植物寡肽运输与硝酸根运输基因家族的研究进展

氮素是植物生长发育的重要营养元素,也是限制植物生物量尤其是经济产量的关键营养元素之一.植物不仅能从外界获取无机氮素(硝酸根、铵根和尿素等),还能以氨基酸、寡肽等形式获取有机氮素.植物已进化出复杂的运输系统来吸收与运输这些含氮化合物.硝酸根运输基因家族分为低亲和力硝酸根运输基因(low-affmity nitrate t...     

A fourth gene from the Candida albicans CDR family of ABC transporters

Using primers derived from a region of the Candida albicans CDR1 (Candida drug resistance) gene that is conserved in other ABC (ATP-binding cassette) transporters, a DNA fragment from a previously unknown CDR gene was obtained by polymerase chain reaction (PCR). After screening a C. albicans genomic library with this fragment as a probe, the complete CDR4 gene was isolated and sequenced. CDR4 codes for a putative ABC transporter of 1490 amino acids with a high degree of homology to Cdr1p, Cdr2p and Cdr3p from C. albicans (62, 59 and 57% amino acid sequence identity, respectively). Cdr4p has a predicted structure typical for cluster I.1 of yeast ABC transporters, characterized by two homologous halves, each comprising an N-terminal hydrophilic domain with consensus sequences for ATP binding and a C-terminal hydrophobic domain with six transmembrane helices. In contrast to the CDR1/CDR2 genes, the genetic structure of the CDR4 gene was conserved in 59 C. albicans isolates from six different patients. Northern hybridization analysis showed that the CDR4 gene was expressed in most isolates, but no correlation between CDR4 mRNA levels and the degree of fluconazole resistance of the isolates was found. In addition, a C. albicans mutant in which both copies of the CDR4 gene were disrupted by insertional mutagenesis was not hypersusceptible to fluconazole as compared to the parent strain. Unlike CDR1 and CDR2, CDR4 does not, therefore, seem to be involved in fluconazole resistance in C. albicans.     

过量表达辣椒CaNPR1提高烟草对青枯菌的抗性

对水稻和拟南芥等模式植物的研究表明,NPR1(nonexpressor of pathogenesis-related genes 1)是依赖于SA通路的防御反应调节基因,但在辣椒和烟草等茄科作物中该蛋白的功能还鲜有报道。研究从辣椒cDNA文库中分离获得一个NPR1的类似物全长cDNA(CaNPR1),并获得了其超表达的转基因烟草T₁代株系。研究结果表明,这些株系与其野生型植株没有明显表型差异,但却表现出较野生型植株更高的抗青枯菌侵染活性。同时,研究还发现CaNPR1的超表达还显著提高了防御相关基因的表达,表明NPR1在不同植物间具有较强的功能保守性。     

Functional characterization of an ammonium transporter gene from Lotus japonicus

NH₄⁺ is the main product of symbiotic nitrogen fixation and the external concentration of combined nitrogen plays a key regulatory role in all the different step of plant-rhizobia interaction. We report the cloning and characterization of the first member of the ammonium transporter family, LjAMT1;1 from a leguminous plant, Lotus japonicus. Sequence analysis reveals a close relationship to plant transporters of the AMT1 family. The wild type and two mutated versions of LjAMT1;1 were expressed and functionally characterized in yeast. LjAMT1;1 is transcribed in roots, leaves and nodules of L. japonicus plants grown under low nitrogen conditions, consistent with a role in uptake of NH₄⁺ by the plant cells.     

FHA2 is a plant-specific ISWI subunit responsible for stamen development and plant fertility

Imitation Switch (ISWI) chromatin remodelers are known to function in diverse multi‐subunit complexes in yeast and animals. However, the constitution and function of ISWI complexes in Arabidopsis thaliana remain unclear. In this study, we identified forkhead‐associated domain 2 (FHA2) as a plant‐specific subunit of an ISWI chromatin‐remodeling complex in Arabidopsis. By in vivo and in vitro analyses, we demonstrated that FHA2 directly binds to RLT1 and RLT2, two redundant subunits of the ISWI complex in Arabidopsis. The stamen filament is shorter in the fha2 and rlt1/2 mutants than in the wild type, whereas their pistil lengths are comparable. The shorter filament, which is due to reduced cell size, results in insufficient pollination and reduced fertility. The rlt1/2 mutant shows an early‐flowering phenotype, whereas the phenotype is not shared by the fha2 mutant. Consistent with the functional specificity of FHA2, our RNA‐seq analysis indicated that the fha2 mutant affects a subset of RLT1/2‐regulated genes that does not include genes involved in the regulation of flowering time. This study demonstrates that FHA2 functions as a previously uncharacterized subunit of the Arabidopsis ISWI complex and is exclusively involved in regulating stamen development and plant fertility.     

Plant sucrose-H+ symporters mediate the transport of vitamin H

A cDNA coding for a vitamin H (biotin) transport protein from Arabidopsis was identified by genetic complementation of a biotin uptake-deficient yeast mutant. Vitamin H transport by this protein was sensitive to the SH-group inhibitor p-chloromercuribenzene sulfonic acid (PCMBS) and to the uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP), suggesting an energy-dependent biotin-H+ symport mechanism. The transport activity could contribute to the so-far uncharacterized plant sucrose-H+ symporter AtSUC5 which mediates the energy-dependent transport of biotin and sucrose, and restores growth of the biotin transport-deficient yeast mutant on medium with low biotin concentrations. Functional comparison of the AtSUC5 transporter with previously characterized plant sucrose or monosaccharide transporters revealed that biotin transport may be a general and specific property of all plant sucrose transporters (sucrose/biotin-H+ symporters). This first report on a transporter with dual substrate specificity for two structurally unrelated molecules has a major impact on general thinking concerning the specificity of membrane transporters. The physiological relevance of this finding is discussed.     

Arabidopsis IRT2 gene encodes a root-periphery iron transporter

Iron uptake from the soil is a tightly controlled process in plant roots, involving specialized transporters. One such transporter, IRT1, was identified in Arabidopsis thaliana and shown to function as a broad-range metal ion transporter in yeast. Here we report the cloning and characterization of the IRT2 cDNA, a member of the ZIP family of metal transporters, highly similar to IRT1 at the amino-acid level. IRT2 expression in yeast suppresses the growth defect of iron and zinc transport yeast mutants and enhances iron uptake and accumulation. However, unlike IRT1, IRT2 does not transport manganese or cadmium in yeast. IRT2 expression is detected only in roots of A. thaliana plants, and is upregulated by iron deficiency. By fusing the IRT2 promoter to the uidA reporter gene, we show that the IRT2 promoter is mainly active in the external cell layers of the root subapical zone, and therefore provide the first tissue localization of a plant metal transporter. Altogether, these data support a role for the IRT2 transporter in iron and zinc uptake from the soil in response to iron-limited conditions.     

The hexose transporter family of Saccharomyces cerevisiae

Saccharomyces cerevisiae accomplishes high rates of hexose transport. The kinetics of hexose transport are complex. The capacity and kinetic complexity of hexose transport in yeast are reflected in the large number of sugar transporter genes in the genome. Twenty hexose transporter genes exist in S. cerevisiae. Some of these have been found by genetic means; many have been discovered by the comprehensive sequencing of the yeast genome. This review codifies the nomenclature of the hexose transporter genes and describes the sequence homology and structural similarity of the proteins they encode. Information about the expression and function of the transporters is presented. Access to the sequences of the genes and proteins at three sequence databases is provided via the World Wide Web. Received: 24 June 1996 / Accepted: 29 July 1996     

Characterization of two Arabidopsis thaliana fructokinases

Two different fructokinase isoforms of Arabidopsis thaliana have been identified and characterized by non-denaturing electrophoresis followed by activity-staining. The two fructokinases, fructokinase1 (FRK1) and fructokinase2 (FRK2), showed a high specificity for fructose and did not stain when glucose or mannose were used as substrate. Fructose and ATP at high concentrations (above 5 mM) induced a substrate inhibition of the two enzymatic activities. Arabidopsis FRK1 and FRK2 were capable of employing GTP, CTP, UTP and TTP as phosphate donors, although with a significantly lower efficiency than ATP. The two fructokinase activities were also activated by K⁺, at around 10–20 mM, and inhibited by ADP and AMP at concentrations above 10 mM. Finally, FRK1 and FRK2 showed a different expression pattern in the plant, with FRK1 being more abundant in the roots and FRK2 in the shoots. The results demonstrate a simple technique that provides important information about fructokinase activities in the plants and which can be useful for the analysis of Arabidopsis mutants.     

Nucleoside transport in Crithidia luciliae

Nucleoside transport was evaluated in the trypanosomatid Crithidia luciliae by a rapid sampling technique. C. luciliae was shown to possess two independent nucleoside transporters, one which transported adenosine, deoxyadenosine, tubercidin, sangivamycin and the pyrimidine nucleoside thymidine, while the second was specific for guanosine, inosine and deoxyguanosine. The rapid influx occurred by a process of facilitated transport. The apparent K_m values for adenosine and guanosine were 9.34 ± 1.30 and 10.6 ± 2.60 μM, respectively. The pyrimidine nucleoside thymidine was transported at a rate approximately 50% lower than the purine nucleosides, whilst uridine, deoxyuridine and deoxycytidine were not transported. The optical isomer, -adenosine entered the organism by simple diffusion rather than by facilitated transport. In contrast to mammalian cells, neither of the nucleoside transporters in C. luciliae were inhibited by nitrobenzylthioinosine, dilazep, or dipyridamole, potent inhibitors of nucleoside transport in mammalian cells, whilst p-chloromercuribenzoate sulphonate inhibited both nucleoside transporters in C. luciliae.     

Genome-wide annotation and functional identification of aphid GLUT-like sugar transporters

Background

Phloem feeding insects, such as aphids, feed almost continuously on plant phloem sap, a liquid diet that contains high concentrations of sucrose (a disaccharide comprising of glucose and fructose). To access the available carbon, aphids hydrolyze sucrose in the gut lumen and transport its constituent monosaccharides, glucose and fructose. Although sugar transport plays a critical role in aphid nutrition, the molecular basis of sugar transport in aphids, and more generally across all insects, remains poorly characterized. Here, using the latest release of the pea aphid, Acyrthosiphon pisum, genome we provide an updated gene annotation and expression profile of putative sugar transporters. Finally, gut expressed sugar transporters are functionally expressed in yeast and screened for glucose and fructose transport activity.

Results

In this study, using a de novo approach, we identified 19 sugar porter (SP) family transporters in the A. pisum genome. Gene expression analysis, based on 214, 834 A. pisum expressed sequence tags, supports 17 sugar porter family transporters being actively expressed in adult female aphids. Further analysis, using quantitative PCR identifies 4 transporters, A. pisum sugar transporter 1, 3, 4 and 9 (ApST1, ApST3, ApST4 and ApST9) as highly expressed and/or enriched in gut tissue. When expressed in a Saccharomyces cerevisiae hexose transporter deletion mutant (strain EBY.VW4000), only ApST3 (previously characterized) and ApST4 (reported here) transport glucose and fructose resulting in functional rescue of the yeast mutant. Here we characterize ApST4, a 491 amino acid protein, with 12 predicted transmembrane regions, as a facilitative glucose/fructose transporter. Finally, phylogenetic reconstruction reveals that ApST4, and related, as yet uncharacterized insect transporters are phylogenetically closely related to human GLUT (SLC2A) class I facilitative glucose/fructose transporters.

Conclusions

The gut enhanced expression of ApST4, and the transport specificity of its product is consistent with ApST4 functioning as a gut glucose/fructose transporter. Here, we hypothesize that both ApST3 (reported previously) and ApST4 (reported here) function at the gut interface to import glucose and fructose from the gut lumen.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-647) contains supplementary material, which is available to authorized users.     

Glutathione utilization by Candida albicans requires a functional glutathione degradation (DUG) pathway and OPT7, an unusual member of the oligopeptide transporter family

Candida albicans lacks the ability to survive within its mammalian host in the absence of endogenous glutathione biosynthesis. To examine the ability of this yeast to utilize exogenous glutathione, we exploited the organic sulfur auxotrophy of C. albicans met15Δ strains. We observed that glutathione is utilized efficiently by the alternative pathway of glutathione degradation (DUG pathway). The major oligopeptide transporters OPT1-OPT5 of C. albicans that were most similar to the known yeast glutathione transporters were not found to contribute to glutathione transport to any significant extent. A genomic library approach to identify the glutathione transporter of C. albicans yielded OPT7 as the primary glutathione transporter. Biochemical studies on OPT7 using radiolabeled GSH uptake revealed a K(m) of 205 μm, indicating that it was a high affinity glutathione transporter. OPT7 is unusual in several aspects. It is the most remote member to known yeast glutathione transporters, lacks the two highly conserved cysteines in the family that are known to be crucial in trafficking, and also has the ability to take up tripeptides. The transporter was regulated by sulfur sources in the medium. OPT7 orthologues were prevalent among many pathogenic yeasts and fungi and formed a distinct cluster quite remote from the Saccharomyces cerevisiae HGT1 glutathione transporter cluster. In vivo experiments using a systemic model of candidiasis failed to detect expression of OPT7 in vivo, and strains disrupted either in the degradation (dug3Δ) or transport (opt7Δ) of glutathione failed to show a defect in virulence.     

Molybdate transport through the plant sulfate transporter SHST1

Molybdenum is an essential micronutrient required by plants. The mechanism of molybdenum uptake in plants is poorly understood, however, evidence has suggested that sulfate transporters may be involved. The sulfate transporter from Stylosanthes hamata, SHST1, restored growth of the sulfate transport yeast mutant, YSD1, on media containing low amounts of molybdate. Kinetic analysis using 99MoO4(2-) demonstrated that SHST1 enhanced the uptake of molybdate into yeast cells at nM concentrations. Uptake was not inhibited by sulfate, but sulfate transport via SHST1 was reduced with molybdate. These results are the first measurement of molybdate transport by a characterised plant sulfate transport protein.     

Loss of Zhf and the tightly regulated zinc-uptake system SpZrt1 in Schizosaccharomyces pombe reveals the delicacy of cellular zinc balance

Zinc is an essential micronutrient, and yet it can be toxic when present in excess. Zinc acquisition and distribution are dependent on tightly controlled transport of Zn²⁺ ions. Schizosaccharomyces pombe represents a second eukaryotic model to study cellular metal homeostasis. In several ways its micronutrient metabolism is fundamentally different from Saccharomyces cerevisiae . We identified the first Zn²⁺-uptake system in S. pombe and named it SpZrt1. Knock-out strains for all three ZIP (Zrt, Irt-like protein) transporters in fission yeast were constructed. Only zrt1 Δ cells were unable to grow at low Zn²⁺ and showed reduced ⁶⁵Zn²⁺ uptake. Elemental profiles revealed a strong decrease in zinc accumulation. Cd²⁺ ions inhibited uptake but Fe²⁺ or Mn²⁺ did not. Both mRNA abundance and protein amount are tightly regulated. Zrt1 activity is rapidly shut down upon transfer of zinc-deficient cells to zinc-replete conditions. In cells lacking Zhf, a transporter mediating endoplasmic reticulum storage of zinc, this response is about 100-fold more sensitive. Thus, removal of excess of zinc from the cytosol is largely Zhf dependent. Moreover, cells deficient for both transporters are no longer able to adjust to changing external Zn²⁺ concentrations. Optimal growth is restricted to a narrow range of Zn²⁺ concentrations, illustrating the fine balance between micronutrient deficiency and toxicity.     

Asparaginase production by a recombinant Pichia pastoris strain harbouring Saccharomyces cerevisiae ASP3 gene

The therapeutic enzyme asparaginase, which is used for the treatment of acute lymphoblastic leukaemia, is industrially produced by the bacteria Escherichia coli or Erwinia crysanthemi. In spite of its effectiveness as a therapeutic agent, the drug causes severe immunological reactions. As asparaginase is also produced by the yeast Saccharomyces cerevisiae, this microorganism could be considered for the production of the enzyme, providing an alternative antitumoral agent. In this study the ASP3 gene, that codes for the periplasmic, nitrogen regulated, asparaginase II from S. cerevisiae, was cloned and expressed in the methylotrophic yeast Pichia pastoris, under the control of the AOX1 gene promoter. Similarly to S. cerevisiae the heterologous enzyme was addressed to the P. pastoris cell periplasmic space. Enzyme yield per dry cell mass reached 800 U g⁻¹, which was seven fold higher than that obtained using a nitrogen de-repressed ure2 dal80 S. cerevisiae strain. High cell density cultures performed with P. pastoris harbouring the ASP3 gene using a 2 l instrumented bioreactor, where biomass concentration reached 107 g l⁻¹, resulted in a dramatic increase in volumetric yield (85,600 U l⁻¹) and global volumetric productivity (1083 U l⁻¹ h⁻¹).