The Arabidopsis heavy metal P-type ATPase HMA5 interacts with metallochaperones and functions in copper detoxification of roots

Since copper (Cu) is essential in key physiological oxidation reactions, organisms have developed strategies for handling Cu while avoiding its potentially toxic effects. Among the tools that have evolved to cope with Cu is a network of Cu homeostasis factors such as Cu-transporting P-type ATPases that play a key role in transmembrane Cu transport. In this work we present the functional characterization of an Arabidopsis Cu-transporting P-type ATPase, denoted heavy metal ATPase 5 (HMA5), and its interaction with Arabidopsis metallochaperones. HMA5 is primarily expressed in roots, and is strongly and specifically induced by Cu in whole plants. We have identified and characterized plants carrying two independent T-DNA insertion alleles, hma5-1 and hma5-2. Both mutants are hypersensitive to Cu but not to other metals such as iron, zinc or cadmium. Interestingly, root tips from Cu-treated hma5 mutants exhibit a wave-like phenotype at early stages and later on main root growth completely arrests whereas lateral roots emerge near the crown. Accordingly, these lines accumulate Cu in roots to a greater extent than wild-type plants under Cu excess. Finally, yeast two-hybrid experiments demonstrate that the metal-binding domains of HMA5 interact with Arabidopsis ATX1-like Cu chaperones, and suggest a regulatory role for the plant-specific domain of the CCH Cu chaperone. Based on these findings, we propose a role for HMA5 in Cu compartmentalization and detoxification.     

PAA1, a P-type ATPase of Arabidopsis,functions in copper transport in chloroplasts

Copper (Cu) is an essential trace element with important roles as a cofactor in many plant functions, including photosynthesis. However, free Cu ions can cause toxicity, necessitating precise Cu delivery systems. Relatively little is known about Cu transport in plant cells, and no components of the Cu transport machinery in chloroplasts have been identified previously. Cu transport into chloroplasts provides the cofactor for the stromal enzyme copper/zinc superoxide dismutase (Cu/ZnSOD) and for the thylakoid lumen protein plastocyanin, which functions in photosynthetic electron transport from the cytochrome b(6)f complex to photosystem I. Here, we characterized six Arabidopsis mutants that are defective in the PAA1 gene, which encodes a member of the metal-transporting P-type ATPase family with a functional N-terminal chloroplast transit peptide. paa1 mutants exhibited a high-chlorophyll-fluorescence phenotype as a result of an impairment of photosynthetic electron transport that could be ascribed to decreased levels of holoplastocyanin. The paa1-1 mutant had a lower chloroplast Cu content, despite having wild-type levels in leaves. The electron transport defect of paa1 mutants was evident on medium containing <1 micro M Cu, but it was suppressed by the addition of 10 micro M Cu. Chloroplastic Cu/ZnSOD activity also was reduced in paa1 mutants, suggesting that PAA1 mediates Cu transfer across the plastid envelope. Thus, PAA1 is a critical component of a Cu transport system in chloroplasts responsible for cofactor delivery to plastocyanin and Cu/ZnSOD.     

Review: Amino acid domains involved in constitutive activation of G-protein-coupled receptors

Guanine nucleotide-binding protein-coupled receptors may attain an active conformation in the absence of agonist by spontaneous isomerization and thus yield constitutive, agonist-independent, activity. This has mainly been demonstrated for isolated membranes and recombinant wild-type receptors, and mutant receptors. They generally show remarkable increases in the sensitivity of a biological response. The location of activating mutations both within a single receptor and across receptors is widespread, with changes reported in the seven-transmembrane domains, the second and third intracellular loop. For most of these receptors, examples of ligands defined as inverse agonists have been documented. Regulation of these receptors by inverse agonists opposite to that observed by agonists, and the therapeutic potential of inverse agonists is underlined.     

Role of a conserved glutamate residue in the Escherichia coli SecA ATPase mechanism

Escherichia coli SecA uses ATP to drive the transport of proteins across cell membranes. Glutamate 210 in the "DEVD" Walker B motif of the SecA ATP-binding site has been proposed as the catalytic base for ATP hydrolysis (Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhofer, J. (2002) Science 297, 2018-2026). Consistent with this hypothesis, we find that mutation of glutamate 210 to aspartate results in a 90-fold reduction of the ATP hydrolysis rate compared with wild type SecA, 0.3 s(-1) versus 27 s(-1), respectively. SecA-E210D also releases ADP at a slower rate compared with wild type SecA, suggesting that in addition to serving as the catalytic base, glutamate 210 might aid turnover as well. Our results contradict an earlier report that proposed aspartate 133 as the catalytic base (Sato, K., Mori, H., Yoshida, M., and Mizushima, S. (1996) J. Biol. Chem. 271, 17439-17444). Re-evaluation of the SecA-D133N mutant used in that study confirms its loss of ATPase and membrane translocation activities, but surprisingly, the analogous SecA-D133A mutant retains full activity, revealing that this residue does not play a key role in catalysis.     

PDE1 encodes a P-type ATPase involved in appressorium-mediated plant infection by the rice blast fungus Magnaporthe grisea

Plant infection by the rice blast fungus Magnaporthe grisea is brought about by the action of specialized infection cells called appressoria. These infection cells generate enormous turgor pressure, which is translated into an invasive force that allows a narrow penetration hypha to breach the plant cuticle. The Magnaporthe pde1 mutant was identified previously by restriction enzyme-mediated DNA integration mutagenesis and is impaired in its ability to elaborate penetration hyphae. Here we report that the pde1 mutation is the result of an insertion into the promoter of a P-type ATPase-encoding gene. Targeted gene disruption confirmed the role of PDE1 in penetration hypha development and pathogenicity but highlighted potential differences in PDE1 regulation in different Magnaporthe strains. The predicted PDE1 gene product was most similar to members of the aminophospholipid translocase group of P-type ATPases and was shown to be a functional homolog of the yeast ATPase gene ATC8. Spatial expression studies showed that PDE1 is expressed in germinating conidia and developing appressoria. These findings implicate the action of aminophospholipid translocases in the development of penetration hyphae and the proliferation of the fungus beyond colonization of the first epidermal cell.     

Ligand-regulated transport of the Menkes copper P-type ATPase efflux pump from the Golgi apparatus to the plasma membrane: a novel mechanism of regulated trafficking.

The Menkes P-type ATPase (MNK), encoded by the Menkes gene (MNK; ATP7A), is a transmembrane copper-translocating pump which is defective in the human disorder of copper metabolism, Menkes disease. Recent evidence that the MNK P-type ATPase has a role in copper efflux has come from studies using copper-resistant variants of cultured Chinese hamster ovary (CHO) cells. These variants have MNK gene amplification and consequently overexpress MNK, the extents of which correlate with the degree of elevated copper efflux. Here, we report on the localization of MNK in these copper-resistant CHO cells when cultured in different levels of copper. Immunofluorescence studies demonstrated that MNK is predominantly localized to the Golgi apparatus of cells in basal medium. In elevated copper conditions there was a rapid trafficking of MNK from the Golgi to the plasma membrane. This shift in steady-state distribution of MNK was reversible and not dependent on new protein synthesis. In media containing basal copper, MNK accumulated in cytoplasmic vesicles after treatment of cells with a variety of agents that inhibit endosomal recycling. We suggest that MNK continuously recycles between the Golgi and the plasma membrane and elevated copper shifts the steady-state distribution from the Golgi to the plasma membrane. These data reveal a novel system of regulated protein trafficking which ultimately leads to the efflux of an essential yet potentially toxic ligand, where the ligand itself appears directly and specifically to stimulate the trafficking of its own transporter.     

Amino acid residues involved in the catalytic mechanism of NAD-dependent glutamate dehydrogenase from Halobacterium salinarum

The pH dependence of kinetic parameters for a competitive inhibitor (glutarate) was determined in order to obtain information on the chemical mechanism for NAD-dependent glutamate dehydrogenase from Halobacterium salinarum. The maximum velocity is pH dependent, decreasing at low pHs giving a pK value of 7.19+/-0.13, while the V/K for l-glutamate at 30 degrees C decreases at low and high pHs, yielding pK values of 7.9+/-0.2 and 9.8+/-0.2, respectively. The glutarate pKis profile decreases at high pHs, yielding a pK of 9. 59+/-0.09 at 30 degrees C. The values of ionization heat calculated from the change in pK with temperature are: 1.19 x 10(4), 5.7 x 10(3), 7 x 10(3), 6.6 x 10(3) cal mol-1, for the residues involved. All these data suggest that the groups required for catalysis and/or binding are lysine, histidine and tyrosine. The enzyme shows a time-dependent loss in glutamate oxidation activity when incubated with diethyl pyrocarbonate (DEPC). Inactivation follows pseudo-first-order kinetics with a second-order rate constant of 53 M-1min-1. The pKa of the titratable group was pK1=6.6+/-0.6. Inactivation with ethyl acetimidate also shows pseudo-first-order kinetics as well as inactivation with TNM yielding second-order constants of 1.2 M-1min-1 and 2.8 M-1min-1, and pKas of 8.36 and 9.0, respectively. The proposed mechanism involves hydrogen binding of each of the two carboxylic groups to tyrosyl residues; histidine interacts with one of the N-hydrogens of the l-glutamate amino group. We also corroborate the presence of a conservative lysine that has a remarkable ability to coordinate a water molecule that would act as general base.     

Role of a strictly conserved active site tyrosine in cofactor genesis in the copper amine oxidase from Hansenula polymorpha

The copper amine oxidases catalyze the O(2)-dependent, two-electron oxidation of amines to aldehydes at an active site that contains Cu(II) and topaquinone (TPQ) cofactor. TPQ arises from the autocatalytic, post-translational oxidation of a tyrosine side chain within the same active site. The contributions of individual active site amino acids to each of these chemical processes are being delineated. Previously, using the amine oxidase from the yeast Hansenula polymorpha (HPAO), mutations of a strictly conserved and structurally pivotal active site tyrosine (Y305) were studied and their effects on the catalytic cycle demonstrated [Hevel, J. M., Mills, S. A., and Klinman, J. P. (1999) Biochemistry 38, 3683-3693]. This study examines mutations at the same position for their effects on cofactor generation. While the Y305A mutation had moderate effects on the kinetics of catalysis (2.5- and 8-fold effects on k(cat) using ethylamine and benzylamine as substrates), the same mutation slows cofactor formation by approximately 45-fold relative to that of the wild-type (WT). Additionally, the Y305A mutant forms at least two species: primarily TPQ at lower pH and a species with a blue-shifted absorbance at high pH (lambda(max) = 400 nm). The 400 nm species does not react with phenylhydrazine or ethylamine and is stable toward pH buffer exchange, long-term storage (>3 weeks), incubation at high temperatures, or incubation with reductants and colorimetric peroxide quenching reagents. A similar species accumulates appreciably even at approximately neutral pH in the Y305F mutant, despite the fact that the rate of TPQ formation is reduced only 3-fold relative to that of WT HPAO. This small impact of Y305F on the rate of biogenesis contracts with a decrease in k(cat) (using ethylamine as the substrate) of 125-fold. The opposing effects of mutations at position 305 in biogenesis versus catalysis indicate that a single residue can be recruited for different roles during these processes.     

Immunochemical and mutational analyses of P-type ATPase Spf1p involved in the yeast secretory pathway.

The yeast SPF1 gene encodes a novel P-type ATPase, the substrate of which specificity has not been identified. It is required for sensitivity to SMKT, a killer toxin produced by the halotolerant yeast Pichia farinosa. To investigate the function of Spf1p, Asp487, the putative phosphorylation site of Spf1p, was replaced by Asn. Expression of the altered SPF1, with Asp487 replaced by Asn, did not suppress the SMKT-resistant phenotype of spf1 mutants, suggesting that the catalytic activity of this ATPase is required for acquisition of sensitivity to SMKT. Subcellular fractionation experiments indicated that the fractionation pattern of Spf1p was similar to that of an early Golgi protein, Och1p. Cells lacking Spf1p had an abnormal fractionation pattern of Sec12p. The spf1 disruptant also showed increased expression of Kar2p and sensitivity to tunicamycin. The glycosylation-defective phenotype and possible role of Spf1p in the secretory pathway are discussed.     

Mutation of a strictly conserved, active-site residue alters substrate specificity and cofactor biogenesis in a copper amine oxidase

The copper amine oxidases (CAOs) catalyze both the single-turnover modification of a peptidyl tyrosine to form the active-site cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ) and the oxidative deamination of primary amines using TPQ. The function of a strictly conserved tyrosine located within hydrogen-bonding distance to TPQ has been explored by employing site-directed mutagenesis on the enzyme from H. polymorpha to form the mutants Y305A, Y305C, and Y305F. Both Y305A and Y305C behave similarly with regard to aliphatic amine oxidase activity, showing 3-7-fold decreases in kinetic parameters relative to WT, while the more conservative substitution of Y305F results in a >100-fold decrease in kcat and >500-fold decrease in kcat/Km relative to WT for the reductive half-reaction. The oxidation of benzylamine by all three mutants is severely impaired, with very significant effects seen in the oxidative half-reaction. CAO activity was studied as a function of pH for WT and Y305A proteins. Profiles for WT-catalyzed methylamine oxidation and Y305A-catalyzed ethylamine oxidation are comparable, while profiles of Y305A-catalyzed methylamine oxidation suggest the pH-dependent build-up of an inhibitory intermediate, which was subsequently observed spectrophotometrically and is attributed to the product Schiff base. The relative effects of mutations at Y305 on catalytic turnover are, thus, concluded to be dependent on the nature of the amino acid which substitutes for tyrosine and the substrate used in amine oxidase assays. TPQ biogenesis experiments demonstrate a approximately 800-fold decrease in kobs for apo-Y305A compared to WT. Despite the strict conservation of Tyr305 in all CAOs, neither biogenesis nor catalytic turnover is abolished upon mutation of this residue. We propose an important, but nonessential, role for Tyr305 in the positioning of the TPQ precursor for biogenesis, and in the maintenance of the correct conformation for TPQ-derived intermediates during catalytic turnover.     

Abscisic acid and transpiration rate are involved in the response to boron toxicity in Arabidopsis plants

Boron (B) is an essential microelement for vascular plant development, but its toxicity is a major problem affecting crop yields in arid and semi‐arid areas of the world. In the literature, several genes involved in abscisic acid (ABA) signalling and responses are upregulated in Arabidopsis roots after treatment with excess B. It is known that the AtNCED3 gene, which encodes a crucial enzyme for ABA biosynthesis, plays a key role in the plant response to drought stress. In this study, root AtNCED3 expression and shoot ABA content were rapidly increased in wild‐type plants upon B‐toxicity treatment. The Arabidopsis ABA‐deficient nced3‐2 mutant had higher transpiration rate, stomatal conductance and accumulated more B in their shoots than wild‐type plants, facts that were associated with the lower levels of ABA in this mutant. However, in wild‐type plants, B toxicity caused a significant reduction in stomatal conductance, resulting in a decreased transpiration rate. This response could be a mechanism to limit the transport of excess B from the roots to the leaves under B toxicity. In agreement with the higher transpiration rate of the nced3‐2 mutant, this genotype showed an increased leaf B concentration and damage upon exposure to 5 mM B. Under B toxicity, ABA application decreased B accumulation in wild‐type and nced3‐2 plants. In summary, this work shows that excess B applied to the roots leads to rapid changes in AtNCED3 expression and gas exchange parameters that would contribute to restrain the B entry into the leaves, this effect being mediated by ABA.