Cellular abundance of sodium phosphate cotransporter SLC20A1/PiT1 and phosphate uptake are controlled post-transcriptionally by ESCRT

Abstract

Inorganic phosphate is essential for human life. The widely expressed mammalian sodium/phosphate cotransporter SLC20A1/PiT1 mediates phosphate uptake into most cell types; however, while SLC20A1 is required for development, and elevated SLC20A1 expression is associated with vascular calcification and aggressive tumor growth, the mechanisms regulating SLC20A1 protein abundance are unknown. Here, we found that SLC20A1 protein expression is low in phosphate-replete cultured cells but is strikingly induced following phosphate starvation, whereas mRNA expression is high in phosphate-replete cells and only mildly increased by phosphate starvation. To identify regulators of SLC20A1 protein levels, we performed a genome-wide CRISPR-based loss-of-function genetic screen in phosphate-replete cells using SLC20A1 protein induction as readout. Our screen revealed that endosomal sorting complexes required for transport (ESCRT) machinery was essential for proper SLC20A1 protein downregulation in phosphate-replete cells. We show that SLC20A1 colocalizes with ESCRT and that ESCRT deficiency increases SLC20A1 protein and phosphate uptake into cells. We also found numerous additional candidate regulators of mammalian phosphate homeostasis, including genes modifying protein ubiquitination and the Krebs cycle and oxidative phosphorylation pathways. Many of these targets have not been previously implicated in this process. We present here a model in which SLC20A1 protein abundance and phosphate uptake are tonically negatively regulated post-transcriptionally in phosphate-replete cells through direct ESCRT-mediated SLC20A1 degradation. Moreover, our screening results provide a comprehensive resource for future studies to elucidate the mechanisms governing cellular phosphate homeostasis. We conclude that genome-wide CRISPR-based genetic screening is a powerful tool to discover proteins and pathways relevant to physiological processes. Inorganic phosphate is essential for human life. The widely expressed mammalian sodium/phosphate cotransporter SLC20A1/PiT1 mediates phosphate uptake into most cell types; however, while SLC20A1 is required for development, and elevated SLC20A1 expression is associated with vascular calcification and aggressive tumor growth, the mechanisms regulating SLC20A1 protein abundance are unknown. Here, we found that SLC20A1 protein expression is low in phosphate-replete cultured cells but is strikingly induced following phosphate starvation, whereas mRNA expression is high in phosphate-replete cells and only mildly increased by phosphate starvation. To identify regulators of SLC20A1 protein levels, we performed a genome-wide CRISPR-based loss-of-function genetic screen in phosphate-replete cells using SLC20A1 protein induction as readout. Our screen revealed that endosomal sorting complexes required for transport (ESCRT) machinery was essential for proper SLC20A1 protein downregulation in phosphate-replete cells. We show that SLC20A1 colocalizes with ESCRT and that ESCRT deficiency increases SLC20A1 protein and phosphate uptake into cells. We also found numerous additional candidate regulators of mammalian phosphate homeostasis, including genes modifying protein ubiquitination and the Krebs cycle and oxidative phosphorylation pathways. Many of these targets have not been previously implicated in this process. We present here a model in which SLC20A1 protein abundance and phosphate uptake are tonically negatively regulated post-transcriptionally in phosphate-replete cells through direct ESCRT-mediated SLC20A1 degradation. Moreover, our screening results provide a comprehensive resource for future studies to elucidate the mechanisms governing cellular phosphate homeostasis. We conclude that genome-wide CRISPR-based genetic screening is a powerful tool to discover proteins and pathways relevant to physiological processes. Phosphate is indispensable for many biological functions, including DNA and RNA synthesis, energy storage, regulation of protein function, formation of plasma membrane lipids, proton buffering, and skeleton formation (1Goldford J.E. Hartman H. Smith T.F. Segre D. Remnants of an ancient metabolism without phosphate.Cell. 2017; 168: 1126-1134.e9Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 2Krebs E.G. Graves J.D. Interactions between protein kinases and proteases in cellular signaling and regulation.Adv. Enzyme Regul. 2000; 40: 441-470Crossref PubMed Scopus (19) Google Scholar, 3Vance J.E. Phospholipid synthesis and transport in mammalian cells.Traffic. 2015; 16: 1-18Crossref PubMed Scopus (319) Google Scholar, 4Chande S. Bergwitz C. Role of phosphate sensing in bone and mineral metabolism.Nat. Rev. Endocrinol. 2018; 14: 637-655Crossref PubMed Scopus (71) Google Scholar). While phosphate homeostasis in humans is tightly regulated, many of the underlying molecular mechanisms are still poorly understood. Phosphate deficiency causes severe diseases, including rhabdomyolysis, seizures, and osteomalacia (5Florenzano P. Cipriani C. Roszko K.L. Fukumoto S. Collins M.T. Minisola S. Pepe J. Approach to patients with hypophosphataemia.Lancet Diabetes Endocrinol. 2020; 8: 163-174Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Phosphate excess is associated with cardiovascular disease in the general population with apparent good health and in patients with chronic kidney disease, and vascular calcification has been identified as a main driver of morbidity and mortality (6Gonzalez-Parra E. Tunon J. Egido J. Ortiz A. Phosphate: A stealthier killer than previously thought?.Cardiovasc. Pathol. 2012; 21: 372-381Crossref PubMed Scopus (55) Google Scholar, 7Ritter C.S. Slatopolsky E. Phosphate toxicity in CKD: The killer among us.Clin. J. Am. Soc. Nephrol. 2016; 11: 1088-1100Crossref PubMed Scopus (78) Google Scholar). All cells require uptake of phosphate, which is mediated by the Na+-coupled inorganic phosphate transporter SLC20A1/PiT1. For a cellular process of such fundamental significance, our knowledge of the regulation of SLC20A1 activity in health and disease is surprisingly limited. Increased transcripts of SLC20A1 have been reported in calcifying vascular tissue, which has been proposed to be pathogenic for soft tissue calcification (8Li X. Yang H.Y. Giachelli C.M. Role of the sodium-dependent phosphate cotransporter, Pit-1, in vascular smooth muscle cell calcification.Circ. Res. 2006; 98: 905-912Crossref PubMed Scopus (348) Google Scholar, 9Masuda M. Miyazaki-Anzai S. Keenan A.L. Shiozaki Y. Okamura K. Chick W.S. Williams K. Zhao X. Rahman S.M. Tintut Y. Adams C.M. Miyazaki M. Activating transcription factor-4 promotes mineralization in vascular smooth muscle cells.JCI Insight. 2016; 1e88646Crossref Google Scholar). Increased SLC20A1 mRNA has been detected in 208F rat fibroblasts following phosphate starvation (10Kavanaugh M.P. Miller D.G. Zhang W. Law W. Kozak S.L. Kabat D. Miller A.D. Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters.Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7071-7075Crossref PubMed Scopus (521) Google Scholar) and in aggressive malignant tumors (11Sato K. Akimoto K. Expression levels of KMT2C and SLC20A1 identified by information-theoretical analysis are powerful prognostic biomarkers in estrogen receptor-positive breast cancer.Clin. Breast Cancer. 2017; 17: e135-e142Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 12Onaga C. Tamori S. Motomura H. Ozaki A. Matsuda C. Matsuoka I. Fujita T. Nozaki Y. Hara Y. Kawano Y. Harada Y. Sato T. Mano Y. Sato K. Akimoto K. High SLC20A1 expression is associated with poor prognoses in claudin-low and basal-like breast cancers.Anticancer Res. 2021; 41: 43-54Crossref PubMed Scopus (4) Google Scholar), presumably to restore cellular phosphate and accommodate rapid growth, respectively. SLC20A1 is a 12 transmembrane-spanning cell surface protein that was initially identified as a retrovirus receptor and is now recognized to mediate uptake of phosphate into cells driven by the inward-directed sodium gradient and negative interior voltage (10Kavanaugh M.P. Miller D.G. Zhang W. Law W. Kozak S.L. Kabat D. Miller A.D. Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters.Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7071-7075Crossref PubMed Scopus (521) Google Scholar, 13Olah Z. Lehel C. Anderson W.B. Eiden M.V. Wilson C.A. The cellular receptor for gibbon ape leukemia virus is a novel high affinity sodium-dependent phosphate transporter.J. Biol. Chem. 1994; 269: 25426-25431Abstract Full Text PDF PubMed Google Scholar, 14Hernando N. Gagnon K. Lederer E. Phosphate transport in epithelial and nonepithelial tissue.Physiol. Rev. 2021; 101: 1-35Crossref PubMed Scopus (25) Google Scholar, 15Johann S.V. Gibbons J.J. O'Hara B. GLVR1, a receptor for gibbon ape leukemia virus, is homologous to a phosphate permease of Neurospora crassa and is expressed at high levels in the brain and thymus.J. Virol. 1992; 66: PubMed Google Scholar, P. M. B. of transport mechanisms and for of the human and J. 2006; PubMed Scopus Google Scholar). SLC20A1 is expressed in a of including and bone (10Kavanaugh M.P. Miller D.G. Zhang W. Law W. Kozak S.L. Kabat D. Miller A.D. Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters.Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7071-7075Crossref PubMed Scopus (521) Google Scholar, 15Johann S.V. Gibbons J.J. O'Hara B. GLVR1, a receptor for gibbon ape leukemia virus, is homologous to a phosphate permease of Neurospora crassa and is expressed at high levels in the brain and thymus.J. Virol. 1992; 66: PubMed Google Scholar, W. T. RNA levels of human retrovirus receptors and not with by PubMed Google Scholar, S. J. C. and regulation of cotransporter genes in murine J. Google Scholar), and is C. S. A. C. N. A. P. D. M. The phosphate transporter revealed as a essential for Scopus Google Scholar). to in phosphate SLC20A1 has been to have a for cell and implicated in sensing of phosphate C. C. C. of a novel of for cell and of phosphate transport Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, N. A. S. S. J. Phosphate of the sodium-dependent and sensing of Biol. Chem. 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). the present we the of phosphate cells to the in phosphate uptake We found that SLC20A1 protein abundance was SLC20A1 mRNA was high at and induction was only a of mechanisms in regulating SLC20A1 protein abundance and phosphate uptake in to phosphate starvation. We that the low SLC20A1 protein levels the mRNA are to a negative of SLC20A1 protein in phosphate-replete cells phosphate be We this and performed a genome-wide protein loss-of-function genetic screen and found that SLC20A1 protein through the endosomal sorting complexes required for transport (ESCRT) is a negative of SLC20A1 protein levels that phosphate uptake into cells. Our screen an of genes and pathways that have not been to ESCRT machinery and phosphate homeostasis that are candidate regulators of SLC20A1 and phosphate homeostasis. to a to mammalian phosphate homeostasis, we SLC20A1 protein abundance following phosphate starvation in human kidney cells. revealed a of SLC20A1 protein levels in induction at the and and of SLC20A1 the of the SLC20A1 that was SLC20A1 protein induction was also in cell a of including bone and presumably cells that this is and performed in cells as this cell SLC20A1 induction following phosphate starvation, is in and is to the molecular biological that for this for SLC20A1 by in cells that phosphate for the and revealed SLC20A1 at the plasma membrane of the SLC20A1 was using cells phosphate starvation in only a of SLC20A1 mRNA levels the of mechanisms for SLC20A1 induction following phosphate starvation. studies that SLC20A1 protein levels be increased through transcription and protein transcription M. Miyazaki-Anzai S. Keenan A.L. Shiozaki Y. Okamura K. Chick W.S. Williams K. Zhao X. Rahman S.M. Tintut Y. Adams C.M. Miyazaki M. Activating transcription factor-4 promotes mineralization in vascular smooth muscle cells.JCI Insight. 2016; 1e88646Crossref Google Scholar). the between the mRNA but SLC20A1 protein following phosphate starvation the of To regulators of SLC20A1 protein levels, we using an SLC20A1 and to in SLC20A1 Phosphate starvation for in a in SLC20A1 that cellular SLC20A1 levels in a and be in a that is for genetic this we performed a loss-of-function genetic screen in phosphate-replete cells to identify genes to increased SLC20A1 protein levels, that genes as negative regulators of SLC20A1 protein abundance of the genetic screening results revealed that cells ESCRT proteins many of the that are in the cells and a of ESCRT genes in the cells The ESCRT protein protein protein and in A.L. A. The ESCRT the plasma membrane to and Rev. Biol. PubMed Scopus Google Scholar), and our screen that only of is in SLC20A1 and that the most in the cells is in the ESCRT these results that ESCRT proteins are negative regulators of SLC20A1 protein of ESCRT in the in are in a in are For of our genetic screening we cells in the ESCRT and the of in cells whereas of in cells with in genetic and cells increased abundance of SLC20A1 protein with cells to increased SLC20A1 protein abundance was a of increased SLC20A1 mRNA we SLC20A1 mRNA in and cells with SLC20A1 mRNA levels between sorting of proteins in the of protein in of the protein using in increased SLC20A1 protein levels which is with our genetic loss-of-function of the protein using the not in increases of SLC20A1 protein levels and protein pathways require ubiquitination to of is to in of proteins A. of Full Text Full Text PDF PubMed Scopus Google Scholar). we increased of proteins following with that To SLC20A1 is associated with the ESCRT we expression of in cultured which to the of proteins that with C. H. S. K. M. K. T. Y. M. a with an and endosomal J. PubMed Scopus Google Scholar). cells for SLC20A1 with and revealed that cells SLC20A1 and A of these with and of which SLC20A1 in the as ESCRT to and of ESCRT proteins cellular phosphate homeostasis in to SLC20A1 protein we phosphate uptake in and cells with cells. phosphate uptake was low in the of in of as most phosphate is through including SLC20A1 of in increased phosphate uptake in cells. phosphate uptake was elevated in and cells with the elevated SLC20A1 protein levels in our ESCRT While phosphate is an essential for cells and in cellular phosphate levels to (1Goldford J.E. Hartman H. Smith T.F. Segre D. Remnants of an ancient metabolism without phosphate.Cell. 2017; 168: 1126-1134.e9Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 2Krebs E.G. Graves J.D. Interactions between protein kinases and proteases in cellular signaling and regulation.Adv. Enzyme Regul. 2000; 40: 441-470Crossref PubMed Scopus (19) Google Scholar, 3Vance J.E. Phospholipid synthesis and transport in mammalian cells.Traffic. 2015; 16: 1-18Crossref PubMed Scopus (319) Google Scholar, 4Chande S. Bergwitz C. Role of phosphate sensing in bone and mineral metabolism.Nat. Rev. Endocrinol. 2018; 14: 637-655Crossref PubMed Scopus (71) Google Scholar, P. Cipriani C. Roszko K.L. Fukumoto S. Collins M.T. Minisola S. Pepe J. Approach to patients with hypophosphataemia.Lancet Diabetes Endocrinol. 2020; 8: 163-174Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 7Ritter C.S. Slatopolsky E. Phosphate toxicity in CKD: The killer among us.Clin. J. Am. Soc. Nephrol. 2016; 11: 1088-1100Crossref PubMed Scopus (78) Google Scholar), is that the mechanisms underlying the regulation of phosphate homeostasis in mammalian cells poorly the of a and a negative interior the SLC20A1 cotransporter the cells with of is for the cell to cell surface SLC20A1 activity for cell a CRISPR-based screening we show that the ESCRT tonically the cellular abundance of phosphate transporter that SLC20A1 protein abundance is regulated at the and that SLC20A1 is for by the ESCRT cellular phosphate uptake to in phosphate screening results numerous novel candidate regulators of mammalian phosphate homeostasis including many of the and oxidative phosphorylation pathways and regulators of protein CRISPR-based screening with a is a powerful tool to such as the mechanisms governing SLC20A1 abundance and activity is of for the ESCRT machinery is in cellular including membrane and the of plasma membrane proteins through M. M. H. The many of Rev. Biol. 2020; 21: PubMed Scopus Google Scholar). For the proteins process formation of the plasma and the of Smith The molecular J. 2021; PubMed Scopus Google Scholar) and protein I. T. P. S. M. A. C.A. N. N. S. J. J. K. in and causes and J. Full Text Full Text PDF PubMed Scopus Google Scholar) among the screening the that SLC20A1 proteins to which to of and C. A. H. and are of a Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. H. formation ESCRT to Biol. PubMed Scopus Google Scholar, M. sorting into the the of a endosomal protein sorting Full Text Full Text PDF PubMed Scopus Google Scholar, M. W.B. B. transport machinery for protein sorting at the Full Text Full Text PDF PubMed Scopus Google Scholar). and and the in as for of M. T. protein required for Full Text Full Text PDF PubMed Scopus Google Scholar, N. Zhang S. A. S. P. of and ESCRT sorting of to the Biol. 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K.L. protein at the Biol. 2017; PubMed Scopus Google Scholar). with this a has been reported for the plasma membrane C. ESCRT surface expression of the Biol. PubMed Google Scholar). While our results that the and the mediate SLC20A1 protein the increased SLC20A1 protein levels following with and the of of proteins among the genes and pathways of our screen that the is the for SLC20A1 protein that be by the Our loss-of-function that SLC20A1 is a direct for however, an is that a of SLC20A1 is through and of ESCRT machinery such a expression results in of ESCRT with of proteins at the ESCRT and with C. H. S. K. M. K. T. Y. M. a with an and endosomal J. PubMed Scopus Google Scholar). The of SLC20A1 and SLC20A1 in the as the ESCRT machinery and is with a in SLC20A1 that SLC20A1 is a direct ESCRT While we increased SLC20A1 protein levels following of ESCRT in our genetic a of this screening is that is an that to the of which of the and not to SLC20A1 was increased at the plasma the increased phosphate uptake in and cells the that SLC20A1 is increased at the plasma membrane of cells and the of an for SLC20A1 to the plasma such mechanisms have been the that the for of endosomal proteins to the plasma membrane M. M. A analysis of and a in and Biol. PubMed Scopus Google Scholar) and the that the M. D. P. N. C.M. H. A. is a for endosomal Biol. 2017; PubMed Scopus Google Scholar). of in the cell surface of cells revealed SLC20A1 cell surface protein levels, and SLC20A1 an that is required for M. D. P. N. C.M. H. A. is a for endosomal Biol. 2017; PubMed Scopus Google Scholar). While these of in of SLC20A1 to the plasma membrane studies are to Our that SLC20A1 in the of phosphate starvation is a candidate for the induction of SLC20A1 is that the induction of SLC20A1 by is than with phosphate starvation. The be by ESCRT in and cells of and of function, by of SLC20A1 of the as is that additional pathways are induced and phosphate studies are to the underlying mechanisms in while many of our screening are to protein we have identified additional genes and pathways among the of our screen without an in mammalian phosphate homeostasis. Our screen revealed regulators of protein ubiquitination of plasma membrane proteins for through the H. S. of and signaling by 2012; Full Text Full Text PDF PubMed Scopus Google Scholar), is an that these genes SLC20A1 protein in to ESCRT-mediated degradation. than of ESCRT machinery many plasma membrane proteins are through the studies are to the mechanisms SLC20A1 with and the candidate regulators identified in our screen provide a for these pathways also of oxidative phosphorylation and the Krebs cycle in analysis as genes that are of the oxidative phosphorylation machinery and the Krebs cycle also identified as phosphate regulated in an genome-wide genetic screen in cells that of high phosphorylation as C. S. J. C. J. A. M. Y. A. M. B. of phosphate in PubMed Scopus Google Scholar). While is that oxidative phosphorylation and the Krebs cycle have been implicated as regulators of phosphate homeostasis in using is these pathways be to phosphate homeostasis. and additional in our screen as a resource for studies into the mechanisms regulating mammalian phosphate homeostasis. and cell using to with cell these the cell not cultured in with and in at For cells with sodium and for with with and in for the for the For for with in cell in for and at the of the For and cells for with in was as genetic was using SLC20A1 was in cells using with as and as negative in with the following with and for and phosphate for For genetic loss-of-function a a a SLC20A1 and a N. M. M. Smith I. Z. C. J. to activity and of 2016; PubMed Scopus Google Scholar, Zhang and genome-wide for 11: PubMed Scopus Google Scholar) into which was a Zhang For virus cells with and using cells with and for virus using for to and and cells in with of human which was a and N. M. M. Smith I. Z. C. 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