Roles of Major Facilitator Superfamily Transporters in Phosphate Response in Drosophila

Shree Ram Singh,Clemens Bergwitz,Mark J Wee,Sumi Sinha,Charles Derobertis,Norbert Perrimon,Hway H Chen,Matthew D Rasmussen,Joanne Huang

doi:10.1371/journal.pone.0031730

Abstract

The major facilitator superfamily (MFS) transporter Pho84 and the type III transporter Pho89 are responsible for metabolic effects of inorganic phosphate in yeast. While the Pho89 ortholog Pit1 was also shown to be involved in phosphate-activated MAPK in mammalian cells, it is currently unknown, whether orthologs of Pho84 have a role in phosphate-sensing in metazoan species. We show here that the activation of MAPK by phosphate observed in mammals is conserved in Drosophila cells, and used this assay to characterize the roles of putative phosphate transporters. Surprisingly, while we found that RNAi-mediated knockdown of the fly Pho89 ortholog dPit had little effect on the activation of MAPK in Drosophila S2R+ cells by phosphate, two Pho84/SLC17A1–9 MFS orthologs (MFS10 and MFS13) specifically inhibited this response. Further, using a Xenopus oocyte assay, we show that MSF13 mediates uptake of [33P]-orthophosphate in a sodium-dependent fashion. Consistent with a role in phosphate physiology, MSF13 is expressed highest in the Drosophila crop, midgut, Malpighian tubule, and hindgut. Altogether, our findings provide the first evidence that Pho84 orthologs mediate cellular effects of phosphate in metazoan cells. Finally, while phosphate is essential for Drosophila larval development, loss of MFS13 activity is compatible with viability indicating redundancy at the levels of the transporters.

Highlights

Inorganic phosphate, the mono- or divalent anion of phosphoric acid [HPO432, H2PO422], is required for cellular functions such as DNA and membrane lipid synthesis, generation of high-energy phosphate esters, and intracellular signaling [1]
To establish an assay for phosphate sensing in Drosophila, we investigated whether, as observed in mammalian cells, phosphate can activate MAPK in Drosophila cells
We show that activation of MAPK is part of the down-stream events stimulated when two Drosophila hemocyte-like cell lines, S2R+ and Kc167, are exposed to phosphate

Summary

Introduction

The mono- or divalent anion of phosphoric acid [HPO432, H2PO422], is required for cellular functions such as DNA and membrane lipid synthesis, generation of high-energy phosphate esters, and intracellular signaling [1]. Hyperphosphatemia is encountered most frequently in patients with chronic kidney disease (CKD), which affects 20 Million Americans today, and the serum phosphate level is an important predictor of mortality in this population [4,5,6]. It is seen in familial hyperphosphatemic tumoral calcinosis, a human disorder that was recently attributed to loss-of function mutations in the genes encoding fibroblast growth factor 23 (FGF23), UDP-GalNAc transferase 3 (GALNT3), and Klotho (KL) [7]. An understanding of the molecular basis underlying the metabolic and endocrine phosphate effects is of great significance for human disease

Methods

Results

Conclusion