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Wolf B Frommer

Publications and source records attributed to Wolf B Frommer.

At least 37 records · Page 2Linked to original sources
PubMed2004

Molecular and functional characterization of a family of amino acid transporters from Arabidopsis.

More than 50 distinct amino acid transporter genes have been identified in the genome of Arabidopsis, indicating that transport of amino acids across membranes is a highly complex feature in plants. Based on sequence similarity, these transporters can be divided into two major superfamilies: the amino acid transporter family and the amino acid polyamine choline transporter family. Currently, mainly transporters of the amino acid transporter family have been characterized. Here, a molecular and functional characterization of amino acid polyamine choline transporters is presented, namely the cationic amino acid transporter (CAT) subfamily. CAT5 functions as a high-affinity, basic amino acid transporter at the plasma membrane. Uptake of toxic amino acid analogs implies that neutral or acidic amino acids are preferentially transported by CAT3, CAT6, and CAT8. The expression profiles suggest that CAT5 may function in reuptake of leaking amino acids at the leaf margin, while CAT8 is expressed in young and rapidly dividing tissues such as young leaves and root apical meristem. CAT2 is localized to the tonoplast in transformed Arabidopsis protoplasts and thus may encode the long-sought vacuolar amino acid transporter.

Amino Acid Transport Systems↗
PubMed2004

Root phloem-specific expression of the plasma membrane amino acid proton co-transporter AAP3.

Amino acids are regarded as the nitrogen 'currency' of plants. Amino acids can be taken up from the soil directly or synthesized from inorganic nitrogen, and then circulated in the plant via phloem and xylem. AtAAP3, a member of the Amino Acid Permease (AAP) family, is mainly expressed in root tissue, suggesting a potential role in the uptake and distribution of amino acids. To determine the spatial expression pattern of AAP3, promoter-reporter gene fusions were introduced into Arabidopsis. Histochemical analysis of AAP3 promoter-GUS expressing plants revealed that AAP3 is preferentially expressed in root phloem. Expression was also detected in stamens, in cotyledons, and in major veins of some mature leaves. GFP-AAP3 fusions and epitope-tagged AAP3 were used to confirm the tissue specificity and to determine the subcellular localization of AtAAP3. When overexpressed in yeast or plant protoplasts, the functional GFP-AAP3 fusion was localized in subcellular organelle-like structures, nuclear membrane, and plasma membrane. Epitope-tagged AAP3 confirmed its localization to the plasma membrane and nuclear membrane of the phloem, consistent with the promoter-GUS study. In addition, epitope-tagged AAP3 protein was localized in endodermal cells in root tips. The intracellular localization suggests trafficking or cycling of the transporter, similar to many metabolite transporters in yeast or mammals, for example, yeast amino acid permease GAP1. Despite the specific expression pattern, knock-out mutants did not show altered phenotypes under various conditions including N-starvation. Microarray analyses revealed that the expression profile of genes involved in amino acid metabolism did not change drastically, indicating potential compensation by other amino acid transporters.

Amino Acid Sequence↗
PubMed2004

UPS1 and UPS2 from Arabidopsis mediate high affinity transport of uracil and 5-fluorouracil.

Salvage pathways play an important role in providing nucleobases to cells, which are unable to synthesize sufficient amounts for their needs. Cellular uptake systems for pyrimidines have been described, but in higher eukaryotes, transporters for thymine and uracil have not been identified. Two plant transporters, AtUPS1 and PvUPS1, were recently identified as transporters for allantoin in Arabidopsis and French bean, respectively. However, Arabidopsis, in contrast to tropical legumes, uses mainly amino acids for long distance transport. Allantoin transport has not been described in the Brassicaceae. Thus, the physiological substrates of ureide permease (UPS) transporters in Arabidopsis may be compounds structurally related to allantoin. A detailed analysis of the substrate specificities of two members of the AtUPS family shows that AtUPS1 and AtUPS2 mediate high affinity uracil and 5-fluorouracil (a toxic uracil analogue) transport when expressed in yeast and Xenopus oocytes. Consistent with a function during germination and early seedling development, AtUPS1 expression is transiently induced during the early stages of seedling development followed by up-regulation of AtUPS2 expression. Arabidopsis ups2 insertion mutants and ups1 lines, in which transcript levels were reduced by post-transcriptional gene silencing, are more tolerant to 5-fluorouracil as compared with wild type plants. The results suggest that in Arabidopsis UPS transporters are the main transporters for uracil and potentially other nucleobases, whereas during evolution legumes may have taken advantage of the low selectivity of UPS proteins for long distance transport of allantoin.

Allantoin↗
PubMed2004

K+ channel interactions detected by a genetic system optimized for systematic studies of membrane protein interactions.

Organization of proteins into complexes is crucial for many cellular functions. However, most proteomic approaches primarily detect protein interactions for soluble proteins but are less suitable for membrane-associated complexes. Here we describe a mating-based split ubiquitin system (mbSUS) for systematic identification of interactions between membrane proteins as well as between membrane and soluble proteins. mbSUS allows in vivo cloning of PCR products into a vector set, detection of interactions via mating, regulated expression of baits, and improved selection of interacting proteins. Cloning is simplified by introduction of lambda attachment sites for GATEWAY. Homo- and heteromeric interactions between Arabidopsis K(+) channels KAT1, AKT1, and AKT2 were identified. Tests with deletion mutants demonstrate that the C terminus of KAT1 and AKT1 is necessary for physical assembly of complexes. Screening of a sorted collection of 84 plant proteins with K(+) channels as bait revealed differences in oligomerization between KAT1, AKT1, and AtKC1, and allowed detection of putative interacting partners of KAT1 and AtKC1. These results show that mbSUS is suited for systematic analysis of membrane protein interactions.

Arabidopsis↗
PubMed2004

Overexpression of GLUTAMINE DUMPER1 leads to hypersecretion of glutamine from Hydathodes of Arabidopsis leaves.

Secretion is a fundamental process providing plants with the means for disposal of solutes, improvement of nutrient acquisition, and attraction of other organisms. Specific secretory organs, such as nectaries, hydathodes, and trichomes, use a combination of secretory and retrieval mechanisms, which are poorly understood at present. To study the mechanisms involved, an Arabidopsis thaliana activation tagged mutant, glutamine dumper1 (gdu1), was identified that accumulates salt crystals at the hydathodes. Chemical analysis demonstrated that, in contrast with the amino acid mixture normally present in guttation droplets, the crystals mainly contain Gln. GDU1 was cloned and found to encode a novel 17-kD protein containing a single putative transmembrane span. GDU1 is expressed in the vascular tissues and in hydathodes. Gln content is specifically increased in xylem sap and leaf apoplasm, whereas the content of several amino acids is increased in leaves and phloem sap. Selective secretion of Gln by the leaves may be explained by an enhanced release of this amino acid from cells. GDU1 study may help to shed light on the secretory mechanisms for amino acids in plants.

Amino Acid Sequence↗
PubMed2004

Expression, purification and characterization of recombinant sucrose synthase 1 from Solanum tuberosum L. for carbohydrate engineering.

The gene sus1 from Solanum tuberosum L. encoding for sucrose synthase 1 was cloned into the plasmid pDR195 under the control of the PMA1 promotor. After transformation of Saccharomyces cerevisiae strain 22574d sus1 was constitutively expressed giving a specific activity of 0.3Umg(-1) protein in the crude extract. A one-step purification by Q-Sepharose resulted in an 14-fold purified enzyme preparation in 74% yield. SuSy1 was subsequently purified by immobilized metal ion affinity chromatography and characterized for its utilization in synthesizing different nucleotide sugars and sucrose analogues. The kinetic constants for the cleavage and synthesis reaction were determined: K(m) (UDP) 4microM; K(iS) (UDP) 0.11mM; K(m) (sucrose) 91.6mM; K(m) (UDP-Glc) 0.5mM; K(iS) (UDP-Glc) 2.3mM; K(m) (D-fructose) 2.1mM; K(iS) (D-fructose) 35.9mM. Different nucleoside diphosphates as well as different donor substrate were accepted as follows: UDP>dTDP>ADP>CDP>GDP in the cleavage reaction and UDP-Glc>dTDP-Glc>ADP-Glc>CDP-Glc in the synthesis reaction. SuSy1 shows also a broad acceptance of D- and L-ketoses and D- and L-aldoses. The acceptance of aldoses was deduced from the binding of the inhibitor 5-deoxy-D-fructose (K(i) 0.3mM), an analogue of the natural substrate D-fructopyranoside. The broad substrate spectrum renders SuSy1 from potato a versatile biocatalyst for carbohydrate engineering.

Chromatography, Affinity↗
PubMed2004

Minimally invasive dynamic imaging of ions and metabolites in living cells.

By 2010, it is expected that biochemical functions will be assigned to many of the products of the approximately 30,000 Arabidopsis genes. Moreover, systematic analysis of mutants will provide insight into the biological function of the gene products. Metabolomic technologies complement these approaches by testing for changes in cellular ion and metabolite patterns, providing essential information for the construction of cellular and whole-plant models of metabolism. However, one important set of information that is especially relevant for multicellular organisms is still lacking, that is, knowledge of the cellular and subcellular variation in metabolite levels. The recent development of protein-based nanosensors for metabolites will help to close this gap by providing a set of tools that can be used to determine cytosolic and subcellular metabolite levels in real time using fluorescence-based microscopy. A major challenge for the future is the application of these nanosensors to quantify metabolite levels in plant cells and tissues.

Diagnostic Imaging↗
PubMed2004

Live imaging of glucose homeostasis in nuclei of COS-7 cells.

Measuring subcellular glucose levels deep in tissues can provide new insights into compartmentalization and specialization of glucose metabolism among different cells. As shown previously, a FRET-based glucose-sensor consisting of two GFP-variants and the Escherichia coli periplasmic glucose/galactose binding protein was successfully expressed in the cytosol of COS7-cells and used to determine cytosolic glucose levels. Recording cytosolic fluorescence intensities in cells located in deeper layers of tissues is often difficult due to loss of signal intensity caused by effects of other cell layers on excitation and emission light. These interfering effects may be reduced by restricting fluorophores to occupy only a fraction of the assayed tissue volume. This can be accomplished by confining fluorophores to a sub-compartment of each cell in the tissue, such as the nucleus. The glucose-sensor was targeted to nuclei of COS7-cells. To determine, whether nuclear glucose levels can be used to track cytosolic changes, nuclear glucose concentrations were quantified as the cells were challenged with external glucose over a range of 0.5 to 10 mM and compared to cytosolic levels. Internal glucose concentrations in both compartments were similar, corresponding to approximately 50% of the external concentration. Taken together, these results indicate that nuclear glucose levels can be used to determine cytosolic levels indirectly, permitting more reliable quantification of fluorescence intensities and providing a tool for measurements not only in cell cultures but also in tissues.

Algorithms↗
PubMed2004

Transport mechanisms for organic forms of carbon and nitrogen between source and sink.

Sugars and amino acids are generated in plants by assimilation from inorganic forms. Assimilated forms cross multiple membranes on their way from production sites to storage or use locations. Specific transport systems are responsible for vacuolar uptake and release, for efflux from the cells, and for uptake into the vasculature. Detailed phylogenetic analyses suggest that only proton-coupled cotransporters involved in phloem loading have been identified to date, whereas systems for vacuolar transport and efflux still await identification. Novel imaging approaches may provide the means to characterize the cellular events and elucidate whole plant control of assimilate partitioning and allocation.

Amino Acids↗
PubMed2003

Fusion to GFP blocks intercellular trafficking of the sucrose transporter SUT1 leading to accumulation in companion cells.

BACKGROUND: Plant phloem consists of an interdependent cell pair, the sieve element/companion cell complex. Sucrose transporters are localized to enucleate sieve elements (SE), despite being transcribed in companion cells (CC). Due to the high turnover of SUT1, sucrose transporter mRNA or protein must traffic from CC to SE via the plasmodesmata. Localization of SUT mRNA at plasmodesmatal orifices connecting CC and SE suggests RNA transport, potentially mediated by RNA binding proteins. In many organisms, polar RNA transport is mediated through RNA binding proteins interacting with the 3'-UTR and controlling localized protein synthesis. To study mechanisms for trafficking of SUT1, GFP-fusions with and without 3'-UTR were expressed in transgenic plants. RESULTS: In contrast to plants expressing GFP from the strong SUC2 promoter, in RolC-controlled expression GFP is retained in companion cells. The 3'-UTR of SUT1 affected intracellular distribution of GFP but was insufficient for trafficking of SUT1, GFP or their fusions to SEs. Fusion of GFP to SUT1 did however lead to accumulation of SUT1-GFP in the CC, indicating that trafficking was blocked while translational inhibition of SUT1 mRNA was released in CCs. CONCLUSION: A fusion with GFP prevents targeting of the sucrose transporter SUT1 to the SE while leading to accumulation in the CC. The 3'-UTR of SUT1 is insufficient for mobilization of either the fusion or GFP alone. It is conceivable that SUT1-GFP protein transport through PD to SE was blocked due to the presence of GFP, resulting in retention in CC particles. Alternatively, SUT1 mRNA transport through the PD could have been blocked due to insertion of GFP between the SUT1 coding sequence and 3'-UTR.

3' Untranslated Regions↗
PubMed2003

Urea transport by nitrogen-regulated tonoplast intrinsic proteins in Arabidopsis.

Urea is the major nitrogen (N) form supplied as fertilizer in agricultural plant production and also an important N metabolite in plants. Because urea transport in plants is not well understood, the aim of the present study was to isolate urea transporter genes from the model plant Arabidopsis. Using heterologous complementation of a urea uptake-defective yeast (Saccharomyces cerevisiae) mutant allowed to isolate AtTIP1;1, AtTIP1;2, AtTIP2;1, and AtTIP4;1 from a cDNA library of Arabidopsis. These cDNAs encode channel-like tonoplast intrinsic proteins (TIPs) that belong to the superfamily of major intrinsic proteins (or aquaporins). All four genes conferred growth of a urea uptake-defective yeast mutant on 2 mm urea in a phloretin-sensitive and pH-independent manner. Uptake studies using 14C-labeled urea into AtTIP2;1-expressing Xenopus laevis oocytes demonstrated that AtTIP2;1 facilitated urea transport also in a pH-independent manner and with linear concentration dependency. Expression studies showed that AtTIP1;2, AtTIP2;1, and AtTIP4;1 genes were up-regulated during early germination and under N deficiency in roots but constitutively expressed in shoots. Subcellular localization of green fluorescent protein-fused AtTIPs indicated that AtTIP1;2, AtTIP2;1, and AtTIP4;1 were targeted mainly to the tonoplast and other endomembranes. Thus, in addition to their role as water channels, TIP transporters may play a role in equilibrating urea concentrations between different cellular compartments.

Animals↗
PubMed2003

Development of a fluorescent nanosensor for ribose.

To analyze ribose uptake and metabolism in living cells, nanosensors were engineered by flanking the Escherichia coli periplasmic ribose binding protein with two green fluorescent protein variants. Following binding of ribose, fluorescence resonance energy transfer decreased with increasing ribose concentration. Five affinity mutants were generated covering binding constants between 400 nM and 11.7 mM. Analysis of nanosensor response in COS-7 cells showed that free ribose accumulates in the cell and is slowly metabolized. Inhibitor studies suggest that uptake is mediated by a monosaccharide transporter of the GLUT family, however, ribose taken up into the cell was not or only slowly released, indicating irreversibility of uptake.

Animals↗
PubMed2003

Homo- and hetero-oligomerization of ammonium transporter-1 NH4 uniporters.

In most organisms, high affinity ammonium uptake is catalyzed by members of the ammonium transporter family (AMT/MEP/Rh). A single point mutation (G458D) in the cytosolic C terminus of the plasma membrane transporter LeAMT1;1 from tomato leads to loss of function, although mutant and wild type proteins show similar localization when expressed in yeast or plant protoplasts. Co-expression of LeAMT1;1 and mutant in Xenopus oocytes inhibited ammonium transport in a dominant negative manner, suggesting homo-oligomerization. In vivo interaction between LeAMT1;1 proteins was confirmed by the split ubiquitin yeast two-hybrid system. LeAMT1;1 is isolated from root membranes as a high molecular mass oligomer, converted to a approximately 35-kDa polypeptide by denaturation. To investigate interactions with the LeAMT1;2 paralog, co-localizing with LeAMT1;1 in root hairs, LeAMT1;2 was characterized as a lower affinity NH4+ uniporter. Co-expression of wild types with the respective G458D/G465D mutants inhibited ammonium transport in a dominant negative manner, supporting the formation of heteromeric complexes in oocytes. Thus, in yeast, oocytes, and plants, ammonium transporters are able to oligomerize, which may be relevant for regulation of ammonium uptake.

Amino Acid Sequence↗
PubMed2003

In vivo imaging of the dynamics of glucose uptake in the cytosol of COS-7 cells by fluorescent nanosensors.

Glucose homeostasis is a function of glucose supply, transport across the plasma membrane, and metabolism. To monitor glucose dynamics in individual cells, a glucose nanosensor was developed by flanking the Escherichia coli periplasmic glucose/galactose-binding protein with two different green fluorescent protein variants. Upon binding of substrate the FLIPglu-170n sensor showed a concentration-dependent decrease in fluorescence resonance energy transfer between the attached chromophores with a binding affinity for glucose of 170 nm. Fluorescence resonance energy transfer measurements with different sugars indicated a broad selectivity for monosaccharides. An affinity mutant with a Kd of approximately 600 microM was generated, which showed higher substrate specificity, and thus allowed specific monitoring of reversible glucose dynamics in COS-7 cells in the physiological range. At external glucose concentrations between 0.5 and 10 mM, reflecting typical blood levels, free cytosolic glucose concentrations remained at approximately 50% of external levels. The removal of glucose lead to reduced glucose levels in the cell, demonstrating reversibility and visualizing homeostasis. Glucose levels dropped even in the presence of the transport inhibitor cytochalasin B, indicating rapid metabolism. Consistently, the addition of 2-deoxyglucose, which is not recognized by the sensor, affects glucose uptake and metabolism rates. Within the physiological range, glucose utilization, i.e. hexokinase activity, was not limiting. Furthermore, the results show that in COS-7 cells, cytosolic glucose concentrations can vary over at least two orders of magnitude. The glucose nanosensor provides a novel tool with numerous scientific, medical, and environmental applications.

Animals↗
PubMed2003

Interactions between co-expressed Arabidopsis sucrose transporters in the split-ubiquitin system.

BACKGROUND: The Arabidopsis genome contains nine sucrose transporter paralogs falling into three clades: SUT1-like, SUT2 and SUT4. The carriers differ in their kinetic properties. Many transport proteins are known to exist as oligomers. The yeast-based split ubiquitin system can be used to analyze the ability of membrane proteins to interact. RESULTS: Promoter-GUS fusions were used to analyze the cellular expression of the three transporter genes in transgenic Arabidopsis plants. All three fusion genes are co-expressed in companion cells. Protein-protein interactions between Arabidopsis sucrose transporters were tested using the split ubiquitin system. Three paralogous sucrose transporters are capable of interacting as either homo- or heteromers. The interactions are specific, since a potassium channel and a glucose transporter did not show interaction with sucrose transporters. Also the biosynthetic and metabolizing enzymes, sucrose phosphate phosphatase and sucrose synthase, which were found to be at least in part bound to the plasma membrane, did not specifically interact with sucrose transporters. CONCLUSIONS: The split-ubiquitin system provides a powerful tool to detect potential interactions between plant membrane proteins by heterologous expression in yeast, and can be used to screen for interactions with membrane proteins as baits. Like other membrane proteins, the Arabidopsis sucrose transporters are able to form oligomers. The biochemical approaches are required to confirm the in planta interaction.

Arabidopsis Proteins↗
PubMed2003

Overexpression of the sucrose transporter SoSUT1 in potato results in alterations in leaf carbon partitioning and in tuber metabolism but has little impact on tuber morphology.

The aim of this work was to examine the consequences of the heterologous expression of a spinach ( Spinacia oleracea L.) sucrose transporter ( SoSUT1) in potato ( Solanum tuberosum L.). Many studies have indicated that reduction of the expression of this class of sucrose transporter has deleterious effects on plant growth and development; however, until now the possibility of improving plant performance by enhancing the expression of this sucrose transporter has not been reported. With this intention we constructed a chimeric construct in which SoSUT1 was cloned in-frame with the myc epitope. We confirmed that this construct, SoSUT1m, was able to mediate sucrose transport by expression in the yeast strain SUSY7. SoSUT1m was expressed in wild-type potato in the sense orientation under the control of the cauliflower mosaic virus 35S promoter to evaluate the effect of an increased constitutive expression of a class-I sucrose transporter. We confirmed that these plants displayed expression of SoSUT1 at both the transcript and protein level and that microsomal fragments isolated from selected lines had an increased sucrose uptake capacity. Analysis of metabolism of these lines indicated that the leaves were characterised by a reduced sucrose level yet exhibited little change in photosynthetic rate. Furthermore, despite the observed increase in sugar (and reduction in amino acid) levels within the tubers, there was little change in either starch content or tuber yield in the transformants. In summary, the genetic manipulation described in this paper resulted in a shift in carbon partitioning in both leaves and tubers and an increased sucrose uptake rate in plasma-membrane vesicles isolated from these lines, but had little impact on tuber metabolism or morphology.

Carbohydrate Metabolism↗
PubMed2003

Identification of an Arabidopsis mitochondrial succinate-fumarate translocator.

Complementation of a yeast acr1 mutant carrying a deletion of the succinate/fumarate carrier gene enabled functional identification of a mitochondrial succinate translocator from Arabidopsis thaliana (AtmSFC1). Thus complementation of yeast mutants is applicable also for identification and characterization of organellar transporters. Reverse transcription polymerase chain reaction and promoter-GUS fusion showed expression of AtmSFC1 in 2 day old dark grown seedlings, which declined in cotyledons during further development, consistent with a role in export of fumarate for gluconeogenesis during lipid mobilization at early germination of Arabidopsis seeds. In mature plants, expression was found in developing and germinating pollen, suggesting a role in ethanolic fermentation.

Amino Acid Sequence↗