Figure 2(A) Model of Rag GTPase-dependent and Rag GTPase-independent pathways that signal to mTORC1. Alanine (Ala), arginine (Arg), histidine (His), leucine (Leu), methionine (Met), serine (Ser), threonine (Thr), and valine (Val) promote mTORC1 lysosomal localization and activation through the Rag GTPase pathway. Glutamine (Gln) and asparagine (Asn) promote mTORC1 lysosomal localization and activation independently of the Rag GTPases, and require Arf1 through an unknown mechanism. Both pathways require growth factor signaling resulting in Rheb GTP-bound allosterically activating mTORC1. (B) Human embryonic kidney 293A (HEK293A) cells were co-transfected with HA-tagged ArfGAP1 or RFP1 (control), together with Myc-tagged S6K1. Cells were starved of amino acids for 1 hour (-AA), and then 1 mM of leucine (+Leu), glutamine (+Gln), or asparagine (+Asn) were added for 2 hours. Immunoprecipitates (IP) or whole cell lysate (WCL) samples were probed for HA, Myc, and phosphorylated S6K1 at Thr 389. Vinculin was a loading control. (C) WT or RagA/B knockout (KO) HEK293A cells were co-transfected with HA-tagged ArfGAP1, or HA-tagged RFP1 (control) with Myc-tagged S6K1. Cells were starved of amino acids (-AA) for 2 hours and then 4 mM of glutamine (+Gln) or asparagine (+Asn) were added for 2 hours. Cell lysates were assed for mTORC1 activity as in by the phosphorylation of S6K1 at Thr 389 (pS6K1) and probed for HA, Myc, S6K1, RagA, RagB, and Vinculin. (D) ArfGAP1 was stably knocked down in HEK293A cells using shRNAs against ArfGAP1 (shArfGAP1) or GFP (shGFP) control. Cells were cultured under normal condition (NC; lanes 1 and 7), or starved of amino acids for 1 hour (-AA 1h; lanes 2 and 8), followed by either maintaining in amino acid starvation media (-AA; lanes 3 and 9), or addition of 1 mM of Leu (+Leu; lanes 4 and 10), Gln (+Gln; lanes 5 and 11), or Asn (+Asn; lanes 6 and 12) for 2 hours. mTORC1 activity was analyzed similar to (B). Protein levels of ArfGAP1 were confirmed by immunoblotting. S6K1 and Actin were probed as controls. (E) RagA/B KO HEK293A cells were co-transfected with HA-tagged ArfGAP1, HA-tagged ArfGAP2, or HA-tagged RFP1 (control) with Myc-tagged S6K1. Cells were starved of amino acids (-AA) for 2 hours and then 4mM of Gln or Asn were added for 2 hours. mTORC1 was analyzed similar to (B). IP or WCL samples were probed for HA, Myc, and phosphorylated S6K1 at Thr 389. Actin serve as a loading control. (F) RagA/B KO HEK293A cells were transfected with HA-tagged ArfGAP1 for 24 hours. Immunofluorescence assay was performed to stain HA-tagged ArfGAP1 (green) and pS6 at Serine 235/236 (red). A representative image of the staining of two channels is shown (scale bar, 20 μm). 8 fields were randomly chosen for quantification analysis and total cell numbers are summarized in the table (Fisher's exact test P <0.0001). Percentages of pS6-positive cells in each field for HA-tagged ArfGAP1 positive or negative cell populations are illustrated in the bar plot (shown as Mean ± SEM; Student's t-test P = 2.1 x 10-6). (G) ArfGAP1 was knocked out using CRISPR/Cas9 in RagA/B KO HEK293A cells. Expression levels of ArfGAP1 in two KO clones (KO-1 and KO-2) were confirmed by immunoblotting, and mTORC1 activity was analyzed as in (B). (H) ArfGAP1 WT or ArfGAP1 KO HEK293A cells were starved of amino acids (-AA) for 2 hours, and stimulated with 4mM glutamine (+Gln) or asparagine (+Asn) for 2 hours. Lysates were analyzed for mTORC1 activity as in (B), and phosphorylation of S6 at Serine 235/236 (pS6) was also analyzed. ArfGAP1 and actin were probed for as controls. (I) RagA/B KO HEK293A cells with WT ArfGAP1 or ArfGAP1 knocked out were starved of amino acids for 1, 2, and 4 hours. NC denotes normal condition. Lysates were analyzed as in (H). "s.e." and "l.e." denote short exposure and long exposure, respectively. (J) RagA/B KO HEK293A cells (lanes 1, 2), RagA/B KO ArfGAP1 KO cells (KO-1) (lanes 3, 4), RagA/B KO ArfGAP1 KO cells with HA-tagged ArfGAP1 overexpressed for 24 hours (lanes 5, 6) were cultured under normal condition (NC) or starved of amino acids (-AA) for 30 minutes. mTORC1 activity was analyzed as in (H). (K) WT HEK293A cells were co-transfected with HA-tagged ArfGAP1, or HA-tagged RFP1 (control) with Myc-tagged S6K1. Cells were cultured under normal condition (NC), or starved of serum (-Serum) for overnight and then treated with 100 nM insulin for 30 minutes. mTORC1 activity was assessed similar to (B). Cell lysates were probed for HA, S6K1, phosphorylated S6K1 at threonine 389 (pS6K1), mTOR, phosphorylated mTOR at Serine 2481 (pmTOR), and Vinculin.