Fig 2: 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).

Fig 3: 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 4: 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 5: GATOR1 and GATOR2 associate with endogenous KICSTOR components in an amino acid insensitive fashiona) An endogenously tagged GATOR1 component co-immunoprecipitates endogenous KICSTOR. Anti-FLAG immunoprecipitates were prepared from HEK-293T cells expressing endogenously FLAG-tagged DEPDC5, a GATOR1 component, that had been starved of amino acids for 50 min or starved and restimulated with amino acids for 10 min. Immunoprecipitates and cell lysates were analyzed by immunoblotting for the indicated proteins.b) An endogenously tagged GATOR2 component co-immunoprecipitates endogenous GATOR1 and KICSTOR. Anti-FLAG immunoprecipitates were prepared from HEK-293T cells expressing endogenously FLAG-tagged WDR59, a GATOR2 component, and treated as in (a). Immunoprecipitates and cell lysates were analyzed by immunoblotting for the indicated proteins.c) An anti-KPTN antibody co-immunoprecipitates endogenous components of KICSTOR, GATOR1, and GATOR2. Anti-KPTN immunoprecipitates were prepared from wild-type HEK-293T treated as in (a) and immunoprecipitates and cell lysates analyzed by immunoblotting for the indicated proteins. Anti-GSK3β immunoprecipitates were used to monitor the non-specific binding of proteins to the beads.d) KPTN and ITFG2 form a heterodimer that requires SZT2 to associate with C12orf66. Anti-FLAG immunoprecipitates and lysates prepared from HEK-293T cells expressing the indicated cDNAs were analyzed by immunoblotting for the relevant epitope tags.e) Loss of KICSTOR components did not have a significant effect on the expression levels of GATOR1 or GATOR2 components. HEK-293T cell clones deficient for indicated KICSTOR components or Nprl3 or WDR24 were generated via the CRISPR/Cas9 system and single cell cloning. Cell lysates were analyzed by immunoblotting for the indicated proteins. DNA sequencing of the C12orf66 gene was used to verify out of frame mutations in the genomic locus of the sgC12orf66 cells because an antibody that detects the C12orf66 protein in cell lysates is not available. The HEK-293T cell clones analyzed here were used in subsequent figures, where indicated.f) KPTN interacts with ITFG2 even in cells lacking other KICSTOR components. Immunoprecipitates and cell lysates prepared from wild-type, SZT2-deficient, or C12orf66-deficient HEK-293T cell clones expressing the indicated proteins were analyzed by immunoblotting.g) Expression levels of KPTN and ITFG2 in HEK-293T cells stably expressing the indicated sgRNAs under amino acid starved or replete conditions. Cell lysates were analyzed by immunoblotting for the levels of the indicated proteins. Raptor serves as a loading control. These same cell lines were also analyzed for phospho-S6K1 and S6K1 levels in Extended Data Fig. 5a–c.h) KPTN-ITFG2 does not compete with C12orf66 for associating with SZT2. HEK-293T cells expressing the indicated cDNAs were treated and analyzed as in (d).