全部 标题 作者
关键词 摘要

PLOS ONE  2012 

Roles of Major Facilitator Superfamily Transporters in Phosphate Response in Drosophila

DOI: 10.1371/journal.pone.0031730

Full-Text   Cite this paper   Add to My Lib


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.


[1]  Bevington A, Kemp GJ, Graham R, Russell G (1992) Phosphate-sensitive enzymes: possible molecular basis for cellular disorders of phosphate metabolism. Clin Chem Enzym Comms 4: 235–257.
[2]  Bringhurst FR, Leder BZ (2006) Regulation of calcium and phosphate homeostasis. In: DeGroot LJ, Jameson JL, editors. Endocrinology. Fifth ed. Philadelphia: W.B. Saunders Co. pp. 805–843.
[3]  Bergwitz C, Juppner H (2009) Disorders of Phosphate Homeostasis and Tissue Mineralisation. Endocr Dev 16: 133–156.
[4]  Razzaque MS (2009) Does FGF23 toxicity influence the outcome of chronic kidney disease? Nephrol Dial Transplant 24: 4–7.
[5]  Gutierrez OM, Mannstadt M, Isakova T, Rauh-Hain JA, Tamez H, et al. (2008) Fibroblast growth factor 23 and mortality among patients undergoing hemodialysis. N Engl J Med 359: 584–592.
[6]  Mizobuchi M, Towler D, Slatopolsky E (2009) Vascular calcification: the killer of patients with chronic kidney disease. J Am Soc Nephrol 20: 1453–1464.
[7]  Sprecher E (2010) Familial tumoral calcinosis: from characterization of a rare phenotype to the pathogenesis of ectopic calcification. J Invest Dermatol 130: 652–660.
[8]  Stubbs JR, Liu S, Tang W, Zhou J, Wang Y, et al. (2007) Role of hyperphosphatemia and 1,25-dihydroxyvitamin d in vascular calcification and mortality in fibroblastic growth factor 23 null mice. J Am Soc Nephrol 18: 2116–2124.
[9]  Morishita K, Shirai A, Kubota M, Katakura Y, Nabeshima Y, et al. (2001) The progression of aging in klotho mutant mice can be modified by dietary phosphorus and zinc. J Nutr 131: 3182–3188.
[10]  Ohnishi M, Razzaque MS (2010) Dietary and genetic evidence for phosphate toxicity accelerating mammalian aging. Faseb J 24: 3562–3571.
[11]  Bun-Ya M, Nishimura M, Harashima S, Oshima Y (1991) The PHO84 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter. Mol Cell Biol 11: 3229–3238.
[12]  Murer H, Forster I, Biber J (2004) The sodium phosphate cotransporter family SLC34. Pflugers Arch 447: 763–767.
[13]  Hsieh YJ, Wanner BL (2010) Global regulation by the seven-component Pi signaling system. Curr Opin Microbiol 13: 198–203.
[14]  Lamarche MG, Wanner BL, Crepin S, Harel J (2008) The phosphate regulon and bacterial virulence: a regulatory network connecting phosphate homeostasis and pathogenesis. FEMS Microbiol Rev 32: 461–473.
[15]  Toh-e A, Tanaka K, Uesono Y, Wickner RB (1988) PHO85, a negative regulator of the PHO system, is a homolog of the protein kinase gene, CDC28, of Saccharomyces cerevisiae. Mol Gen Genet 214: 162–164.
[16]  Mouillon JM, Persson BL (2006) New aspects on phosphate sensing and signalling in Saccharomyces cerevisiae. FEMS Yeast Res 6: 171–176.
[17]  Saier MH Jr, Beatty JT, Goffeau A, Harley KT, Heijne WH, et al. (1999) The major facilitator superfamily. J Mol Microbiol Biotechnol 1: 257–279.
[18]  Reimer RJ, Edwards RH (2004) Organic anion transport is the primary function of the SLC17/type I phosphate transporter family. Pflugers Arch 447: 629–635.
[19]  Collins JF, Bai L, Ghishan FK (2004) The SLC20 family of proteins: dual functions as sodium-phosphate cotransporters and viral receptors. Pflugers Arch 447: 647–652.
[20]  Khoshniat S, Bourgine A, Julien M, Weiss P, Guicheux J, et al. (2010) The emergence of phosphate as a specific signaling molecule in bone and other cell types in mammals. Cell Mol Life Sci 68: 205–218.
[21]  Bergwitz C, Juppner H (2011) Phosphate sensing. Adv Chronic Kidney Dis 18: 132–144.
[22]  Werner A, Kinne RK (2001) Evolution of the Na-P(i) cotransport systems. Am J Physiol Regul Integr Comp Physiol 280: R301–312.
[23]  Hubbard TJ, Aken BL, Beal K, Ballester B, Caccamo M, et al. (2007) Ensembl 2007. Nucleic Acids Res 35: D610–617.
[24]  Beck GR Jr, Knecht N (2003) Osteopontin regulation by inorganic phosphate is ERK1/2?, protein kinase C?, and proteasome-dependent. J Biol Chem 278: 41921–41929.
[25]  Nair D, Misra RP, Sallis JD, Cheung HS (1997) Phosphocitrate inhibits a basic calcium phosphate and calcium pyrophosphate dihydrate crystal-induced mitogen-activated protein kinase cascade signal transduction pathway. J Biol Chem 272: 18920–18925.
[26]  Julien M, Magne D, Masson M, Rolli-Derkinderen M, Chassande O, et al. (2007) Phosphate stimulates matrix Gla protein expression in chondrocytes through the extracellular signal regulated kinase signaling pathway. Endocrinology 148: 530–537.
[27]  Yamazaki M, Ozonoa K, Okada T, Tachikawa K, Kondou H, et al. (2010) Both FGF23 and extracellular phosphate activate Raf/MEK/ERK pathway via FGF receptors in HEK293 cells. J Cell Biochem.
[28]  Chang SH, Yu KN, Lee YS, An GH, Beck GR Jr, et al. (2006) Elevated inorganic phosphate stimulates Akt-ERK1/2-Mnk1 signaling in human lung cells. Am J Respir Cell Mol Biol 35: 528–539.
[29]  Mansfield K, Teixeira CC, Adams CS, Shapiro IM (2001) Phosphate ions mediate chondrocyte apoptosis through a plasma membrane transporter mechanism. Bone 28: 1–8.
[30]  Yoshiko Y, Candeliere GA, Maeda N, Aubin JE (2007) Osteoblast autonomous Pi regulation via Pit1 plays a role in bone mineralization. Mol Cell Biol 27: 4465–4474.
[31]  Beck L, Leroy C, Salaun C, Margall-Ducos G, Desdouets C, et al. (2009) Identification of a novel function of PiT1 critical for cell proliferation and independent of its phosphate transport activity. J Biol Chem 284: 31363–31374.
[32]  Suzuki A, Ammann P, Nishiwaki-Yasuda K, Sekiguchi S, Asano S, et al. (2010) Effects of transgenic Pit-1 overexpression on calcium phosphate and bone metabolism. J Bone Miner Metab 28: 139–148.
[33]  Beck L, Leroy C, Beck-Cormier S, Forand A, Salaun C, et al. (2010) The phosphate transporter PiT1 (Slc20a1) revealed as a new essential gene for mouse liver development. PLoS One 5: e9148.
[34]  Festing MH, Speer MY, Yang HY, Giachelli CM (2009) Generation of mouse conditional and null alleles of the type III sodium-dependent phosphate cotransporter PiT-1. Genesis 47: 858–863.
[35]  Yanagawa S, Lee JS, Ishimoto A (1998) Identification and characterization of a novel line of Drosophila Schneider S2 cells that respond to wingless signaling. J Biol Chem 273: 32353–32359.
[36]  Segal D, Cherbas L, Cherbas P (1996) Genetic transformation of Drosophila cells in culture by P element-mediated transposition. Somat Cell Mol Genet 22: 159–165.
[37]  Hubbard TJ, Aken BL, Ayling S, Ballester B, Beal K, et al. (2009) Ensembl 2009. Nucleic Acids Res 37: D690–697.
[38]  Finn RD, Tate J, Mistry J, Coggill PC, Sammut SJ, et al. (2008) The Pfam protein families database. Nucleic Acids Res 36: D281–288.
[39]  Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574.
[40]  Friedman A, Perrimon N (2006) A functional RNAi screen for regulators of receptor tyrosine kinase and ERK signalling. Nature 444: 230–234.
[41]  Kulkarni MM, Booker M, Silver SJ, Friedman A, Hong P, et al. (2006) Evidence of off-target effects associated with long dsRNAs in Drosophila melanogaster cell-based assays. Nat Methods 3: 833–838.
[42]  DRSC (2010) Drosophila RNAi Screening Center.
[43]  Jaureguiberry G, Carpenter TO, Forman S, Juppner H, Bergwitz C (2008) A novel missense mutation in SLC34A3 that causes hereditary hypophosphatemic rickets with hypercalciuria in humans identifies threonine 137 as an important determinant of sodium-phosphate cotransport in NaPi-IIc. Am J Physiol Renal Physiol 295: F371–379.
[44]  Ponton F, Chapuis MP, Pernice M, Sword GA, Simpson SJ (2011) Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster. J Insect Physiol 57: 840–850.
[45]  Celniker SE, Dillon LA, Gerstein MB, Gunsalus KC, Henikoff S, et al. (2009) Unlocking the secrets of the genome. Nature 459: 927–930.
[46]  Sims D, Bursteinas B, Gao Q, Zvelebil M, Baum B (2006) FLIGHT: database and tools for the integration and cross-correlation of large-scale RNAi phenotypic datasets. Nucleic Acids Res 34: D479–483.
[47]  Chintapalli VR, Wang J, Dow JA (2007) Using FlyAtlas to identify better Drosophila melanogaster models of human disease. Nat Genet 39: 715–720.
[48]  Chertow GM, Dillon M, Burke SK, Steg M, Bleyer AJ, et al. (1999) A randomized trial of sevelamer hydrochloride (RenaGel) with and without supplemental calcium. Strategies for the control of hyperphosphatemia and hyperparathyroidism in hemodialysis patients. Clin Nephrol 51: 18–26.
[49]  Tenenhouse HS, Klugerman AH, Neal JL (1989) Effect of phosphonoformic acid, dietary phosphate and the Hyp mutation on kinetically distinct phosphate transport processes in mouse kidney. Biochim Biophys Acta 984: 207–213.
[50]  Mozar A, Haren N, Chasseraud M, Louvet L, Maziere C, et al. (2008) High extracellular inorganic phosphate concentration inhibits RANK-RANKL signaling in osteoclast-like cells. J Cell Physiol 215: 47–54.
[51]  Julien M, Khoshniat S, Lacreusette A, Gatius M, Bozec A, et al. (2009) Phosphate-dependent regulation of MGP in osteoblasts: role of ERK1/2 and Fra-1. J Bone Miner Res 24: 1856–1868.
[52]  Wittrant Y, Bourgine A, Khoshniat S, Alliot-Licht B, Masson M, et al. (2009) Inorganic phosphate regulates Glvr-1 and -2 expression: role of calcium and ERK1/2. Biochem Biophys Res Commun 381: 259–263.


comments powered by Disqus