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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.

SUBMITTER: Zechner C

PROVIDER: S-EPMC9123275 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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

Zechner Christoph C Henne W Mike WM Sathe Adwait A AA Xing Chao C Hernandez Genaro G Sun Shengyi S Cheong Mi Cheong MC

The Journal of biological chemistry 20220418 6

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 ...[more]

PMID: 35447110

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Project description:Syncytins are envelope genes of retroviral origin that play a critical role in the formation of a syncytial structure at the fetomaternal interface via their fusogenic activity. The mouse placenta is unique among placental mammals since the fetomaternal interface comprises two syncytiotrophoblast layers (ST-I and ST-II) instead of one observed in all other hemochorial placentae. Each layer specifically expresses a distinct mouse syncytin, namely syncytin-A (SynA) for ST-I and syncytin-B (SynB) for ST-II, which have been shown to be essential to placentogenesis and embryonic development. The cellular receptor for SynA has been identified as the membrane protein LY6E and is not the receptor for SynB. Here, by combining a cell-cell fusion assay with the screening of a human ORFeome-derived expression library, we identified the transmembrane multipass sodium-dependent phosphate transporter 1 PiT1/SLC20A1 as the receptor for SynB. Transfection of cells with the cloned receptor, but not the closely related PiT2/SLC20A2, leads to their fusion with cells expressing SynB, with no cross-reactive fusion activity with SynA. The interaction between the two partners was further demonstrated by immunoprecipitation. PiT1/PiT2 chimera and truncation experiments identified the PiT1 N-terminus as the major determinant for SynB-mediated fusion. RT-qPCR analysis of PiT1 expression on a panel of mouse adult and fetal tissues revealed a concomitant increase of PiT1 and SynB specifically in the developing placenta. Finally, electron microscopy analysis of the placenta of PiT1 null embryo before they die (E11.5) disclosed default of ST-II formation with lack of syncytialization, as previously observed in cognate SynB null placenta, and consistent with the present identification of PiT1 as the SynB partner.IMPORTANCESyncytins are envelope genes of endogenous retroviruses, coopted for a physiological function in placentation. They are fusogenic proteins that mediate cell-cell fusion by interacting with receptors present on the partner cells. Here, by devising an in vitro fusion assay that enables the screening of an ORFeome-derived expression library, we identified the long-sought receptor for syncytin-B (SynB), a mouse syncytin responsible for syncytiotrophoblast formation at the fetomaternal interface of the mouse placenta. This protein - PiT1/SLC20A1 - is a multipass transmembrane protein, also known as the receptor for a series of infectious retroviruses. Its profile of expression is consistent with a role in both ancestral endogenization of a SynB founder retrovirus and present-day mouse placenta formation, with evidence-in PiT1 knockout mice-of unfused cells at the level of the cognate placental syncytiotrophoblast layer.

Project description:BackgroundWhile searching by microarray for sugar-responsive genes, we inadvertently discovered that sodium-phosphate cotransporter 2B (NaPi-2b) mRNA concentrations were much lower in fructose-perfused than in glucose-perfused intestines of neonatal rats. Changes in NaPi-2b mRNA abundance by sugars were accompanied by similar changes in NaPi-2b protein abundance and in rates of inorganic phosphate (Pi) uptake.ObjectiveWe tested the hypothesis that luminal fructose regulates NaPi-2b.DesignWe perfused into the intestine fructose, glucose, and nonmetabolizable or poorly transported glucose analogs as well as phlorizin.ResultsNaPi-2b mRNA concentrations and Pi uptake rates in fructose-perfused intestines were approximately 30% of those in glucose and its analogs. NaPi-2b inhibition by fructose is specific because the mRNA abundance and activity of the fructose transporter GLUT5 (glucose transporter 5) increased with fructose perfusion, whereas those of other transporters were independent of the perfusate. Plasma Pi after 4 h of perfusion was independent of the perfusate, probably because normal kidneys can maintain normophosphatemia. Inhibiting glucose-6-phosphatase, another fructose-responsive gene, with tungstate or vanadate nonspecifically inhibited NaPi-2b mRNA expression and Pi uptake in both glucose- or fructose-perfused intestines. The AMP kinase (AMPK)-activator AICAR (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside) enhanced and the fatty acid synthase-AMPK inhibitor C75 (3-carboxy-4-octyl-2-methylenebutyrolactone trans-4-carboxy-5-octyl-3-methylenebutyrolactone) prevented fructose inhibition of NaPi-2b but had no effect on expression of other transporters. NaPi-2b expression decreased markedly with age and was inhibited by fructose in all age groups.ConclusionsEnergy levels in enterocytes may play a role in NaPi-2b inhibition by luminal fructose. Consumption of fructose that supplies approximately 10% of caloric intake by Americans clearly affects absorption of Pi and may promote Pi homeostasis in patients with impaired renal function.

Dataset Information

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

Publications

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

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