Roles Of Phosphate Transporters Research Articles

Characterization of TNSALP expression, localization, and activity in ovine utero-placental tissues†.

Tissue-nonspecific alkaline phosphatase (TNSALP; encoded by ALPL gene) has a critical role in the regulation of phosphate homeostasis postnatally. However, the utero-placental expression of TNSALP and the role in phosphate transport in pregnancy is poorly understood. Estrous cycles of ewes were synchronized, and ewes were euthanized and hysterectomized on Days 1, 9, or 14 of the estrous cycle or bred to fertile rams and euthanized and hysterectomized on Days 9, 12, 17, 30, 50, 70, 90, 110, or 125 of pregnancy. The expression of ALPL mRNA, immunolocalization of TNSALP protein, and quantification and localization of TNSALP enzymatic activity was performed on ovine endometria and placentomes. Day of the estrous cycle did not alter ALPL mRNA expression or enzymatic activity of TNSALP. TNSALP protein localized to uterine epithelial and stromal cells, blood vessels, myometrium, caruncular, and cotyledonary stroma. TNSALP activity was localized to uterine epithelia, blood vessels, caruncular stroma (from Day 70 of gestation), and the apical surface of chorionic epithelia (from Day 50 of gestation). TNSALP protein and activity localized to the apical surface of uterine epithelia during the estrous cycle and in early pregnancy. Endometrial TNSALP enzymatic activity was downregulated on Days 17 and 30 of gestation (P< 0.05). Expression of ALPL mRNA decreased in late gestation in endometria and placentomes (P< 0.05). TNSALP activity peaked in placentomes on Days 70 and 90 of gestation. Collectively, these results suggest a potential role of TNSALP in the regulation of phosphate transport and homeostasis at the maternal-conceptus interface in ruminants.

Role of Silicon in Mediating Phosphorus Imbalance in Plants.

The soil bioavailability of phosphorus (P) is often low because of its poor solubility, strong sorption and slow diffusion in most soils; however, stress due to excess soil P can occur in greenhouse production systems subjected to high levels of P fertilizer. Silicon (Si) is a beneficial element that can alleviate multiple biotic and abiotic stresses. Although numerous studies have investigated the effects of Si on P nutrition, a comprehensive review has not been published. Accordingly, here we review: (1) the Si uptake, transport and accumulation in various plant species; (2) the roles of phosphate transporters in P acquisition, mobilization, re-utilization and homeostasis; (3) the beneficial role of Si in improving P nutrition under P deficiency; and (4) the regulatory function of Si in decreasing P uptake under excess P. The results of the reviewed studies suggest the important role of Si in mediating P imbalance in plants. We also present a schematic model to explain underlying mechanisms responsible for the beneficial impact of Si on plant adaption to P-imbalance stress. Finally, we highlight the importance of future investigations aimed at revealing the role of Si in regulating P imbalance in plants, both at deeper molecular and broader field levels.

Disruption in phosphate transport affects membrane lipid and lipid droplet homeostasis in Saccharomyces cerevisiae.

Phosphate plays a crucial role in phospholipid metabolism and it is transported by the phosphate (Pi) transporters. Phospholipids are building blocks of the cell membrane, and essential for cell growth; however, the role of phosphate transporters in lipid metabolism remains elusive. The present study shows that the deletion of Pi transporters exhibited an increase in both phospholipid and neutral lipid levels when compared to wild type. The mRNA expressions of genes involved in phospholipid synthesis (CKI1, EKI1, CHO2, and OPI3) were increased due to de-repression of the transcription factors (INO2 and INO4). Neutral lipid levels (triacylglycerol and sterol ester) and their synthesizing genes (LRO1, ARE2, ACC1, and FAS1) were also increased, resulting in lipid droplet accumulation in Pi transporter mutants. Interestingly, phospholipase (PLC1) and histone acetyltransferase genes (ESA1, EAF1, YNG1, YNG2, and GCN5) were also found to be significantly increased, leading to dysregulation of lipid levels in Pi transporter mutants. In summary, our results suggest that the Pi transporters are involved in lipid droplet and membrane lipid homeostasis.

A Phosphate Transporter from the Root Endophytic Fungus Piriformospora indica Plays a Role in Phosphate Transport to the Host Plant

Because pure cultures and a stable transformation system are not available for arbuscular mycorrhizal fungi, the role of their phosphate transporters for the symbiotic interaction with the plant up till now could not be studied. Here we report the cloning and the functional analysis of a gene encoding a phosphate transporter (PiPT) from the root endophytic fungus Piriformospora indica, which can be grown axenically. The PiPT polypeptide belongs to the major facilitator superfamily. Homology modeling reveals that PiPT exhibits twelve transmembrane helices divided into two halves connected by a large hydrophilic loop in the middle. The function of the protein encoded by PiPT was confirmed by complementation of a yeast phosphate transporter mutant. The kinetic analysis of PiPT (K(m) 25 mum) reveals that it belongs to the high affinity phosphate transporter family (Pht1). Expression of PiPT was localized to the external hyphae of P. indica colonized with maize plant root, which suggests that external hyphae are the initial site of phosphate uptake from the soil. To understand the physiological role of PiPT, knockdown transformants of the gene were prepared using electroporation and RNA interference. Knockdown transformants transported a significantly lower amount of phosphate to the host plant than wild-type P. indica. Higher amounts of phosphate were found in plants colonized with wild-type P. indica than that of non-colonized and plants colonized with knockdown PiPT P. indica. These observations suggest that PiPT is actively involved in the phosphate transportation and, in turn, P. indica helps improve the nutritional status of the host plant.

Role of NHERF and scaffolding proteins in proximal tubule transport

Eukaryotic cells coordinate specific responses to hormones and growth factors by spatial and temporal organization of "signaling components." Through the formation of multiprotein complexes, cells are able to generate "signaling components" that transduce hormone signals through proteins, such as PSD-95/Dlg/ZO-1(PDZ)-containing proteins that associate by stable and dynamic interactions. The PDZ homology domain is a common protein interaction domain in eukaryotes and with greater than 500 PDZ domains identified, it is the most abundant protein interaction domain in eukaryotic cells. The NHERF (sodium hydrogen exchanger regulatory factor) proteins are PDZ domain-containing proteins that play an important role in maintaining and regulating cell function. NHERF-1 was initially identified as a brush border membrane-associated phosphoprotein essential for the cAMP/PKA-induced inhibition of the sodium hydrogen exchanger isoform 3 (NHE3). Mouse, rabbit and human renal proximal tubules also express NHERF-2 (E3KARP), a structurally related protein, which in model cell systems also binds NHE3 and mediates its inhibition by cAMP. PDZK1 (NHERF-3) and IKEPP (NHERF-4) were later identified and found to have similar homology domains, leading to their recent reclassification. Although studies have revealed similar binding partners and overlapping functions for the NHERF proteins, it is clear that there is a significant amount of specificity between them. This review focuses primarily on NHERF-1, as the prototypical PDZ protein and will give a brief summary of its role in phosphate transport and the development of some forms of nephrolithiasis.

Structure and Function of the GTP Binding Protein Gtr1 and Its Role in Phosphate Transport in Saccharomyces cerevisiae

The Pho84 high-affinity phosphate permease is the primary phosphate transporter in the yeast Saccharomyces cerevisiae under phosphate-limiting conditions. The soluble G protein, Gtr1, has previously been suggested to be involved in the derepressible Pho84 phosphate uptake function. This idea was based on a displayed deletion phenotype of Deltagtr1 similar to the Deltapho84 phenotype. As of yet, the mode of interaction has not been described. The consequences of a deletion of gtr1 on in vivo Pho84 expression, trafficking and activity, and extracellular phosphatase activity were analyzed in strains synthesizing either Pho84-green fluorescent protein or Pho84-myc chimeras. The studies revealed a delayed response in Pho84-mediated phosphate uptake and extracellular phosphatase activity under phosphate-limiting conditions. EPR spectroscopic studies verified that the N-terminal G binding domain (residues 1-185) harbors the nucleotide responsive elements. In contrast, the spectra obtained for the C-terminal part (residues 186-310) displayed no evidence of conformational changes upon GTP addition.

Organic anion transport is the primary function of the SLC17/type I phosphate transporter family.

Recently, molecular studies have determined that the SLC17/type I phosphate transporters, a family of proteins initially characterized as phosphate carriers, mediate the transport of organic anions. While their role in phosphate transport remains uncertain, it is now clear that the transport of organic anions facilitated by this family of proteins is involved in diverse processes ranging from the vesicular storage of the neurotransmitter glutamate to the degradation and metabolism of glycoproteins.

The spatial expression patterns of a phosphate transporter (MtPT1) from Medicago truncatula indicate a role in phosphate transport at the root/soil interface.

The movement of phosphate from the soil into plant root cells is the first of many crucial transport events required to supply phosphorous (P) to cells throughout the plant. In addition to the ability to acquire phosphate from the soil, the majority of the vascular plants are able to form arbuscular mycorrhizal associations in which phosphate may be delivered to the cortex via a fungus. Previously, we cloned two phosphate transporter genes, MtPT1 and MtPT2 from Medicago truncatula roots. Complementation of a yeast phosphate transport mutant revealed that MtPT1 is a functional phosphate transporter and Northern analyses revealed that MtPT1 is expressed exclusively in roots (Liu et al., 1998, Mol. Plant-Microbe Interact. 11, 14--22). Utilising an antibody specific for MtPT1, we have analysed the accumulation and spatial expression patterns of the MtPT1 transporter. MtPT1 transcript and protein levels show close correlation and increase dramatically in the roots in response to phosphate starvation. MtPT1 protein levels decrease in roots during development of a symbiosis with arbuscular mycorrhizal (AM) fungi, indicating that this transporter is not involved in symbiotic phosphate transport. Membrane fractionation and analysis of a MtPT1/GFP fusion protein revealed that MtPT1 is located in the plasma membrane, while in situ hybridisation and immunolocalisation demonstrate the presence of MtPT1 transcripts and protein in the epidermal cells and root hairs of M. truncatula roots. MtPT1 shows expression patterns consistent with a role specifically in the acquisition of phosphate from the soil and is distinct from the other phosphate transporter of this class described to date.

Phorbol myristate acetate activates protein kinase C, stimulates the phosphorylation of endogenous proteins and inhibits phosphate transport in mouse renal tubules

Calcium-activated, phospholipid-dependent protein kinase (protein kinase C) has been implicated in the regulation of transport processes in a variety of tissues and cell lines. To establish whether protein kinase C participates in the regulation of renal phosphate transport, we examined the effect of phorbol myristate acetate (PMA), a potent activator of protein kinase C, on phosphate uptake in fresh preparations of mouse renal tubules, and we correlated the changes in transport activity with protein kinase C activation and phosphorylation of endogenous proteins. PMA inhibited Na+-dependent phosphate transport, elicited a rapid translocation of protein kinase C from the cytosolic to the particulate fraction and stimulated the phosphorylation of endogenous substrates in the cytosolic and brush border membrane fractions. Effects of PMA were maximal after a 10 min incubation of the tubules with the activator. 4 alpha-Phorbol, an inert analogue of PMA, did not elicit any of these effects. The present results demonstrate a temporal correlation between inhibition of Na+-dependent phosphate transport, translocation and activation of protein kinase C, and phosphorylation of endogenous proteins in mouse renal tubules. These data suggest that protein kinase C may play a regulatory role in phosphate transport in mammalian kidney.

Alpha2-Adrenoceptor Regulation of Parathyroid Hormone Function in the Isolated Perfused Kidney

We investigated physiological interactions between alpha 2-adrenoceptors and parathyroid hormone (PTH) in the isolated buffer-perfused kidney. PTH infusion (10nM) caused a rapid and significant rise in cAMP excretion which diminished despite continued hormone infusion. PTH also caused a delayed phosphaturia which peaked 20-30 minutes following the start of PTH infusion. alpha 2-Adrenoceptor stimulation with epinephrine (28nM) diminished PTH-stimulated cAMP accumulation by 68-71% (p less than 0.05) but had no effect on PTH-induced phosphaturia. None of the experimental interventions affected renal hemodynamics. In additional studies, we used lithium (1mM) as a marker of proximal tubular sodium transport. PTH caused a rapid rise in lithium excretion which was temporally distinct (maximum response in 0-10 minutes) from the phosphaturia. alpha 2-Adrenoceptor stimulation completely blocked the inhibitory effect of PTH on lithium reabsorption. These results suggest that alpha 2-adrenoceptors regulate the stimulatory effect of PTH at proximal tubular adenylate cyclase. However, alpha 2-adrenoceptors play no role in phosphate transport in this segment. alpha 2-Adrenoceptor stimulation reverses PTH-induced lithium excretion, suggesting physiological antagonism in the proximal tubule, most likely involving Na/H exchange.

Alkaline phosphatase activity does not mediate phosphate transport in the renal-cortical brush-border membrane.

We studied (1) the effect of primary modulators of phosphate transport, namely the hypophosphataemic mouse mutant (Hyp) and low-phosphorus diet, on alkaline phosphatase activity in mouse renal-cortex brush-border membrane vesicles and (2) the effect of several primary inhibitors of alkaline phosphatase on phosphate transport. Brush-border membrane vesicles from Hyp-mouse kidney had 50% loss of Na+-dependent phosphate transport, but only 18% decrease in alkaline phosphatase activity. The low-phosphorus diet effectively stimulated Na+/phosphate co-transport in brush-border membrane vesicles (+ 118%), but increased alkaline phosphatase activity only slightly (+13%). Levamisole (0.1 mM) and EDTA (1.0 mM) inhibited brush-border membrane-vesicle alkaline phosphatase activity of 82% and 93% respectively, but had no significant effect on Na+/phosphate co-transport. We conclude that alkaline phosphatase does not play a direct role in phosphate transport across the brush-border membrane of mouse kidney.

Inhibition of CO 2 fixation by adenosine 5′-diphosphate and the role of phosphate transport in isolated pea chloroplasts

CO 2-dependent oxygen evolution by isolated pea ( Pisum sativum cv Massey Gem) chloroplasts was inhibited by ADP. AMP inhibited oxygen evolution to a greater extent than ADP while no inhibition was observed with ATP. Oxygen evolution with ADP or AMP was more sensitive to phosphate inhibition than in the absence of added adenine nucleotides. Photosynthetic intermediates (ribose 5-phosphate or 3-phosphoglycerate) partially relieved the inhibition by ADP. Pyrophosphate also inhibited oxygen evolution, yet, with PP 1 plus ADP, oxygen evolution was stimulated. Inhibition of the phosphate transporter using p-chloromercuriphenylsulfonic acid did not reverse ADP inhibition but the subsequent reversal of ADP inhibition by PP 1 was prevented. Pyrophosphate relieved inhibition of oxygen evolution by P i both in the presence and in the absence of added adenine nucleotides. Uptake of ADP into pea chloroplasts apparently results in an inhibition of 3-phosphoglycerate kinase, and the subsequent accumulation of 3-phosphoglycerate and decrease in internal phosphate make the chloroplasts highly susceptible to inhibition by phosphate. We propose that PP i enters chloroplasts via the phosphate transporter and is hydrolyzed internally by the chloroplast inorganic pyrophosphatase. The increase in internal phosphate concentration would prevent loss of photosynthetic intermediates from the chloroplast, under these conditions, and thus stimulate oxygen evolution.