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.     

Induction of an extracellular cyclic nucleotide phosphodiesterase as an accessory ribonucleolytic activity during phosphate starvation of cultured tomato cells

During growth under conditions of phosphate limitation, suspension-cultured cells of tomato (Lycopersicon esculentum Mill.) secrete phosphodiesterase activity in a similar fashion to phosphate starvation-inducible ribonuclease (RNase LE), a cyclizing endoribonuclease that generates 2':3'-cyclic nucleoside monophosphates (NMP) as its major monomeric products (T. Nürnberger, S. Abel, W. Jost, K. Glund [1990] Plant Physiol 92: 970-976). Tomato extracellular phosphodiesterase was purified to homogeneity from the spent culture medium of phosphate-starved cells and was characterized as a cyclic nucleotide phosphodiesterase. The purified enzyme has a molecular mass of 70 kD, a pH optimum of 6.2, and an isoelectric point of 8.1. The phosphodiesterase preparation is free of any detectable deoxyribonuclease, ribonuclease, and nucleotidase activity. Tomato extracellular phosphodiesterase is insensitive to EDTA and hydrolyzes with no apparent base specificity 2':3'-cyclic NMP to 3'-NMP and the 3':5'-cyclic isomers to a mixture of 3'-NMP and 5'-NMP. Specific activities of the enzyme are 2-fold higher for 2':3'-cyclic NMP than for 3':5'-cyclic isomers. Analysis of monomeric products of sequential RNA hydrolysis with purified RNase LE, purified extracellular phosphodiesterase, and cleared -Pi culture medium as a source of 3'-nucleotidase activity indicates that cyclic nucleotide phosphodiesterase functions as an accessory ribonucleolytic activity that effectively hydrolyzes primary products of RNase LE to substrates for phosphate-starvation-inducible phosphomonoesterases. Biosynthetical labeling of cyclic nucleotide phopshodiesterase upon phosphate starvation suggests de novo synthesis and secretion of a set of nucleolytic enzymes for scavenging phosphate from extracellular RNA substrates.     

Ddi1p and Rad23p play a cooperative role as negative regulators in the PHO pathway in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the PHO pathway regulates expression of phosphate-responsive genes such as PHO5, which encodes repressible acid phosphatase (rAPase). In this pathway, Pho81p functions as an inhibitor of the cyclin-cyclin-dependent kinase (CDK) complex Pho80p-Pho85p. However, the mechanism regulating the inhibitory activity of Pho81p is poorly understood. Through use of the yeast two-hybrid system, we identified the UbL-UbA protein Ddi1p as a Pho81p-binding protein. Further, Pho81p levels were found to be low under high-phosphate condition and high during phosphate starvation, indicating that Pho81p is regulated by phosphate concentration. However, our results revealed that Ddi1p and its associated protein Rad23p are not involved in the decrease in Pho81p level under high-phosphate condition. Significantly, the Δddi1Δrad23 strain exhibited a remarkable increase in rAPase activity at an intermediate-phosphate concentration of 0.4 mM, suggesting that Ddi1p and Rad23p play a cooperative role as negative regulators in the PHO pathway.     

Phosphate transport and sensing in Saccharomyces cerevisiae.

Cellular metabolism depends on the appropriate concentration of intracellular inorganic phosphate; however, little is known about how phosphate concentrations are sensed. The similarity of Pho84p, a high-affinity phosphate transporter in Saccharomyces cerevisiae, to the glucose sensors Snf3p and Rgt2p has led to the hypothesis that Pho84p is an inorganic phosphate sensor. Furthermore, pho84Delta strains have defects in phosphate signaling; they constitutively express PHO5, a phosphate starvation-inducible gene. We began these studies to determine the role of phosphate transporters in signaling phosphate starvation. Previous experiments demonstrated a defect in phosphate uptake in phosphate-starved pho84Delta cells; however, the pho84Delta strain expresses PHO5 constitutively when grown in phosphate-replete media. We determined that pho84Delta cells have a significant defect in phosphate uptake even when grown in high phosphate media. Overexpression of unrelated phosphate transporters or a glycerophosphoinositol transporter in the pho84Delta strain suppresses the PHO5 constitutive phenotype. These data suggest that PHO84 is not required for sensing phosphate. We further characterized putative phosphate transporters, identifying two new phosphate transporters, PHO90 and PHO91. A synthetic lethal phenotype was observed when five phosphate transporters were inactivated, and the contribution of each transporter to uptake in high phosphate conditions was determined. Finally, a PHO84-dependent compensation response was identified; the abundance of Pho84p at the plasma membrane increases in cells that are defective in other phosphate transporters.     

The SPX domain of the yeast low‐affinity phosphate transporter Pho90 regulates transport activity

Yeast has two phosphate‐uptake systems that complement each other: the high‐affinity transporters (Pho84 and Pho89) are active under phosphate starvation, whereas Pho87 and Pho90 are low‐affinity transporters that function when phosphate is abundant. Here, we report new regulatory functions of the amino‐terminal SPX domain of Pho87 and Pho90. By studying truncated versions of Pho87 and Pho90, we show that the SPX domain limits the phosphate‐uptake velocity, suppresses phosphate efflux and affects the regulation of the phosphate signal transduction pathway. Furthermore, split‐ubiquitin assays and co‐immunoprecipitation suggest that the SPX domain of both Pho90 and Pho87 interacts physically with the regulatory protein Spl2. This work suggests that the SPX domain inhibits low‐affinity phosphate transport through a physical interaction with Spl2.     

Inhibition of migration and invasion of colorectal cancer cells via deletion of Rac1 with RNA interference

Nucleotides and nucleosides represent an important and ubiquitous class of molecules that interact with specific receptors, regulate a variety of activities within the liver, and play a role in the pathogenesis of hepatic fibrosis. Ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs) are ecto-enzymes that are located on the cell surface. NPP1, NPP2, and NPP3 (abbreviated as NPP1–3 hereafter) have been implicated in the hydrolysis of nucleotides; together with other ecto-nucleotidases, they control the events induced by extracellular nucleotides. We have identified and compared the expression of E-NPP family members in two different phenotypes of the mouse hepatic stellate cell line (GRX). In quiescent-like hepatic stellate cells (HSCs), E-NPP activity was significantly higher, NPP2 mRNA expression decreased and NPP3 mRNA increased. The differential NPP activity and expression in two phenotypes of GRX cells suggests that they are involved in the regulation of extracellular nucleotide metabolism in HSCs. However, the role of E-NPPs in the liver remains to be clarified.     

Terminal oxidases are essential to bypass the requirement for ResD for full Pho induction in Bacillus subtilis

The Bacillus subtilis Pho signal transduction network, which regulates the cellular response to phosphate starvation, integrates the activity of three signal transduction systems to regulate the level of the Pho response. This signal transduction network includes a positive feedback loop between the PhoP/PhoR and ResD/ResE two-component systems. Within this network, ResD is responsible for 80% of the Pho response. To date, the role of ResD in the generation of the Pho response has not been understood. Expression of two terminal oxidases requires ResD function, and expression of at least one terminal oxidase is needed for the wild-type Pho response. Previously, our investigators have shown that strains bearing mutations in resD are impaired for growth and acquire secondary mutations which compensate for the loss of the a-type terminal oxidases by allowing production of cytochrome bd. We report here that the expression of cytochrome bd in a DeltaresDE background is sufficient to compensate for the loss of ResD for full Pho induction. A ctaA mutant strain, deficient in the production of heme A, has the same Pho induction phenotype as a DeltaresDE strain. This demonstrates that the production of a-type terminal oxidases is the basis for the role of ResD in Pho induction. Terminal oxidases affect the redox state of the quinone pool. Reduced quinones inhibit PhoR autophosphorylation in vitro, consistent with a requirement for terminal oxidases for full Pho induction in vivo.     

The phosphate regulon and bacterial virulence: a regulatory network connecting phosphate homeostasis and pathogenesis

Bacterial pathogens regulate virulence factor gene expression coordinately in response to environmental stimuli, including nutrient starvation. The phosphate (Pho) regulon plays a key role in phosphate homeostasis. It is controlled by the PhoR/PhoB two-component regulatory system. PhoR is an integral membrane signaling histidine kinase that, through an interaction with the ABC-type phosphate-specific transport (Pst) system and a protein called PhoU, somehow senses environmental inorganic phosphate (P(i)) levels. Under conditions of P(i) limitation (or in the absence of a Pst component or PhoU), PhoR activates its partner response regulator PhoB by phosphorylation, which, in turn, up- or down-regulates target genes. Single-cell profiling of PhoB activation has shown recently that Pho regulon gene expression exhibits a stochastic, "all-or-none" behavior. Recent studies have also shown that the Pho regulon plays a role in the virulence of several bacteria. Here, we present a comprehensive overview of the role of the Pho regulon in bacterial virulence. The Pho regulon is clearly not a simple regulatory circuit for controlling phosphate homeostasis; it is part of a complex network important for both bacterial virulence and stress response.