The Saccharomyces cerevisiae high affinity phosphate transporter encoded by PHO84 also functions in manganese homeostasis

In the bakers' yeast Saccharomyces cerevisiae, high affinity manganese uptake and intracellular distribution involve two members of the Nramp family of genes, SMF1 and SMF2. In a search for other genes involved in manganese homeostasis, PHO84 was identified. The PHO84 gene encodes a high affinity inorganic phosphate transporter, and we find that its disruption results in a manganese-resistant phenotype. Resistance to zinc, cobalt, and copper ions was also demonstrated for pho84Delta yeast. When challenged with high concentrations of metals, pho84Delta yeast have reduced metal ion accumulation, suggesting that resistance is due to reduced uptake of metal ions. Pho84p accounted for virtually all the manganese accumulated under metal surplus conditions, demonstrating that this transporter is the major source of excess manganese accumulation. The manganese taken in via Pho84p is indeed biologically active and can not only cause toxicity but can also be incorporated into manganese-requiring enzymes. Pho84p is essential for activating manganese enzymes in smf2Delta mutants that rely on low affinity manganese transport systems. A role for Pho84p in manganese accumulation was also identified in a standard laboratory growth medium when high affinity manganese uptake is active. Under these conditions, cells lacking both Pho84p and the high affinity Smf1p transporter accumulated low levels of manganese, although there was no major effect on activity of manganese-requiring enzymes. We conclude that Pho84p plays a role in manganese homeostasis predominantly under manganese surplus conditions and appears to be functioning as a low affinity metal transporter.     

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.     

PHO2-dependent degradation of PHO1 modulates phosphate homeostasis in Arabidopsis

The Arabidopsis thaliana pho2 mutant, which is defective in a ubiquitin-conjugating E2 enzyme, displays inorganic phosphate (Pi) toxicity as a result of enhanced uptake and root-to-shoot translocation of Pi. To elucidate downstream components of the PHO2-dependent regulatory pathway, we identified two pho2 suppressors as carrying missense mutations in PHO1, which has been implicated in Pi loading to the xylem. In support of the genetic interaction between PHO1 and PHO2, we found that the protein level of PHO1 is increased in pho2, whereas such accumulation is ameliorated in both pho2 suppressors. Results from cycloheximide and endosomal Cys protease inhibitor E-64d treatments further suggest that PHO1 degradation is PHO2 dependent and involves multivesicular body-mediated vacuolar proteolysis. Using the transient expression system of tobacco (Nicotiana tabacum) leaves, we demonstrated that PHO1 and PHO2 are partially colocalized and physically interact in the endomembranes, where the ubiquitin conjugase activity of PHO2 is required for PHO1 degradation. In addition, reduced PHO1 expression caused by PHO1 mutations impede Pi uptake, indicating a functional association between xylem loading and acquisition of Pi. Together, our findings uncover a pivotal molecular mechanism by which PHO2 modulates the degradation of PHO1 in the endomembranes to maintain Pi homeostasis in plants.     

Structure and function of the PHO82-pho4 locus controlling the synthesis of repressible acid phosphatase of Saccharomyces cerevisiae.

pho4 mutants of Saccharomyces cerevisiae, although rare among phosphatase-negative mutants isolated from wild-type strains, were isolated efficiently from pho80, pho85, or pho80 pho85 strains. The distribution of these pho4 mutants over the pho4 locus was determined by analyzing random spores of two- and three-factor crosses. The pho4-4 mutation confers temperature-sensitive synthesis of repressible acid phosphatase. An intragenic suppressor for the pho4-12 allele results in the temperature-sensitive synthesis of repressible acid phosphatase. Recombination between these sites occurs at 1.0 to 3.0%, the highest for any pair of sites within the pho4 locus. All these results strongly indicate that the information of the pho4 locus is translated into a protein. The PHO82 site was mapped inside the pho4 locus by random spore analysis. The order met10-pho4-1PHO82-1-pho4-9 on the right arm of chromosome VI was confirmed by tetrad analysis. Doubly heterozygous diploids, pho3 PHO82c PHO4+/pho3 pho82+ pho4, produce variable amounts of repressible acid phosphatase under repressive conditions depending on the combination of PHO82c and pho4 alleles. This phenomenon may reflect the constitutive production of the pho82+-pho4 product in the repressed condition, which interferes with the function of the PHO82c-PHO4+ product. The earlier model for the function of the PHO82-pho4 cluster, in which the PHO82 site acts as an operator of the pho4 gene, has been revised to a model in which the PHO82 site codes for the part of the pho4 protein that has affinity for the regulatory protein encoded by the pho80 and pho85 genes.     

The role of the miR399-<Emphasis Type="Italic">PHO2</Emphasis> module in the regulation of flowering time in response to different ambient temperatures in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A moderate change in ambient temperature significantly affects plant physiology including flowering time. MiR399 and its target gene PHOSPHATE 2 (PHO2) are known to play a role in the maintenance of phosphate homeostasis. However, the regulation of flowering time by the miR399-PHO2 module has not been investigated. As we have previously identified miR399 as an ambient temperature-responsive miRNA, we further investigated whether a change in expression of the miR399-PHO2 module affects flowering time in response to ambient temperature changes. Here, we showed that miR399b-overexpressing plants and a loss-of-function allele of PHO2 (pho2) exhibited an early flowering phenotype only at normal temperature (23°C). Interestingly, their flowering time at lower temperature (16°C) was similar to that of wild-type plants, suggesting that alteration in flowering time by miR399 and its target PHO2 was seen only at normal temperature (23°C). Flowering time ratio (16°C/23°C) revealed that miR399b-overexpressing plants and pho2 mutants showed increased sensitivity to ambient temperature changes. Expression analysis indicated that expression of TWIN SISTER OF FT (TSF) was increased in miR399b-overexpressing plants and pho2 mutants at 23°C, suggesting that their early flowering phenotype is associated with TSF upregulation. Taken together, our results suggest that miR399, an ambient temperature-responsive miRNA, plays a role in ambient temperature-responsive flowering in Arabidopsis.