Sugar transporters in higher plants--a diversity of roles and complex regulation

Sugar-transport proteins play a crucial role in the cell-to-cell and long-distance distribution of sugars throughout the plant. In the past decade, genes encoding sugar transporters (or carriers) have been identified, functionally expressed in heterologous systems, and studied with respect to their spatial and temporal expression. Higher plants possess two distinct families of sugar carriers: the disaccharide transporters that primarily catalyse sucrose transport and the monosaccharide transporters that mediate the transport of a variable range of monosaccharides. The tissue and cellular expression pattern of the respective genes indicates their specific and sometimes unique physiological tasks. Some play a purely nutritional role and supply sugars to cells for growth and development, whereas others are involved in generating osmotic gradients required to drive mass flow or movement. Intriguingly, some carriers might be involved in signalling. Various levels of control regulate these sugar transporters during plant development and when the normal environment is perturbed. This article focuses on members of the monosaccharide transporter and disaccharide transporter families, providing details about their structure, function and regulation. The tissue and cellular distribution of these sugar transporters suggests that they have interesting physiological roles.     

Arabidopsis INOSITOL TRANSPORTER4 mediates high-affinity H+ symport of myoinositol across the plasma membrane

Four genes of the Arabidopsis (Arabidopsis thaliana) monosaccharide transporter-like superfamily share significant homology with transporter genes previously identified in the common ice plant (Mesembryanthemum crystallinum), a model system for studies on salt tolerance of higher plants. These ice plant transporters had been discussed as tonoplast proteins catalyzing the inositol-dependent efflux of Na(+) ions from vacuoles. The subcellular localization and the physiological role of the homologous proteins in the glycophyte Arabidopsis were unclear. Here we describe Arabidopsis INOSITOL TRANSPORTER4 (AtINT4), the first member of this subgroup of Arabidopsis monosaccharide transporter-like transporters. Functional analyses of the protein in yeast (Saccharomyces cerevisiae) and Xenopus laevis oocytes characterize this protein as a highly specific H(+) symporter for myoinositol. These activities and analyses of the subcellular localization of an AtINT4 fusion protein in Arabidopsis and tobacco (Nicotiana tabacum) reveal that AtINT4 is located in the plasma membrane. AtINT4 promoter-reporter gene plants demonstrate that AtINT4 is strongly expressed in Arabidopsis pollen and phloem companion cells. The potential physiological role of AtINT4 is discussed.     

Sugar transport across the plasma membranes of higher plants

The fluxes of carbohydrates across the plasma membranes of higher-plant cells are catalysed mainly by monosaccharide and disaccharide-H⁺ symporters. cDNAs encoding these different transporters have been cloned recently and the functions and properties of the encoded proteins have been studied extensively in heterologous expression systems. Several of the proteins have been identified biochemically in these expression systems and their location in plants has been shown immunohistochemically or with transgenic plants which were transformed with reporter genes, expressed under the control of the promoters of individual transporter genes. In this paper we summarize the current knowledge on the molecular biology and biochemistry of higher-plant sugar transport proteins.     

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.     

Functional analysis of the Arabidopsis PHT4 family of intracellular phosphate transporters

The transport of phosphate (Pi) between subcellular compartments is central to metabolic regulation. Although some of the transporters involved in controlling the intracellular distribution of Pi have been identified in plants, others are predicted from genetic, biochemical and bioinformatics studies. Heterologous expression in yeast, and gene expression and localization in plants were used to characterize all six members of an Arabidopsis thaliana membrane transporter family designated here as PHT4. PHT4 proteins share similarity with SLC17/type I Pi transporters, a diverse group of animal proteins involved in the transport of Pi, organic anions and chloride. All of the PHT4 proteins mediate Pi transport in yeast with high specificity. Bioinformatic analysis and localization of PHT4-GFP fusion proteins indicate that five of the proteins are targeted to the plastid envelope, and the sixth resides in the Golgi apparatus. PHT4 genes are expressed in both roots and leaves, although two of the genes are expressed predominantly in leaves and one mostly in roots. These expression patterns, together with Pi transport activities and subcellular locations, suggest roles for PHT4 proteins in the transport of Pi between the cytosol and chloroplasts, heterotrophic plastids and the Golgi apparatus.     

Functional survey for heterologous sugar transport proteins, using Saccharomyces cerevisiae as a host

Molecular transport is a key process in cellular metabolism. This step is often limiting when using a nonnative carbon source, as exemplified by xylose catabolism in Saccharomyces cerevisiae. As a step toward addressing this limitation, this study seeks to characterize monosaccharide transport preference and efficiency. A group of 26 known and putative monosaccharide transport proteins was expressed in a recombinant Saccharomyces cerevisiae host unable to transport several monosaccharides. A growth-based assay was used to detect transport capacity across six different carbon sources (glucose, xylose, galactose, fructose, mannose, and ribose). A mixed glucose-and-xylose cofermentation was performed to determine substrate preference. These experiments identified 10 transporter proteins that function as transporters of one or more of these sugars. Most of these proteins exhibited broad substrate ranges, and glucose was preferred in all cases. The broadest transporters confer the highest growth rates and strongly prefer glucose. This study reports the first molecular characterization of the annotated XUT genes of Scheffersomyces stipitis and open reading frames from the yeasts Yarrowia lipolytica and Debaryomyces hansenii. Finally, a phylogenetic analysis demonstrates that transporter function clusters into three distinct groups. One particular group comprised of D. hansenii XylHP and S. stipitis XUT1 and XUT3 demonstrated moderate transport efficiency and higher xylose preferences.     

Protein-protein interactions between sucrose transporters of different affinities colocalized in the same enucleate sieve element

Suc represents the major transport form for carbohydrates in plants. Suc is loaded actively against a concentration gradient into sieve elements, which constitute the conduit for assimilate export out of leaves. Three members of the Suc transporter family with different properties were identified: SUT1, a high-affinity Suc proton cotransporter; SUT4, a low-affinity transporter; and SUT2, which in yeast is only weakly active and shows features similar to those of the yeast sugar sensors RGT2 and SNF3. Immunolocalization demonstrated that all three SUT proteins are localized in the same enucleate sieve element. Thus, the potential of Suc transporters to form homooligomers was tested by the yeast-based split-ubiquitin system. The results show that both SUT1 and SUT2 have the potential to form homooligomers. Moreover, all three Suc transporters have the potential to interact with each other. As controls, a potassium channel and a monosaccharide transporter, expressed in the plasma membrane, did not interact with the SUTs. The in vivo interaction between the functionally different Suc transporters indicates that the membrane proteins are capable of forming oligomeric structures that, like mammalian Glc transporter complexes, might be of functional significance for the regulation of transport.     

Molecular cloning of two Arabidopsis UDP-galactose transporters by complementation of a deficient Chinese hamster ovary cell line

Nucleotide-sugar transporters (NSTs) form a family of structurally related transmembrane proteins that transport nucleotide-sugars from the cytoplasm to the endoplasmic reticulum and Golgi lumen. In these organelles, activated sugars are substrates for various glycosyltransferases involved in oligo- and polysaccharide biosynthesis. The Arabidopsis thaliana genome contains more than 40 members of this transporter gene family, of which only a few are functionally characterized. In this study, two Arabidopsis UDP-galactose transporter cDNAs (UDP-GalT1 and UDP-GalT2) are isolated by expression cloning using a Chinese hamster ovary cell line (CHO-Lec8) deficient in UDP-galactose transport. The isolated genes show only 21% identity to each other and very limited sequence identity with human and yeast UDP-galactose transporters and other NSTs. Despite this low overall identity, the two proteins clearly belong to the same gene family. Besides complementing Lec8 cells, the two NSTs are shown to transport exclusively UDP-galactose by an in vitro NST assay. The most homologous proteins with known function are plant transporters that locate in the inner chloroplast membrane and transport triose-phosphate, phosphoenolpyruvate, glucose-6-phosphate, and xylulose 5-phosphate. Also, the latter proteins are members of the same family, which therefore has been named the NST/triose-phosphate transporter family.     

A sink-specific H+/monosaccharide co-transporter from Nicotiana tabacum: cloning and heterologous expression in baker's yeast

A cDNA clone for a monosaccharide transporter (MST1) was isolated from tobacco, which is most strongly expressed in the various sink tissues of mature tobacco plants: roots, flowers, and young leaves. An open reading frame of 1569 bp codes for a protein with 523 amino acids and a calculated molecular weight of 57 717 Da. The protein is homologous to a group of other plant monosaccharide transport proteins from Arabidopsis thaliana and Chlorella kessleri , to human glucose transporters and to Saccharomyces cerevisiae and several bacterial sugar transport proteins. As with these other transporters, the MST1 protein is extremely lipophilic and has 12-putative membrane-spanning domains. Heterologous expression of the MST1 cDNA clone in Saccharomyces cerevisiae allowed its characterization as a putative H⁺/monosaccharide co-transporter, catalyzing the uptake of hexoses (e.g. d -glucose and d -galactose) or pentoses (e.g. d -xylose) and the energy dependent and uncoupler sensitive accumulation of non-metabolizable substrates (e.g. d -xylose or 3- O -methyl-glucose). Polyclonal antibodies were raised against a fusion protein of β-galactosidase and the last 27 amino acids of the C-terminus of the MST1 protein. In SDS extracts of transformed yeast cells these antibodies recognize a polypeptide with an apparent molecular weight of 42 kDa, which is absent in extracts from untransformed control cells.     

Arabidopsis POLYOL TRANSPORTER5, a new member of the monosaccharide transporter-like superfamily, mediates H+-Symport of numerous substrates, including myo-inositol, glycerol, and ribose

Six genes of the Arabidopsis thaliana monosaccharide transporter-like (MST-like) superfamily share significant homology with polyol transporter genes previously identified in plants translocating polyols (mannitol or sorbitol) in their phloem (celery [Apium graveolens], common plantain [Plantago major], or sour cherry [Prunus cerasus]). The physiological role and the functional properties of this group of proteins were unclear in Arabidopsis, which translocates sucrose and small amounts of raffinose rather than polyols. Here, we describe POLYOL TRANSPORTER5 (AtPLT5), the first member of this subgroup of Arabidopsis MST-like transporters. Transient expression of an AtPLT5–green fluorescent protein fusion in plant cells and functional analyses of the AtPLT5 protein in yeast and Xenopus oocytes demonstrate that AtPLT5 is located in the plasma membrane and characterize this protein as a broad-spectrum H⁺-symporter for linear polyols, such as sorbitol, xylitol, erythritol, or glycerol. Unexpectedly, however, AtPLT5 catalyzes also the transport of the cyclic polyol myo-inositol and of different hexoses and pentoses, including ribose, a sugar that is not transported by any of the previously characterized plant sugar transporters. RT-PCR analyses and AtPLT5 promoter-reporter gene plants revealed that AtPLT5 is most strongly expressed in Arabidopsis roots, but also in the vascular tissue of leaves and in specific floral organs. The potential physiological role of AtPLT5 is discussed.     

Substrate recognition by UDP-galactose and CMP-sialic acid transporters. Different sets of transmembrane helices are utilized for the specific recognition of UDP-galactose and CMP-sialic acid

Human UDP-galactose transporter (hUGT1) and CMP-sialic acid transporter (hCST) are related Golgi membrane proteins with 10 transmembrane helices. We have constructed chimeras between these proteins in order to identify submolecular regions responsible for the determination of substrate specificity. To assess the UGT and CST activities, chimeric cDNAs were transiently expressed in either UGT-deficient mutant Lec8 cells or CST-deficient mutant Lec2 cells, and the binding of plant lectins, GS-II or PNA, respectively, to these cells was examined. During the course of analysis of various chimeric transporters, we found that chimeras whose submolecular regions contained helices 1, 8, 9, and 10, and helices 2, 3, and 7 derived from hUGT1 and hCST sequences, respectively, exhibited both UGT and CST activities. The dual substrate specificity for UDP-galactose and CMP-sialic acid of one such representative chimera was directly confirmed by in vitro measurement of the nucleotide sugar transport activity using a heterologous expression system in the yeast Saccharomyces cerevisiae. These findings indicated that the regions which are critical for determining the substrate specificity of UGT and CST resided in different submolecular sites in the two transporters, and that these different determinants could be present within one protein without interfering with each other's function.     

Differential expression of sucrose transporter and polyol transporter genes during maturation of common plantain companion cells

The cDNAs of two sorbitol transporters, common plantain (Plantago major) polyol transporter (PLT) 1 and 2 (PmPLT1 and PmPLT2), were isolated from a vascular bundle-specific cDNA library from common plantain, a dicot plant transporting Suc plus sorbitol in its phloem. Here, we describe the kinetic characterization of these sorbitol transporters by functional expression in Brewer's yeast (Saccharomyces cerevisiae) and in Xenopus sp. oocytes and for the first time the localization of plant PLTs in specific cell types of the vascular tissue. In the yeast system, both proteins were shown to be uncoupler sensitive and could be characterized as low-affinity and low-specificity polyol symporters. The Km value for the physiological substrate sorbitol is 12 mm for PmPLT1 and even higher for PmPLT2, which showed an almost linear increase in sorbitol transport rates up to 20 mm. These data were confirmed in the Xenopus sp. system, where PmPLT1 was analyzed in detail and characterized as a H+ symporter. Using peptide-specific polyclonal antisera against PmPLT1 or PmPLT2 and simultaneous labeling with the monoclonal antiserum 1A2 raised against the companion cell-specific PmSUC2 Suc transporter, both PLTs were localized to companion cells of the phloem in common plantain source leaves. These analyses revealed two different types of companion cells in the common plantain phloem: younger cells expressing PmSUC2 at higher levels and older cells expressing lower levels of PmSUC2 plus both PLT genes. The putative role of these low-affinity transporters in phloem loading is discussed.     

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.     

Comparative molecular biological analysis of membrane transport genes in organisms

Comparative analyses of membrane transport genes revealed many differences in the features of transport homeostasis in eight diverse organisms, ranging from bacteria to animals and plants. In bacteria, membrane-transport systems depend mainly on single genes encoding proteins involved in an ATP-dependent pump and secondary transport proteins that use H⁺ as a co-transport molecule. Animals are especially divergent in their channel genes, and plants have larger numbers of P-type ATPase and secondary active transporters than do other organisms. The secondary transporter genes have diverged evolutionarily in both animals and plants for different co-transporter molecules. Animals use Na⁺ ions for the formation of concentration gradients across plasma membranes, dependent on secondary active transporters and on membrane voltages that in turn are dependent on ion transport regulation systems. Plants use H⁺ ions pooled in vacuoles and the apoplast to transport various substances; these proton gradients are also dependent on secondary active transporters. We also compared the numbers of membrane transporter genes in Arabidopsis and rice. Although many transporter genes are similar in these plants, Arabidopsis has a more diverse array of genes for multi-efflux transport and for response to stress signals, and rice has more secondary transporter genes for carbohydrate and nutrient transport. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

PmSUC3: characterization of a SUT2/SUC3-type sucrose transporter from Plantago major

Higher plants possess medium-sized gene families that encode plasma membrane-localized sucrose transporters. For several plant species, it has been shown that at least one of these genes (e.g., AtSUC3 in Arabidopsis and LeSUT2 in tomato) differs from all other family members in several features, such as the length of the open reading frame, the number of introns, and the codon usage bias. For these reasons, and because two of these proteins did not rescue a yeast mutant defective in sucrose utilization, it had been speculated that this subgroup of transporters might have sensor functions. Here, we describe the detailed functional characterization and cellular localization of PmSUC3, the orthologous transporter from the Plantago major transporter family. The PmSUC3 protein is localized in the sieve elements of the Plantago phloem and mediates the energy-dependent transport of sucrose and maltose. In contrast to the situation in solanaceous plants, PmSUC3 is not colocalized with PmSUC2, the source-specific, phloem-loading sucrose transporter of Plantago. Moreover, PmSUC3 also was identified in sieve elements of sink leaves and in several nonphloem cells and tissues. Arguments for and against a potential sensor function for this type of sucrose transporter are presented, and the role of this type of transporter in the regulation of sucrose fluxes is discussed.     

The conservation of polyol transporter proteins and their involvement in lichenized Ascomycota

In lichen symbiosis, polyol transfer from green algae is important for acquiring the fungal carbon source. However, the existence of polyol transporter genes and their correlation with lichenization remain unclear. Here, we report candidate polyol transporter genes selected from the genome of the lichen-forming fungus (LFF) Ramalina conduplicans. A phylogenetic analysis using characterized polyol and monosaccharide transporter proteins and hypothetical polyol transporter proteins of R. conduplicans and various ascomycetous fungi suggested that the characterized yeast’ polyol transporters form multiple clades with the polyol transporter-like proteins selected from the diverse ascomycetous taxa. Thus, polyol transporter genes are widely conserved among Ascomycota, regardless of lichen-forming status. In addition, the phylogenetic clusters suggested that LFFs belonging to Lecanoromycetes have duplicated proteins in each cluster. Consequently, the number of sequences similar to characterized yeast’ polyol transporters were evaluated using the genomes of 472 species or strains of Ascomycota. Among these, LFFs belonging to Lecanoromycetes had greater numbers of deduced polyol transporter proteins. Thus, various polyol transporters are conserved in Ascomycota and polyol transporter genes appear to have expanded during the evolution of Lecanoromycetes.