共查询到20条相似文献,搜索用时 15 毫秒
1.
Mei H Zhao J Pittman JK Lachmansingh J Park S Hirschi KD 《Journal of experimental botany》2007,58(12):3419-3427
Vacuolar localized Ca(2+)/H(+) exchangers such as Arabidopsis thaliana cation exchanger 1 (CAX1) play important roles in Ca(2+) homeostasis. When expressed in yeast, CAX1 is regulated via an N-terminal autoinhibitory domain. In yeast expression assays, a 36 amino acid N-terminal truncation of CAX1, termed sCAX1, and variants with specific mutations in this N-terminus, show CAX1-mediated Ca(2+)/H(+) antiport activity. Furthermore, transgenic plants expressing sCAX1 display increased Ca(2+) accumulation and heightened activity of vacuolar Ca(2+)/H(+) antiport. Here the properties of N-terminal CAX1 variants in plants and yeast expression systems are compared and contrasted to determine if autoinhibition of CAX1 is occurring in planta. Initially, using ionome analysis, it has been demonstrated that only yeast cells expressing activated CAX1 transporters have altered total calcium content and fluctuations in zinc and nickel. Tobacco plants expressing activated CAX1 variants displayed hypersensitivity to ion imbalances, increased calcium accumulation, heightened concentrations of other mineral nutrients such as potassium, magnesium and manganese, and increased activity of tonoplast-enriched Ca(2+)/H(+) transport. Despite high in planta gene expression, CAX1 and N-terminal variants of CAX1 which were not active in yeast, displayed none of the aforementioned phenotypes. Although several plant transporters appear to contain N-terminal autoinhibitory domains, this work is the first to document clearly N-terminal-dependent regulation of a Ca(2+) transporter in transgenic plants. Engineering the autoinhibitory domain thus provides a strategy to enhance transport function to affect agronomic traits. 相似文献
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Pittman JK Shigaki T Cheng NH Hirschi KD 《The Journal of biological chemistry》2002,277(29):26452-26459
Regulation of Ca(2+)/H(+) antiporters may be an important function in determining the duration and amplitude of cytosolic Ca(2+) oscillations. Previously the Arabidopsis Ca(2+)/H(+) transporter, CAX1 (cation exchanger 1), was identified by its ability to suppress yeast mutants defective in vacuolar Ca(2+) transport. Recently, a 36-amino acid N-terminal regulatory region on CAX1 has been identified that inhibits CAX1-mediated Ca(2+)/H(+) antiport. Here we show that a synthetic peptide designed against the CAX1 36 amino acids inhibited Ca(2+)/H(+) transport mediated by an N-terminal-truncated CAX1 but did not inhibit Ca(2+) transport by other Ca(2+)/H(+) antiporters. Ca(2+)/H(+) antiport activity measured from vacuolar-enriched membranes of Arabidopsis root was also inhibited by the CAX1 peptide. Through analyzing CAX chimeric constructs the region of interaction of the N-terminal regulatory region was mapped to include 7 amino acids (residues 56-62) within CAX1. The CAX1 N-terminal regulatory region was shown to physically interact with this 7-amino acid region by yeast two-hybrid analysis. Mutagenesis of amino acids within the N-terminal regulatory region implicated several residues as being essential for regulation. These findings describe a unique mode of antiporter autoinhibition and demonstrate the first detailed mechanisms for the regulation of a Ca(2+)/H(+) antiporter from any organism. 相似文献
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Zinc transporter of Arabidopsis thaliana AtMTP1 is localized to vacuolar membranes and implicated in zinc homeostasis 总被引:6,自引:0,他引:6
Kobae Y Uemura T Sato MH Ohnishi M Mimura T Nakagawa T Maeshima M 《Plant & cell physiology》2004,45(12):1749-1758
Cation diffusion facilitator (CDF) proteins belong to a family of heavy metal efflux transporters that might play an essential role in homeostasis and tolerance to metal ions. We investigated the subcellular localization of Arabidopsis thaliana AtMTP1, a member of the CDF family, and its physiological role in the tolerance to Zn using MTP1-deficient mutant plants. AtMTP1 was immunochemically detected as a 43 kDa protein in the vacuolar membrane fractioned by sucrose density gradient centrifugation. The expression level of AtMTP1 in suspension-cultured cells was not affected by the Zn concentration in the medium. When AtMTP1 fused with green fluorescent protein was transiently expressed in protoplasts prepared from Arabidopsis suspension-cultured cells, green fluorescence was clearly observed in the vacuolar membrane. A T-DNA insertion mutant line for AtMTP1 displays enhanced sensitivity to high Zn concentrations ranging from 200 to 500 microM, but not to Zn-deficient conditions. Mesophyll cells of the mtp1-1 mutant plants grown in the presence of 500 microM Zn were degraded, suggesting that Zn at high concentrations causes serious damage to leaves and that AtMTP1 plays a crucial role in preventing this damage in plants. Thus we propose that AtMTP1 is localized in the vacuolar membrane and is involved in sequestration of excess Zn in the cytoplasm into vacuoles to maintain Zn homeostasis. 相似文献
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Arabidopsis thaliana MTP1 is a Zn transporter in the vacuolar membrane which mediates Zn detoxification and drives leaf Zn accumulation 总被引:7,自引:0,他引:7
Desbrosses-Fonrouge AG Voigt K Schröder A Arrivault S Thomine S Krämer U 《FEBS letters》2005,579(19):4165-4174
The Arabidopsis thaliana metal tolerance protein 1 (MTP1) of the cation diffusion facilitator family of membrane transport proteins can mediate the detoxification of Zn in Arabidopsis and yeast. Xenopus laevis oocytes expressing AtMTP1 accumulate more Zn than oocytes expressing the AtMTP1(D94A) mutant or water-injected oocytes. An AtMTP1-GFP fusion protein localizes to the vacuolar membrane in root and leaf cells. The analysis of Arabidopsis transformed with a promoter-GUS construct suggests that AtMTP1 is not produced throughout the plant, but primarily in the subpopulation of dividing, differentiating and expanding cells. RNA interference-mediated silencing of AtMTP1 causes Zn hypersensitivity and a reduction in Zn concentrations in vegetative plant tissues. 相似文献
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HUA LIU RENJIE TANG YUE ZHANG CUITING WANG QUNDAN LV XIAOSHU GAO WENBIN LI HONGXIA ZHANG 《Plant, cell & environment》2010,33(11):1989-1999
Three vacuolar cation/H+ antiporters, AtNHX1 (At5g27150), 2 (At3g05030) and 5 (At1g54370), have been characterized as functional Na+/H+ antiporters in Arabidopsis. However, the physiological functions of AtNHX3 (At5g55470) still remain unclear. In this study, the physiological functions of AtNHX3 were studied using T‐DNA insertion mutant and 35S‐driven AtNHX3 over‐expression Arabidopsis plants. RT‐PCR analyses revealed that AtNHX3 is highly expressed in germinating seeds, flowers and siliques. Experiments with AtNHX3::YFP fusion protein in tobacco protoplasts indicated that AtNHX3 is mainly localized to vacuolar membrane, with a minor localization to pre‐vacuolar compartments (PVCs) and endoplasmic reticulum (ER). Seedlings of null nhx3 mutants were hypersensitive to K+‐deficient conditions. Expression of AtNHX3 complemented the sensitivity to K+ deficiency in nhx3 seedlings. Tonoplast vesicles isolated from transgenic plants over‐expressing AtNHX3 displayed significantly higher K+/H+ exchange rates than those isolated from wild‐type plants. Furthermore, nhx3 seeds accumulated less K+ and more Na+ when both wild‐type and nhx3 were grown under normal growth condition. The overall results indicate that AtNHX3 encodes a K+/H+ antiporter required for low‐potassium tolerance during germination and early seedling development, and may function in K+ utilization and ion homeostasis in Arabidopsis. 相似文献
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Using a reactive molecular dynamics simulation methodology, the free energy barrier for water-mediated proton transport between the two proton gating residues Glu203 and Glu148 in the ClC-ec1 antiporter, including the Grotthuss mechanism of proton hopping, was calculated. Three different chloride-binding states, with 1), both the central and internal Cl−, 2), the central Cl− only, and 3), the internal Cl− only, were considered and the coupling to the H+ transport studied. The results show that both the central and internal Cl− are essential for the proton transport from Glu203 to Glu148 to have a favorite free energy driving force. The rotation of the Glu148 side chain was also found to be independent of the internal chloride binding state. These results emphasize the importance of the 2:1 stoichiometry of this well-studied Cl−/H+ antiporter. 相似文献
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Hirschi Kendal D. Miranda Maricar L. Wilganowski Nathaniel L. 《Plant molecular biology》2001,46(1):57-65
In plants, cytosolic Ca2+ levels are tightly regulated, and changes in cytosolic Ca2+ have been implicated in converting numerous signals into adapted responses. Vacuolar ion transporters are thought to be key mediators of cytosolic Ca2+ concentrations. In an attempt to interpret the role of vacuolar Ca2+ transport in plant processes, we have expressed the yeast vacuolar Ca2+/H+ antiporter, VCX1, in Arabidopsis and tobacco. This transporter localizes to the plant vacuolar membrane. VCX1-expressing Arabidopsis plants displayed increased sensitivity to sodium and other ions. These ion sensitivities could be suppressed by addition of calcium to the media. VCX1-expressing plants demonstrated increased tonoplast-enriched Ca2+/H+ antiport activity as well as increased Ca2+ accumulation. These results suggest that VCX1 expression in Arabidopsis could be a valuable tool with which to experimentally dissect the role of Ca2+ transport around the plant vacuole. 相似文献
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拟南芥液泡膜Na+/H+逆向转运蛋白的研究进展 总被引:2,自引:0,他引:2
拟南芥液泡膜Na /H 逆向转运蛋白是由AtNHX1基因编码的一个在盐胁迫中起重要作用的蛋白。本文综述了AtNHX1的基本结构、功能及作用机制,展望其作为有效植物耐盐基因的前景,并对拟南芥液泡膜Na /H 逆向转运蛋白基因家族其他成员的研究,也做了相应的概括。 相似文献
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Shigaki T Cheng NH Pittman JK Hirschi K 《The Journal of biological chemistry》2001,276(46):43152-43159
Ca(2+) levels in plants, fungi, and bacteria are controlled in part by H(+)/Ca(2+) exchangers; however, the relationship between primary sequence and biological activity of these transporters has not been reported. The Arabidopsis H(+)/cation exchangers, CAX1 and CAX2, were identified by their ability to suppress yeast mutants defective in vacuolar Ca(2+) transport. CAX1 has a much higher capacity for Ca(2+) transport than CAX2. An Arabidopsis thaliana homolog of CAX1, CAX3, is 77% identical (93% similar) and, when expressed in yeast, localized to the vacuole but did not suppress yeast mutants defective in vacuolar Ca(2+) transport. Chimeric constructs and site-directed mutagenesis showed that CAX3 could suppress yeast vacuolar Ca(2+) transport mutants if a nine-amino acid region of CAX1 was inserted into CAX3 (CAX3-9). Biochemical analysis in yeast showed CAX3-9 had 36% of the H(+)/Ca(2+) exchange activity as compared with CAX1; however, CAX3-9 and CAX1 appear to differ in their transport of other ions. Exchanging the nine-amino acid region of CAX1 into CAX2 doubled yeast vacuolar Ca(2+) transport but did not appear to alter the transport of other ions. This nine-amino acid region is highly variable among the plant CAX-like transporters. These findings suggest that this region is involved in CAX-mediated Ca(2+) specificity. 相似文献
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AtNHX1, a vacuolar Na+/H+ antiporter gene from Arabidopsis thaliana, was introduced into tobacco genome via Agrobacterium tumefaciens-mediated transformation to evaluate the role of vacuolar energy providers in plants salt stress response. Compared to the
wild-type plants, over-expression of AtNHX1 increased salt tolerance in the transgenic tobacco plants, allowing higher germination rates of seeds and successful seedling
establishment in the presence of toxic concentrations of NaCl. More importantly, the induced Na+/H+ exchange activity in the transgenic plants was closely correlated to the enhanced activity of vacuolar H+-ATPase (V-ATPase) when exposed to 200 mM NaCl. In addition, inhibition of V-ATPase activity led to the malfunction of Na+/H+ exchange activity, placing V-ATPase as the dominant energy provider for the vacuolar Na+/H+ antiporter AtNHX1. V-ATPase and vacuolar Na+/H+ antiporter thus function in an additive or synergistic way. Simultaneous overexpression of V-ATPase and vacuolar Na+/H+ antiporter might be appropriate for producing plants with a higher salt tolerance ability. 相似文献
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A 1034 bp cDNA encoding the full length sequence of subunit D of the vacuolar H+-ATPase was cloned from Arabidopsis thaliana. The open reading frame of the cDNA clone vatpD contains 780 bp and codes for a protein of 29.1 kDa with a pI of 9.52. Structural predictions show similarities to subunit gamma of the F-ATP synthases. Identity between subunit D of the vacuolar H+-ATPase of A. thaliana and subunits D from other eukaryotic organisms is in the range of 57% (Bos taurus) to 48% (Candida albicans). Hybridization of genomic DNA with vatpD indicates the existence of one gene copy of subunit D in A. thaliana. Northern blot hybridization and in situ hybridization showed expression of vatpD in all cell types. The expression of subunit D was not modified by salt stress or abscisic acid treatment in A. thaliana. 相似文献
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Ahmed SU Rojo E Kovaleva V Venkataraman S Dombrowski JE Matsuoka K Raikhel NV 《The Journal of cell biology》2000,149(7):1335-1344
Many soluble plant vacuolar proteins are sorted away from secreted proteins into small vesicles at the trans-Golgi network by transmembrane cargo receptors. Cleavable vacuolar sorting signals include the NH(2)-terminal propeptide (NTPP) present in sweet potato sporamin (Spo) and the COOH-terminal propeptide (CTPP) present in barley lectin (BL). These two proteins have been found to be transported by different mechanisms to the vacuole. We examined the ability of the vacuolar cargo receptor AtELP to interact with the sorting signals of heterologous and endogenous plant vacuolar proteins in mediating vacuolar transport in Arabidopsis thaliana. AtELP extracted from microsomes was found to interact with the NTPPs of barley aleurain and Spo, but not with the CTPPs of BL or tobacco chitinase, in a pH-dependent and sequence-specific manner. In addition, EM studies revealed the colocalization of AtELP with NTPP-Spo at the Golgi apparatus, but not with BL-CTPP in roots of transgenic Arabidopsis plants. Further, we found that AtELP interacts in a similar manner with the NTPP of the endogenous vacuolar protein AtALEU (Arabidopsis thaliana Aleu), a protein highly homologous to barley aleurain. We hypothesize that AtELP functions as a vacuolar sorting receptor involved in the targeting of NTPP-, but not CTPP-containing proteins in Arabidopsis. 相似文献
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Aranda-Sicilia MN Cagnac O Chanroj S Sze H Rodríguez-Rosales MP Venema K 《Biochimica et biophysica acta》2012,1818(9):2362-2371
KEA genes encode putative K(+) efflux antiporters that are predominantly found in algae and plants but are rare in metazoa; however, nothing is known about their functions in eukaryotic cells. Plant KEA proteins show homology to bacterial K(+) efflux (Kef) transporters, though two members in the Arabidopsis thaliana family, AtKEA1 and AtKEA2, have acquired an extra hydrophilic domain of over 500 residues at the amino terminus. We show that AtKEA2 is highly expressed in leaves, stems and flowers, but not in roots, and that an N-terminal peptide of the protein is targeted to chloroplasts in Arabidopsis cotyledons. The full-length AtKEA2 protein was inactive when expressed in yeast; however, a truncated AtKEA2 protein (AtsKEA2) lacking the N-terminal domain complemented disruption of the Na(+)(K(+))/H(+) antiporter Nhx1p to confer hygromycin resistance and tolerance to Na(+) or K(+) stress. To test transport activity, purified truncated AtKEA2 was reconstituted in proteoliposomes containing the fluorescent probe pyranine. Monovalent cations reduced an imposed pH gradient (acid inside) indicating AtsKEA2 mediated cation/H(+) exchange with preference for K(+)=Cs(+)>Li(+)>Na(+). When a conserved Asp(721) in transmembrane helix 6 that aligns to the cation binding Asp(164) of Escherichia coli NhaA was replaced with Ala, AtsKEA2 was completely inactivated. Mutation of a Glu(835) between transmembrane helix 8 and 9 in AtsKEA2 also resulted in loss of activity suggesting this region has a regulatory role. Thus, AtKEA2 represents the founding member of a novel group of eukaryote K(+)/H(+) antiporters that modulate monovalent cation and pH homeostasis in plant chloroplasts or plastids. 相似文献
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O Shaul D W Hilgemann J de-Almeida-Engler M Van Montagu D Inz G Galili 《The EMBO journal》1999,18(14):3973-3980
Cellular functions require adequate homeostasis of several divalent metal cations, including Mg(2+) and Zn(2+). Mg(2+), the most abundant free divalent cytoplasmic cation, is essential for many enzymatic reactions, while Zn(2+) is a structural constituent of various enzymes. Multicellular organisms have to balance not only the intake of Mg(2+) and Zn(2+), but also the distribution of these ions to various organs. To date, genes encoding Mg(2+) transport proteins have not been cloned from any multicellular organism. We report here the cloning and characterization of an Arabidopsis thaliana transporter, designated AtMHX, which is localized in the vacuolar membrane and functions as an electrogenic exchanger of protons with Mg(2+) and Zn(2+) ions. Functional homologs of AtMHX have not been cloned from any organism. Ectopic overexpression of AtMHX in transgenic tobacco plants render them sensitive to growth on media containing elevated levels of Mg(2+) or Zn(2+), but does not affect the total amounts of these minerals in shoots of the transgenic plants. AtMHX mRNA is mainly found at the vascular cylinder, and a large proportion of the mRNA is localized in close association with the xylem tracheary elements. This localization suggests that AtMHX may control the partitioning of Mg(2+) and Zn(2+) between the various plant organs. 相似文献
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Pittman JK Sreevidya CS Shigaki T Ueoka-Nakanishi H Hirschi KD 《Plant physiology》2002,130(2):1054-1062
The regulation of intracellular Ca(2+) levels is achieved in part by high-capacity vacuolar Ca(2+)/H(+) antiporters. An N-terminal regulatory region (NRR) on the Arabidopsis Ca(2+)/H(+) antiporter CAX1 (cation exchanger 1) has been shown previously to regulate Ca(2+) transport by a mechanism of N-terminal auto-inhibition. Here, we examine the regulation of other CAX transporters, both within Arabidopsis and from another plant, mung bean (Vigna radiata), to ascertain if this mechanism is commonly used among Ca(2+)/H(+) antiporters. Biochemical analysis of mung bean VCAX1 expressed in yeast (Saccharomyces cerevisiae) showed that N-terminal truncated VCAX1 had approximately 70% greater antiport activity compared with full-length VCAX1. A synthetic peptide corresponding to the NRR of CAX1, which can strongly inhibit Ca(2+) transport by CAX1, could not dramatically inhibit Ca(2+) transport by truncated VCAX1. The N terminus of Arabidopsis CAX3 was also shown to contain an NRR. Additions of either the CAX3 or VCAX1 regulatory regions to the N terminus of an N-terminal truncated CAX1 failed to inhibit CAX1 activity. When fused to N-terminal truncated CAX1, both the CAX3 and VCAX1 regulatory regions could only auto-inhibit CAX1 after mutagenesis of specific amino acids within this NRR region. These findings demonstrate that N-terminal regulation is present in other plant CAX transporters, and suggest distinct regulatory features among these transporters. 相似文献