Division of Endocrinology, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA; Charles and Jane Pak Center for Mineral Metabolism and Clinical Research, University of Texas Southwestern Medical Center, Dallas, Texas, USA; Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
J Biol Chem. 2022 Jun;298(6):101945. doi: 10.1016/j.jbc.2022.101945. Epub 2022 Apr 18.
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.
无机磷酸盐对人类生命至关重要。广泛表达的哺乳动物钠/磷酸盐协同转运蛋白 SLC20A1/PiT1 介导磷酸盐进入大多数细胞类型；然而，虽然 SLC20A1 是发育所必需的，并且 SLC20A1 的表达升高与血管钙化和侵袭性肿瘤生长有关，但调节 SLC20A1 蛋白丰度的机制尚不清楚。在这里，我们发现磷酸盐充足的培养细胞中 SLC20A1 蛋白表达水平较低，但在磷酸盐饥饿后显著诱导，而磷酸盐充足的细胞中 mRNA 表达水平较高，仅轻度增加磷酸盐饥饿。为了鉴定 SLC20A1 蛋白水平的调节剂，我们在磷酸盐充足的细胞中使用 SLC20A1 蛋白诱导作为读出物进行了基于全基因组 CRISPR 的功能丧失遗传筛选。我们的筛选表明，内体分选复合物所需的运输（ESCRT）机制对于在磷酸盐充足的细胞中正确下调 SLC20A1 蛋白是必不可少的。我们表明 SLC20A1 与 ESCRT 共定位，并且 ESCRT 缺陷会增加 SLC20A1 蛋白和磷酸盐进入细胞。我们还发现了许多其他调节哺乳动物磷酸盐稳态的候选调节剂，包括修饰蛋白质泛素化和三羧酸循环和氧化磷酸化途径的基因。这些靶标中有许多以前没有涉及到这个过程。我们在这里提出了一个模型，即在磷酸盐充足的细胞中，通过直接 ESCRT 介导的 SLC20A1 降解，SLC20A1 蛋白丰度和磷酸盐摄取在后转录水平受到紧张的负调节。此外，我们的筛选结果为未来阐明细胞磷酸盐稳态的机制提供了一个全面的资源。我们得出结论，基于全基因组 CRISPR 的遗传筛选是发现与生理过程相关的蛋白质和途径的有力工具。
