Reagents were obtained from the following sources: antibodies to ATP6V1B2 and LAMP2 from Abcam; antibodies to phospho-T389 S6K1, S6K1, RagA, RagC, p14, p18, MP1, C7orf59, HBXIP mTOR, phospho-T398 dS6K, and the FLAG epitope from Cell Signaling Technology; HRP-labeled anti-mouse, and anti-rabbit secondary antibodies from Santa Cruz Biotechnology; antibody to the HA tag from Bethyl laboratories; RPMI, FLAG M2 affinity gel, GTPγS, GDP, Chaps, Triton, and amino acids from Sigma Aldrich; [3H]GDP and [35S]GTPγS from Perkin Elmer; protein G-sepharose and immobilized glutathione beads from Pierce; FuGENE 6 and Complete Protease Cocktail from Roche; Alexa 488 and 568-conjugated secondary antibodies, Schneider's media, Express Five Drosophila-SFM, and Inactivated Fetal Calf Serum (IFS) from Invitrogen; amino acid-free RPMI, and amino acid free Schneider's media from US Biological; siRNAs targeting indicated genes and siRNA transfection reagent from Dharmacon; human cDNA encoding HBXIP from Open Biosystems; Concanamycin A from A. G. Scientific; nitrocellulose membrane filters from Advantec; calf-alkaline phosphatase from NEB. The dS6K antibody was a generous gift from Mary Stewart (North Dakota State University). Salicylihalamide A (SalA) was a generous gift from Jeff DeBrabander (UT Southwestern).
Cell lysis and immunoprecipitation
Cells were rinsed once with ice-cold PBS and lysed with Chaps lysis buffer (0.3% Chaps, 10 mM β-glycerol phosphate, 10 mM pyrophosphate, 40 mM Hepes pH 7.4, 2.5 mM MgCl2
and 1 tablet of EDTA-free protease inhibitor [Roche] per 25 ml). Where specified in the figures, Chaps lysis buffer was supplemented with 12.5 mM EDTA. When only cell lysates were required (i.e., no immunoprecipitation was to be performed), 1% Triton X-100 was substituted for Chaps. When the interaction between Ragulator and mTORC1 was interrogated, in cell cross-linking with DSP was preformed as described in (Sancak et al., 2008
) prior to cell lysis. The soluble fractions of cell lysates were isolated by centrifugation at 13,000 rpm in a microcentrifuge for 10 minutes. For immunoprecipitations, primary antibodies were added to the cleared lysates and incubated with rotation for 1.5 hours at 4°C. 60 μl of a 50% slurry of protein G-sepharose was then added and the incubation continued for an additional 1 hour. Immunoprecipitates were washed three times with lysis buffer containing 150 mM NaCl. Immunoprecipitated proteins were denatured by the addition of 20 μl of sample buffer and boiling for 5 minutes, resolved by 8%–16% SDS-PAGE, and analyzed by immunoblotting as described (Kim et al., 2002
). For anti-FLAG-immunoprecipitations, the FLAG-M2 affinity gel was washed with lysis buffer 3 times. 20 μl of a 50% slurry of the affinity gel was then added to cleared cell lysates and incubated with rotation for 2 hours at 4°C. The beads were washed 3 times with lysis buffer containing 150 mM NaCl. Immunoprecipitated proteins were denatured by the addition of 50 μl of sample buffer and boiling for 5 minutes.
For co-transfection experiments, 2,000,000 HEK293T cells were plated in 10 cm culture dishes. Twenty-four hours later, cells were transfected with the pRK5-based cDNA expression plasmids indicated in the figures in the following amounts: 100 ng or 1000 ng FLAG- or HA-HBXIP; 100 ng or 1000 ng FLAG- or HA-HBXIP; 100 ng or 1000 ng FLAG-p14; 100 ng HA-MP1; 100 ng or 1000 ng FLAG- or HA-p18; 100 ng or 1000 ng FLAG-Rap2a; 300 ng Flag-Metap2; 300 ng Flag-VPS39; 100 ng Flag- or HA-RagB and 100 ng HA- or HA-GST-RagC. The total amount of plasmid DNA in each transfection was normalized to 2 μg with empty pRK5. Thirty-six hours after transfection, cells were lysed as described above.
Identification of HBXIP and C7orf59
Immunoprecipitates from HEK-293T cells stably expressing FLAG-p18, FLAG-p14, FLAG-RagB or FLAG-Metap2 were prepared using Chaps lysis buffer as described above. Proteins were eluted with the FLAG peptide (sequence DYKDDDDK) from the FLAG-M2 affinity gel, resolved on 4-12% NuPage gels (Invitrogen), and stained with simply blue stain (Invitrogen). Each gel lane was sliced into 10-12 pieces and the proteins in each gel slice digested overnight with trypsin. The resulting digests were analyzed by mass spectrometry as described (Sancak et al., 2008
). Peptides corresponding to HBXIP and C7orf59 were identified in the FLAG-p14, FLAG-p18 and FLAG-RagB immunoprecipitates, while no peptides were detected in negative control immunoprecipitates of FLAG-Metap2.
Total amino acid starvation and stimulation, leucine starvation and stimulation, Salicylihalamide A and Concanamycin A treatment
HEK-293T cells in culture dishes or coated glass cover slips were rinsed with and incubated in amino acid-free RPMI or leucine-free RPMI for either 50 minutes or 2 hours, and stimulated with a 10X mixture of total amino acids or 10X leucine for 10-20 minutes, respectively. After stimulation, the final concentration of amino acids in the media was the same as in RPMI. The 10X mixture of total amino acids was prepared from individual powders of amino acids. Where drug treatment was performed, cells were incubated with 2.5 μm of Salicylihalamide A or 2.5 μm of Concanamycin A during the 2 hour starvation period and the 15 min stimulation period.
RNAi in mammalian cells
On day one, 200,000 HEK-293T cells were plated in a 6 well plate. Twenty-fours hours later, the cells were transfected with 250 nM of a pool of siRNAs [Dharmacon] targeting HBXIP or C7orf59, a non-targeting pool, or 125 nM of siRNAs targeting p14 or p18. On day four, the cells were transfected again but this time with double the amount of siRNAs. On day five, the cells were either split onto coated glass cover slips or rinsed with ice-cold PBS, lysed and subjected to immunobloting as described above.
RNAi in Drosophila S2 cells
dsRNAs against Drosophila HBXIP
genes were designed as described in (Sancak et al., 2008
). Primer sequences used to amplify DNA templates for dsRNA synthesis for dHBXIP and, dC7orf59 including underlined 5’ and 3’ T7 promoter sequences, are as follows:
Forward primer: GAATTAATACGACTCACTATAGGGAGAGGAGAAAGTCCTAGCGGAAATC
Reverse primer: GAATTAATACGACTCACTATAGGGAGAGCTTGAAGATAACGCCTGTGAT
Forward primer: GAATTAATACGACTCACTATAGGGAGACTGATACTAAAGGAAGATGGAGCAG
Reverse primer: GAATTAATACGACTCACTATAGGGAGAGTATATTCTACGGTTGGACATGCAG
dsRNAs targeting GFP and dRagC were used as positive and negative controls, respectively. On day one, 4,000,000 S2 cells were plated in 6-cm culture dishes in 5 ml of Express Five SFM media. Cells were transfected with 1 μg of dsRNA per million cells using Fugene (Roche). Two days later, a second round of dsRNA transfection was performed. On day five, cells were rinsed once with amino acid-free Schneider's medium, and starved for amino acids by replacing the media with amino acid-free Schneider's medium for 1.5 hours. To stimulate with amino acids, the amino acid-free medium was replaced with complete Schneider's medium for 30 minutes. Cells were then washed with ice cold PBS, lysed, and subjected to immunoblotting for phospho-T398 dS6K and total dS6K.
Immunofluorescence assays were performed as described in (Sancak et al., 2010
). Briefly, 200,000 HEK-293T cells were plated on fibronectin-coated glass coverslips in 12-well tissue culture plates. Twenty-four hours later, the slides were rinsed with PBS once and fixed for 15 min with 4% paraformaldehyde in PBS at room temperature. The slides were rinsed twice with PBS and cells were permeabilized with 0.05% Triton X-100 in PBS for 5 min. After rinsing twice with PBS, the slides were incubated with primary antibody in 5% normal donkey serum for 1 hr at room temperature, rinsed four times with PBS, and incubated with secondary antibodies produced in donkey (diluted 1:1000 in 5% normal donkey serum) for 45 min at room temperature in the dark and washed four times with PBS. Slides were mounted on glass coverslips using Vectashield (Vector Laboratories) and imaged on a spinning disk confocal system (Perkin Elmer) or a Zeiss Laser Scanning Microscope (LSM) 710.
In immunofluorescence assays where HBXIP or C7orf59 were co-localized with p18, HEK-293T cells were seeded and processed as described above with the following exceptions. Immediately after seeding, cells were transfected with the following constructs (all cDNAs were expressed from pRK5 expression plasmid): 50 ng Flag-HBXIP, 50 ng HA-p14, 50 ng HA-MP1, 50 ng HA-C7orf59 and 50 ng HA-p18; or 50 ng Flag-C7orf59, 50 ng HA-p14, 50 ng HA-MP1, 50 ng HA-HBXIP and 50 ng HA-p18. The cells were processed the following day.
Cell size determinations
For measurements of cell size, HEK-293T cells treated with siRNAs as described above were harvested by trypsinization in a 4 ml volume and diluted 1:20 with counting solution (Isoton II Diluent, Beckman Coulter). Cell diameters were determined with a particle size counter (Coulter Z2, Beckman Coulter) running Coulter Z2 AccuComp software.
Protein purification of recombinant Rag heterodimers and Ragulator
To produce protein complexes used for GEF or in vitro binding assays, 4,000,000 HEK-293T cells were plated in 15 cm culture dishes. Forty-eight hours later, cells were transfected separately with the following constructs (all cDNAs were expressed from pRK5 expression plasmid). For pentameric Ragulator: 4 μg Flag-p14, 8 μg HA-MP1, 8 μg HA-p18G2A (a lipidation defective mutant), 8 μg HA-HBXIP, and 8 μg HA-C7orf59. For trimeric Ragulator complexes: 8 μg FLAG-p14, 16 μg HA-MP1 and 16 μg HA-p18G2A; or 8 μg FLAG-HBXIP, 16 μg HA-C7orf59 and 16 μg HA-p18G2A. For dimeric complexes: 8 μg FLAG-p14 and 16 μg HA-MP1; 8 μg FLAG-HBXIP and 16 μg HA-C7orf59; 8 μg of HA-GST-p14 and 16 μg of MP1; 8 μg FLAG-RagBD163N and 16 μg HA-RagC; 8 μg FLAG-RagCD181N and 16 μg HA-RagB; or 8 μg FLAG-RagB and 16 μg HA-RagC. For individual proteins: 10 μg Flag-p18G2A; 10 μg Flag-Metap2; 15 μg Flag-VPS39; 10 μg HA-GST-HBXIP, 10 μg HA-GST-C7orf59; or 10 μg HAGST-Rap2a
Thirty-six hours post transfection cell lysates were prepared as described above and either 200 μl of a 50% slurry of glutathione affinity beads or 200 μl of a 50% slurry of FLAG-M2 affinity gel were added to lysates from cells expressing HA-GST-tagged or FLAG-tagged proteins, respectively. Recombinant proteins were immunoprecipitated for 3 hours at 4°C. Each sample was washed once with Triton lysis buffer, followed by 3 washes with Triton lysis buffer supplemented with 500 mM NaCl. Samples containing FLAG-tagged proteins were eluted from the FLAG-M2 affinity gel with a competing FLAG peptide as described above.
In vitro binding assays
For the binding reactions, 20 μl of a 50% slurry containing immobilized HA-GST-tagged proteins were incubated in binding buffer (1% Triton X-100, 2.5mM MgCl2, 40 mM Hepes pH 7.4, 2 mM DTT and 1mg/ml BSA) with 2 μg of FLAG-tagged proteins in a total volume of 50 μl for 1 hour and 30 minutes at 4°C. In binding assays where HA-GST-Ragulator was used, HA-GST-p14-MP1 was pre-bound to FLAG-HBXIP-HA-C7orf59 and FLAG-p18 for 5 minutes at 4°C prior to the addition of other FLAG-tagged proteins. In experiments where the Flag-RagB-HA-RagC heterodimer was loaded with nucleotides, 2 μg of FLAG-RagB-HA-RagC was incubated at 25°C for 10 minutes in Rag loading buffer (0.3% Chaps, 40 mM Hepes pH 7.4, 5 mM EDTA, 2 mM DTT and 1 mg/ml BSA) supplemented with either 1 mM GTPγS or 1 mM GDP in a total volume of 10 μl. The Rag-nucleotide complex was stabilized by the addition of 20 mM MgCl2 and incubated for an additional 5 minutes at 25°C. In assays with nucleotide free Rags, 2 μg of FLAG-RagB-HA-RagC was added to the binding assay with 3 μl of Calf-alkaline phosphatase (NEB). Binding assays in which Ragulator was incubated with nucleotide-loaded or -free Rags were conducted at 4°C for 45 minutes. For the nucleotide competition assay, 2 μg FLAG-RagB-HA-RagC was pre-bound to Ragulator proteins for 30 minutes followed by the addition of 1 mM GTPγS and further incubated for 1 hour and 30 minutes at 4°C. To terminate all binding assays, samples were washed 3 times with 1 ml of ice-cold binding buffer supplemented with 150 mM NaCl followed by the addition of 50 μl of sample buffer.
Nucleotide exchange (GEF) assays
40 pmols of FLAG-RagBD163N-HA-RagC, FLAG-RagCD181N-HA-RagB or FLAG-Rap2a were loaded with either 2 μM of [3H]GDP (25-50 Ci/mmol), 10 μCi of [35S]GTPγS (1250 Ci/mmol), 2 mM GDP (for GTP binding assays), or co-loaded with guanine nucleotides and either 50 nM of XTPγS or 50 nM XDP (Ragulator GEF activity was maintained between a range of 5-500 nM xanthine nucleotide) in a total volume of 100 μl of Rag loading buffer as described above. The GTPase-[3H]GDP-XDP/ XTPγS or GTPase-[35S]GTPγS-XDP/ XTPγS and GTPase-GDP complexes were stabilized by addition of 20 mM MgCl2 followed by a further incubation at 4°C for 12 hours or 25°C for 5 minutes, respectively. To initiate the GEF assay, 40 pmols of pentameric Ragulator, the indicated Ragulator subcomplexes or a control (FLAG-Metap2, FLAG-VPS39, or FLAG-HBXIP-HA-C7orf59) were added along with 200 μM GTPγS or 5 μCi of [35S]GTPγS (for GTP binding assays) and incubated at 25°C. Samples were taken every 2 minutes and spotted on nitrocellulose filters, which were washed with 2 ml of wash buffer (40 mM Hepes pH 7.4, 150 mM NaCl and 5 mM MgCl2). Filter-associated radioactivity was measured using a TriCarb scintillation counter (Perkin Elmer).
Monomeric Rag GDP loading
40 pmols of FLAG-RagB, FLAG-RagBS54N, FLAG-RagBD163N, FLAG-RagC, FLAG-RagCT75N or FLAG-RagCD181N were loaded with 2 μM of [3H]GDP as described for Rag heterodimers, but MgCl2 stabilization lasted for 5 min at 25°C. The amount of [3H]GDP bound to monomeric Rags was determined with a filter binding assay and was normalized to [3H]GDP binding by wild type RagB or RagC.