Phosphate transport in mitochondria: Past accomplishments,present problems,and future challenges

The requirement of inorganic phosphate (P_i) for oxidative phosphorylation in eukaryotic cells is fulfilled through specific P_i transport systems. The mitochondrial proton/phosphate symporter (P_ic) is a membrane-embedded protein which translocates P_i from the cytosol into the mitochondrial matrix. P_ic is responsible for the very rapid transport of most of the P_i used in ATP synthesis. During the past five years there have been advances on several fronts. Genomic and cDNA clones for yeast, bovine, rat, and human P_ic have been isolated and sequenced. Functional expression of yeast P_ic in yeast strains deficient in P_i transport and expression inEscherichia coli of a chimera protein involving P_ic and ATP synthase subunit have been accomplished. P_ic, in contrast to other members of the family of transporters involved in energy metabolism, was demonstrated to have a presequence, which optimizes the import of the precursor protein into mitochondria. Six transmembrane segments appear to be a structural feature shared between P_ic and other mitochondrial anion carriers, and recent-site directed mutagenesis studies implicate structure-functional relationships to bacteriorhodopsin. These recent advances on P_ic will be assessed in light of a more global interpretation of transport mechanism across the inner mitochondrial membrane.     

Phosphate transport in plants

Transport of inorganic phosphate (P_i) through plant membranes is mediated by a number of families of transporter proteins. Studies on the topology, function, regulation and sites of expression of the genes that encode the members of these transporter families are enabling roles to be ascribed to each of them. The Pht1 family, of which there are nine members in the Arabidopsis genome, includes proteins involved in the uptake of P_i from the soil solution and the redistribution of P_i within the plant. Members of this family are H₂PO₄ ^–/H⁺ symporters. Most of the genes of the Pht1 family that are expressed in roots are up-regulated in P-stressed plants. Two members of the Pht1 family have been isolated from the cluster roots of white lupin. These same genes are expressed in non-cluster roots. The evidence available to date suggests that there are no major differences between the types of transport systems that cluster roots and non-cluster roots use to acquire P_i. Differences in uptake rates between cluster and non-cluster roots can be ascribed to more high-affinity P_i transporters in the plasma membranes of cluster roots, rather than any difference in the characteristics of the transporters. The efficient acquisition of P_i by cluster roots arises primarily from their capacity to increase the availability of soil P_i immediately adjacent to the rootlets by excretion of carboxylates, protons and phosphatases within the cluster. This paper reviews P_i transport processes, concentrating on those mediated by the Pht1 family of transporters, and attempts to relate those processes involved in P_i acquisition to likely P_i transport processes in cluster roots.     

The renal type IIa Na/Pi cotransporter: structure-function relationships

The type IIa Na/P_i cotransporter mediates proximal tubular brush-border membrane secondary active phosphate (P_i) flux. It is rate limiting in tubular P_i reabsorption and, thus, a final target in many physiological and pathophysiological situations of altered renal P_i handling (1–4). In the present short review, we will briefly summarize our current knowledge about the transport mechanism (cycle) as well as particular regions of the transporter protein (“molecular domains”) that potentially determine transport characteristics.     

A Biophysical Model of the Mitochondrial ATP-Mg/Pi Carrier

Mitochondrial adenine nucleotide (AdN) content is regulated through the Ca²⁺-activated, electroneutral ATP-Mg/P_i carrier (APC). The APC is a protein in the mitochondrial carrier super family that localizes to the inner mitochondrial membrane (IMM). It is known to modulate a number of processes that depend on mitochondrial AdN content, such as gluconeogenesis, protein synthesis, and citrulline synthesis. Despite this critical role, a kinetic model of the underlying mechanism has not been developed and validated. Here, a biophysical model of the APC is developed that is thermodynamically balanced and accurately reproduces a number of reported data sets from isolated rat liver and rat kidney mitochondria. The model is based on an ordered bi-bi mechanism for heteroexchange of ATP and P_i and includes homoexchanges of ATP and P_i to explain both the initial rate and time course data on ATP and P_i transport via the APC. The model invokes seven kinetic parameters regarding the APC mechanism and three parameters related to matrix pH regulation by external P_i. These parameters are estimated based on 19 independent data curves; the estimated parameters are validated using six additional data curves. The model takes into account the effects of pH, Mg²⁺, and Ca²⁺ on ATP and P_i transport via the APC, and supports the conclusion that the pH gradient across the IMM serves as the primary driving force for AdN uptake or efflux. Moreover, computer simulations demonstrate that extramatrix Ca²⁺ modulates the turnover rate of the APC and not the binding affinity of ATP, as previously suggested.     

Swelling and contraction of heart mitochondria suspended in ammonium phosphate

Bovine heart mitochondria which have been allowed to swell in isotonic NH ₄ ⁺ phosphate contract in response to initiation of oxidative phosphorylation. The contraction occurs optimally at pH 6.0 and appears from inhibition studies to result from P_i uptake being slower than removal of internal P_i via phosphorylation of external ADP. Similar results are obtained when K⁺ + nigericin is substituted for NH ₄ ⁺ . Mersalyl inhibition of P_i transport in respiring, nonphosphorylating mitochondria which have been allowed to swell in NH ₄ ⁺ phosphate reveals a contractile process having an alkaline pH optimum. This contraction resembles closely the contraction observed in salts of strong acids and presumably occurs by electrophoretic ejection of P_i anions driven by electrogenic H⁺ ejection.     

Parathyroid hormone inhibition of phosphate transport in renal brush border vesicles from phosphate-depleted dogs

Dietary phosphate (P_i) restriction increases renal P_i reabsorption and induces resistance to the phosphaturic action of parathyroid hormone. Na⁺-gradient-stimulated P_i transport in membrane vesicles isolated from the renal brush border of experimental animals has been shown to parallel changes in renal P_i reabsorption induced by dietary P_i restriction and in vivo administration of parathyroid hormone. Dietary P_i restriction has been shown to markedly inhibit the phosphaturic response to parathyroid hormone in rats and dogs. Parathyroid hormone has been reported not to decrease the Na⁺-gradient-stimulated transport of P_i in brush border membrane vesicles isolated from dietary P_i restricted rats unless the rats were administered an acute P_i load prior to killing, however, thyroparathyroidectomy of rats fed a low P_i diet has been reported to increase Na⁺-gradient-stimulated P_i transport. Using the dietary P_i restricted dog, we demonstrated no significant decrease in renal reabsorption of P_i in response to parathyroid hormone administration. However, significant decreases in P_i transport in brush border membrane vesicles isolated from the kidneys of dietary P_i restricted dogs were observed in response to in vivo parathyroid hormone administration. These data demonstrate that the resistance to the phosphaturic action of parathyroid hormone observed in vivo does not include resistance to the inhibitory effect of parathyroid hormone on P_i transport in brush border membrane vesicles. Thus, the data suggest that parathyroid hormone continues to alter P_i transport characteristics of the brush border membrane in states of P_i depletion despite the resistance to parathyroid hormone seen in vivo.     

Regulation of bacterial phosphate taxis and polyphosphate accumulation in response to phosphate starvation stress

Phosphorus (P) is an essential constituent in all types of living organisms. Bacteria, which use inorganic phosphate (P_i), as the preferred P source, have evolved complex systems to survive during P_i starvation conditions. Recently, we found thatPseudomonas aeruginosa, a monoflagellated, obligately aerobic bacterium, is attracted to P_i. The evidence that the chemotactic response to P_i (P_i taxis) was observed only with cells grown in P_i-limiting medium suggests that P_i taxis plays an important role in scavenging P_i residues under conditions of P_i starvation. Many bacteria also exhibit rapid and extensive accumulation of polyphosphate (polyP), when P_i is added to cells previously subjected to P_i starvation stress. Since polyP can serve as a P source during P_i starvation conditions, it is likely that polyP accumulation is a protective mechanism for survival during P_i starvation. In the present review, we summarize our current knowledge on regulation of bacterial P_i taxis and polyP accumulation in response to P_i starvation stress.     

Influence of dietary calcium and phosphorus supply on epithelial phosphate transport in preruminant goats

P homeostasis affected by high or low Ca and/or P supply in preruminant goats was characterized by balance studies in vivo. The main excretion pathway was the renal P_i excretion whose extent was modulated by variations in dietary P and/or Ca supply. Faecal P excretion remained low irrespective of dietary regimen. The balance data were combined with respective in vitro data on P_i transport properties and their adaptation in response to changes in dietary Ca and/or P intake. Therefore, P_i transport capacities were determined by P_i uptake into brush border membrane vesicles of jejunum and kidney. Epithelial P_i transporters were determined semiquantitatively by northern and western blot analyses in jejunum, kidney and salivary gland. Renal P_i transport was downregulated by doubling dietary P supply while doubling both, Ca and P as well as restrictive Ca at unchanged P led to slight, but not significant reductions in renal P_i transport. Jejunal P_i transport was reduced by P excess (doubling P and doubling both, Ca and P), but only NaPi IIb protein expression was significantly diminished. In conclusion, the significance of epithelial adaptation to dietary Ca and P supply for P homeostasis is discussed in preruminant goats.     

Does Spirodela punctata break P-C bonds?

Spirodela punctata was cultivated on phosphate-deficient medium (–P_i) with racemic 1-amino-2-phenylethylphosphonic acid (PheP) as a source of P_i. The growth of duckweed was inversely correlated with PheP concentration. The growth of plants on medium –P_i with 0.1 M PheP was accelerated whereas with 0.001 mM PheP was slower than in –P_i control. PheP at low concentrations decreased loss of chlorophyll in comparison with –P_i plants. Content of anthocyanins decreased but activity of the extractable constitutive phosphatases of pH 6.0 and pH 7.5 increased along with increasing concentration of PheP in the medium. We suggest that S. punctata does not break P-C bonds but probably PheP interrupts processes involved in the regulation of P_i-starvation response.     

Thyroid hormones stimulate Na+–Pi transport activity in rat renal brush-border membranes: Role of membrane lipid composition and fluidity

Antecedent studies have suggested that lipid composition and fluidity of cellular membranes of various organs are altered in response to thyroid hormone status. To date, the effects of thyroid hormone status on these parameters have not been examined in rat renal apical membrane in regard to sodium-dependent phosphate transport. In the present study, we determined the potential role of alterations in cortical brush-border membrane lipid composition and fluidity in modulation of Na⁺–P_i transport activity in response to thyroid hormone status. Thyroid hormone status influences the fractional excretion of Pi, which is associated with alteration in renal brush-border membrane phosphate transport. The increment in Na⁺–P_i transport in renal BBMV isolated from Hyper-T rats is manifested as an increase in the maximal velocity (V_max) of Na⁺–P_i transport. Further, the cholesterol content was significantly increased in renal BBM of Hypo-T rats and decreased in Hyper-T rats as compared to the Eu-T rats. The molar ratio of cholesterol/phospholipids was also higher in renal BBM from hypo-T rats. Subsequently, fluorescence anisotropy of diphenyl hexatriene (rDPH) and microviscosity were significantly decreased in the renal BBM of the Hyper-T rats and increased in the Hypo-T rats as compared to Eu-T rats. The result of this study, therefore, suggest that alteration in renal BBM cholesterol, cholesterol/phospholipid molar ratio, and membrane fluidity play an important role in the modulation of renal BBM Na⁺–P_i transport in response to thyroid hormone status of animals. (Mol Cell Biochem 268: 75–82, 2005)     

Proton- and sodium-coupled phosphate transport systems and energy status of Yarrowia lipolytica cells grown in acidic and alkaline conditions

In this study we have used a newly isolated Yarrowia lipolytica yeast strain with a unique capacity to grow over a wide pH range (3.5–10.5), which makes it an excellent model system for studying H⁺- and Na⁺-coupled phosphate transport systems. Even at extreme growth conditions (low concentrations of extracellular phosphate, alkaline pH values) Y. lipolytica preserved tightly-coupled mitochondria with the fully competent respiratory chain containing three points of energy conservation. This was demonstrated for the first time for cells grown at pH 9.5–10.0. In cells grown at pH 4.5, inorganic phosphate (P_i) was accumulated by two kinetically discrete H⁺/P_i-cotransport systems. The low-affinity system is most likely constitutively expressed and operates at high P_i concentrations. The high-affinity system, subjected to regulation by both extracellular P_i availability and intracellular polyphosphate stores, is mobilized during P_i-starvation. In cells grown at pH 9.5–10, P_i uptake is mediated by several kinetically discrete Na⁺-dependent systems that are specifically activated by Na⁺ ions and insensitive to the protonophore CCCP. One of these, a low-affinity transporter operative at high P_i concentrations is kinetically characterized here for the first time. The other two, high-affinity, high-capacity systems, are derepressible and functional during P_i-starvation and appear to be controlled by extracellular P_i. They represent the first examples of high-capacity, Na⁺-driven P_i transport systems in an organism belonging to neither the animal nor bacterial kingdoms. The contribution of the H⁺- and Na⁺-coupled P_i transport systems in Y. lipolytica cells grown at different pH values was quantified. In cells grown at pH values of 4.5 and 6.0, the H⁺-coupled P_i transport systems are predominant. The contribution of the Na⁺/P_i cotransport systems to the total cellular P_i uptake activity is progressively increased with increasing pH, reaching its maximum at pH 9 and higher. Received: 15 December 2000/Revised: 14 May 2001     

Characterization of orthophosphate-induced active cation transport in isolated liver mitochondria

The kinetics of the P_i-induced active transport of ions by isolated liver mitochondria were studied by monitoring photometrically mitochondrial volume changes. In a previous communication, these volume changes were shown to correlate quantitatively with the net uptake or release of ions. In the present study the specificity of the P_i role was further characterized. The data support the contention that cations are actively transported. Anions follow the transfer of cations to maintain electrical neutrality. The relationship of the transport system to oxidative phosphorylation was investigated by simultaneously monitoring both processes under different experimental conditions. The results of the experiments are quantitatively consistent with a model proposed for the P_i-induced active transport in isolated rat liver mitochondria. The model includes the following features. 1. P_i induces an inwardly directed, carrier-mediated active transport of cations. 2. The transport is coupled to the energy-conserving reactions of the cytochrome chain. 3. The efflux of ions accumulated in the presence of low P_i concentrations occurs by passive diffusion. 4. Net accumulation ceases when the rates of active transport and passive diffusion become equal. 5. The active transport competes with oxidative phosphorylation for a common, nonphosphorylated, high-energy intermediate.     

Molecular biological approaches to plant nutrition

In the last decade, understanding of ion transport has grown sufficiently to pose sensible questions about the molecular nature of the processes and their regulation. Techniques for identifying and cloning genes and for genetic transformation provide the means for answering these questions.Transport of ions across membranes is obviously a major aspect of mineral nutrition since it occurs during initial absorption, compartmentation and mobilisation of nutrients. Here, we will briefly review the types of transport protein involved and show how molecular biology and recombinant DNA technology have revealed something of their structure. Strategies used to identify the genes for transporters are discussed and reference is made to areas in which the availability of cloned genes will facilitate future studies.Mineral nutrition involves, however, more than membrane transport. The absorption rates of major nutrients are quite strictly regulated by biochemical factors which vary with the rate at which nutrients are used in growth. Nitrogen, sulphur and phosphate nutrition in micro-organisms are regulated by the interaction of various DNA-binding proteins with the promoter regions of genes for key enzymes in the assimilatory pathways and the specific ion permeases. The expression of the regulatory protein or its activity can be modified by metabolites, such as glutamine. Some evidence supports the idea that higher plants also have groups of genes with a common regulation of expression.An attempt is made to identify some reasonable objectives, which should increase understanding of the regulation of nutrient transport.Abbreviations and conventions cDNA complementary strand of DNA prepared from a messenger RNA - NR nitrate reductase - NiR nitrite reductase - P_i inorganic phosphate - PPase pyrophosphate - RUBISCO rubulose bisphosphate carboxylase oxygenase. Genes are referred to as follows e.g. cys-3 and scon-1. The protein products of these genes, where they have no familiar name are referred to as CYS3 and SCON-1 respectively     

Intracellular phosphate and its possible role as an exchange anion for active transport of methotrexate in L1210 cells

L1210 cells transport P_i in the absence of added Na⁺. Uptake shows saturation kinetics (K_t = 1.7 mM), is temperature-dependent, and can be reduced 80% by high levels of unlabeled P_i, and thus has the characteristics of a carrier-mediated process. This transport process is also inhibited by methotrexate. The methotrexate-sensitive component constitutes half of total P_i uptake, and is reduced by 50% at a concentration of methotrexate (2 μM) that is comparable to its K_t (1.5 μM) for transport into the cells. An impermeable fluorescent analog of methotrexate and an irreversible inhibitor of the methotrexate transport system (carbodiimide-activated methotrexate) also inhibit this same P_i uptake component. It is concluded that methotrexate and P_i can be transported by the same carrier system. The basis for this shared uptake is suggested to be that the methotrexate carrier protein facilitates the obligatory exchange of extracellular folate compounds for intracellular divalent anions, and that a primary exchange anion is P_i. A principal energy source for active transport of methotrexate might then be the concentration gradient for P_i that is maintained by the Na⁺-dependent, P_i transport system of these cells.     

Phosphate: Known and potential roles during development and regeneration of teeth and supporting structures

Inorganic phosphate (P_i) is abundant in cells and tissues as an important component of nucleic acids and phospholipids, a source of high‐energy bonds in nucleoside triphosphates, a substrate for kinases and phosphatases, and a regulator of intracellular signaling. The majority of the body's P_i exists in the mineralized matrix of bones and teeth. Systemic P_i metabolism is regulated by a cast of hormones, phosphatonins, and other factors via the bone‐kidney‐intestine axis. Mineralization in bones and teeth is in turn affected by homeostasis of P_i and inorganic pyrophosphate (PPi), with further regulation of the P_i/PP_i ratio by cellular enzymes and transporters. Much has been learned by analyzing the molecular basis for changes in mineralized tissue development in mutant and knock‐out mice with altered P_i metabolism. This review focuses on factors regulating systemic and local P_i homeostasis and their known and putative effects on the hard tissues of the oral cavity. By understanding the role of P_i metabolism in the development and maintenance of the oral mineralized tissues, it will be possible to develop improved regenerative approaches. Birth Defects Research (Part C) 84:281–314, 2008. © 2008 Wiley‐Liss, Inc.     

Identification of a Novel Function of PiT1 Critical for Cell Proliferation and Independent of Its Phosphate Transport Activity

PiT1 is a Na⁺-phosphate (P_i) cotransporter located at the plasma membrane that enables P_i entry into the cell. Its broad tissue expression pattern has led to the idea that together with the closely related family member PiT2, PiT1 is the ubiquitous supplier of P_i to the cell. Moreover, the role of P_i in phosphorylation reactions, ATP production, DNA structure, and synthesis has led to the view that P_i availability could be an important determinant of cell growth. However, these issues have not been clearly addressed to date, and the role of either P_i or PiT proteins in cell proliferation is unknown. Using RNA interference in HeLa and HepG2 cells, we show that transient or stable PiT1 depletion markedly reduces cell proliferation, delays cell cycle, and impairs mitosis and cytokinesis. In vivo, PiT1 depletion greatly reduced tumor growth when engineered HeLa cells were injected into nude mice. We provide evidence that this effect on cell proliferation is specific to PiT1 and not shared by PiT2 and is not the consequence of impaired membrane Na⁺-P_i transport. Moreover, we show that modulation of cell proliferation by PiT1 is independent from its transport function because the proliferation of PiT1-depleted cells can be rescued by non-transporting PiT1 mutants. PiT1 depletion leads to the phosphorylation of p38 mitogen-activated protein (MAP) kinase, whereas other MAP kinases and downstream targets of mammalian target of rapamycin (mTOR) remain unaffected. This study is the first to describe the effects of a P_i transporter in cell proliferation, tumor growth, and cell signaling.     

Expression of the Na/P<Subscript>i</Subscript>-cotransporter Type IIb in Sf9 Cells: Functional Characterization and Purification

In mammals the type IIb Na/P_i-cotransporter is expressed in various tissues such as intestine, brain, lung and testis. The type IIb cotransporter shows 51% homology with the renal type IIa Na/P_i-cotransporter, for which a detailed model of the secondary structure has emerged based on recent structure/function studies. To make the type IIb Na/P_i-cotransporter available for future structural studies, we have expressed this cotransporter in Sf9 cells. Sf9 cells were infected with recombinant baculovirus containing 6His NaPi-IIb. Infected cells expressed a polypeptide of ~90 kDa, corresponding to a partially glycosylated form of the type IIb cotransporter. Transport studies demonstrated that the type IIb protein expressed in Sf9 cells mediates transport of phosphate in a Na-dependent manner with similar kinetic characteristics (apparent K _ms for sodium and phosphate and pH dependence) as previously described. Solubilization experiments demonstrated that, in contrast to the type IIa cotransporter, the type IIb can be solubilized by nonionic detergents and that solubilized type IIb Na/P_i-cotransporter can be purified by Ni-NTA chromatography.