PROJECT SUMMARY / ABSTRACT The mechanisms by which mammalian cells sense phosphate availability to maintain homoestasis of this critical nutrient are unknown. Phosphate is indispensable for many biological functions including DNA and RNA synthesis, bone formation, and preservation of energy as high energy phosphates. Consequently, serious complications develop during phosphate deficiency, e. g. rhabdomyolysis, and during phosphate excess, e. g. cardiovascular disease and vascular calcification. Genetic mutations that lead to massive phosphate excess are associated with a premature aging syndrome. A hallmark of vascular calcification is induction of the sodium- coupled phosphate transporter SLC20A1/PiT1, and SLC20A1 is also induced in aggressive cancers. SLC20A1 is 1) widely expressed, 2) provides phosphate for basic cellular functions, and is 3) massively upregulated in phosphate-starved cells. To interrogate the mechanisms underlying SLC20A1 regulation and more broadly phosphate homeostasis and sensing, we performed a set of complementary CRISPR-based genome-wide loss- of-function genetic screens in phosphate-replete and in phosphate-starved cultured mammalian cells. We found that proteostasis plays a crucial role in the regulation of SLC20A1 protein abundance through regulation of protein degradation, recycling, and synthesis. We hypothesize that phosphate starvation is detected by thus far unidentified phosphate sensing machinery, which leads to coordinated regulation of SLC20A1 degradation, recycling, and synthesis resulting in increased SLC20A1 protein abundance. We will examine this hypothesis through three independent yet synergistic projects. In Project 1, we will examine the mechanisms governing SLC20A1 internalization and degradation. Interestingly, loss of one of our candidate negative regulators of SLC20A1 degradation leads to a premature aging syndrome that closely resembles genetic syndromes with phosphate excess. Therefore, we will examine if decreased SLC20A1 internalization and degradation with dysregulated cellular phosphate uptake also leads to a premature aging phenotype. In Project 2, we will examine the mechanisms for SLC20A1 recycling to the plasma membrane, which will also be informative for many other proteins as this process is generally not well understood. In Project 3, we will examine the role of protein synthesis in the regulation of SLC20A1 protein abundance. Each Project is designed to also identify members of the underlying phosphate sensing machinery. Our ultimate goal is identification of novel pharmacological targets for the treatment of hypo- and hyperphosphatemic patients, which makes these studies highly clinically relevant.