植物Na+/H+反向转运蛋白的研究进展

盐胁迫是限制植物生长发育的主要因素之一,植物Na+/H+反向转运蛋白可通过将Na+逆向转运出细胞外或将Na+区隔化于液泡中来抵制环境中过高的Na+浓度.植物中Na+/H+反向转运蛋白存在于细胞质膜和液泡膜上,现在已得到多种编码这些Na+/H+反向转运蛋白的基因,对其结构功能特性进行了大量研究,并发现将这些基因转入非抗盐植物中过量表达可提高转基因植物的抗盐性.概述了Na+/H+反向转运蛋白及其编码基因的最新研究进展.     

植物液泡膜阳离子/H+反向转运蛋白结构和功能研究进展

阳离子转运蛋白在调节细胞质阳离子浓度过程中发挥关键作用。液泡是一个储存多种离子的重要细胞器,阳离子 (Ca2+)/H+反向转运蛋白CAXs定位在液泡膜上,主要参与Ca2+向液泡的转运,也参与其他阳离子的转运。近年来,植物中分离鉴定了多个CAX基因,植物CAXs主要有4个功能域：NRR通过自抑制机制调节Ca2+转运活性,CaD和C功能域分别赋予CAXs的Ca2+和Mn2+专一性转运活性,D功能域可调节细胞质pH。拟南芥AtCAXs参与植物的生长发育和胁迫适应过程,AtCAX3主要在盐胁迫下转运Ca2+,At     

拟南芥内膜Na+,K+/H+反向转运体研究进展

拟南芥内膜Na,K~+/H~+反向转运体(Endosomal NHX)的亚细胞定位、离子转运特性及生物学功能阐释取得了重要进展。拟南芥内膜Na~+,K~+/H~+反向转运体包含AtNHX5和AtNHX6两个成员,它们的氨基酸序列相似性为78.7%。研究表明,AtNHX5和AtNHX6具有功能冗余,它们都定位在高尔基体(Golgi)、反面高尔基体管网状结构(TGN)、内质网(ER)和液胞前体(PVC),参与调控耐盐胁迫、pH平衡和K~+平衡等。有报道显示内膜NHXs跨膜结构域存在能够调控自身离子活性的酸性保守氨基酸残基,对其自身功能具有决定性作用。最新研究结果表明,拟南芥内膜NHXs影响囊泡运输和蛋白存贮,并参与生长素介导的植物生长和发育。文中主要对拟南芥内膜NHXs的亚细胞定位、离子转运、功能及应用进展进行了概述。     

NaCl胁迫对盐芥质膜和液泡膜ATPase活性的影响

以盐生植物盐芥和中生植物拟南芥幼苗为材料,研究了盐胁迫对它们叶片和根质膜、液泡膜H+-ATPase、Ca2+-ATPases和K+-ATPase活性以及H+-ATPase、Na+/H+ 逆向转运蛋白表达的影响.结果显示:在NaCl胁迫下,盐芥叶片和根质膜的H+-ATPase活性分别比对照显著升高41%～212%和35%～53%,液泡膜的H+-ATPase分别显著升高281%～373%和4%～38%,而拟南芥却比相应对照都显著降低;相同盐浓度胁迫下,盐芥叶片的H+-ATPase活性比根部高4～8倍,盐芥根也远高于拟南芥.在NaCl胁迫下,盐芥叶片和根的液泡膜H+-ATPase蛋白质β亚基含量变化与其酶活性变化趋势一致,质膜Na+/H+ 逆向转运蛋白的表达量与Na+含量变化趋势一致.盐胁迫下盐芥根中Ca2+-ATPases和K+-ATPase活性的增加与根中Ca2+和K+含量呈显著正相关.研究发现,在盐胁迫条件下,盐芥能有效增强H+-ATPase蛋白和Na+/H+逆向转运蛋白表达,显著提高其根系与叶片质膜和液泡膜的H+-ATPase、Ca2+-ATPase和K+-ATPase活性,维持细胞质中较高的Ca2+和K+水平,从而缓解盐胁迫的伤害,增强耐盐性.     

Na+/H+ 逆向转运蛋白与植物耐盐性关系

Na+/H+ 逆向转运蛋白与植物的耐盐性有密切的关系。在高等植物体内,主要存在两种Na+/H+ 逆向转运蛋白,分别为位于细胞质膜上的逆向转运蛋白SOS1,以及存在于液泡膜上的AtNHX1。质膜Na+/H+ 逆向转运蛋白主要负责Na+ 的外排,液泡膜Na+/H+ 逆向转运蛋白主要负责把Na+ 区隔化入液泡。过量表达质膜Na+/H+ 逆向转运蛋白SOS1或液泡膜Na+/H+ 逆向转运蛋白AtNHX1能够明显提高植物的耐盐性。本文对植物中Na+/H+ 逆向转运蛋白及其与植物耐盐性之间的关系研究最新进展作一概述。     

7种植物K+/H+逆向转运蛋白的生物信息学分析

K+/H+逆向转运是普遍存在于几乎所有生物体内的重要离子平衡机制之一.该机制主要由多基因家族KEA (K efflux antiporter)介导.然而,长期以来对KEA的功能特征和生理意义的认识,除了在大肠杆菌中有零星的报道之外,绝大部分尚为空白.本文通过生物信息学分析,比较了7个植物物种KEA的同源和进化关系,发现KEA家族中的部分成员在物种进化过程中具有极高的保守性.以模式植物拟南芥为例,进一步研究了KEA的蛋白质跨膜结构、关键结构域和亚细胞定位预测等.结果表明,AtKEA大多具有10～12个跨膜结构,是典型的膜蛋白,在跨膜区的N端有多个丝氨酸磷酸化调控位点;都含有K+/H+交换结构域和相应的调控域,推测其能够介导K+/H+交换.基因芯片研究表明,AtKEA在根、茎、叶、花和荚果等不同组织器官的表达丰度不同,表明KEA基因家族各成员的生理功能具有时空分异性.上述结果可为进一步深入研究植物K+/H+逆向转运系统的功能提供借鉴.     

花烟草NaNHX1基因的克隆及其在非生物胁迫下的表达模式

植物NHX家族基因,在植物的生长发育以及生物与非生物胁迫的应答反应中发挥着十分重要的作用。为了探究花烟草Na+/H+逆向转运蛋白的生理功能,为花烟草耐盐分子机制的研究提供参考。采用同源克隆的方法进行基因克隆,对花烟草进行非生物胁迫,并运用qPCR的方法进行基因表达模式分析。结果表明,从花烟草(Nicotiana alata)中克隆了一个属于Na+/H+逆向转运蛋白家族的基因NaNHX1。该基因的开放阅读框全长为1 599 bp,编码了532个氨基酸残基。生物信息学分析结果表明,该基因编码的蛋白分子量为58.4 kD,等电点为5.66;具有Na+/H+逆向转运蛋白家族典型的保守结构域NhaP2;该蛋白属于疏水性蛋白,包含10个跨膜区。NaNHX1基因主要定位于细胞质膜,并含有多个磷酸化位点。同源性分析的结果显示,NaNHX1基因与美花烟草(Nicotiana sylvestris)、茸毛烟草(Nicotiana tomentosiformis)以及番茄(Solanum lycoperisicum)NHX基因的亲缘关系最近,而与拟南芥的NHX基因同源性最低。NaNHX1基因的表达具有组织表达特异性,花中表达量最高,茎中次之,根和叶中表达量较低。在高盐、干旱、低温、ABA、低钾及H2O2等非生物胁迫下,NaNHX1的表达呈现3种不同的表达模式。其中,对高盐及低钾胁迫的响应强烈。本研究的结果表明,NaNHX1基因属于Na+/H+逆向转运蛋白家族,可能参与了花烟草高盐和低钾胁迫,以及其它非生物胁迫响应在内的众多生理过程。     

生物信息学对番茄LeNHX1基因的研究

应用生物信息学的方法和工具对番茄LeNHX1蛋白质的理化性质、跨膜区域、疏水性/亲水性、二级结构、结构功能域、功能分类和同源性进行分析.结果表明此蛋白为疏水性稳定蛋白,包含一个保守的氨氯吡嗪咪结合位点LFFIYVLPPI区域,相对分子量为59.0 kD,等电点为6.60,存在10个跨膜区域,蛋白质二级结构中的主要构成元件是α-螺旋和不规则卷曲,功能分类和蛋白质同源性分析表明番茄LeNHX1属于液泡膜Na+/H+反向转运蛋白.     

黄花草木樨MoSOS1基因克隆及表达分析

植物质膜Na+/H+逆向转运蛋白基因SOS1是植物耐盐性必需的基因之一,在抵御盐胁迫过程中发挥十分重要的作用。以黄花草木樨叶片总RNA为模板,通过RT-PCR结合RACE方法克隆得到黄花草木樨MoSOS1基因全长序列,命名为MoSOS1。序列分析表明该基因全长为3 931 bp,开放阅读框(ORF)为2 874 bp,编码957个氨基酸,分子量为112.8 k D,等电点为5.31。TMHAM软件跨膜区的预测分析表明,黄花草木樨MoSOS1蛋白具有8个跨膜结构区域,N端和C端都位于细胞外。氨基酸序列分析表明,MoSOS1蛋白含有1个Na+/H+Exchanger superfamily和一个c NMP(Cyclic nucleotide-monophosphate)结合位点以及1个CAP_ED(Catabolite gene activator protein-effector domain)superfamily结构域。生物信息预测显示,MoSOS1的编码蛋白为不稳定酸性蛋白,不存在信号肽,二级结构多为α-螺旋和无规则卷曲。荧光实时定量RT-PCR分析表明:随着Na Cl浓度的增加,黄花草木樨地上部和根中MoSOS1基因表达水平呈增加趋势,根中表达量大于地上部,表明MoSOS1基因的表达受盐胁迫诱导和调节。     

植物Na^+,K^+/H^+反向转运体:pH平衡与囊泡运输

细胞内pH和囊泡运输是影响细胞功能的重要影响因子,也是决定细胞是否死亡的重要因素。植物Na^+,K^+/H^+反向转运体是位于细胞膜结构上的跨膜反向转运蛋白,介导Na^+、K^+与质子(H^+)的跨膜反向转运,影响胞内pH的动态平衡。研究表明,NHX缺失造成细胞pH失衡的同时,将影响囊泡运输,从而对生长发育产生不利影响。主要对植物NHX在pH调节、囊泡运输中的功能进展进行了概述,并对其关系进行探讨。     

Isolation and characterization of a new Na+/H+ antiporter gene OsNHA1 from rice (Oryza sativa L.).

The full-length cDNA (3612 bp) of OsNHA1 was cloned by RT-PCR approach from rice (Oryza sativa L.), which encodes a putative plasma membrane Na+/H+ antiporter. Its deduced protein, OsNHA1, has 11 transmembrane domains and a significant similarity to a plasma membrane Na+/H+ antiporter AtNHA1 from Arabidopsis thaliana. Phylogenetic analysis showed that the OsNHA1 clusters with the plasma membrane Na+/H+ antiporters from various organisms. The semi-quantitative RT-PCR assay revealed that the expression of OsNHA1 was up-regulated in both shoots and roots of rice seedlings under salt stress, whereas it was not induced in the rice seedlings treated by drought stress.     

Importance of the seryl and threonyl residues of the fifth transmembrane domain to the substrate specificity of yeast plasma membrane Na+/H+ antiporters

The Zygosaccharomyces rouxii Na+/H+ antiporter Sod2-22p is a member of the subfamily of yeast plasma membrane Nha/Sod antiporters that do not recognize potassium as their substrate. A functional study of two ZrSod2-22p mutated versions that improved the tolerance of a S. cerevisiae alkali-metal-cation sensitive strain to high extracellular concentration of KCl identified two polar non-charged amino-acid residues in the fifth transmembrane domain, Thr141 and Ser150, as being involved in substrate recognition and transport in yeast Nha/Sod antiporters. A reciprocal substitution of amino-acid residues with a hydroxyl group at these positions, T141S or S150T, produced a broadened cation selectivity of the antiporter for K+, in addition to Na+ and Li+. Site-directed mutagenesis of Ser150 showed that while the replacement of Ser150 with a small hydrophobic (valine) or negatively charged (aspartate) amino acid did not produce a significant change in ZrSod2-22p substrate specificity, the introduction of a positive charge at this position stopped the activity of the antiporter. This data demonstrates that the amino-acid composition of the fifth transmembrane domain, mainly the presence of amino acids containing hydroxyl groups in this part of the protein, is critical for the recognition and transport of substrates and could participate in conformational movements during the binding and/or cation transport cycle in yeast plasma membrane Na+/H+ antiporters.     

Structure of a plasma membrane H+-ATPase gene from the plant Arabidopsis thaliana

Physiological and biochemical studies have suggested that the plant plasma membrane H+-ATPase controls many important aspects of plant physiology, including growth, development, nutrient transport, and stomata movements. We have started the genetic analysis of this enzyme by isolating both genomic and cDNA clones of an H+-ATPase gene from Arabidopsis thaliana. The cloned gene is interrupted by 15 introns, and there is partial conservation of exon boundaries with respect to animal (Na+/K+)- and Ca2+-ATPases. In general, the relationship between exons and the predicted secondary and transmembrane structure of different ATPases with phosphorylated intermediate support a somewhat degenerate correspondence between exons and structural modules. The predicted amino acid sequence of the plant H+-ATPase is more closely related to fungal and protozoan H+-ATPases than to bacterial K+-ATPases or to animal (Na+/K+)-, (H+/K+)-, and Ca2+-ATPases. There is evidence for the existence of at least three isoforms of the plant H+-ATPase gene. These results open the way for a molecular approach to the structure and function of the plant proton pump.     

高等植物Na+ 吸收、转运及细胞内Na+ 稳态平衡研究进展

盐胁迫是影响农业生产的重要环境因素之一。本文对植物Na+吸收的机制和途径、Na+在植物体内的长距离转运以及细胞内Na+稳态平衡的研究进展进行了概述。参与植物Na+吸收与转运的蛋白和通道可能包括HKT、LCT1、AKT和NSCC等。其中, HKT是植物体内普遍存在的一类转运蛋白, 能够介导Na+的吸收, 其结构中的带电氨基酸残基对于其离子选择性有着非常明显的影响。LCT1是从小麦中发现的一类能够介导低亲和性阳离子吸收的蛋白, 然而在典型的土壤Ca2+浓度下LCT1并不能发挥吸收Na+的功能。AKT家族的成员在高盐环境下可能也参与了Na+的吸收。目前虽然还没有克隆到编码NSCC蛋白的基因, 但是NSCC作为植物吸收Na+的主要途径的观点已被广泛接受。SOS1和HKT参与了Na+在根部与植株地上部的长距离转运过程, 它们在木质部和韧皮部的Na+装载和卸载中发挥重要作用, 从而影响植物的抗盐性。另外, 由质膜Na+/H+逆向转运蛋白SOS1、蛋白激酶SOS2以及Ca2+结合蛋白SOS3组成的SOS复合体对细胞的Na+稳态具有重要的调节作用, 单子叶和双子叶植物之间的这种调节机制在结构和功能上具有保守性。SOS复合体与其它位于质膜或液泡膜上的Na+/H+逆向转运蛋白以及H+泵一起调节着细胞的Na+稳态。     

Cation/proton antiport systems in Escherichia coli. Solubilization and reconstitution of delta pH-driven sodium/proton and calcium/proton antiporters

Uptake of 22Na+ and 45Ca2+ into everted membrane vesicles from Escherichia coli was measured with imposed transmembrane pH gradients, acid interior, as driving force. Vesicles loaded with 0.5 M KCl were diluted into 0.5 M choline chloride to create a potassium gradient. Addition of nigericin to produce K+/H+ exchange resulted in formation of a pH gradient. This imposed gradient was capable of driving 45Ca2+ accumulation. In another method vesicles loaded with 0.5 M NH4Cl were diluted into 0.5 M choline chloride, creating an ammonium diffusion potential. A gradient of H+ was produced by passive efflux of NH3. With an ammonium gradient as driving force, everted vesicles accumulated both 45Ca2+ and 22Na+. The data suggest that 22Na+ uptake was via the sodium/proton antiporter and 45Ca2+ via the calcium/proton antiporter. Uptake of both cations required alkaline pHout. A minimum pH gradient of 0.9 unit was needed for transport of either ion, suggesting gating of the antiporters. Octyl glucoside extracts of inner membrane were reconstituted with E. coli phospholipids in 0.5 M NH4Cl. NH4+-loaded proteoliposomes accumulated both 22Na+ and 45Ca2+, demonstrating that the sodium/proton and calcium/proton antiporters could be solubilized and reconstituted in a functional form.     

Isolation and functional characterization of Ca2+/H+ antiporters from cyanobacteria

Genome sequences of cyanobacteria, Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and Thermosynechococcus elongatus BP-1 revealed the presence of a single Ca2+/H+ antiporter in these organisms. Here, we isolated the putative Ca2+/H+ antiporter gene from Synechocystis sp. PCC 6803 (synCAX) as well as a homologous gene from a halotolerant cyanobacterium Aphanothece halophytica (apCAX). In contrast to plant vacuolar CAXs, the full-length apCAX and synCAX genes complemented the Ca2+-sensitive phenotype of an Escherichia coli mutant. ApCAX and SynCAX proteins catalyzed specifically the Ca2+/H+ exchange reaction at alkaline pH. Immunological analysis suggested their localization in plasma membranes. The Synechocystis sp. PCC 6803 cells disrupted of synCAX exhibited lower Ca2+ efflux activity and a salt-sensitive phenotype. Overexpression of ApCAX and SynCAX enhanced the salt tolerance of Synechococcus sp. PCC 7942 cells. Mutagenesis analyses indicate the importance of two conserved acidic amino acid residues, Glu-74 and Glu-324, in the transmembrane segments for the exchange activity. These results clearly indicate that cyanobacteria contain a Ca2+/H+ antiporter in their plasma membranes, which plays an important role for salt tolerance.     

Characterization of the ion transport activity of the budding yeast Na+/H+ antiporter, Nha1p, using isolated secretory vesicles

The Saccharomyces cerevisiae Nha1p, a plasma membrane protein belonging to the monovalent cation/proton antiporter family, plays a key role in the salt tolerance and pH regulation of cells. We examined the molecular function of Nha1p by using secretory vesicles isolated from a temperature sensitive secretory mutant, sec4-2, in vitro. The isolated secretory vesicles contained newly synthesized Nha1p en route to the plasma membrane and showed antiporter activity exchanging H+ for monovalent alkali metal cations. An amino acid substitution in Nha1p (D266N, Asp-266 to Asn) almost completely abolished the Na+/H+ but not K+/H+ antiport activity, confirming the validity of this assay system as well as the functional importance of Asp-266, especially for selectivity of substrate cations. Nha1p catalyzes transport of Na+ and K+ with similar affinity (12.7 mM and 12.4 mM), and with lower affinity for Rb+ and Li+. Nha1p activity is associated with a net charge movement across the membrane, transporting more protons per single sodium ion (i.e., electrogenic). This feature is similar to the bacterial Na+/H+ antiporters, whereas other known eukaryotic Na+/H+ antiporters are electroneutral. The ion selectivity and the stoichiometry suggest a unique physiological role of Nha1p which is distinct from that of other known Na+/H+ antiporters.     

Transfection of the CD20 cell surface molecule into ectopic cell types generates a Ca2+ conductance found constitutively in B lymphocytes

CD20 is a plasma membrane phosphoprotein expressed exclusively by B lymphocytes. mAb binding to CD20 alters cell cycle progression and differentiation, indicating that CD20 plays an essential role in B lymphocyte function. Whole-cell patch clamp and fluorescence microscopy measurements of plasma membrane ionic conductance and cytosolic-free Ca2+ activity, respectively, were used to directly examine CD20 function. Transfection of human T and mouse pre-B lymphoblastoid cell lines with CD20 cDNA and subsequent stable expression of CD20 specifically increased transmembrane Ca2+ conductance. Transfection of CD20 cDNA and subsequent expression of CD20 in nonlymphoid cells (human K562 erythroleukemia cells and mouse NIH-3T3 fibroblasts) also induced the expression of an identical transmembrane Ca2+ conductance. The binding of a CD20-specific mAb to CD20+ lymphoblastoid cells also enhanced the transmembrane Ca2+ conductance. The mAb-enhanced Ca2+ currents had the same conductance characteristics as the CD20- associated Ca2+ currents in CD20 cDNA-transfected cells. C20 is structurally similar to several ion channels; each CD20 monomer possesses four membrane spanning domains, and both the amino and carboxy termini reside within the cytoplasm. Biochemical cross-linking of cell-surface molecules with subsequent immunoprecipitation analysis of CD20 suggests that CD20 may be present as a multimeric oligomer within the membrane, as occurs with several known membrane channels. Taken together, these findings indicate that CD20 directly regulates transmembrane Ca2+ conductance in B lymphocytes, and suggest that multimeric complexes of CD20 may form Ca2+ conductive ion channels in the plasma membrane of B lymphoid cells.