With the large number of cellular processes that rely on phosphate, the uptake and storage of phosphate must be tightly controlled. Based on the natural affinity of phosphate compounds with metals, one might predict a linkage between phosphate and metal metabolism. This prediction was directly tested in these studies using yeast pho80 mutants that hyper-accumulate phosphate. We find that loss of phosphate control in pho80 mutants has wide spread effects on homeostasis of biologically relevant metals when the cells are grow in rich medium. These effects range from dramatic (10–100 fold) rises in steady state levels of the hard metal cations calcium and sodium, to increases in toxicity from the transition metals manganese, cobalt, zinc and copper, to activation of an iron starvation response.
We observed that a subset of effects of pho80
mutations on metal cations is due to up-regulation of Pho84p, the metal-phosphate transporter. Pho84p has previously been shown to transport phosphate complexes of manganese and cobalt [10
] and our earlier genetic studies suggested a similar role for Pho84p in transport of zinc and copper as well [11
]. However, a role for Pho84p in uptake of hard metal cations including magnesium, potassium, sodium and calcium has not been previously noted, and in fact magnesium has been shown to inhibit Pho84p transport activity [10
]. Pho84p may not be transporting complexes of calcium and sodium, rather the elevation of these elements in pho80
mutants could represent a secondary consequence of high phosphate. These cations may enter the cell as a counter ion to balance the negative charge imposed by increased phosphate, although this is not sufficient to increase total cell volume as monitored by microscopy (not shown). The sequestration of calcium and sodium by high intracellular phosphate could be sensed as a calcium and/or sodium starvation state, stimulating transport systems for these cations. This is analogous to what we observe for phosphate effects on iron. Despite relatively normal levels of iron, pho80
mutants appear starved for iron, and genes involved in iron uptake and storage are induced. We find that pho80
mutants accumulate elevated levels of ortho- and polyphosphate in the cytosol, but not in the vacuole. It is therefore likely that phosphate binding to metal cations in the cytosol decreases the bioavailability of these ions, resulting in a cation starvation stress response.
Although many effects of pho80
mutations can be ascribed to Pho4p induction of Pho84p metal-phosphate transport, there exists a PHO4
-independent component to the increased toxicity from manganese, cobalt and copper. In our search thus far for this Pho4/Pho84 independent pathway, we have excluded a number of candidates. For example, pho80
mutations still confer toxicity in a Pho4/Pho84 independent manner when the genes that affect synthesis (VTC4
]), degradation (PPN1
]) and intracellular localization (PHO91
]) of polyphosphate are deleted. We have likewise excluded another downstream transcription factor phosphorylated by Pho80p, Rim15p [37
], various manganese and iron transporters (Fet4p [39
], Smf1p and Smf2p [40
]), as well as the aforementioned Aft1p and Aft2p iron regulators (data not shown). New as of yet unidentified target(s) of Pho80p must be involved and this is under current investigation.
Finally it is worth mentioning that previous studies in bacteria and plants have indicated that high phosphate can be protective against metal toxicity presumably by phosphate sequestration of the metals [6
]. By contrast, our studies in S. cerevisae
argue that phosphate can augment metal toxicity both by promoting metal uptake (via Pho84p) and by another effect on metal homeostasis that is independent of metal uptake. Cellular phosphate could influence metal toxicity through formation of metal-phosphate complexes that directly cause toxicity, as has been proposed for the potent Mn+3
-phosphate oxidant [41
]. In any case, these studies underscore the importance of maintaining a balance of charge in the cell and that disruptions in an abundant anion such as phosphate can have a dramatic and widespread impact on homeostasis of biologically important metals.