with recognizable assembly domains. (4) The lonely alpha-subunits also lack the major site for ouabain binding that apparently arose before the separation of vertebrates and invertebrates and thus well before the separation of vertebrate Na,K-ATPases and H,K-ATPases. These findings support the idea that a relaxation of functional constraints would increase the rate of evolution and provide clues for identifying the origins of inhibitor sensitivity, subunit assembly, and separation of Na,K- and H,K-ATPases.

Trichomonas vaginalis: characterization of a family of P-type ATPase genes

P-type ATPases are ion-transporting pumps that enable organisms to control cellular functions and survive changing environmental conditions by regulating internal ion concentrations. Eight P-type ATPases were identified in the amitochondriate protist Trichomonas vaginalis using polymerase chain reaction (PCR) amplification with oligonucleotide primers that recognize conserved motifs present in all P-type ATPases, the ATP phosphorylation site (DKTGTLT) and the ATP binding site (TGDGVND). Phylogenetic analysis and the presence of conserved motifs in predicted peptide sequences identify the Trichomonas ATPases as a sarcoplasmic-endoplasmic reticulum calcium pump (TVCA1); three additional Ca 2+ transporting pumps (TVCA2-4), three phospholipid translocases (TVAPLT1-3), and one P-type ATPase of unknown transport specificity (TVPATP8). Southern blot analyses indicate that the P-type ATPase genes are not linked and are present in single copy, except TVCA2 and TVCA4 which contain additional copies or closely related homologues within the genome. Transcripts of 3.1 kb for TVCA1, 3.0 kb for TVCA2, 2.9 kb for TVCA3, 4.0 kb for TVAPLT1, 4.2 kb for TVAPLT2, 3.9 kb for TVAPLT3, and 3.1 kb for TVPATP8 were detected by Northern blot analysis. No TVCA4 transcript was observed, however, RT-PCR amplification of a transcript product indicates that TVCA4 is expressed. RNA expression of the Trichomonas ATPases, except TVCA3, was constitutive over a range of environmental conditions. TVCA1, TVAPLT3 and TVPATP8 had the highest levels of RNA expression while TVAPLT1 and TVAPLT2 expression was the lowest.

Primary structure and evolution of the ATP-binding domains of the P-type ATPases in Tetrahymena thermophila.

The P-type ATPases (e.g., Na+-K+-ATPase and Ca2+-ATPase) occur widely in living cells of fungi, Protozoa, plants, and animals. These ion pumps show a high degree of divergence in their primary structures but share a limited number of common amino acid residues for their ATP-catalytic function. Particularly, the amino acid sequences for the phosphorylation site (DKTGTLT) and the binding site for ATP (and its analogs; GDGVND) are conserved throughout evolution. Using two degenerate oligonucleotides corresponding to these regions, we applied a polymerase chain reaction (PCR) technique to the search for P-type ATPase isoforms, which will provide a clue to the evolutionary mechanisms of ion pumps in Tetrahymena thermophila. A total of 12 distinct P-type ATPase genes were identified. Sequence comparisons revealed that seven of them can be compiled into a multigene family, which is similar to animal Na+-K+- and H+-K+-ATPase genes. One of them is close to the sarco(endo)plasmic reticulum Ca2+-ATPase gene, and the other four share a significant homology with the gene encoding Plasmodium ATPase-1 whose function is unknown. A Northern blot analysis and reverse transcriptase-PCR demonstrated that all identified genes are expressed, but the expression levels vary widely under different culture conditions. A Southern blot analysis after pulse-field gel electrophoresis showed that all of these genes exist in T. thermophila macronuclei. The Na+-K+- and H+-K+-ATPase gene family has a high multiplicity (at least 10 different genes detected on genomic Southern blot analysis) and is distributed on four different macronuclear chromosomes. On the basis of a calculation with the amino acid sequences of the cloned cytoplasmic loop region (between the phosphorylation and the gamma-[4-(N-2-chloroethyl-N-methylamino)]-benzylamido ATP sites), the genes with >80% identity form a cognate linkage group within the same macronuclei chromosome, whereas the genes with <70% identity are separated in different chromosomes. The phylogenetic analysis showed that this multigene family is the result of a series of gene duplications.

Molecular cloning and sequence of two novel P-type adenosinetriphosphatases from Plasmodium falciparum.

We have identified two novel P-type ATPase genes from Plasmodium falciparum and report the full-length nucleotide and derived amino acid sequence of the ATPase2 gene from P. falciparum (PfATPase2). PfATPase2 is phylogenetically remote from the different members of prokaryotic and mammalian P-type ATPases but shares features with a putative membrane-spanning Ca2+ ATPase involved in ribosome function in yeast. PfATPase2 is expressed during the intraerythrocytic life cycle of the parasite and appears to be required in the late stages of its asexual development. We also present the partial sequence of another malarial gene displaying sequence similarity with the family of P-type transporting ATPases (PfATPase4). We have analysed the organisation of the genes encoding the P-type ATPases of P. falciparum and show that they are a highly dispersed gene family.

A copper‐transporting P‐type ATPase found in the thylakoid membrane of the cyanobacterium Synechococcus species PCC7942

P-type ATPases constitute a large family of cation pumps that play crucial physiological roles in many organisms, including bacteria, plants and mammals. They are postulated to play important roles in a variety of environmental adaptation systems. Recently, we cloned two distinct putative P-type ATPase genes (pacS and pacL) from a photosynthetic cyanobacterium, Synechococcus species PCC7942. In this study, one of the gene products (named PacS) was found to possess a putative metal-binding motif (Gly-Met-X-Cys-X-X-Cys) in its N-terminal portion. Thus we supposed that this ATPase may function as a metal pump. Indeed, the results of Northern blotting analysis showed that pacS-mRNA specifically increases upon addition of copper or silver to the growth medium. The results of Western blotting analysis confirmed the view that PacS accumulates in copper-treated Synechococcus cells. Thus we concluded that the expression of PacS ATPase is regulated in response to the change in concentration of external metals, namely copper and silver. Consistent with this, an insertional inactivation mutant of pacS exhibited hypersensitivity in terms of growth to these potentially toxic metals. It was also revealed that PacS was mainly located in the thylakoid membrane, in which the photosynthetic reactions take place. This P-type ATPase in the thylakoid membrane is implicated as a copper-transporting system that may be involved in copper-homeostasis crucial to the photosynthetic thylakoid function.

Molecular Cloning of a P-type ATPase Gene from the Cyanobacterium Synechocystis sp. PCC 6803 : Homology to Eukaryotic Ca 2+-ATPases

With oligonucleotide primers derived from P-type ATPase genes of different sources, a part of Synechocystis sp. PCC 6803 genomic DNA was amplified and used as hybridization probe for the Synechocystis gone. A 4·7 kb HindIII fragment was cloned and sequenced; it contains the open reading frame of the E 1E 2-ATPase. The Synechocystis ATPase (named PM A1) consists of 915 amino acids with a M r of 98,902; it has ten putative transmembrane domains and contains the conserved regions a to j common to all P-type ATPases. Its amino acid sequence shows less than 20% identity to prokaryotic ATPases but about 30% identity to eukaryotic Ca 2+-ATPases. An alignment to rat kidney and yeast Ca 2+-ATPase protein sequences shows homology in stalk regions and transmembrane domains which are thought to be involved in calcium binding and transport; these three ATPases reveal very similar hydropathy plots and form a separate group in the phylogenetic tree of P-type ATPases. The results strongly support the assumption that PM A1 of Synechocystis is a calcium translocating ATPase, possibly involved in regulatory processes with calcium as second messenger.