Characterization of the histidine-rich loop of Arabidopsis vacuolar membrane zinc transporter AtMTP1 as a sensor of zinc level in the cytosol.

The vacuolar Zn(2+)/H(+) antiporter of Arabidopsis thaliana, AtMTP1, has a long cytosolic histidine-rich loop. A mutated AtMTP1 in which the first half of the loop (His-half) was deleted exhibited a 11-fold higher transport velocity in yeast cells. Transgenic lines overexpressing the His-half-deleted AtMTP1 in the loss-of-function mutant were evaluated for growth and metal content in the presence of various zinc concentrations. These overexpressing lines (35S-AtMTP1 and 35S-His-half lines) showed high tolerance to excess concentrations of zinc at 150 µM, as did the wild type, compared with the loss-of-function line. The His-half AtMTP1 transported cobalt in a heterologous expression assay in yeast, but the cumulative amount of cobalt in 35S-His-half plants was not increased. Moreover, the accumulation of calcium and iron was not changed in plants. Under zinc-deficient conditions, growth of 35S-His-half lines was markedly suppressed. Under the same conditions, the 35S-His-half lines accumulated larger amounts of zinc in roots and smaller amounts of zinc in shoots compared with the other lines, suggesting an abnormal accumulation of zinc in the roots of 35S-His-half lines. As a result, the shoots may exhibit zinc deficiency. Taken together, these results suggest that the His-loop acts as a sensor of cytosolic zinc to maintain an essential level in the cytosol and that the dysfunction of the loop results in an uncontrolled accumulation of zinc in the vacuoles of root cells.

Vacuolar nicotianamine has critical and distinct roles under iron deficiency and for zinc sequestration in Arabidopsis.

The essential micronutrients Fe and Zn often limit plant growth but are toxic in excess. Arabidopsis thaliana ZINC-INDUCED FACILITATOR1 (ZIF1) is a vacuolar membrane major facilitator superfamily protein required for basal Zn tolerance. Here, we show that overexpression of ZIF1 enhances the partitioning into vacuoles of the low molecular mass metal chelator nicotianamine and leads to pronounced nicotianamine accumulation in roots, accompanied by vacuolar buildup of Zn. Heterologous ZIF1 protein localizes to vacuolar membranes and enhances nicotianamine contents of yeast cells engineered to synthesize nicotianamine, without complementing a Zn-hypersensitive mutant that additionally lacks vacuolar membrane Zn(2+)/H(+) antiport activity. Retention in roots of Zn, but not of Fe, is enhanced in ZIF1 overexpressors at the expense of the shoots. Furthermore, these lines exhibit impaired intercellular Fe movement in leaves and constitutive Fe deficiency symptoms, thus phenocopying nicotianamine biosynthesis mutants. Hence, perturbing the subcellular distribution of the chelator nicotianamine has profound, yet distinct, effects on Zn and Fe with respect to their subcellular and interorgan partitioning. The zif1 mutant is also hypersensitive to Fe deficiency, even in media lacking added Zn. Therefore, accurate levels of ZIF1 expression are critical for both Zn and Fe homeostasis. This will help to advance the biofortification of crops.

Up-regulation of genes involved in N-acetylglucosamine uptake and metabolism suggests a recycling mode of chitin in intraradical mycelium of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal (AM) fungi colonize roots and form two kinds of mycelium, intraradical mycelium (IRM) and extraradical mycelium (ERM). Arbuscules are characteristic IRM structures that highly branch within host cells in order to mediate resource exchange between the symbionts. They are ephemeral structures and at the end of their life span, arbuscular branches collapse from the tip, fungal cytoplasm withdraws, and the whole arbuscule shrinks into fungal clumps. The exoskeleton of an arbuscule contains structured chitin, which is a polymer of N-acetylglucosamine (GlcNAc), whereas a collapsed arbuscule does not. The molecular mechanisms underlying the turnover of chitin in AM fungi remain unknown. Here, a GlcNAc transporter, RiNGT, was identified from the AM fungus Rhizophagus irregularis. Yeast mutants defective in endogenous GlcNAc uptake and expressing RiNGT took up (14)C-GlcNAc, and the optimum uptake was at acidic pH values (pH 4.0-4.5). The transcript levels of RiNGT in IRM in mycorrhizal Lotus japonicus roots were over 1000 times higher than those in ERM. GlcNAc-6-phosphate deacetylase (DAC1) and glucosamine-6-phosphate isomerase (NAG1) genes, which are related to the GlcNAc catabolism pathway, were also induced in IRM. Altogether, data suggest the existence of an enhanced recycling mode of GlcNAc in IRM of AM fungi.

A mutant strain Arabidopsis thaliana that lacks vacuolar membrane zinc transporter MTP1 revealed the latent tolerance to excessive zinc.

A mutant line of Arabidopsis thaliana that lacks a vacuolar membrane Zn(2+)/H(+) antiporter MTP1 is sensitive to zinc. We examined the physiological changes in this loss-of-function mutant under high-Zn conditions to gain an understanding of the mechanism of adaptation to Zn stress. When grown in excessive Zn and observed using energy-dispersive X-ray analysis, wild-type roots were found to accumulate Zn in vacuolar-like organelles but mutant roots did not. The Zn content of mutant roots, determined by chemical analysis, was one-third that of wild-type roots grown in high-Zn medium. Severe inhibition of root growth was observed in mtp1-1 seedlings in 500 muM ZnSO(4). Suppression of cell division and elongation by excessive Zn was reversible and the cells resumed growth in normal medium. In mutant roots, a marked formation of reactive oxygen species (ROS) appeared in the meristematic zone, where the MTP1 gene was highly expressed. Zn treatment enhanced the expression of several genes involved in Zn tolerance: namely, the plasma membrane Zn(2+)-export ATPase, HMA4, and plasma and vacuolar membrane proton pumps. CuZn-superoxide dismutases, involved in the detoxification of ROS, were also induced. The expression of plasma membrane Zn-uptake transporter, ZIP1, was suppressed. The up- or down-regulation of these genes might confer the resistance to Zn toxicity. These results indicate an essential role of MTP1 in detoxification of excessive Zn and provide novel information on the latent adaptation mechanism to Zn stress, which is hidden by MTP1.

Synchrony between flower opening and petal-color change from red to blue in morning glory, Ipomoea tricolor cv. Heavenly Blue.

Petal color change in morning glory Ipomoea tricolor cv. Heavenly Blue, from red to blue, during the flower-opening period is due to an unusual increase in vacuolar pH (pHv) from 6.6 to 7.7 in colored epidermal cells. We clarified that this pHv increase is involved in tonoplast-localized Na+/H+ exchanger (NHX). However, the mechanism of pHv increase and the physiological role of NHX1 in petal cells have remained obscure. In this study, synchrony of petal-color change from red to blue, pHv increase, K+ accumulation, and cell expansion growth during flower-opening period were examined with special reference to ItNHX1. We concluded that ItNHX1 exchanges K+, but not Na+, with H+ to accumulate an ionic osmoticum in the vacuole, which is then followed by cell expansion growth. This function may lead to full opening of petals with a characteristic blue color.

A high molecular mass zinc transporter MTP12 forms a functional heteromeric complex with MTP5 in the Golgi in Arabidopsis thaliana.

UNLABELLED: Zinc (Zn) is an essential micronutrient required for plant growth and development. In Arabidopsis thaliana, several families of Zn transporters engaged in Zn import, export and intracellular compartmentalization play important roles in Zn homeostasis. We describe a novel Zn transporter, A. thaliana metal tolerance protein 12 (AtMTP12), which belongs to the cation diffusion facilitator family. AtMTP12 is predicted to consist of 798 amino acids and have 14 transmembrane segments. The expression of AtMTP12 in suspension-cultured cells was not affected by Zn deficiency or excess. Heterologous expression in a mutant of budding yeast (Saccharomyces cerevisiae) that lacks Msc2p, an orthologue of AtMTP12, revealed that AtMTP12 complements the growth phenotype of the msc2 mutant when AtMTP5t1, one of the splicing variants of AtMTP5, is coexpressed. Transient expression of AtMTP12-fused green fluorescent protein in A. thaliana mesophyll protoplasts demonstrated that AtMTP12 is localized to the Golgi apparatus. Moreover, AtMTP12 and AtMTP5t1 interact in the Golgi, as determined by a bimolecular fluorescence complementation assay. These results suggest that AtMTP12 forms a functional complex with AtMTP5t1 to transport Zn into the Golgi. DATABASE: Nucleotide sequence data for full-length of AtMTP12 is available in the DDBJ/EMBL/GenBank database under accession number AB986563.

Zinc-binding and structural properties of the histidine-rich loop of Arabidopsis thaliana vacuolar membrane zinc transporter MTP1.

The vacuolar Zn(2+)/H(+) antiporter of Arabidopsis thaliana, AtMTP1, has a cytosolic histidine-rich loop (His-loop). We characterized the structures and Zn(2+)-binding properties of the His-loop and other domains. Circular dichroism analyses revealed that the His-loop partly consists of a polyproline type II structure and that its conformational change is induced by Zn(2+) as well as the C-terminal domain. Isothermal titration calorimetry of the His-loop revealed a binding number of four Zn(2+) per molecule. Numbers of Ni and Co associated with the His-loop were approximately one ion per molecule and the thermodynamic parameters of the association with these ions were different from that of Zn(2+). These results suggest the involvement of the His-loop in sensing cytosolic Zn(2+) and in the regulation of zinc transport activity through Zn(2+)-induced structural change.

Amino acid screening based on structural modeling identifies critical residues for the function, ion selectivity and structure of Arabidopsis MTP1.

Arabidopsis thaliana MTP1 is a vacuolar membrane Zn(2+)/H(+) antiporter of the cation diffusion facilitator family. Here we present a structure-function analysis of AtMTP1-mediated transport and its remarkable Zn(2+) selectivity by functional complementation tests of more than 50 mutant variants in metal-sensitive yeast strains. This was combined with homology modeling of AtMTP1 based on the crystal structure of the Escherichia coli broad-specificity divalent cation transporter YiiP. The Zn(2+)-binding sites of EcYiiP in the cytoplasmic C-terminus, and the pore formed by transmembrane helices TM2 and TM5, are conserved in AtMTP1. Although absent in EcYiiP, Cys31 and Cys36 in the extended N-terminal cytosolic domain of AtMTP1 are necessary for complementation of a Zn-sensitive yeast strain. On the cytosolic side of the active Zn(2+)-binding site inside the transmembrane pore, Ala substitution of either Asn258 in TM5 or Ser101 in TM2 non-selectively enhanced the metal tolerance conferred by AtMTP1. Modeling predicts that these residues obstruct the movement of cytosolic Zn(2+) into the intra-membrane Zn(2+)-binding site of AtMTP1. A conformational change in the immediately preceding His-rich cytosolic loop may displace Asn258 and permit Zn(2+) entry into the pore. This would allow dynamic coupling of Zn(2+) transport to the His-rich loop, thus acting as selectivity filter or sensor of cytoplasmic Zn(2+) levels. Individual mutations at diverse sites within AtMTP1 conferred Co and Cd tolerance in yeast, and included deletions in N-terminal and His-rich intra-molecular cytosolic domains, and mutations of single residues flanking the transmembrane pore or participating in intra- or inter-molecular domain interactions, all of which are not conserved in the non-selective EcYiiP.

Deletion of a histidine-rich loop of AtMTP1, a vacuolar Zn(2+)/H(+) antiporter of Arabidopsis thaliana, stimulates the transport activity.

Arabidopsis thaliana AtMTP1 belongs to the cation diffusion facilitator family and is localized on the vacuolar membrane. We investigated the enzymatic kinetics of AtMTP1 by a heterologous expression system in the yeast Saccharomyces cerevisiae, which lacked genes for vacuolar membrane zinc transporters ZRC1 and COT1. The yeast mutant expressing AtMTP1 heterologously was tolerant to 10 mm ZnCl(2). Active transport of zinc into vacuoles of living yeast cells expressing AtMTP1 was confirmed by the fluorescent zinc indicator FuraZin-1. Zinc transport was quantitatively analyzed by using vacuolar membrane vesicles prepared from AtMTP1-expressing yeast cells and radioisotope (65)Zn(2+). Active zinc uptake depended on a pH gradient generated by endogenous vacuolar H(+)-ATPase. The activity was inhibited by bafilomycin A(1), an inhibitor of the H(+)-ATPase. The K(m) for Zn(2+) and V(max) of AtMTP1 were determined to be 0.30 microm and 1.22 nmol/min/mg, respectively. We prepared a mutant AtMTP1 that lacked the major part (32 residues from 185 to 216) of a long histidine-rich hydrophilic loop in the central part of AtMTP1. Yeast cells expressing the mutant became hyperresistant to high concentrations of Zn(2+) and resistant to Co(2+). The K(m) and V(max) values were increased 2-11-fold. These results indicate that AtMTP1 functions as a Zn(2+)/H(+) antiporter in vacuoles and that a histidine-rich region is not essential for zinc transport. We propose that a histidine-rich loop functions as a buffering pocket of Zn(2+) and a sensor of the zinc level at the cytoplasmic surface. This loop may be involved in the maintenance of the level of cytoplasmic Zn(2+).

Identification of trypsin I as a candidate for influenza A virus and Sendai virus envelope glycoprotein processing protease in rat brain.

Extracellular cleavage of virus envelope fusion glycoprotein hemagglutinin (HA0) by host trypsin-like proteases is a prerequisite for the infectivity and pathogenicity of human influenza A viruses and Sendai virus. The common epidemic influenza A viruses are pneumotropic, but occasionally cause encephalopathy or encephalitis, although the HA0 processing enzyme in the brain has not been identified. In searching for the brain processing proteases, we identified a processing enzyme in rat brain that was inducible by infection with these viruses. The purified enzyme exhibited an apparent molecular mass of approximately 22 kDa on SDS-PAGE and the N-terminal amino acid sequence was consistent with that of rat pancreatic trypsin I. Its substrate specificities and inhibition profiles were the same as those of pancreatic trypsin I. In situ hybridization and immunohistochemical studies on trypsin I distribution revealed heavy deposits in the brain capillaries, particularly in the allocortex, as well as in clustered neuronal cells of the hippocampus. The purified enzyme efficiently processed the HA0 of human influenza A virus and the fusion glycoprotein precursor of Sendai virus. Our results suggest that trypsin I in the brain potentiates virus multiplication in the pathogenesis and progression of influenza-associated encephalopathy or encephalitis.

The involvement of tonoplast proton pumps and Na+(K+)/H+ exchangers in the change of petal color during flower opening of Morning Glory, Ipomoea tricolor cv. Heavenly Blue.

The petal color of morning glory, Ipomoea tricolor cv. Heavenly Blue, changes from purplish red to blue during flower opening. This color change is caused by an unusual increase in vacuolar pH from 6.6 to 7.7 in the colored adaxial and abaxial cells. To clarify the mechanism underlying the alkalization of epidermal vacuoles in the open petals, we focused on vacuolar H+-ATPase (V-ATPase), H+-pyrophosphatase (V-PPase) and an isoform of Na+/H+ exchanger (NHX1). We isolated red and blue protoplasts from the petals in bud and fully open flower, respectively, and purified vacuolar membranes. The membranes contained V-ATPase, V-PPase and NHX1, which were immunochemically detected, with relatively high transport activity. NHX1 could be detected only in the vacuolar membranes prepared from flower petals and its protein level was the highest in the colored petal epidermis of the open flower. These results suggest that the increase of vacuolar pH in the petals during flower opening is due to active transport of Na+ and/or K+ from the cytosol into vacuoles through a sodium- or potassium-driven Na+(K+)/H+ exchanger NXH1 and that V-PPase and V-ATPase may prevent the over-alkalization. This systematic ion transport maintains the weakly alkaline vacuolar pH, producing the sky-blue petals.

Identification of ectopic anionic trypsin I in rat lungs potentiating pneumotropic virus infectivity and increased enzyme level after virus infection.

Extracellular cleavage of virus envelope fusion glycoproteins by host cellular proteases is a prerequisite for the infectivity of mammalian and nonpathogenic avian influenza viruses, and Sendai virus. In search of such target processing proteases in the airway, we recently found a new candidate trypsin-like processing protease in rat lungs, which was induced by Sendai virus infection, and identified as ectopic rat anionic trypsin I. On SDS/PAGE under reducing and nonreducing conditions, the purified enzyme gave protein bands corresponding to 29 and 22 kDa, respectively, i.e. at the same positions as rat pancreatic anionic trypsin I. It exhibited an apparent molecular mass of 31 kDa on molecular sieve chromatography and its isoelectric point was pH 4.7. The amino-acid sequences of the N-terminus and proteolytic digest peptides of the purified enzyme were consistent with those of rat pancreatic anionic trypsin I. Its substrate specificities and inhibitor sensitivities were the same as those of the pancreatic enzyme. The purified enzyme efficiently processed the fusion glycoprotein precursor of Sendai virus and hemagglutinin of human influenza A virus, and potentiated the infectivity of Sendai virus in the same dose-dependent manner as the pancreatic one. Immunohistochemical studies revealed that this protease is located in the stromal cells in peri-bronchiolar regions. These results suggest that ectopic anionic trypsin I in rat lungs induced by virus infection may trigger virus spread in rat lungs.