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PLoS One
2012 Jan 01;72:e31730. doi: 10.1371/journal.pone.0031730.
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Roles of major facilitator superfamily transporters in phosphate response in Drosophila.
Bergwitz C
,
Rasmussen MD
,
DeRobertis C
,
Wee MJ
,
Sinha S
,
Chen HH
,
Huang J
,
Perrimon N
.
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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 [(33)P]-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.
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Figure 1. Western blot analysis of phosphate-induced MAPK in S2R+ and Kc167 cells.A: Dose response and time course of phosphate-activated MAPK in S2R+ cells. B: Dose response of phosphate-activated MAPK in S2R+ cells. C: Time course of phosphate-activated MAPK in S2R+ cells. D: Time course of phosphate-activated MAPK in Kc167 cells. E: Effect of PFA on activation of MAPK in S2R+ cells Shown are one representative Western blot autoradiogram (A), or pooled densitometic data of at least three independent Western blot experiments (BâE). Abbreviations: Piâ=âinorganic phosphate, Insâ=âhuman insulin, Lyâ=âLy294002 (PI3K-inhibitor, 50 uM), PFAâ=âphosphonoformic acid (30 mM, unless otherwise noted), S10â=âsodium sulfate (10 mM), P10â=âsodium phosphate (10 mM).
Figure 2. Blast and Bayes analysis of MFS transporters.Heatmap of pairwise BLAST bit scores for all known yeast, Drosophila and human proteins containing the MFS protein domain PF07690 [38] (left panel) sorted by a hierarchical clustering (middle panel). Bayesian phylogenetic reconstruction (dendogram) was used to identify 29 fly orthologs that are most closely related to yeast Pho84 (YML123C) and human SLC17A1â9. Posterior probabilities are indicated above each branch. Fly transporters found to be expressed in S2R+ cells are shown in bold/italic script.
Figure 3. Effect of RNAi knockdown of MFS transporters and dPit on MAPK.A: mRNA expression of MFS and Pit transporters in S2R+ cells. Data of three replicate experiments are shown as mean±SEM expression relative to actin 5 C. B: Effect of RNAi knockdown of MFS transporters and dPit on MAPK. Data of three replicate experiments are shown as mean±SEM relative to cells transfected with dsRNA targeting lucifierase (luc). C: RNAi knockdown efficiency. To calculate efficiency of knockdown, parallel wells prepared for pERK1/2 Western analysis above (Fig. 2B) were used for total RNA extraction and quantitative RT-PCR. Shown are mean±SEM of three replicate experiments after expression was corrected for actin 5 C mRNA. Cells treated with dsRNA targeting luciferase are set 100% for each specific primer pair.
Figure 4. Phosphate transport after expression of MFS and dPit transporters in X. oocytes.Phosphate uptake of Xenopus oocytes injected with capped RNA encoding MFS10 (FBgn0030452), MFS13 (FBgn0010497), and dPit (FBgn0260795), was measured in ND100+33P, or ND0+33P, in the presence or absence of 5 mM PFA at pH7.4 or at pH5.5 or 8.5 where indicated. 33P-uptake is expressed in multiples over basal seen with non-injected oocytes as mean±SEM from at least three different batches of oocytes (nâ=â10/bath).
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