Heavy metal transporters in Hemiascomycete yeasts

We have compiled all known heavy metal transporters of the yeast Saccharomyces cerevisiae and identified their orthologs in four other species spanning the entire Hemiascomycete phylum. The 213 transporters belong to 27 distinct phylogenetic families distributed within the three classes: channels, secondary porters (permeases) and transport ATPases. They are present in all cellular membranes: plasma membranes, vacuoles, mitochondria, endoplasmic reticulum, nucleus, Golgi and various cytoplasmic vesicles. The major physiological heavy metals transported are: iron, manganese, zinc, copper, arsenite and cadmium. The major subfamilies that comprise the highest number of transporters are Siderophore-Iron Transporters (SIT) and CT2 (conjugated ABC transporters). They transport heavy metals (iron or cadmium, respectively) conjugated to organic chelators such as siderophores or glutathione. Both subfamilies are considerably amplified in the yeast Yarrowia lipolytica. The pattern of expansion and restriction of the subfamilies during the evolution of the different species is highly variable. The phylogenetic trees of the major transporters subfamilies distinguish homogenous clusters of transporters suggesting that possible different physiological or mechanistic functions evolved independently. We also validated the use of the Hemiascomycetes heavy metal transporters for identification of orthologs transporters in the pathogenic Basidiomycetes Cryptococcus neoformans.     

Cadmium uptake and sequestration kinetics in individual leaf cell protoplasts of the Cd/Zn hyperaccumulator Thlaspi caerulescens

Hyperaccumulators store accumulated metals in the vacuoles of large leaf epidermal cells (storage cells). For investigating cadmium uptake, we incubated protoplasts obtained from leaves of Thlaspi caerulescens (Ganges ecotype) with a Cd-specific fluorescent dye. A fluorescence kinetic microscope was used for selectively measuring Cd-uptake and photosynthesis in different cell types, so that physical separation of cell types was not necessary. Few minutes after its addition, cadmium accumulated in the cytoplasm before its transport into the vacuole. This demonstrated that vacuolar sequestration is the rate-limiting step in cadmium uptake into protoplasts of all leaf cell types. During accumulation in the cytoplasm, Cd-rich vesicle-like structures were observed. Cd uptake rates into epidermal storage cells were higher than into standard-sized epidermal cells and mesophyll cells. This shows that the preferential heavy metal accumulation in epidermal storage cells, previously observed for several metals in intact leaves of various hyperaccumulator species, is due to differences in active metal transport and not differences in passive mechanisms like transpiration stream transport or cell wall adhesion. Combining this with previous studies, it seems likely that the transport steps over the plasma and tonoplast membranes of leaf epidermal storage cells are driving forces behind the hyperaccumulation phenotype.     

Molecular mechanisms of heavy metal hyperaccumulation and phytoremediation

A relatively small group of hyperaccumulator plants is capable of sequestering heavy metals in their shoot tissues at high concentrations. In recent years, major scientific progress has been made in understanding the physiological mechanisms of metal uptake and transport in these plants. However, relatively little is known about the molecular bases of hyperaccumulation. In this paper, current progresses on understanding cellular/molecular mechanisms of metal tolerance/hyperaccumulation by plants are reviewed. The major processes involved in hyperaccumulation of trace metals from the soil to the shoots by hyperaccumulators include: (a) bioactivation of metals in the rhizosphere through root–microbe interaction; (b) enhanced uptake by metal transporters in the plasma membranes; (c) detoxification of metals by distributing to the apoplasts like binding to cell walls and chelation of metals in the cytoplasm with various ligands, such as phytochelatins, metallothioneins, metal-binding proteins; (d) sequestration of metals into the vacuole by tonoplast-located transporters. The growing application of molecular-genetic technologies led to the well understanding of mechanisms of heavy metal tolerance/accumulation in plants, and subsequently many transgenic plants with increased resistance and uptake of heavy metals were developed for the purpose of phytoremediation. Once the rate-limiting steps for uptake, translocation, and detoxification of metals in hyperaccumulating plants are identified, more informed construction of transgenic plants would result in improved applicability of the phytoremediation technology.     

Hyperaccumulation of cadmium and zinc in Thlaspi caerulescens and Arabidopsis halleri at the leaf cellular level

Vacuolar compartmentalization or cell wall binding in leaves could play a major role in hyperaccumulation of heavy metals. However, little is known about the physiology of intracellular cadmium (Cd) sequestration in plants. We investigated the role of the leaf cells in allocating metal in hyperaccumulating plants by measuring short-term (109)Cd and (65)Zn uptake in mesophyll protoplasts of Thlaspi caerulescens "Ganges" and Arabidopsis halleri, both hyperaccumulators of zinc (Zn) and Cd, and T. caerulescens "Prayon," accumulating Cd at a lower degree. The effects of low temperature, several divalent cations, and pre-exposure of the plants to metals were investigated. There was no significant difference between the Michaelis-Menten kinetic constants of the three plants. It indicates that differences in metal uptake cannot be explained by different constitutive transport capacities at the leaf protoplast level and that plasma and vacuole membranes of mesophyll cells are not responsible for the differences observed in heavy metal allocation. This suggests the existence of regulation mechanisms before the plasma membrane of leaf mesophyll protoplasts. However, pre-exposure of the plants to Cd induced an increase in Cd accumulation in protoplasts of "Ganges," whereas it decreased Cd accumulation in A. halleri protoplasts, indicating that Cd-permeable transport proteins are differentially regulated. The experiment with competitors has shown that probably more than one single transport system is carrying Cd in parallel into the cell and that in T. caerulescens "Prayon," Cd could be transported by a Zn and Ca pathway, whereas in "Ganges," Cd could be transported mainly by other pathways.     

Research Advances on the Mechanisms of Heavy Metal Tolerance in Plants

Heavy metals impact on the cytoplasmic function in a number of different ways, principally by their binding to protein sulflhdryl groups, by producing a deficiency of essential ions and, eventually, by substituting the essemial ions. Other modes of toxicity are possible, including disruption of cell transport processes and oxidative damage by free radicals generated by metal redox cycling. Plants have developed a variety of biochemical defense strategies to prevent heavy metal poisoning. The possible defense mechanism in plant may involve: metal binding to cell walls, avoidance of uptake these toxic metal ions, reduction of heavy metal transport across the cell membrane, active efflux, compartmentalization and metal chelation. Phytochelatins that can tightly bind and sequester metals may play an important role in the accumulation of heavy metals and preventing them from entering the cell metabolic pathway, the rates of high molecular weight (HMW) metal phytochelatin complexes (Cd-Sa-complex) formation may be an important determinant of the plant tolerance. In addition, plants possess several antioxidant defense systems to protect themselves from the oxidative stress by heavy metals.     

植物重金属转运蛋白研究进展

土壤中的有毒重金属不仅对植物有害,也可通过食物链危害人类和动物的健康.重金属转运蛋白在植物吸收、抵抗重金属的复杂机制中起着关键作用.植物重金属转运蛋白分为吸收蛋白和排出蛋白,其中,吸收蛋白转运必需重金属进入细胞,同时也会因为必需重金属的缺乏或离子之间的竞争而运载有毒重金属;排出蛋白是一类解毒蛋白,可将过量的或有毒的重金属逆向转运出细胞,或区室化于液泡中.目前,细胞内多种重金属转运蛋白基因的转录水平与重金属离子积累之间的联系已被揭示,并分离克隆出诸多相关蛋白家族成员.本文综述了近年来发现并鉴定的主要重金属转运蛋白的金属亲和性、器官表达特异性及细胞内定位等的研究进展.     

Cellular localization and developmental changes of Zip8, Zip14 and transferrin receptor 1 in the inner ear of rats

Prior studies have demonstrated that the inner ear can accumulate a variety of essential and potentially toxic heavy metals including manganese, lead, cobalt and cadmium. Metal accumulation is regulated in part by the functionality and affinity of these metals for the different transport systems responsible for uptake across the blood-cochlea barrier and their subsequent uptake into the different cells within the inner ear. Transport of these metals across cell membranes occurs by many of the same transport systems which include DMT1, Zip8 and Zip14. All three metal transporters have been identified in the cochlea based on quantitative PCR analysis. Prior studies in our laboratory examined the localization and developmental changes of DMT1 in rat cochlea and since the two Zip proteins are also likely to contribute to the transport of essential and non-essential divalent cations, we performed immunolabeling experiments in postnatal day three rat pups and adult rats. For comparison, we also immunolabeled the specimens with antibody against transferrin receptor 1 (TfR1) which is important in DMT1-mediated transport of Fe and Mn. Results presented in this paper demonstrate that the cellular and subcellular distribution of both Zip8 and Zip14 within the different components of the inner ear are distinct from that of DMT1. Nuclear localization for both Zip transporters as well as TfR1 was observed. The findings also reveal that the selective distribution of the three proteins was altered during development presumably to meet the changing needs of the cells to maintain normal and functional levels of iron and other essential metals.     

Uptake of cadmium and zinc by the alga Chlorella vulgaris: II. Multi-ion situation

Many microorganisms are capable of sequestering and concentrating heavy metals from their aqueous environment. While much research has beep carried out on the uptake of single species of metal ions, little attention seems to have been given to the study of multimetal ion systems. A mathematical model has previously been developed to describe the uptake of individual metal species by a microorganism. The model proposes two sequential processes: an initial rapid uptake due to cellular surface adsorption and a subsequent slow uptake due to membrane transport of the metal into the cells. This article extends the treatment by considering the uptake of two metal species together, cadmium and zinc, under different experimental conditions. The results are discussed in terms of possible mechanistic interactions.     

Inhibition of the plastidial phosphatidylcholine synthesis by silver, copper, lead and mercury induced by formation of mercaptides with the lyso-PC acyltransferase

Plastids greatly rely on the import of extraplastidial precursors for the synthesis of their own lipids, and several studies have shown that a lyso-PC acyltransferase located in the envelope may be involved in the import process. Because the presence of heavy metals in soil or in nutrient solutions induces changes in the lipid composition of plastid membranes (and therefore greatly reduces the photosynthetic capability of plants), we analysed the effect of several metal salts on plastidial lyso-PC acyltransferase activity. Among the 12 heavy metals studied, silver, copper, mercury and lead inhibited this activity. Metal bound to the enzyme was not - or only very slightly - released from the protein except when thiol-reducing agents (and not imidazole) were added. The results strongly suggest that the inhibitory effect is due to a formation of mercaptide between metal and cysteine(s). The relationship between the inhibition of the plastidial lyso-PC acyltransferase activity and the in vivo effects of metal salts on the plastid membranes is discussed.     

植物对重金属耐性的分子生态机理

植物适应重金属元素胁迫的机制包括阻止和控制重金属的吸收、体内螯合解毒、体内区室化分隔以及代谢平衡等。近年来,随着分子生物学技术在生态学研究中的深入应用,控制这些过程的分子生态机理逐渐被揭示出来。菌根、根系分泌物以及细胞膜是控制重金属进入植物根系细胞的主要生理单元。外生菌根能显著提高寄主植物的重金属耐性,根系分泌物通过改变根际pH、改变金属物质的氧化还原状态和形成络合物等机理减少植物对重金属的吸收。目前,控制菌根和根系分泌物重金属抗性的分子生态机理还不清楚。但细胞膜跨膜转运器已得到深入研究,相关金属离子转运器被鉴定和分离,一些控制基因如铁锌控制运转相关蛋白(ZIP)类、自然抵抗相关巨噬细胞蛋白(Nramp)类、P_1B-type ATPase类基因已被发现和克隆。金属硫蛋白(MTs)、植物螯合素(PCs)、有机酸及氨基酸等是植物体内主要的螯合物质,它们通过螯合作用固定金属离子,降低其生物毒性或改变其移动性。与MTs合成相关的MT-like基因已经被克隆,PCs合成必需的植物螯合素合酶(PCS), 即γ-Glu-Cys二肽转肽酶(γ-ECS) 的编码基因已经被克隆,控制麦根酸合成的氨基酸尼克烟酰胺(NA)在重金属耐性中的作用和分子机理也被揭示出来。ATP 结合转运器(ABC)和阳离子扩散促进器(CDF) 是植物体内两种主要膜转运器,通过它们和其它跨膜方式,重金属被分隔贮藏于液泡内。控制这些蛋白转运器合成的基因也已经被克隆,在植物中的表达证实其与重金属的体内运输和平衡有关。热休克蛋白(HSP)等蛋白类物质的产生是一种重要的体内平衡机制,其分子机理有待进一步研究。重金属耐性植物在这些环节产生了相关响应基因或功能蛋白质,分子克隆和转基因技术又使它们在污染治理上得到了初步的应用。     

植物耐重金属机理研究进展

由于工业“三废”和机动车尾气的排放、污水灌溉及农药、除草剂和化肥的使用,严重地污染了土壤、水质和大气,其中土壤中的重金属（Ｈｇ、Ｃｄ、Ａｓ、Ｃｕ和Ａｌ）污染更为严重［１］。重金属在植物根、茎、叶及籽粒中的大量累积,不仅严重地影响植物的生长和发育［１～...     

A novel heavy metal ATPase peptide from <Emphasis Type="Italic">Prosopis juliflora</Emphasis> is involved in metal uptake in yeast and tobacco

Heavy metal pollution of agricultural soils is one of the most severe ecological problems in the world. Prosopis juliflora, a phreatophytic tree species, grows well in heavy metal laden industrial sites and is known to accumulate heavy metals. Heavy Metal ATPases (HMAs) are ATP driven heavy metal pumps that translocate heavy metals across biological membranes thus helping the plant in heavy metal tolerance and phytoremediation. In the present study we have isolated and characterized a novel 28.9 kDa heavy metal ATPase peptide (PjHMT) from P. juliflora which shows high similarity to the C-terminal region of P_1B ATPase HMA1. It also shows the absence of the invariant signature sequence DKTGT, and the metal binding CPX motif but the presence of conserved regions like MVGEGINDAPAL (ATP binding consensus sequence), HEGGTLLVCLNS (metal binding domain) and MLTGD, GEGIND and HEGG motifs which play important roles in metal transport or ATP binding. PjHMT, was found to be upregulated under cadmium and zinc stress. Heterologous expression of PjHMT in yeast showed a higher accumulation and tolerance of heavy metals in yeast. Further, transgenic tobacco plants constitutively expressing PjHMT also showed increased accumulation and tolerance to cadmium. Thus, this study suggests that the transport peptide from P. juliflora may have an important role in Cd uptake and thus in phytoremediation.     

H(+)-coupled heavy metal transport in plants

It has been recently well documented that metal transport systems play a crucial role in the uptake, distribution and detoxification of heavy metals throughout the plant. A range of gene families that are likely to be involved in essential and non-essential metal transport has been now identified and their plasma membrane and/or tonoplast localization in plant cells has been recently confirmed. These include the primary metal transporters, using ATP as the source of energy and H(+)-coupling transporters, utilizing the electrochemical gradient previously generated by plasma membrane and tonoplast proton pumps. As the presence of nucleotide binding domains in the protein sequence may indicate its ATP-hydrolytic activity, it is more difficult to determine the H(+)-coupling activity of protein on the base of its structure. Thus, the H(+)-coupling activity of protein may be only proved by functional analysis of the protein. In this work, we briefly review the structure, regulation and function of the metal transporters operating as H(+)/metal cotransporters.     

Transition metal transporters in plants

Transition metals such as Fe, Cu, Mn, and Zn are essential minerals for normal plant growth and development, although they can be toxic when present in excess. Thus, for healthy plant growth, a range of transition metals must be acquired from the soil, distributed around the plant, and their concentrations carefully regulated within different cells and organelles. Membrane transport systems are likely to play a central role in these processes. The application of powerful genetic and molecular techniques has now identified a range of gene families that are likely to be involved in transition metal transport. These include the heavy metal ATPases (HMAs), the Nramps, the cation diffusion facilitator (CDF) family, the ZIP family, and the cation antiporters. This review provides a broad overview of the range of potential transport systems now thought to be involved in the uptake, distribution and homeostasis of transition metals in plants.     

Mechanisms of heavy-metal sequestration and detoxification in crustaceans: a review

This review is an update of information recently obtained about the physiological, cellular, and molecular mechanisms used by crustacean organ systems to regulate and detoxify environmental heavy metals. It uses the American lobster, Homarus americanus, and other decapod crustaceans as model organisms whose cellular detoxification processes may be widespread among both invertebrates and vertebrates alike. The focus of this review is the decapod hepatopancreas and its complement of metallothioneins, membrane metal transport proteins, and vacuolar sequestration mechanisms, although comparative remarks about potential detoxifying roles of gills, integument, and kidneys are included. Information is presented about the individual roles of hepatopancreatic mitochondria, lysosomes, and endoplasmic reticula in metal sequestration and detoxification. Current working models for the involvement of mitochondrial and endoplasmic reticulum calcium-transport proteins in metal removal from the cytoplasm and the inhibitory interactions between the metals and calcium are included. In addition, copper transport proteins and V-ATPases associated with lysosomal membranes are suggested as possible sequestration processes in these organelles. Together with several possible cytoplasmic divalent and trivalent anions such as sulfate, oxalate, or phosphate, accumulations of metals in lysosomes and their complexation into detoxifying precipitation granules may be regulated by variations in lysosomal pH brought about by bafilomycin-sensitive proton ATPases. Efflux processes for metal transport from hepatopancreatic epithelial cells to the hemolymph are described, as are the possible roles of hemocytes as metal sinks. While some of the cellular processes for isolating heavy metals from general circulation occur in the hepatopancreas and are beginning to be understood, very little is currently known about the roles of the gills, integument, and kidneys in metal regulation. Therefore, much remains to be clarified about the organs and mechanisms involved in metal homeostasis in decapod crustaceans.Abbreviations ER endoplasmic reticulum - SERCA sarco/endoplasmic reticulum calcium ATPase - V-ATPase vacuolar ATPase - PMCA plasma membrane calcium ATPaseCommunicated by I.D. Hume     

植物对重金属的吸收和分布

植物修复是利用植物来清除污染土壤中重金属的一项技术。该技术成功与否取决于植物从土壤中吸取金属以及向地上部运输金属的能力。植物对金属的吸收主要取决于自由态离子活度。许多螯合剂能诱导植物对重金属的吸收。金属离子在液泡中的区域化分布是植物耐重金属的主要原因。同时,细胞内的金属硫蛋白、植物螯合脓等蛋白质以及有机酸、氨基酸等在金属贮存和解毒方面也起重要作用。本文还论述了重金属在植物体内运输的生理及分子方面的研究进展。     

植物对重金属的吸收和分布

植物修复是利用植物来清除污染土壤中重金属的一项技术。该技术成功与否取决于植 物从土壤中吸取金属以及向地上部运输金属的能力。植物对金属的吸收主要取决于自由态离子活度。许多螯合剂能诱导植物对重金属的吸收。金属离子在液泡中的区域化分布是植物耐 重金属的主要原因。同时,细胞内的金属硫蛋白、植物螯合肽等蛋白质以及有机酸、氨基酸等在金属贮存和解毒方面也起重要作用。本文还论述了重金属在植物体内运输的生理及分子 方面的研究进展。     

Microbial heavy-metal resistance

We are just beginning to understand the metabolism of heavy metals and to use their metabolic functions in biotechnology, although heavy metals comprise the major part of the elements in the periodic table. Because they can form complex compounds, some heavy metal ions are essential trace elements, but, essential or not, most heavy metals are toxic at higher concentrations. This review describes the workings of known metal-resistance systems in microorganisms. After an account of the basic principles of homoeostasis for all heavy-metal ions, the transport of the 17 most important (heavy metal) elements is compared. Received: 25 November 1998 / Received revision: 18 February 1999 / Accepted: 20 February 1999