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M G Palmgren

Publications and source records attributed to M G Palmgren.

At least 19 recordsLinked to original sources
PubMed2003

Mechanism of proton transport by plant plasma membrane proton ATPases.

The mechanism of proton translocation by P-type proton ATPases is poorly defined. Asp684 in transmembrane segment M6 of the Arabidopsis thaliana AHA2 plasma membrane P-type proton pump is suggested to act as an essential proton acceptor during proton translocation. Arg655 in transmembrane segment M5 seems to be involved in this proton translocation too, but in contrast to Asp684, is not essential for transport. Asp684 may participate in defining the E(1) proton-binding site, which could possibly exist as a hydronium ion coordination center. A model of proton translocation of AHA2 involving the side chains of amino acids Asp684 and Arg655 is discussed.

Arabidopsis↗
PubMed2003

Mechanism of proton pumping by plant plasma membrane H+-ATPase: role of residues in transmembrane segments 5 and 6.

The mechanism of proton pumping by P-type plasma membrane H(+)-ATPases is not well clarified. Site-directed mutagenesis studies suggest that Asp684, situated in transmembrane segment M6, is involved in coordination of proton(s) in plant plasma membrane H(+)-ATPase. This hypothesis is supported by atomic models of H(+)-ATPases built on the basis of the crystal structure of the related SERCA1a Ca(2+)-ATPase. However, more biochemical, genetic, and structural studies are required before we will be able to understand the nature of the proton binding site(s) in P-type H(+)-ATPases and the mechanism of action of these pumps.

Amino Acid Sequence↗
PubMed2002

Phosphorylation-independent interaction between 14-3-3 protein and the plant plasma membrane H+-ATPase.

14-3-3 proteins interact with a novel phosphothreonine motif (Y(946)pTV) at the extreme C-terminal end of the plant plasma membrane H(+)-ATPase molecule. Phosphorylation-independent binding of 14-3-3 protein to the YTV motif can be induced by the fungal phytotoxin fusicoccin. The molecular basis for the phosphorylation-independent interaction between 14-3-3 and H(+)-ATPase in the presence of fusicoccin has been investigated in more detail. Fusicoccin binds to a heteromeric receptor that involves both 14-3-3 protein and H(+)-ATPase. Binding of fusicoccin is dependent upon the YTV motif in the H(+)-ATPase and, in addition, requires residues further upstream of this motif. Apparently, 14-3-3 proteins interact with the unusual epitope in H(+)-ATPase via its conserved amphipathic groove. This implies that very diverse epitopes bind to a common structure in the 14-3-3 protein.

14-3-3 Proteins↗
PubMed2001

Large scale expression, purification and 2D crystallization of recombinant plant plasma membrane H+-ATPase.

P-type ATPases convert chemical energy into electrochemical gradients that are used to energize secondary active transport. Analysis of the structure and function of P-type ATPases has been limited by the lack of active recombinant ATPases in quantities suitable for crystallographic studies aiming at solving their three-dimensional structure. We have expressed Arabidopsis thaliana plasma membrane H+-ATPase isoform AHA2, equipped with a His(6)-tag, in the yeast Saccharomyces cerevisiae. The H+-ATPase could be purified both in the presence and in the absence of regulatory 14-3-3 protein depending on the presence of the diterpene fusicoccin which specifically induces formation of the H+-ATPase/14-3-3 protein complex. Amino acid analysis of the purified complex suggested a stoichiometry of two 14-3-3 proteins per H+-ATPase polypeptide. The purified H(+)-ATPase readily formed two-dimensional crystals following reconstitution into lipid vesicles. Electron cryo-microscopy of the crystals yielded a projection map at approximately 8 A resolution, the p22(1)2(1) symmetry of which suggests a dimeric protein complex. Three distinct regions of density of approximately equal size are apparent and may reflect different domains in individual molecules of AHA2.

14-3-3 Proteins↗
PubMed2001

Two-dimensional crystallization of a membrane protein on a detergent-resistant lipid monolayer.

Two-dimensional crystals of a membrane protein, the proton ATPase from plant plasma membranes, have been obtained by a new strategy based on the use of functionalized, fluorinated lipids spread at the air-water interface. Monolayers of the fluorinated lipids are stable even in the presence of high concentrations of various detergents as was established by ellipsometry measurements. A nickel functionalized fluorinated lipid was spread into a monolayer at the air-water interface. The overexpressed His-tagged ATPase solubilized by detergents was added to the subphase. 2D crystals of the membrane protein, embedded in a lipid bilayer, formed as the detergent was removed by adsorption. Electron microscopy indicated that the 2D crystals were single layers with dimensions of 10 microm or more. Image processing yielded a projection map at 9 A resolution, showing three well-separated domains of the membrane-embedded proton ATPase.

Adsorption↗
PubMed2001

A putative proton binding site of plasma membrane H(+)-ATPase identified through homology modelling.

We have used the 2.6 A structure of the rabbit sarcoplasmic reticulum Ca(2+)-ATPase isoform 1a, SERCA1a [Toyoshima, C., Nakasako, M., Nomura, H. and Ogawa, H. (2000) Nature 405, 647-655], to build models by homology modelling of two plasma membrane (PM) H(+)-ATPases, Arabidopsis thaliana AHA2 and Saccharomyces cerevisiae PMA1. We propose that in both yeast and plant PM H(+)-ATPases a strictly conserved aspartate in transmembrane segment (M)6 (D684(AHA2)/D730(PMA1)), and three backbone carbonyls in M4 (I282(AHA2)/I331(PMA1), G283(AHA2)/I332(PMA1) and I285(AHA2)/V334(PMA1)) comprise a binding site for H3O(+), suggesting a previously unknown mechanism for transport of protons. Comparison with the structure of the SERCA1a made it feasible to suggest a possible receptor region for the C-terminal auto-inhibitory domain extending from the phosphorylation and anchor domains into the transmembrane region.

Animals↗
PubMed2001

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.

Adenosine Triphosphatases↗
PubMed2000

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.

Adenosine Triphosphatases↗
PubMed2000

Molecular aspects of higher plant P-type Ca(2+)-ATPases.

Recent genomic data in the model plant Arabidopsis thaliana reveal the existence of at least 11 Ca(2+)-ATPase genes, and an analysis of expressed sequence tags suggests that the number of calcium pumps in this organism might be even higher. A phylogenetic analysis shows that 11 Ca(2+)-ATPases clearly form distinct groups, type IIA (or ECA for ER-type Ca(2+)-ATPase) and type IIB (ACA for autoinhibited Ca(2+)-ATPase). While plant IIB calcium pumps characterized so far are localized to internal membranes, their animal homologues are exclusively found in the plasma membrane. However, Arabidopsis type IIB calcium pump isoforms ACA8, ACA9 and ACA10 form a separate outgroup and, based on the high molecular masses of the encoded proteins, are good candidates for plasma membrane bound Ca(2+)-ATPases. All known plant type IIB calcium ATPases seem to employ an N-terminal calmodulin-binding autoinhibitor. Therefore it appears that the activity of type IIB Ca(2+)-ATPases in plants and animals is controlled by N-terminal and C-terminal autoinhibitory domains, respectively. Possible functions of plant calcium pumps are described and - beside second messenger functions directly linked to calcium homeostasis - new data on a putative involvement in secretory and salt stress functions are discussed.

Amino Acid Sequence↗
PubMed2000

At-ACA8 encodes a plasma membrane-localized calcium-ATPase of Arabidopsis with a calmodulin-binding domain at the N terminus.

A Ca(2+)-ATPase was purified from plasma membranes (PM) isolated from Arabidopsis cultured cells by calmodulin (CaM)-affinity chromatography. Three tryptic fragments from the protein were microsequenced and the corresponding cDNA was amplified by polymerase chain reaction using primers designed from the microsequences of the tryptic fragments. At-ACA8 (Arabidopsis-autoinhibited Ca(2+)-ATPase, isoform 8, accession no. AJ249352) encodes a 1,074 amino acid protein with 10 putative transmembrane domains, which contains all of the characteristic motifs of Ca(2+)-transporting P-type Ca(2+)-ATPases. The identity of At-ACA8p as the PM Ca(2+)-ATPase was confirmed by immunodetection with an antiserum raised against a sequence (valine-17 through threonine-31) that is not found in other plant CaM-stimulated Ca(2+)-ATPases. Confocal fluorescence microscopy of protoplasts immunodecorated with the same antiserum confirmed the PM localization of At-ACA8. At-ACA8 is the first plant PM localized Ca(2+)-ATPase to be cloned and is clearly distinct from animal PM Ca(2+)-ATPases due to the localization of its CaM-binding domain. CaM overlay assays localized the CaM-binding domain of At-ACA8p to a region of the N terminus of the enzyme around tryptophan-47, in contrast to a C-terminal localization for its animal counterparts. Comparison between the sequence of At-ACA8p and those of endomembrane-localized type IIB Ca(2+)-ATPases of plants suggests that At-ACA8 is a representative of a new subfamily of plant type IIB Ca(2+)-ATPases.

Amino Acid Sequence↗
PubMed2000

The ACA4 gene of Arabidopsis encodes a vacuolar membrane calcium pump that improves salt tolerance in yeast.

Several lines of evidence suggest that regulation of intracellular Ca(2+) levels is crucial for adaptation of plants to environmental stress. We have cloned and characterized Arabidopsis auto-inhibited Ca(2+)-ATPase, isoform 4 (ACA4), a calmodulin-regulated Ca(2+)-ATPase. Confocal laser scanning data of a green fluorescent protein-tagged version of ACA4 as well as western-blot analysis of microsomal fractions obtained from two-phase partitioning and Suc density gradient centrifugation suggest that ACA4 is localized to small vacuoles. The N terminus of ACA4 contains an auto-inhibitory domain with a binding site for calmodulin as demonstrated through calmodulin-binding studies and complementation experiments using the calcium transport yeast mutant K616. ACA4 and PMC1, the yeast vacuolar Ca(2+)-ATPase, conferred protection against osmotic stress such as high NaCl, KCl, and mannitol when expressed in the K616 strain. An N-terminally modified form of ACA4 specifically conferred increased NaCl tolerance, whereas full-length ATPase had less effect.

Adaptation, Physiological↗
PubMed2000

Chilling tolerance in Arabidopsis involves ALA1, a member of a new family of putative aminophospholipid translocases.

The lipid composition of membranes is a key determinant for cold tolerance, and enzymes that modify membrane structure seem to be important for low-temperature acclimation. We have characterized ALA1 (for aminophospholipid ATPase1), a novel P-type ATPase in Arabidopsis that belongs to the gene family ALA1 to ALA11. The deduced amino acid sequence of ALA1 is homologous with those of yeast DRS2 and bovine ATPase II, both of which are putative aminophospholipid translocases. ALA1 complements the deficiency in phosphatidylserine internalization into intact cells that is exhibited by the drs2 yeast mutant, and expression of ALA1 results in increased translocation of aminophospholipids in reconstituted yeast membrane vesicles. These lines of evidence suggest that ALA1 is involved in generating membrane lipid asymmetry and probably encodes an aminophospholipid translocase. ALA1 complements the cold sensitivity of the drs2 yeast mutant. Downregulation of ALA1 in Arabidopsis results in cold-affected plants that are much smaller than those of the wild type. These data suggest a link between regulation of transmembrane bilayer lipid asymmetry and the adaptation of plants to cold.

Adaptation, Physiological↗
PubMed1999

Binding of 14-3-3 protein to the plasma membrane H(+)-ATPase AHA2 involves the three C-terminal residues Tyr(946)-Thr-Val and requires phosphorylation of Thr(947).

14-3-3 proteins play a regulatory role in a diverse array of cellular functions such as apoptosis, regulation of the cell cycle, and regulation of gene transcription. The phytotoxin fusicoccin specifically induces association of virtually any 14-3-3 protein to plant plasma membrane H(+)-ATPase. The 14-3-3 binding site in the Arabidopsis plasma membrane H(+)-ATPase AHA2 was localized to the three C-terminal residues of the enzyme (Tyr(946)-Thr-Val). Binding of 14-3-3 protein to this target was induced by phosphorylation of Thr(947) (K(D) = 88 nM) and was in practice irreversible in the presence of fusicoccin (K(D) = 7 nM). Mass spectrometry analysis demonstrated that AHA2 expressed in yeast was phosphorylated at Thr(947). We conclude that the extreme end of AHA2 contains an unusual high-affinity binding site for 14-3-3 protein.

14-3-3 Proteins↗
PubMed1999

Molecular dissection of the C-terminal regulatory domain of the plant plasma membrane H+-ATPase AHA2: mapping of residues that when altered give rise to an activated enzyme.

The plasma membrane H+-ATPase is a proton pump belonging to the P-type ATPase superfamily and is important for nutrient acquisition in plants. The H+-ATPase is controlled by an autoinhibitory C-terminal regulatory domain and is activated by 14-3-3 proteins which bind to this part of the enzyme. Alanine-scanning mutagenesis through 87 consecutive amino acid residues was used to evaluate the role of the C-terminus in autoinhibition of the plasma membrane H+-ATPase AHA2 from Arabidopsis thaliana. Mutant enzymes were expressed in a strain of Saccharomyces cerevisiae with a defective endogenous H+-ATPase. The enzymes were characterized by their ability to promote growth in acidic conditions and to promote H+ extrusion from intact cells, both of which are measures of plasma membrane H+-ATPase activity, and were also characterized with respect to kinetic properties such as affinity for H+ and ATP. Residues that when altered lead to increased pump activity group together in two regions of the C-terminus. One region stretches from K863 to L885 and includes two residues (Q879 and R880) that are conserved between plant and fungal H+-ATPases. The other region, incorporating S904 to L919, is situated in an extension of the C-terminus unique to plant H+-ATPases. Alteration of residues in both regions led to increased binding of yeast 14-3-3 protein to the plasma membrane of transformed cells. Taken together, our data suggest that modification of residues in two regions of the C-terminal regulatory domain exposes a latent binding site for activatory 14-3-3 proteins.

14-3-3 Proteins↗
PubMed1998

14-3-3 proteins activate a plant calcium-dependent protein kinase (CDPK).

Plants and protozoa contain a unique family of calcium-dependent protein kinases (CDPKs) which are defined by the presence of a carboxyl-terminal calmodulin-like regulatory domain. We present biochemical evidence indicating that at least one member of this kinase family can be stimulated by 14-3-3 proteins. Isoform CPK-1 from the model plant Arabidopsis thaliana was expressed as a fusion protein in E. coli and purified. The calcium-dependent activity of this recombinant CPK-1 was shown to be stimulated almost twofold by three different 14-3-3 isoforms with 50% activation around 200 nM. 14-3-3 proteins bound to the purified CPK-1, as shown by binding assays in which either the 14-3-3 or CPK-1 were immobilized on a matrix. Both the 14-3-3 binding and activation of CPK-1 were specifically disrupted by a known 14-3-3 binding peptide LSQRQRSTpSTPNVHMV (IC50 = 30 microM). These results raise the question of whether 14-3-3 can modulate the activity of CDPK signal transduction pathways in plants.

14-3-3 Proteins↗
PubMed1998

A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain.

To study transporters involved in regulating intracellular Ca2+, we isolated a full-length cDNA encoding a Ca2+-ATPase from a model plant, Arabidopsis, and named it ACA2 (Arabidopsis Ca2+-ATPase, isoform 2). ACA2p is most similar to a "plasma membrane-type" Ca2+-ATPase, but is smaller (110 kDa), contains a unique N-terminal domain, and is missing a long C-terminal calmodulin-binding regulatory domain. In addition, ACA2p is localized to an endomembrane system and not the plasma membrane, as shown by aqueous-two phase fractionation of microsomal membranes. ACA2p was expressed in yeast as both a full-length protein (ACA2-1p) and an N-terminal truncation mutant (ACA2-2p; Delta residues 2-80). Only the truncation mutant restored the growth on Ca2+-depleted medium of a yeast mutant defective in both endogenous Ca2+ pumps, PMR1 and PMC1. Although basal Ca2+-ATPase activity of the full-length protein was low, it was stimulated 5-fold by calmodulin (50% activation around 30 nM). In contrast, the truncated pump was fully active and insensitive to calmodulin. A calmodulin-binding sequence was identified within the first 36 residues of the N-terminal domain, as shown by calmodulin gel overlays on fusion proteins. Thus, ACA2 encodes a novel calmodulin-regulated Ca2+-ATPase distinguished by a unique N-terminal regulatory domain and a non-plasma membrane localization.

Amino Acid Sequence↗