Inositol Transport Research Articles

A systematic approach to the amplified expression, functional characterization and purification of inositol transporters fromBacillus subtilis

Abstract A systematic approach was used for the cloning and amplified expression in Escherichia coli of the genes for each of three inositol transport proteins (IolF, IolT, YfiG) from Bacillus subtilis that are evolutionarily-related to human transporters. Inducible amplified expression of each was achieved to levels of ∼ 10-15% of total protein in E. coli inner membrane preparations. The functional integrity of each heterologously-expressed protein was demonstrated by measuring the kinetics of (3)H-myo-inositol transport into energized whole cells; this confirmed that IolT is the major inositol transporter, IolF is an inefficient transporter of this substrate and demonstrated that YfiG is an inositol transport protein for the first time. Competition for (3)H-myo-inositol transport by 17 unlabelled compounds revealed all three proteins to be highly specific in recognizing inositols over sugars. IolT was confirmed to be highly specific for both myo- and D-chiro-inositol and IolF was confirmed to prefer D-chiro-inositol over myo-inositol. YfiG selectively recognized myo-inositol, D-chiro-inositol and, uniquely, L-chiro-inositol. All three proteins were successfully solubilized and purified in milligram quantities from inner membrane preparations and their suitability for inclusion in crystallization trials was assessed by analysis of structural integrity and thermal stability using circular dichroism spectroscopy followed by examination for monodispersity using gel filtration chromatography.

Routes to the tonoplast: the sorting of tonoplast transporters in Arabidopsis mesophyll protoplasts.

Vacuoles perform a multitude of functions in plant cells, including the storage of amino acids and sugars. Tonoplast-localized transporters catalyze the import and release of these molecules. The mechanisms determining the targeting of these transporters to the tonoplast are largely unknown. Using the paralogous Arabidopsis thaliana inositol transporters INT1 (tonoplast) and INT4 (plasma membrane), we performed domain swapping and mutational analyses and identified a C-terminal di-leucine motif responsible for the sorting of higher plant INT1-type transporters to the tonoplast in Arabidopsis mesophyll protoplasts. We demonstrate that this motif can reroute other proteins, such as INT4, SUCROSE TRANSPORTER2 (SUC2), or SWEET1, to the tonoplast and that the position of the motif relative to the transmembrane helix is critical. Rerouted INT4 is functionally active in the tonoplast and complements the growth phenotype of an int1 mutant. In Arabidopsis plants defective in the β-subunit of the AP-3 adaptor complex, INT1 is correctly localized to the tonoplast, while sorting of the vacuolar sucrose transporter SUC4 is blocked in cis-Golgi stacks. Moreover, we demonstrate that both INT1 and SUC4 trafficking to the tonoplast is sensitive to brefeldin A. Our data show that plants possess at least two different Golgi-dependent targeting mechanisms for newly synthesized transporters to the tonoplast.

Down-Regulation of the Myoinositol Transporter SMIT by JAK2

Background/Aims: Janus-activated kinase-2 JAK2 is activated by energy depletion and hyperosmotic shock and modifies the activity of several Na⁺ coupled transporters. The Na⁺ coupled osmolyte transporter SMIT (myoinositol transporter) is upregulated by osmotic shock and downregulated by energy depletion. The present study thus explored whether JAK2 contributes to the regulation of SMIT activity. Methods: To this end, cRNA encoding SMIT was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild type JAK2, constitutively active ^V617FJAK2 or inactive ^K882EJAK2. Inositol-induced current (I_SMIT) was determined by dual electrode voltage clamp and taken as measure for electrogenic inositol transport. Results: No appreciable I_SMIT was observed in water injected oocytes. In SMIT expressing oocytes I_SMIT was significantly decreased by additional coexpression of JAK2 or ^V617FJAK2, but not by coexpression of ^K882EJAK2. According to kinetic analysis coexpression of JAK2 decreased maximal I_SMIT without significantly modifying the concentration required for halfmaximal I_SMIT (K_M). In oocytes expressing both, SMIT and JAK2, ISMIT was gradually increased by JAK2 inhibitor AG490 (40 µM). Disruption of carrier insertion with brefeldin A (5 µM) was followed by a decline of I_SMIT to a similar extent in Xenopus oocytes expressing SMIT with JAK2 and in Xenopus oocytes expressing SMIT alone, suggesting that JAK2 did not affect carrier stability in the cell membrane. Conclusion: JAK2 contributes to the regulation of the inositol transporter SMIT.

Sodium/myo-Inositol Transporters: Substrate Transport Requirements and Regional Brain Expression in the TgCRND8 Mouse Model of Amyloid Pathology

Inositol stereoisomers, myo- and scyllo-inositol, are known to enter the brain and are significantly elevated following oral administration. Elevations in brain inositol levels occur across a concentration gradient as a result of active transport from the periphery. There are two sodium/myo-inositol transporters (SMIT1, SMIT2) that may be responsible for regulating brain inositol levels. The goals of this study were to determine the effects of aging and Alzheimer's disease (AD)-like amyloid pathology on transporter expression, to compare regional expression and to analyze substrate requirements of the inositol transporters. QPCR was used to examine expression of the two transporters in the cortex, hippocampus and cerebellum of TgCRND8 mice, a mouse model of amyloid pathology, in comparison to non-transgenic littermates. In addition, we examined the structural features of inositol required for active transport, utilizing a cell-based competitive uptake assay. Disease pathology did not alter transporter expression in the cortex or hippocampus (p>0.005), with only minimal effects of aging observed in the cerebellum (SMIT1: F2,26 = 12.62; p = 0.0002; SMIT2: F2,26 = 8.71; p = 0.0015). Overall, brain SMIT1 levels were higher than SMIT2, however, regional differences were observed. For SMIT1, at 4 and 6 months cerebellar SMIT1 levels were significantly higher than cortical and hippocampal levels (p<0.05). For SMIT2, at all three ages both cortical and cerebellar SMIT2 levels were significantly higher than hippocampal levels (p<0.05) and at 4 and 6 months of age, cerebellar SMIT2 levels were also significantly higher than cortical levels (p<0.05). Inositol transporter levels are stably expressed as a function of age, and expression is unaltered with disease pathology in the TgCRND8 mouse. Given the fact that scyllo-inositol is currently in clinical trials for the treatment of AD, the stable expression of inositol transporters regardless of disease pathology is an important finding.

Phosphotransferase system-independent glucose utilization in corynebacterium glutamicum by inositol permeases and glucokinases.

Phosphoenolpyruvate-dependent glucose phosphorylation via the phosphotransferase system (PTS) is the major path of glucose uptake in Corynebacterium glutamicum, but some growth from glucose is retained in the absence of the PTS. The growth defect of a deletion mutant lacking the general PTS component HPr in glucose medium could be overcome by suppressor mutations leading to the high expression of inositol utilization genes or by the addition of inositol to the growth medium if a glucokinase is overproduced simultaneously. PTS-independent glucose uptake was shown to require at least one of the inositol transporters IolT1 and IolT2 as a mutant lacking IolT1, IolT2, and the PTS component HPr could not grow with glucose as the sole carbon source. Efficient glucose utilization in the absence of the PTS necessitated the overexpression of a glucokinase gene in addition to either iolT1 or iolT2. IolT1 and IolT2 are low-affinity glucose permeases with K(s) values of 2.8 and 1.9 mM, respectively. As glucose uptake and phosphorylation via the PTS differs from glucose uptake via IolT1 or IolT2 and phosphorylation via glucokinase by the requirement for phosphoenolpyruvate, the roles of the two pathways for l-lysine production were tested. The l-lysine yield by C. glutamicum DM1729, a rationally engineered l-lysine-producing strain, was lower than that by its PTS-deficient derivate DM1729Δhpr, which, however, showed low production rates. The combined overexpression of iolT1 or iolT2 with ppgK, the gene for PolyP/ATP-dependent glucokinase, in DM1729Δhpr enabled l-lysine production as fast as that by the parent strain DM1729 but with 10 to 20% higher l-lysine yield.

Lysosomal degradation of Leishmania hexose and inositol transporters is regulated in a stage-, nutrient- and ubiquitin-dependent manner

Leishmania parasites experience variable nutrient levels as they cycle between the extracellular promastigote stage in the sandfly vector and the obligate intracellular amastigote stage in the mammalian host. Here we show that the surface expression of three Leishmania mexicana hexose and myo-inositol transporters is regulated in both a stage-specific and nutrient-dependent manner. GFP-chimeras of functionally active hexose transporters, LmGT2 and LmGT3, and the myo-inositol transporter, MIT, were primarily expressed in the cell body plasma membrane in rapidly dividing promastigote stages. However MIT-GFP was mostly rerouted to the multivesicular tubule (MVT)-lysosome when promastigotes reached stationary phase growth and all three nutrient transporters were targeted to the amastigote lysosome following transformation to in vitro differentiated or in vivo imaged amastigote stages. This stage-specific decrease in surface expression of GFP-tagged transporters correlated with decreased hexose or myo-inositol uptake in stationary phase promastigotes and amastigotes. The MVT-lysosme targeting of the MIT-GFP protein was reversed when promastigotes were deprived of myo-inositol, indicating that nutrient signals can override stage-specific changes in transporter distribution. The surface expression of the hexose and myo -inositol transporters was not regulated by interactions with the subpellicular cytoskeleton, as both classes of transporters associated with detergent-resistant membranes. LmGT3-GFP and MIT-GFP proteins C-terminally modified with mono-ubiquitin were constitutively transported to the MVT-lysosome, suggesting that ubiquitination may play a key role in regulating the subcellular distribution of these transporters and parasite adaptation to different nutrient conditions.

Two Major Inositol Transporters and Their Role in Cryptococcal Virulence

Cryptococcus neoformans is an AIDS-associated human fungal pathogen and the most common cause of fungal meningitis, with a mortality rate over 40% in AIDS patients. Significant advances have been achieved in understanding its disease mechanisms. Yet the underlying mechanism of a high frequency of cryptococcal meningitis remains unclear. The existence of high inositol concentrations in brain and our earlier discovery of a large inositol transporter (ITR) gene family in C. neoformans led us to investigate the potential role of inositol in Cryptococcus-host interactions. In this study, we focus on functional analyses of two major ITR genes to understand their role in virulence of C. neoformans. Our results show that ITR1A and ITR3C are the only two ITR genes among 10 candidates that can complement the growth defect of a Saccharomyces cerevisiae strain lacking inositol transporters. Both S. cerevisiae strains heterologously expressing ITR1A or ITR3C showed high inositol uptake activity, an indication that they are major inositol transporters. Significantly, itr1a itr3c double mutants showed significant virulence attenuation in murine infection models. Mutating both ITR1A and ITR3C in an ino1 mutant background activates the expression of several remaining ITR candidates and does not show more severe virulence attenuation, suggesting that both inositol uptake and biosynthetic pathways are important for inositol acquisition. Overall, our study provides evidence that host inositol and fungal inositol transporters are important for Cryptococcus pathogenicity.

Role of an Expanded Inositol Transporter Repertoire in Cryptococcus neoformans Sexual Reproduction and Virulence

ABSTRACTCryptococcus neoformans and Cryptococcus gattii are globally distributed human fungal pathogens and the leading causes of fungal meningitis. Recent studies reveal that myo-inositol is an important factor for fungal sexual reproduction. That C. neoformans can utilize myo-inositol as a sole carbon source and the existence of abundant inositol in the human central nervous system suggest that inositol is important for Cryptococcus development and virulence. In accord with this central importance of inositol, an expanded myo-inositol transporter (ITR) gene family has been identified in Cryptococcus. This gene family contains two phylogenetically distinct groups, with a total of 10 or more members in C. neoformans and at least six members in the sibling species C. gattii. These inositol transporter genes are differentially expressed under inositol-inducing conditions based on quantitative real-time PCR analyses. Expression of ITR genes in a Saccharomyces cerevisiae itr1 itr2 mutant lacking inositol transport can complement the slow-growth phenotype of this strain, confirming that ITR genes are bona fide inositol transporters. Gene mutagenesis studies reveal that the Itr1 and Itr1A transporters are important for myo-inositol stimulation of mating and that functional redundancies among the myo-inositol transporters likely exist. Deletion of the inositol 1-phosphate synthase gene INO1 in an itr1 or itr1a mutant background compromised virulence in a murine inhalation model, indicating the importance of inositol sensing and acquisition for fungal infectivity. Our study provides a platform for further understanding the roles of inositol in fungal physiology and virulence.

Investigation of the H+–myo-inositol transporter (HMIT) as a neuronal regulator of phosphoinositide signalling

Phosphoinositide signalling regulates a series of important neuronal processes that are thought to be altered in mood disorders. Furthermore, mood-stabilizing drugs inhibit key enzymes that regulate phosphoinositide production and alter neuronal growth cone morphology in an inositol-reversible manner. Inositol is taken up by neurons from the extracellular fluid, presumably via membrane transporters; it can also be synthesized by the enzyme MIP-synthase (myo-inositol-1-phosphate synthase) and, in addition, it is generated by inositol phospholipid hydrolysis. The neuronal-specific HMIT (H(+)-myo-inositol transporter) represents a potential regulator of inositol signalling in neurons that warrants further investigation.

Knockout mice in understanding the mechanism of action of lithium

Lithium inhibits IMPase (inositol monophosphatase) activity, as well as inositol transporter function. To determine whether one or more of these mechanisms might underlie lithium's behavioural effects, we studied Impa1 (encoding IMPase) and Smit1 (sodium-myo-inositol transporter 1)-knockout mice. In brains of adult homozygous Impa1-knockout mice, IMPase activity was found to be decreased; however, inositol levels were not found to be altered. Behavioural analysis indicated decreased immobility in the forced-swim test as well as a strongly increased sensitivity to pilocarpine-induced seizures. These are behaviours robustly induced by lithium. In homozygous Smit1-knockout mice, free inositol levels were decreased in the frontal cortex and hippocampus. These animals behave like lithium-treated animals in the model of pilocarpine seizures and in the Porsolt forced-swim test model of depression. In contrast with O'Brien et al. [O'Brien, Harper, Jove, Woodgett, Maretto, Piccolo and Klein (2004) J. Neurosci. 24, 6791-6798], we could not confirm that heterozygous Gsk3b (glycogen synthase kinase 3beta)-knockout mice exhibit decreased immobility in the Porsolt forced-swim test or decreased amphetamine-induced hyperactivity in a manner mimicking lithium's behavioural effects. These data support the role of inositol-related processes rather than GSK3beta in the mechanism of the therapeutic action of lithium.

Folate Receptor Alpha Defect Causes Cerebral Folate Transport Deficiency: A Treatable Neurodegenerative Disorder Associated with Disturbed Myelin Metabolism

Sufficient folate supplementation is essential for a multitude of biological processes and diverse organ systems. At least five distinct inherited disorders of folate transport and metabolism are presently known, all of which cause systemic folate deficiency. We identified an inherited brain-specific folate transport defect that is caused by mutations in the folate receptor 1 (FOLR1) gene coding for folate receptor alpha (FRalpha). Three patients carrying FOLR1 mutations developed progressive movement disturbance, psychomotor decline, and epilepsy and showed severely reduced folate concentrations in the cerebrospinal fluid (CSF). Brain magnetic resonance imaging (MRI) demonstrated profound hypomyelination, and MR-based in vivo metabolite analysis indicated a combined depletion of white-matter choline and inositol. Retroviral transfection of patient cells with either FRalpha or FRbeta could rescue folate binding. Furthermore, CSF folate concentrations, as well as glial choline and inositol depletion, were restored by folinic acid therapy and preceded clinical improvements. Our studies not only characterize a previously unknown and treatable disorder of early childhood, but also provide new insights into the folate metabolic pathways involved in postnatal myelination and brain development.

Evaluation of expression and function of the H+/myo-inositol transporter HMIT

BackgroundThe phosphoinositide (PIns) signalling pathway regulates a series of neuronal processes, such as neurotransmitter release, that are thought to be altered in mood disorders. Furthermore, mood-stabilising drugs have been shown to inhibit key enzymes that regulate PIns production and alter neuronal growth cone morphology in an inositol-reversible manner. Here, we describe analyses of expression and function of the recently identified H+/myo-inositol transporter (HMIT) investigated as a potential regulator of PIns signalling.ResultsWe show that HMIT is primarily a neuronal transporter widely expressed in the rat and human brain, with particularly high levels in the hippocampus and cortex, as shown by immunohistochemistry. The transporter is localised at the Golgi apparatus in primary cultured neurones. No HMIT-mediated electrophysiological responses were detected in rat brain neurones or slices; in addition, inositol transport and homeostasis were unaffected in HMIT targeted null-mutant mice.ConclusionTogether, these data do not support a role for HMIT as a neuronal plasma membrane inositol transporter, as previously proposed. However, we observed that HMIT can transport inositol triphosphate, indicating unanticipated intracellular functions for this transporter that may be relevant to mood control.

Characterization of inositol transporters as a method for drug delivery to the CNS

Characterization of inositol transporters as a method for drug delivery to the CNS

Strategies for acquiring the phospholipid metabolite inositol in pathogenic bacteria, fungi and protozoa: making it and taking it

myo-Inositol (inositol) is an essential nutrient that is used for building phosphatidylinositol and its derivatives in eukaryotes and even in some eubacteria such as the mycobacteria. As a consequence, fungal, protozoan and mycobacterial pathogens must be able to acquire inositol in order to proliferate and cause infection in their hosts. There are two primary mechanisms for acquiring inositol. One is to synthesize inositol from glucose 6-phosphate using two sequentially acting enzymes: inositol-3-phosphate synthase (Ino1p) converts glucose 6-phosphate to inositol 3-phosphate, and then inositol monophosphatase (IMPase) dephosphorylates inositol 3-phosphate to generate inositol. The other mechanism is to import inositol from the environment via inositol transporters. Inositol is readily abundant in the bloodstream of mammalian hosts, providing a source from which many pathogens could potentially import inositol. However, despite this abundance of inositol in the host, some pathogens such as the bacterium Mycobacterium tuberculosis and the protist parasite Trypanosoma brucei must be able to make inositol de novo in order to cause disease (M. tuberculosis) or even grow (T. brucei). Other pathogens such as the fungus Candida albicans are equally adept at causing disease by importing inositol or by making it de novo. The role of inositol acquisition in the biology and pathogenesis of the parasite Leishmania and the fungus Cryptococcus are being explored as well. The specific strategies used by these pathogens to acquire inositol while in the host are discussed in relation to each pathogen's unique metabolic requirements.

Hypertonicity stimulates PGE2 signaling in the renal medulla by promoting EP3 and EP4 receptor expression

Hypertonicity in the renal medulla stimulates local cyclooxygenase 2 expression, leading to abundant PGE(2) production. Here we found that mRNA expression by the PGE(2)-activated G-protein-coupled receptors, EP3 and EP4 in the renal medulla was decreased by furosemide treatment, a procedure that reduces medullary hypertonicity. When HepG2 cells were cultured in hypertonic conditions by addition of salt or sorbitol, EP3 expression was induced. A specific EP3 agonist inhibited cAMP production, indicating receptor functionality, and this led to a substantial increase in cell survival in hypertonic media. Survival was independent of the SLC5A3 inositol transporter and aldose reductase expression, suggesting that EP3 promoted cell survival under hypertonic conditions independent of cellular organic osmolyte accumulation. Reduced cAMP production did not contribute to increased survival. EP4 expression was stimulated by hypertonicity in MDCK and HepG2 cells, which was associated with increased cAMP production in response to an EP4 agonist. Our study shows that local hypertonicity promotes PGE(2) signaling in the renal medulla by stimulating cognate receptor and cyclooxygenase 2 expression that likely regulates local hemodynamics and tubular transport.Kidney International (2009) 75, 278-284. doi:10.1038/ki.2008.498.

Astrocytic alkalinization by therapeutically relevant lithium concentrations: implications for myo-inositol depletion

One theory for therapeutic effects of the lithium ion (Li+) in bipolar disorder is that myo-inositol, needed for phospholipase C-mediated signaling, is depleted by Li(+)-induced inhibition of inositolphosphate hydrolysis or of myo-inositol uptake, an effect demonstrated in cultured mouse astrocytes at high myo-inositol concentrations. In contrast, myo-inositol uptake is inhibited at low concentrations, reflecting that it occurs both by the high-affinity Na(+)-dependent myo-inositol transporter (SMIT) and the lower-affinity H(+)-dependent inositol transporter (HMIT). Increased intracellular pH (pHi) stimulates SMIT but inhibits HMIT, suggesting that the effect of Li+ could be caused by intracellular alkalinization. In this study, we therefore investigated Li+ effects on intracellular pH in astrocytes, measured by 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) fluorescence. Chronic treatment with the therapeutically relevant Li+ concentration of 1 mM for 2 or 3 weeks increased pHi by approximately 0.10, whereas 0.5 mM was ineffective, and 2 mM caused a larger increase. The alkalinization resulted from acute stimulation of the Na+/H+ exchanger (NHE) by extracellular Li+, demonstrated after acid load with NH4Cl. In response to continuous stimulation, NHE1 mRNA was down-regulated, but protein was not. Chronic treatment with pharmacologically relevant Li+ concentrations increases pHi in astrocytes, creating conditions for decreased uptake of high myo-inositol concentrations and increased uptake of low concentrations. The pharmacological relevance of this effect is supported by literature data suggesting brain acidosis in bipolar patients and by preliminary observations that carbamazepine and valproate also increase pHi in astrocytes. Stimulation of NHE1-stimulated sodium ion uptake might also trigger uptake of chloride ions and osmotically obliged water.

Homozygote inositol transporter knockout mice show a lithium‐like phenotype

Lithium inhibits inositol monophosphatase and also reduces inositol transporter function. To determine if one or more of these mechanisms might underlie the behavioral effects of lithium, we studied inositol transporter knockout mice. We previously reported that heterozygous knockout mice with reduction of 15-37% in brain inositol had no abnormalities of pilocarpine sensitivity or antidepressant-like behavior in the Porsolt forced swim test. We now report on studies of homozygous inositol transporter knockout mice. Homozygote knockout mice were rescued by 2% inositol supplementation to the drinking water of the dam mice through pregnancy and lactation. Genotyping was carried out by polymerase chain reaction followed by agarose electrophoresis. Brain free myo-inositol levels were determined gas-chromatographically. Motor activity and coordination were assessed by the rotarod test. Behavior of the mice was studied in lithium-pilocarpine seizure models for lithium action and in the Porsolt forced swim test model for depression. In homozygote knockout mice, free inositol levels were reduced by 55% in the frontal cortex and by 60% in the hippocampus. There were no differences in weight or motor coordination by the rotarod test. They behaved similarly to lithium-treated animals in the model of pilocarpine seizures and in the Porsolt forced swimming test model of depression. Reduction of brain inositol more than 15-37% may be required to elicit lithium-like neurobehavioral effects.

Candida albicans Uses Multiple Mechanisms To Acquire the Essential Metabolite Inositol during Infection

Candida albicans is an important cause of life-threatening systemic bloodstream infections in immunocompromised patients. In order to cause infections, C. albicans must be able to synthesize the essential metabolite inositol or acquire it from the host. Based on the similarity of C. albicans to Saccharomyces cerevisiae, it was predicted that C. albicans may generate inositol de novo, import it from the environment, or both. The C. albicans inositol synthesis gene INO1 (orf19.7585) and inositol transporter gene ITR1 (orf19.3526) were each disrupted. The ino1Delta/ino1Delta mutant was an inositol auxotroph, and the itr1Delta/itr1Delta mutant was unable to import inositol from the medium. Each of these mutants was fully virulent in a mouse model of systemic infection. It was not possible to generate an ino1Delta/ino1Delta itr1Delta/itr1Delta double mutant, suggesting that in the absence of these two genes, C. albicans could not acquire inositol and was nonviable. A conditional double mutant was created by replacing the remaining wild-type allele of ITR1 in an ino1Delta/ino1Delta itr1Delta/ITR1 strain with a conditionally expressed allele of ITR1 driven by the repressible MET3 promoter. The resulting ino1Delta/ino1Delta itr1Delta/P(MET3)::ITR1 strain was found to be nonviable in medium containing methionine and cysteine (which represses the P(MET3) promoter), and it was avirulent in the mouse model of systemic candidiasis. These results suggest a model in which C. albicans has two equally effective mechanisms for obtaining inositol while in the host. It can either generate inositol de novo through Ino1p, or it can import it from the host through Itr1p.

Arabidopsis INOSITOL TRANSPORTER2 Mediates H+ Symport of Different Inositol Epimers and Derivatives across the Plasma Membrane

Of the four genes of the Arabidopsis (Arabidopsis thaliana) INOSITOL TRANSPORTER family (AtINT family) so far only AtINT4 has been described. Here we present the characterization of AtINT2 and AtINT3. cDNA sequencing revealed that the AtINT3 gene is incorrectly spliced and encodes a truncated protein of only 182 amino acids with four transmembrane helices. In contrast, AtINT2 codes for a functional transporter. AtINT2 localization in the plasma membrane was demonstrated by transient expression of an AtINT2-GREEN FLUORESCENT PROTEIN fusion in Arabidopsis and tobacco (Nicotiana tabacum) epidermis cells and in Arabidopsis protoplasts. Its functional and kinetic properties were determined by expression in yeast (Saccharomyces cerevisiae) cells and Xenopus laevis oocytes. Expression of AtINT2 in a Deltaitr1 (inositol uptake)/Deltaino1 (inositol biosynthesis) double mutant of bakers' yeast complemented the deficiency of this mutant to grow on low concentrations of myoinositol. In oocytes, AtINT2 mediated the symport of H(+) and several inositol epimers, such as myoinositol, scylloinositol, d-chiroinositol, and mucoinositol. The preference for individual epimers differed from that found for AtINT4. Moreover, AtINT2 has a lower affinity for myoinositol (K(m) = 0.7-1.0 mm) than AtINT4 (K(m) = 0.24 mm), and the K(m) is slightly voltage dependent, which was not observed for AtINT4. Organ and tissue specificity of AtINT2 expression was analyzed in AtINT2 promoter/reporter gene plants and showed weak expression in the anther tapetum, the vasculature, and the leaf mesophyll. A T-DNA insertion line (Atint2.1) and an Atint2.1/Atint4.2 double mutant were analyzed under different growth conditions. The physiological roles of AtINT2 are discussed.

Cloning, localization and expression analysis of vacuolar sugar transporters in the CAM plant Ananas comosus (pineapple)

In photosynthetic tissues of the CAM plant pineapple (Ananas comosus), storage of soluble sugars in the central vacuole during the daytime and their remobilization at night is required to provide carbon skeletons for nocturnal CO(2) fixation. However, soluble sugars produced photosynthetically must also be exported to support growth processes in heterotrophic tissues. To begin to address how vacuolar sugar storage and assimilate partitioning are regulated in A. comosus, degenerate PCR and cDNA library screening were used to clone three candidate sugar transporters from the leaves of this species. Subcellular localization of the three transporters was investigated via expression of YFP-fusion proteins in tobacco epidermal cells and their co-localization with subcellular markers by confocal microscopy. Using this strategy, a putative hexose transporter (AcMST1) and a putative inositol transporter (AcINT1) were identified that both localized to the tonoplast, whereas a putative sucrose transporter (AcSUT1) was found to localize to prevacuolar compartments. A cDNA (AcMST2) with high similarity to a recently characterized tonoplast hexose transporter in Arabidopsis was also identified from an A. comosus fruit EST database. Analyses of transcript abundance indicated that AcMST1 was more highly expressed in fruits compared to leaves of A. comosus, whilst transcripts of AcINT1, AcSUT1, and AcMST2 were more abundant in leaves. Transcript abundance of AcINT1, the putative inositol transporter, showed day-night changes comparable to those of other CAM-related transcripts described in Mesembryanthemum crystallinum. The results are discussed in terms of the role of vacuolar sugar transporters in regulating carbon flow during the diel cycle in CAM plants.