Fig 2: PRMT1 regulates mTORC1 activation upstream of Rag GTPases(A) The key complexes of the mTORC1 pathway in amino-acid-sensing branch.(B) IB analysis of WCLs derived from HEK293T cells stably expressing FLAG-Rap2a or FLAG-RagBQ99L with or without PRMT1 depletion. Cells were starved of aa for 50 min or starved for 50 min and then restimulated with aa for 10 min before harvesting. Similar results were obtained in three independent experiments.(C) IB analysis of WCL derived from HEK293T cells depleted of PRMT1 or PRMT1 and NPRL2. Cells were starved of aa for 50 min or starved for 50 min and then restimulated with aa for 10 min before harvesting. Similar results were obtained in three independent experiments.(D) IB analysis of WCL derived from HEK293T cells depleted of PRMT1, KPTN, or both. Cells were starved of aa for 50 min or starved for 50 min and then restimulated with aa for 10 min before harvesting. Similar results were obtained in three independent experiments.(E and F) IB analysis of WCLs derived from cells stably expressing Tet-on-FLAG-PRMT1 and depleted of WDR24 (E) or WDR59 (F). 1 μg/mL doxycycline was added for 48 hours to induce PRMT1 expression. Cells were starved of aa for 50 min or starved for 50 min and then restimulated with aa for 10 min before harvesting. Similar results were obtained in three independent experiments.See also Figure S4 and Table S1.

Fig 3: NGFRETD-nLAP-tagged WDR59 expression in HEK spCas9+ single cell clones. A nLaP-tagged WDR59 protein expression visualized on Western blots probed with anti-WDR59 antibodies and anti-GFP antibodies. GAPDH serves as loading control. B Representative FACS profile of selected nLAP-tagged WDR59 expressing HEK WDR59-NGFRETD-nLAP cell clones (D5, upper panel and B3, lower panel). GFP fluorescence (Ex488nm) was measured through 530/30 nm bandpass filter. The flow cytometry results were analyzed using FlowJo™ v7.6.5 Software (BD Life Sciences). MFI, mean fluorescence intensity; WT, wild type

Fig 4: Binding of native GATOR2 interacting proteins to nLAP-tagged WDR59. nLAP-WDR59 was immunoprecipitated from WDR59-NGFRETD-nLAP clone D5 using anti-EGFP antibodies (EGFP-TrapA beads). Endogenous WDR59 and GATOR2 interactors were identified on Western blots with high affinity anti-WDR59, anti-WDR24, anti-MIOS, anti-NPRL3 and anti-SESN2 antibodies, respectively. A Known interactors (WDR24, SESN2) were identified by Western bloting using high affinity anti-WDR24 and anti-SESN2 antibodies, respectively. GAPDH was used as control. B CoIP of WDR24, MIOS (GATOR2), NPRL3 (GATOR1) as well as SESN2 with nLAP-tagged WDR59 IP in the absence of amino acids and after stimulation with amino acids or leucine for 60 min. Stimulation with all amino acids or leucine decreases interaction between WDR59 (GATOR2) and other GATOR2 subunits (WDR24 or MIOS), as well as between WDR59 and SESN2 as indicated with percentages, normalized to WDR59-GFP signals and to amino acid-starved samples. The interaction with NPRL3 (GATOR1) is increased by stimulation. β-Actin was used as loading control. C Amino acid-/ leucine-dependent mTORC1 activity was determined by phosphorylation of 4EPB1 (T37/T46). β-Actin was used as loading control D Co-immunoprecipitation (CoIP) of WDR24 and MIOS (GATOR2), NPRL3 (GATOR1) as well as leucine-sensing SESN2 with nLAP-tagged WDR59 IP in the absence of leucine and after stimulation with leucine for 60 min. Leucine stimulation increases binding of WDR59 to WDR24, MIOS, SESN2 and NPRL3 (GATOR1). β-Actin was used as control. E Leucine-dependent mTORC1 activity was determined by phosphorylation of 4EPB1 (T37/T46). β-Actin was used as loading control F Quantification of CoIP (D), shown is the relative intensity normalized to nLAP-tagged WDR59 signal and to leucine-starved samples; n=2, data presented as mean ± SEM. Abbreviations: DEPDC5, DEP Domain Containing 5; GATOR, GAP activity towards RAG; MIOS, Meiosis Regulator For Oocyte Development (Homolog (Drosophila)); NPRL2/3, Nitrogen Permease Regulator-Like Protein 2/3; SEC13 Homolog, Nuclear Pore And COPII Coat Complex Component; SEH1L, SEH1 Like Nucleoporin; SESN2, Sestrin-2; WDR24/59, WD Repeat Domain 24/59

Fig 5: Comparison of anti-NGFR APC flow cytometry versus GFP autofluorescence. A Sorting of HEK WDR59-NGFRETD-nLAP cells by anti-NGFR-APC staining. Comparison between NGFR-APC signal and endogenous GFP expression of co-transfected cells before NGFR-APC sorting. Left panel: HEK (Lenti-X 293T) spCas9+ cells (wild type (WT)) with and without NGFR-APC staining. After successful tagging, HEK WDR59-NGFRETD-nLAP cells, stained with APC-conjugated anti-NGFR antibodies, showed high expression of NGFR, of which the highest 5% NGFR-APC positive cells were sorted and sub-cultivated in single clones. In contrast, endogenous GFP expression (right panel) is weaker and rather unsuitable to guarantee sorting with appropriate purity and accuracy. B Western Blot for pre-expressed spCas9-FLAG in the Lenti-XTM 293T cells used for endogenous tagging of WDR59. β-Actin serves as loading control. The flow cytometry results were analyzed using FlowJo™ v7.6.5 Software (BD Life Sciences).