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1 Figure S1 Nucleotide binding status of RagA mutants. Wild type and mutant forms of MycRagA was transfected into HEK293 cells and the transfected cells were labeled with 32 Pphosphate. MycRagA was immunoprecipitated and the bound nucleotides were eluted and analyzed by thin layer chromatography. Transfection of pcdna3 vector was included as a negative control. 1

2 a RagA QL RagA TN DNA (ng) b A QLC SN Osm. stress AA AA AA HARagC SN FlagRagA HARagA QL 34 Figure S2 Rag is specifically involved in nutrient signaling. (a) RagA T21N inhibits S6K phosphorylation in a dose dependent manner. Each indicated amount (ng) of RagA Q66L or RagA T21N was cotransfected with. RagA Q66L transfected cells were AA starved for 1 h and RagA T21N transfected cells were AA starved for 1 h and stimulated with AA for 30 min. Phosphorylation and protein levels were determined by immunoblotting with appropriate antibodies, as indicated. A low protein level of RagA T21N could be detected with 300 ng of transfection. (b) Rag cannot reverse the inhibitory effect by osmotic stress. 200 ng of pcdna3 or each indicated Rag construct was cotransfected with HA S6K into HEK293 cells. For osmotic stress, cells were treated with 600 mm of sorbitol for 30 min before harvest. Phosphorylation and protein levels were determined by immunoblotting with appropriate antibodies, as indicated. 2

3 Medium AA No AA Readd AA Insulin S6K pakt (S473) AKT Figure S3 Amino acid starvation diminishes the stimulatory effect of insulin on TORC1 but not TORC2. HeLa cells were serum starved for 16 h. Cells were then remained in serum starvation media or switched to AA starvation media (No AA). After 1 h of AA starvation, cells were either remained in AA starvation media or stimulated with AA containing media (readd AA) for 30 min. Insulin (400 nm) was added for 30 min before harvest. Phosphorylation and expression levels of endogenous proteins were detected by immunoblotting with the indicated antibodies. 3

4 a draga Q61L b draga T16N c 10 Suppression of Tsc1 lethality. 8 percent viable Tsc1 / dragc/; Tsc1 / 0 Tsc1/ dragc/; Tsc1/ Figure S4 Genetic interactions and growth effects of drag GTPases in Drosophila. (a, b) draga expression affects wing growth. Expression of draga Q61L (a) or draga T16N (b) in the dorsal epithelial cells of the wing using the apgal4 driver results in downward (increased rate of growth) or upward (decreased rate of growth) curvature of the wing blade, respectively, reflecting an altered rate of growth in these cells. (c, d) dragc mutation dominantly suppresses the lethality of Tsc1 null homozygous mutant animals. Tsc1 29 mutants die at the early second instar larval stage. Mutation of a single copy of dragc in the Tsc1 29 null background partially rescues the lethality, allowing survival to the pupal stage in 7.4% of animals (n = 474). 4

5 Amino acid import (CPM) Vec Rheb Rag Figure S5 Rag does not affect amino acid import. Rheb S16H (labeled as Rheb) or RagA Q66L and RagC S75N (labeled as Rag) expressing stable clones of HeLa cells were incubated with [ 3 H]labeled amino acid mixture in the presence of 2 mm cold amino acids. Cells were washed with PBS before lysis and radioactivity in the lysates were counted. The amino acid import is specific because the increase of radioactivity inside cells could be completely competed by a high concentration of amino acid in the culture medium in a dose dependent manner (data not shown). 5

6 A B A T21N A Q66L B T54N B Q99L C D C S75N C Q120L D S76N D Q121L 1a dsrna AA draga dragc Cdc42 Rab2 Rac2 RhoL Rheb AA AA 1b dsrna NC Rheb draga dragc AA AA AA draga drag C 1C 2a Rag MycRagD MycRagC FlagRagB FlagRagA Figure S6 Full scans of original blots for data in figure 1, 2, 3, 5, and 7e. Panels corresponding to the figures in the paper are indicated. 6

7 2b Rag A AC A TN A TN C A TN C SN A TN C QL A QL A QL C A QL C SN A QL C QL e HARag MycLC3 AA AA AQLCSN ATNCQL AQLCSN ATNCQL 2C MycRagC FlagRagA 3a Rag A C AC A TN A QL C SN C QL 3b AA AA AA AA AA AA A TN C QL MycRagC FlagRagA HARagC QL HARagA TN Figure S6 continued 7

8 3c A TN C QL Insulin 5a A QL C SN mtorkd Rapa. HARagC QL AA HARagA TN 5b A QL C SN TSC1/2 AA 5c RNAi TSC2 TSC2 AB AB (Rag) HATSC1/2 HARagC SN HARagA QL 5d A TN 7e HARag MycLC3 AA AA AQLCSN ATNCQL AQLCSN ATNCQL C QL Rheb HARagC QL HARagA TN MycRheb HA Figure S6 continued 8

9 Supplementary Methods GTP Labeling Assay GTP labeling assay was performed as described 1. Briefly, HEK293 cells were cultured in sixwell plates and transfected with either pcdna3 or MycRagA constructs using Lipofectamine reagent (Invitrogen). Thirty six hours later, cells were washed with phosphatefree DMEM (GIBCO Cat. No ) and incubated with 1 ml of phosphatefree DMEM for 90 min. Cells were then incubated with 25 µci of [ 32 P]phosphate/ml for 4 hr. After labeling, cells were lysed with prechilled lysis buffer (0.5% NP40, 50 mm Tris [ph 7.5], 100 mm NaCl, 10 mm MgCl 2, 1 mm dithiothreitol(dtt), 1 mm phenylmethylsulfonyl fluoride, 10 µg of leupeptin/ml, 10 µg of aprotinin/ml) for just 30 sec on ice. The lysates were then centrifuged at 12,000 X g for 15 min at 4 C. The supernatant (160 µl) was transferred to a fresh tube and 16 µl of NaCl (500 mm) was added to inhibit GAP activity. MycRagA was then immunoprecipitated with antimyc antibody and proteing sepharose bead slurry (50%, Amersham Biosciences) for 1 hr at 4 C. The beads were washed with wash buffer 1 (50 mm Tris [ph 8.0], 500 mm NaCl, 5 mm MgCl 2, 1 mm DTT, 0.5% Triton X100) three times at 4 C and then washed with wash buffer 2 (50 mm Tris [ph 8.0], 100 mm NaCl, 5 mm MgCl 2, 1 mm DTT, 0.1% Triton X100) three times at 4 C. The RagAbound nucleotides were eluted with 20 µl of elution buffer (2 mm EDTA, 0.2% sodium dodecyl sulfate, 1 mm GDP, 1 mm GTP) at 68 C for 10 min. The eluted nucleotides were applied onto polyethyleneimine cellulose plates (Bakerflex) and developed in 0.75 M KH 2 PO 4 [ph 3.4] solution. GTP and GDP was visualized and quantified by a phosphoimager.

10 Amino Acids Incorporation Assay Tritiatedamino acids uptake was determined as described with modifications 2. Rheb S16H or RagA Q66L and RagC S75N expressing stable clones of HeLa cells were cultured in 12well plates. When cells reached approximately 7080% confluency, the culture medium was replaced by amino acid deprived media (DMEMKAA) for 2 h at 37 C. The medium was then replaced by 1 ml of amino acids sufficient media (DMEMK, total amino acids concentration: 2 mm) containing [ 3 H]labeled amino acids mixture (GE Health Care, TRK440) at a concentration of 2 µci/ml (~ 0.02 µm amino acid mixture) for 15 min at 37 C. Cells were washed three times with icecold PBS on ice and 250 µl of 1 M NaOH were added to lyse cells. After incubation for 10 min at 4 C, 250 µl of 1 M HCl was added to neutralize ph. Cell lysates were centrifuged at 13,200 rpm for 10 min at 4 C and the supernatant was used for protein quantification as well as for radioactivity counting.

11 Supplementary references