Regulation of Hematopoiesis and Methionine Homeostasis by mTORC1 Inhibitor NPRL2

Abstract

SUMMARY Nitrogen permease regulator-like 2 (NPRL2) is a component of a conserved complex that inhibits mTORC1 (mammalian Target Of Rapamycin Complex 1) in response to amino acid insufficiency. Here we show that NPRL2 is required for mouse viability and its absence significantly compromises fetal liver hematopoiesis in developing embryos. Moreover, NPRL2 KO embryos have significantly reduced methionine levels and exhibit phenotypes reminiscent of cobalamin (vitamin B12)-deficiency. Consistent with this idea, NPRL2 KO liver and mouse embryonic fibroblasts (MEFs) show defective processing of the cobalamin-transport protein transcobalamin 2, along with impaired lysosomal acidification and lysosomal gene expression. NPRL2 KO MEFs exhibit a significant defect in the cobalamin-dependent synthesis of methionine from homocysteine, which can be rescued by supplementation with cyanocobalamin. Taken together, these findings demonstrate a role for NPRL2 and mTORC1 in the regulation of lysosomal-dependent cobalamin processing, methionine synthesis and maintenance of cellular re-methylation potential, which are important during hematopoiesis.

The GATOR1 Complex Regulates Metabolic Homeostasis and the Response to Nutrient Stress inDrosophila melanogaster

“…Thus, unlike what is observed in single-celled eukaryotes, in Drosophila the GATOR1 complex is required to maintain baseline levels of TORC1 activity in vivo under nutrient-replete conditions. Similarly, rat and mouse cells cultured from nprl2 , nprl3 , or iml1/Depdc5 mutants have increased TORC1 activity relative to controls (Kowalczyk et al 2012; Dutchak et al 2015; Marsan et al 2016). Thus, in both Drosophila and mammals, the GATOR1 complex is required to maintain baseline levels of TORC1 activity independent of nutrient status.…”

“…In contrast, in mice and rats, components of the GATOR1 complex are required for viability, strongly suggesting that, in metazoans, the GATOR1 complex has evolved roles beyond the response to nutrient stress (Table 1) (Kowalczyk et al 2012; Dutchak et al 2015; Marsan et al 2016). In order to determine if the GATOR1 complex is required to maintain baseline levels of TORC1 activity in vivo in Drosophila , we measured phosphorylation of S6 Kinase (S6K), a direct readout for TORC1 activity, in well-fed whole animal lysates by western blot using an anti-phosopho-Thr398 S6K antibody (Hara et al 1998).…”

“…In contrast, in mammalian and Drosophila tissue culture cells, depleting GATOR1 components results in a dramatic increase in TORC1 activity under standard culture conditions (Bar-Peled et al 2013; Wei and Lilly 2014). Notably, recent reports have shown that GATOR1 knockouts of iml1/Depdc5 (rat), nprl2 (mouse), and nprl3 (mouse) result in late embryonic lethality, with embryos exhibiting developmental defects in the heart, liver, and brain (Kowalczyk et al 2012; Dutchak et al 2015; Marsan et al 2016). Additionally, in Drosophila , the GATOR1 complex regulates entry into the meiotic cycle during oogenesis (Wei et al 2014).…”

A Metabolic Function for Phospholipid and Histone Methylation

“…Cells equivalent to one OD 600 unit were spread onto a YPD plate. Individual methyltransferase mutants were patched onto the plate and then incubated at 30°C for about 7 days.Spotting assayCells were pre-cultured in YPL overnight to stationary phase, and 3 μL aliquots of a series of 10-fold dilutions from 0.5 unit of OD 600 were spotted onto YPL plates and incubated for 2 days at 30°C.Metabolite extraction and quantitationIntracellular metabolites were extracted from yeast and mammalian cell lines using a previous established method (Dutchak et al, 2015; Tu et al, 2007). Care was taken to quench cells quickly and maintain metabolites in acid to minimize oxidation.…”

“…Intracellular metabolites were extracted from yeast and mammalian cell lines using a previous established method (Dutchak et al, 2015; Tu et al, 2007). Care was taken to quench cells quickly and maintain metabolites in acid to minimize oxidation.…”