Inventory of the superfamily of P-type ion pumps in Arabidopsis

A total of 45 genes encoding for P-type ATPases have been identified in the complete genome sequence of Arabidopsis. Thus, this plant harbors a primary transport capability not seen in any other eukaryotic organism sequenced so far. The sequences group in all five subfamilies of P-type ATPases. The most prominent subfamilies are P(1B) ATPases (heavy metal pumps; seven members), P(2A) and P(2B) ATPases (Ca(2+) pumps; 14 in total), P(3A) ATPases (plasma membrane H(+) pumps; 12 members including a truncated pump, which might represent a pseudogene or an ATPase-like protein with an alternative function), and P(4) ATPases (12 members). P(4) ATPases have been implicated in aminophosholipid flipping but it is not known whether this is a direct or an indirect effect of pump activity. Despite this apparent plethora of pumps, Arabidopsis appears to be lacking Na(+) pumps and secretory pathway (PMR1-like) Ca(2+)-ATPases. A cluster of Arabidopsis heavy metal pumps resembles bacterial Zn(2+)/Co(2+)/Cd(2+)/Pb(2+) transporters. Two members of the cluster have extended C termini containing putative heavy metal binding motifs. The complete inventory of P-type ATPases in Arabidopsis is an important starting point for reverse genetic and physiological approaches aiming at elucidating the biological significance of these pumps.     

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.     

The plant Ca2+ -ATPase repertoire: biochemical features and physiological functions

Ca(2+)-ATPases are P-type ATPases that use the energy of ATP hydrolysis to pump Ca(2+) from the cytoplasm into intracellular compartments or into the apoplast. Plant cells possess two types of Ca(2+) -pumping ATPase, named ECAs (for ER-type Ca(2+)-ATPase) and ACAs (for auto-inhibited Ca(2+)-ATPase). Each type comprises different isoforms, localised on different membranes. Here, we summarise available knowledge of the biochemical characteristics and the physiological role of plant Ca(2+)-ATPases, greatly improved after gene identification, which allows both biochemical analysis of single isoforms through heterologous expression in yeast and expression profiling and phenotypic analysis of single isoform knock-out mutants.     

P-type proton ATPases are involved in intracellular calcium and proton uptake in the plant parasite Phytomonas francai

The use of digitonin to permeabilize the plasma membrane of promastigotes of Phytomonas francai allowed the identification of two non-mitochondrial Ca(2+) compartments; one sensitive to ionomycin and vanadate (neutral or alkaline), possibly the endoplasmic reticulum, and another sensitive to the combination of nigericin plus ionomycin (acidic), possibly the acidocalcisomes. A P-type (phospho-intermediate form) Ca(2+)-ATPase activity was found to be responsible for intracellular Ca(2+) transport in these cells, with no evidence of a mitochondrial Ca(2+) transport activity. ATP-driven acidification of internal compartments in cell lysates and cells mechanically permeabilized was assayed spectrophotometrically with acridine orange. This activity was inhibited by low concentrations of vanadate and digitonin, was insensitive to bafilomycin A(1), and stimulated by Na(+) ions. Taken together, our results indicate that P-type ATPases are involved in intracellular Ca(2+) and H(+) transport in promastigotes of P. francai.     

Crystal structure of phosphoserine phosphatase from Methanococcus jannaschii, a hyperthermophile, at 1.8 A resolution

BACKGROUND: D-Serine is a co-agonist of the N-methyl-D-aspartate subtype of glutamate receptors, a major neurotransmitter receptor family in mammalian nervous systems. D-Serine is converted from L-serine, 90% of which is the product of the enzyme phosphoserine phosphatase (PSP). PSP from M. jannaschii (MJ) shares significant sequence homology with human PSP. PSPs and P-type ATPases are members of the haloacid dehalogenase (HAD)-like hydrolase family, and all members share three conserved sequence motifs. PSP and P-type ATPases utilize a common mechanism that involves Mg(2+)-dependent phosphorylation and autodephosphorylation at an aspartyl side chain in the active site. The strong resemblance in sequence and mechanism implies structural similarity among these enzymes. RESULTS: The PSP crystal structure resembles the NAD(P) binding Rossmann fold with a large insertion of a four-helix-bundle domain and a beta hairpin. Three known conserved sequence motifs are arranged next to each other in space and outline the active site. A phosphate and a magnesium ion are bound to the active site. The active site is within a closed environment between the core alpha/beta domain and the four-helix-bundle domain. CONCLUSIONS: The crystal structure of MJ PSP was determined at 1.8 A resolution. Critical residues were assigned based on the active site structure and ligand binding geometry. The PSP structure is in a closed conformation that may resemble the phosphoserine bound state or the state after autodephosphorylation. Compared to a P-type ATPase (Ca(2+)-ATPase) structure, which is in an open state, this PSP structure appears also to be a good model for the closed conformation of P-type ATPase.     

Cloning and Characterization of an ATPase Gene from Pneumocystis carinii which Closely Resembles Fungal H+ ATPases

ABSTRACT. A gene encoding a P-type cation translocating ATPase was cloned from a genomic library of rat-derived Pneumocystis carinii. The nucleotide sequence of the gene contains a 2781 base-pair open reading frame that is predicted to encode a 101, 401 dalton protein composed of 927 amino acids. The P. carinii ATPase protein (pcal) is 69–75% identical when compared with eight proton pumps from six fungal species. The Pneumocystis ATPase is less than 34% identical to ATPase proteins from protozoans, vertebrates or the Ca⁺⁺ ATPases of yeast. The P. carinii ATPase contains 115 of 121 residues previously identified as characteristic of H⁺ ATPases. Alignment of the Pneumocystis and fungal proton pumps reveals five homologous domains specific for fungal H⁺ ATPases.     

Abolishment of proton pumping and accumulation in the E1P conformational state of a plant plasma membrane H+-ATPase by substitution of a conserved aspartyl residue in transmembrane segment 6

The plasma membrane H(+)-ATPase AHA2 of Arabidopsis thaliana, which belongs to the P-type ATPase superfamily of cation-transporting ATPases, pumps protons out of the cell. To investigate the mechanism of ion transport by P-type ATPases we have mutagenized Asp(684), a residue in transmembrane segment M6 of AHA2 that is conserved in Ca(2+)-, Na(+)/K(+)-, H(+)/K(+)-, and H(+)-ATPases and which coordinates Ca(2+) ions in the SERCA1 Ca(2+)-ATPase. We describe the expression, purification, and biochemical analysis of the Asp(684) --> Asn mutant, and provide evidence that Asp(684) in the plasma membrane H(+)-ATPase is required for any coupling between ATP hydrolysis, enzyme conformational changes, and H(+)-transport. Proton pumping by the reconstituted mutant enzyme was completely abolished, whereas ATP was still hydrolyzed. The mutant was insensitive to the inhibitor vanadate, which preferentially binds to P-type ATPases in the E(2) conformation. During catalysis the Asp(684) --> Asn enzyme accumulated a phosphorylated intermediate whose stability was sensitive to addition of ADP. We conclude that the mutant enzyme is locked in the E(1) conformation and is unable to proceed through the E(1)P-E(2)P transition.     

Autoinhibition of a calmodulin-dependent calcium pump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain

The regulation of Ca(2+)-pumps is important for controlling [Ca(2+)] in the cytosol and organelles of all eukaryotes. Here, we report a genetic strategy to identify residues that function in autoinhibition of a novel calmodulin-activated Ca(2+)-pump with an N-terminal regulatory domain (isoform ACA2 from Arabidopsis). Mutant pumps with constitutive activity were identified by complementation of a yeast (K616) deficient in two Ca(2+)-pumps. Fifteen mutations were found that disrupted a segment of the N-terminal autoinhibitor located between Lys(23) and Arg(54). Three mutations (E167K, D219N, and E341K) were found associated with the stalk that connects the ATPase catalytic domain (head) and with the transmembrane domain. Enzyme assays indicated that the stalk mutations resulted in calmodulin-independent activity, with V(max), K(mATP), and K(mCa(2+)) similar to that of a pump in which the N-terminal autoinhibitor had been deleted. A highly conservative substitution at Asp(219) (D219E) still produced a deregulated pump, indicating that the autoinhibitory structure in the stalk is highly sensitive to perturbation. In plasma membrane H(+)-ATPases from yeast and plants, similarly positioned mutations resulted in hyperactive pumps. Together, these results suggest that a structural feature of the stalk is of general importance in regulating diverse P-type ATPases.     

CadA, the Cd2+-ATPase from Listeria monocytogenes, can use Cd2+ as co-substrate

CadA is a membrane protein of the P-type ATPase family which is the major determinant of the resistance to Cd2+ in Listeria monocytogenes. During its catalytic cycle, CadA undergoes auto-phosphorylation from ATP at Asp398, which allows Cd2+ translocation across the membrane. In the reverse mode, Asp398 is phosphorylated from Pi. From the data obtained so far, the CadA catalytic mechanism is similar to that proposed for the sarcoplasmic reticulum Ca2+-ATPase, the model of the P-type ATPase family. We show here that CadA is sensitive to two different ranges of Cd2+ concentration. The 0.1-10 microM range of added CdCl2 corresponds to Cd2+ binding at the transport site of unphosphorylated CadA which induces the reaction of the enzyme with ATP and impairs its reaction with Pi. The 0.1-1 mM range of added CdCl2 could correspond to Cd2+ binding to the transport site accessible from the extracellular medium. In addition, although it is widely accepted that the actual substrate of P-type ATPases is the MgATP complex, we show here that CadA can also perform its cycle in the absence of Mg2+, using CdATP in the place of MgATP at the catalytic site.     

Molecular cloning and tissue distribution of alternatively spliced mRNAs encoding possible mammalian homologues of the yeast secretory pathway calcium pump.

Rat stomach and testis cDNAs corresponding to two alternatively spliced mRNAs encoding variants of a P-type ion-transport ATPase that closely resembles the yeast secretory pathway Ca2+ pump have been isolated and characterized. A partial kidney cDNA was identified previously using an oligonucleotide probe corresponding to part of the sarcoplasmic reticulum Ca(2+)-ATPase [Gunteski-Hamblin, A., Greeb, J., & Shull, G.E. (1988) J. Biol. Chem. 263, 15032-15040]. In the present study, we first isolated and characterized a stomach cDNA that contains the entire coding sequence. The 919 amino acid enzyme has the same apparent transmembrane organization and contains all of the conserved domains present in other P-type ATPases. Northern blot analyses demonstrate that 3.9- and 5-kilobase mRNAs corresponding to the cDNA were present in all tissues examined, suggesting that the protein it encodes performs a housekeeping function. Rat testis also contained a 3.7-kilobase mRNA that hybridized with a probe from the 5' end of the stomach cDNA but did not hybridize with a probe from the 3' end. Cloning and characterization of cDNAs corresponding to the smaller testis mRNA revealed that it is derived from the same gene but encodes a variant of the enzyme in which the C-terminal residue, Val-919, is replaced by the sequence Phe-919-Tyr-Pro-Lys-Ile-923. Similarity comparisons show that the two enzymes are more closely related to the known Ca2+ pumps than to other P-type ATPases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anticipating antiport in P-type ATPases

Cation-transporting P-type ATPases show a high degree of structural and functional homology. Nevertheless, for many members of this large family, the molecular mechanism of transport is unclear; namely, whether transport is electrogenic or not and if countertransport is involved remains to be established. In a few well-studied cases such as the Na(+)-K(+)-ATPase, plasma membrane Ca(2+) ATPase (PMCA) and sarcoplasmic reticulum Ca(2+) ATPase (SERCA) countertransport has been clearly demonstrated. New data based on the crystal structure of SERCA now strongly indicate that countertransport could be mandatory for all P-type ATPases. This concept should be verified for other known and for all newly characterized P-type ATPases.     

Molecular mechanism of the P-type ATPases

The recent determination of the structure of the Ca²⁺-ATPase of sarcoplasmic reticulum to atomic resolution in the Ca²⁺-bound state and to near atomic resolution in the Ca²⁺-free, decavanadate-bound state has paved the way for an ultimate complete understanding of the molecular mechanism of the P-type ATPases. Analysis of this new structure information together with the large amount of biochemical information about these enzymes that preceded it has produced important new revelations about how the P-type ATPases work. Most models propose that these transporters operate by a strictly conformational energy coupling mechanism in which conformational changes in the large cytoplasmic head region mechanically drive the ions to be transported from their binding sites in the transmembrane helix region 50 Å away. However, while these enzymes do indeed undergo profound conformational changes, the available evidence suggests that they do not mechanically transduce the chemical energy of ATP hydrolysis into transmembrane ion gradients via these conformational changes. As an alternative, it is proposed that the effects of the chemical events that occur at the phosphorylation/dephosphorylation site in the cytoplasmic region are exerted on the ion-binding sites via two well-defined charge transfer pathways that electronically connect the chemical reaction site with the site of ion binding. The recognition of these charge transfer pathways provides rational explanations of all of the key biochemical features of the P-type ATPase catalytic cycle. Thus, although a few details await elucidation, a nearly complete understanding of the P-type ATPase reaction mechanism may be at hand.     

Flexible P-type ATPases interacting with the membrane

Cation pumps and lipid flippases of the P-type ATPase family maintain electrochemical gradients and asymmetric lipid distributions across membranes, and offer significant insight of protein:membrane interactions. The sarcoplasmic reticulum Ca(2+)-ATPase features flexible and adaptive interactions with the surrounding membrane, while the Na(+),K(+)-ATPase complex is modulated by membrane components and a role for the γ-subunit as a stabilizer of a specific lipid interaction with the α-subunit has been proposed. The first crystal structure of a heavy-metal transporting ATPase shows a markedly amphipathic helix at the cytoplasmic membrane surface, highlighting this structure as a general motif of all P-type ATPases although with specialization to different membranes. Residues of central importance for the lipid flippase activity of the P4-type ATPase subfamily have been pinpointed by mutational studies, but the transport pathway and mechanism remain unknown.     

The p-type ATPase superfamily

P-type ATPases function to provide homeostasis in higher eukaryotes, but they are essentially ubiquitous, being found in all domains of life. Thever and Saier [J Memb Biol 2009;229:115-130] recently reported analyses of eukaryotic P-type ATPases, dividing them into nine functionally characterized and 13 functionally uncharacterized (FUPA) families. In this report, we analyze P-type ATPases in all major prokaryotic phyla for which complete genome sequence data are available, and we compare the results with those for eukaryotic P-type ATPases. Topological type I (heavy metal) P-type ATPases predominate in prokaryotes (approx. tenfold) while type II ATPases (specific for Na(+),K(+), H(+) Ca(2+), Mg(2+) and phospholipids) predominate in eukaryotes (approx. twofold). Many P-type ATPase families are found exclusively in prokaryotes (e.g. Kdp-type K(+) uptake ATPases (type III) and all ten prokaryotic FUPA familes), while others are restricted to eukaryotes (e.g. phospholipid flippases and all 13 eukaryotic FUPA families). Horizontal gene transfer has occurred frequently among bacteria and archaea, which have similar distributions of these enzymes, but rarely between most eukaryotic kingdoms, and even more rarely between eukaryotes and prokaryotes. In some bacterial phyla (e.g. Bacteroidetes, Flavobacteria and Fusobacteria), ATPase gene gain and loss as well as horizontal transfer occurred seldom in contrast to most other bacterial phyla. Some families (i.e. Kdp-type ATPases) underwent far less horizontal gene transfer than other prokaryotic families, possibly due to their multisubunit characteristics. Functional motifs are better conserved across family lines than across organismal lines, and these motifs can be family specific, facilitating functional predictions. In some cases, gene fusion events created P-type ATPases covalently linked to regulatory catalytic enzymes. In one family (FUPA Family 24), a type I ATPase gene (N-terminal) is fused to a type II ATPase gene (C-terminal) with retention of function only for the latter. Several pseudogene-encoded nonfunctional ATPases were identified. Genome minimalization led to preferential loss of P-type ATPase genes. We suggest that in prokaryotes and some unicellular eukaryotes, the primary function of P-type ATPases is protection from extreme environmental stress conditions. The classification of P-type ATPases of unknown function into phylogenetic families provides guides for future molecular biological studies.     

Functional modules of KdpB, the catalytic subunit of the Kdp-ATPase from Escherichia coli

The large cytoplasmic domain (H4H5) of KdpB of the KdpFABC complex (P-type ATPase) from Escherichia coli consists of two separate modules, the phosphorylation domain (KdpBP) and the nucleotide binding domain (KdpBN). The H4H5 and the KdpBN domains were purified as soluble 10His-tagged fusion proteins. Both proteins exhibit a mainly alpha-helical secondary structure as judged by CD spectroscopy. Fluorescein 5-isothiocyanate (FITC) labeling studies revealed that both proteins form a proper nucleotide binding site. Adenosine nucleotides protect the H4H5 loop but not KdpBN against FITC modification. Trinitrophenyl (TNP)-nucleotide binding studies revealed that both H4H5 and KdpBN bind nucleotides with high affinity. Furthermore, the H4H5 loop was still able to hydrolyze ATP, as well as p-nitrophenyl phosphate (pNPP). These results lend support to the notion that the separately synthesized H4H5 and KdpBN domains retain their native structure and that they reveal properties of both P2-type ATPases (e.g., Na(+),K(+)-ATPase and Ca(2+)-ATPase) and P1b-type ATPases (e.g., heavy metal transporting ATPases). Furthermore, this report also emphasizes the unique position of the Kdp-ATPase within the P-type ATPase family.     

Molecular dissection of functional domains of the E1E2-ATPase using sodium and calcium pump chimeric molecules.

Proposed models for the catalytic subunit of the E1E2-ATPases (ion pumps) predict that the first four transmembrane domains (M1 - M4) reside in the NH2 terminal one-third of the molecule, and the remainder (M5 - M10) in the COOH terminal one-third. The amino-acid sequences for the 5'-(p-fluorosulfonyl)-benzoyl-adenosine (FSBA) binding region residing just before M5 segment are very well conserved among distinct ion pumps. Taking advantage of these models, we have constructed a set of chicken chimeric ion pumps between the (Na++ K+)-ATPase alpha-subunit and the Ca(2+)-ATPase using the FSBA-binding site as an exchange junction, thereby preserving overall topological structure as E1E2 ATPases. From various functional assays on these chimeric ion pumps, including ouabain-inhibitable ATPase activity, Ca2+ binding, Ca2+ uptake, and subunit assembly based on immuno-coprecipitation, the following conclusions were obtained: (a) A (Na++ K+)-ATPase inhibitor, ouabain, binds to the regions before M4 in the alpha-subunit and exerts its inhibitory effect. (b) The regions after M5 of the (Na++ K+)-ATPase alpha-subunit bind the beta-subunit, even when these regions are incorporated into the corresponding domains in the Ca(2+)-ATPase. (c) The corresponding domains of the Ca(2+)-ATPase, the regions after M5, bind 45Ca even when it is incorporated into the corresponding position of the (Na++ K+)-ATPase alpha-subunit.