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• W2067109110 abstract "The TORC1 signaling pathway plays a major role in the control of cell growth and response to stress. Here we demonstrate that the SEA complex physically interacts with TORC1 and is an important regulator of its activity. During nitrogen starvation, deletions of SEA complex components lead to Tor1 kinase delocalization, defects in autophagy, and vacuolar fragmentation. TORC1 inactivation, via nitrogen deprivation or rapamycin treatment, changes cellular levels of SEA complex members. We used affinity purification and chemical cross-linking to generate the data for an integrative structure modeling approach, which produced a well-defined molecular architecture of the SEA complex and showed that the SEA complex comprises two regions that are structurally and functionally distinct. The SEA complex emerges as a platform that can coordinate both structural and enzymatic activities necessary for the effective functioning of the TORC1 pathway. The TORC1 signaling pathway plays a major role in the control of cell growth and response to stress. Here we demonstrate that the SEA complex physically interacts with TORC1 and is an important regulator of its activity. During nitrogen starvation, deletions of SEA complex components lead to Tor1 kinase delocalization, defects in autophagy, and vacuolar fragmentation. TORC1 inactivation, via nitrogen deprivation or rapamycin treatment, changes cellular levels of SEA complex members. We used affinity purification and chemical cross-linking to generate the data for an integrative structure modeling approach, which produced a well-defined molecular architecture of the SEA complex and showed that the SEA complex comprises two regions that are structurally and functionally distinct. The SEA complex emerges as a platform that can coordinate both structural and enzymatic activities necessary for the effective functioning of the TORC1 pathway. The highly conserved Target of Rapamycin Complex 1 (TORC1) 1The abbreviations used are:TORC1target of rapamycin complex 1EGOescape from rapamycin growth arrest (complex)GAPGTPase activating proteinGEFguanine nucleotide exchange factorSEASeh1 associated complexSEACATSEA subcomplex activating TORC1SEACITSEA subcomplex inhibiting TORC1. 1The abbreviations used are:TORC1target of rapamycin complex 1EGOescape from rapamycin growth arrest (complex)GAPGTPase activating proteinGEFguanine nucleotide exchange factorSEASeh1 associated complexSEACATSEA subcomplex activating TORC1SEACITSEA subcomplex inhibiting TORC1. controls eukaryotic cell growth and cellular responses to a variety of signals, including nutrients, hormones, and stresses (1Loewith R. Hall M.N. Target of rapamycin (TOR) in nutrient signaling and growth control.Genetics. 2011; 189: 1177-1201Crossref PubMed Scopus (609) Google Scholar, 2Laplante M. Sabatini D.M. mTOR signaling in growth control and disease.Cell. 2012; 149: 274-293Abstract Full Text Full Text PDF PubMed Scopus (6172) Google Scholar). In a nutrient-rich environment, TORC1 promotes anabolic processes including ribosome biogenesis and translation. Nutrient limitation or treatment with rapamycin inhibits the Tor1 kinase and initiates autophagy, a catabolic process that mediates the degradation and recycling of cytoplasmic components. However, the nutrient-sensing function of TORC1 is not fully understood, and the mechanisms of TORC1 modulation by amino acid and nitrogen availability are not yet clear. target of rapamycin complex 1 escape from rapamycin growth arrest (complex) GTPase activating protein guanine nucleotide exchange factor Seh1 associated complex SEA subcomplex activating TORC1 SEA subcomplex inhibiting TORC1. target of rapamycin complex 1 escape from rapamycin growth arrest (complex) GTPase activating protein guanine nucleotide exchange factor Seh1 associated complex SEA subcomplex activating TORC1 SEA subcomplex inhibiting TORC1. In the yeast Saccharomyces cerevisiae, the TOR1 complex is composed of four subunits (Tor1, Kog1, Tco89, and Lst8) and is localized to the vacuole membrane. Amino acid levels are signaled to TORC1 (at least partially) via the EGO complex (Ragulator-Rag in mammals), which consists of Ego1, Ego3, Gtr1 (RagA/RagB), and Gtr2 (RagC/RagD) (3Sancak Y. Peterson T.R. Shaul Y.D. Lindquist R.A. Thoreen C.C. Bar-Peled L. Sabatini D.M. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1.Science. 2008; 320: 1496-1501Crossref PubMed Scopus (1939) Google Scholar, 4Sancak Y. Bar-Peled L. Zoncu R. Markhard A.L. Nada S. Sabatini D.M. Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids.Cell. 2010; 141: 290-303Abstract Full Text Full Text PDF PubMed Scopus (1698) Google Scholar, 5Bar-Peled L. Schweitzer L.D. Zoncu R. Sabatini D.M. Ragulator is a GEF for the rag GTPases that signal amino acid levels to mTORC1.Cell. 2012; 150: 1196-1208Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar, 6Binda M. Peli-Gulli M.P. Bonfils G. Panchaud N. Urban J. Sturgill T.W. Loewith R. De Virgilio C. The Vam6 GEF controls TORC1 by activating the EGO complex.Mol. Cell. 2009; 35: 563-573Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). The small GTPases Gtr1 and Gtr2 function as heterodimers and in their active form exist as the Gtr1-GTP/Gtr2-GDP complex. Amino acid sensing via the EGO complex involves the conserved vacuolar membrane protein Vam6, a member of the HOPS tethering complex. Vam6 is a GDP exchange factor that regulates the nucleotide-binding status of Gtr1 (6Binda M. Peli-Gulli M.P. Bonfils G. Panchaud N. Urban J. Sturgill T.W. Loewith R. De Virgilio C. The Vam6 GEF controls TORC1 by activating the EGO complex.Mol. Cell. 2009; 35: 563-573Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). At the same time, the GTP-bound state of Gtr1 is controlled by a leucyl t-RNA synthetase (7Bonfils G. Jaquenoud M. Bontron S. Ostrowicz C. Ungermann C. De Virgilio C. Leucyl-tRNA synthetase controls TORC1 via the EGO complex.Mol. Cell. 2012; 46: 105-110Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). In mammals, amino acids promote interaction of Ragulator-Rag with mTORC1 and its translocation to the lysosomal membrane (3Sancak Y. Peterson T.R. Shaul Y.D. Lindquist R.A. Thoreen C.C. Bar-Peled L. Sabatini D.M. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1.Science. 2008; 320: 1496-1501Crossref PubMed Scopus (1939) Google Scholar, 4Sancak Y. Bar-Peled L. Zoncu R. Markhard A.L. Nada S. Sabatini D.M. Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids.Cell. 2010; 141: 290-303Abstract Full Text Full Text PDF PubMed Scopus (1698) Google Scholar). Ragulator interacts with the v-ATPase complex at the lysosomal membrane (8Zoncu R. Bar-Peled L. Efeyan A. Wang S. Sancak Y. Sabatini D.M. mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H(+)-ATPase.Science. 2011; 334: 678-683Crossref PubMed Scopus (1157) Google Scholar), and leucyl t-RNA synthetase binds to RagD to activate mTORC1 (9Han J.M. Jeong S.J. Park M.C. Kim G. Kwon N.H. Kim H.K. Ha S.H. Ryu S.H. Kim S. Leucyl-tRNA synthetase is an intracellular leucine sensor for the mTORC1-signaling pathway.Cell. 2012; 149: 410-424Abstract Full Text Full Text PDF PubMed Scopus (586) Google Scholar). A genome-wide screen for TORC1 regulators in yeast identified two proteins, Npr2 and Npr3, as proteins that mediate amino acid starvation signal to TORC1 (10Neklesa T.K. Davis R.W. A genome-wide screen for regulators of TORC1 in response to amino acid starvation reveals a conserved Npr2/3 complex.PLoS Genet. 2009; 5: e1000515Crossref PubMed Scopus (111) Google Scholar). Npr2 and Npr3 are both members of the SEA complex that we discovered recently (11Dokudovskaya S. Waharte F. Schlessinger A. Pieper U. Devos D.P. Cristea I.M. Williams R. Salamero J. Chait B.T. Sali A. Field M.C. Rout M.P. Dargemont C. A conserved coatomer-related complex containing Sec13 and Seh1 dynamically associates with the vacuole in Saccharomyces cerevisiae.Mol. Cell. Proteomics. 2011; 10 (M110.006478)Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 12Dokudovskaya S. Rout M.P. A novel coatomer-related SEA complex dynamically associates with the vacuole in yeast and is implicated in the response to nitrogen starvation.Autophagy. 2011; 7: 1392-1393Crossref PubMed Scopus (16) Google Scholar, 13Algret R. Dokudovskaya S. The SEA complex—the beginning.Biopolymers Cell. 2012; 28: 281-284Crossref Scopus (5) Google Scholar). Besides Npr2 and Npr3, the SEA complex also contains four previously uncharacterized proteins (Sea1–Sea4) and two proteins also found in the nuclear pore complex, Seh1 and Sec13, the latter of which is additionally a component of the endoplasmic-reticulum-associated COPII coated vesicle. However, the SEA complex localizes to the vacuole membrane, and not to the nuclear pore complex or endoplasmic reticulum. The Sea proteins contain numerous structural elements present in intracellular structural trafficking complexes (11Dokudovskaya S. Waharte F. Schlessinger A. Pieper U. Devos D.P. Cristea I.M. Williams R. Salamero J. Chait B.T. Sali A. Field M.C. Rout M.P. Dargemont C. A conserved coatomer-related complex containing Sec13 and Seh1 dynamically associates with the vacuole in Saccharomyces cerevisiae.Mol. Cell. Proteomics. 2011; 10 (M110.006478)Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). For example, proteins Sea2–Sea4 are predicted to possess β-propeller/α-solenoid folds and contain RING domains, architectural combinations characteristic to protein complexes that form coats around membranes (e.g. coated vesicles, nuclear pore complexes) or participate in membrane tethering (e.g. HOPS, CORVET complexes). Npr2 and Npr3 possess a longin domain, found in many guanine nucleotide exchange factors (GEFs) (14Nookala R.K. Langemeyer L. Pacitto A. Ochoa-Montano B. Donaldson J.C. Blaszczyk B.K. Chirgadze D.Y. Barr F.A. Bazan J.F. Blundell T.L. Crystal structure of folliculin reveals a hidDENN function in genetically inherited renal cancer.Open Biol. 2012; 2: 120071Crossref PubMed Scopus (86) Google Scholar, 15Zhang D. Iyer L.M. He F. Aravind L. Discovery of novel DENN proteins: implications for the evolution of eukaryotic intracellular membrane structures and human disease.Front. Genet. 2012; 3: 283Crossref PubMed Scopus (192) Google Scholar, 16Levine T.P. Daniels R.D. Wong L.H. Gatta A.T. Gerondopoulos A. Barr F.A. Discovery of new Longin and Roadblock domains that form platforms for small GTPases in Ragulator and TRAPP-II.Small GTPases. 2013; 4: 1-8Crossref PubMed Scopus (66) Google Scholar), and Sea1/Iml1 is a GTPase activating protein (GAP) for Gtr1 (17Panchaud N. Peli-Gulli M.P. De Virgilio C. Amino acid deprivation inhibits TORC1 through a GTPase-activating protein complex for the Rag family GTPase Gtr1.Sci. Signal. 2013; 6: ra42Crossref PubMed Scopus (200) Google Scholar). These structural characteristics, taken together with functional data, indicate a role for the SEA complex in intracellular trafficking, amino acid biogenesis, regulation of the TORC1 pathway, and autophagy (11Dokudovskaya S. Waharte F. Schlessinger A. Pieper U. Devos D.P. Cristea I.M. Williams R. Salamero J. Chait B.T. Sali A. Field M.C. Rout M.P. Dargemont C. A conserved coatomer-related complex containing Sec13 and Seh1 dynamically associates with the vacuole in Saccharomyces cerevisiae.Mol. Cell. Proteomics. 2011; 10 (M110.006478)Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 12Dokudovskaya S. Rout M.P. A novel coatomer-related SEA complex dynamically associates with the vacuole in yeast and is implicated in the response to nitrogen starvation.Autophagy. 2011; 7: 1392-1393Crossref PubMed Scopus (16) Google Scholar, 13Algret R. Dokudovskaya S. The SEA complex—the beginning.Biopolymers Cell. 2012; 28: 281-284Crossref Scopus (5) Google Scholar, 17Panchaud N. Peli-Gulli M.P. De Virgilio C. Amino acid deprivation inhibits TORC1 through a GTPase-activating protein complex for the Rag family GTPase Gtr1.Sci. Signal. 2013; 6: ra42Crossref PubMed Scopus (200) Google Scholar, 18Graef M. Nunnari J. Mitochondria regulate autophagy by conserved signalling pathways.EMBO J. 2011; 30: 2101-2114Crossref PubMed Scopus (149) Google Scholar, 19Panchaud N. Peli-Gulli M.P. De Virgilio C. SEACing the GAP that nEGOCiates TORC1 activation: evolutionary conservation of Rag GTPase regulation.Cell Cycle. 2013; 12: 1-5Crossref PubMed Scopus (80) Google Scholar, 20Wu X. Tu B.P. Selective regulation of autophagy by the Iml1-Npr2-Npr3 complex in the absence of nitrogen starvation.Mol. Biol. Cell. 2011; 22: 4124-4133Crossref PubMed Scopus (65) Google Scholar). A mammalian analog of the SEA complex, termed GATOR1/GATOR2, has recently been identified (21Bar-Peled L. Chantranupong L. Cherniack A.D. Chen W.W. Ottina K.A. Grabiner B.C. Spear E.D. Carter S.L. Meyerson M. Sabatini D.M. A tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1.Science. 2013; 340: 1100-1106Crossref PubMed Scopus (706) Google Scholar). GATORS are localized at the lysosome membrane and serve as upstream regulators of mammalian TORC1 via GATOR1 GAP activity toward RagA and RagB (21Bar-Peled L. Chantranupong L. Cherniack A.D. Chen W.W. Ottina K.A. Grabiner B.C. Spear E.D. Carter S.L. Meyerson M. Sabatini D.M. A tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1.Science. 2013; 340: 1100-1106Crossref PubMed Scopus (706) Google Scholar). In this study, we characterized the structural and functional organization of the yeast SEA complex. We present here a well-defined molecular architecture of the SEA complex obtained via an integrative modeling approach based on a variety of biochemical data. The structure reveals the relative positions and orientations of two SEA subcomplexes, Sea1/Npr2/Npr3 (or SEACIT (19Panchaud N. Peli-Gulli M.P. De Virgilio C. SEACing the GAP that nEGOCiates TORC1 activation: evolutionary conservation of Rag GTPase regulation.Cell Cycle. 2013; 12: 1-5Crossref PubMed Scopus (80) Google Scholar)) and Sea2/Sea3/Sea4/Sec13/Seh1 (or SEACAT (19Panchaud N. Peli-Gulli M.P. De Virgilio C. SEACing the GAP that nEGOCiates TORC1 activation: evolutionary conservation of Rag GTPase regulation.Cell Cycle. 2013; 12: 1-5Crossref PubMed Scopus (80) Google Scholar)), and identifies the Sea3/Sec13 dimer as a major interacting hub within the complex. We describe how the SEA complex interacts physically with TORC1 and the vacuole and is required for the relocalization of Tor1, and how every member of the Sea1/Npr2/Npr3 subcomplex is required for general autophagy. The following materials were used in this study: Dynabeads M-270 Epoxy (Invitrogen/LifeTechnologies, 143.02D), rabbit IgG (Sigma, 15006), protease inhibitor mixture (Sigma, P-8340), disuccinimidyl suberate (Creative Molecules, 001S), HRP-mouse IgG (Jackson ImmunoResearch Laboratories West Grove, PA), anti-GFP antibody (Roche, 11814460001), anti-PGK1 antibody (Sigma, 459250), and concanavalin A (Sigma, C7275). Yeast strains used in this study are listed in supplemental Table S1. Yeast were grown to mid-log phase in Wickerham media for immunoprecipitation experiments (0.3% Bacto malt extract, 0.3% yeast extract, 0.5% Bacto Peptone, and 1% glucose), in yeast synthetic complete media for imaging (0.67% yeast nitrogen base without amino acids and carbohydrates, 0.2% complete drop-out mix, and 2% glucose), and in YPD (2% Bacto-Peptone, 1% yeast extract, and 2% glucose) or an appropriate drop-out media for all other purposes. Starvation experiments were conducted in synthetic media lacking nitrogen (0,17% yeast nitrogen base without ammonium and amino acids, 2% glucose). Three types of SEA members were used for immunopurifications: (i) PrA tagged proteins expressed in the wild-type background; (ii) PrA tagged proteins expressed in cells where a gene of another component of the SEA complex was deleted; and (iii) PrA tagged C-terminal truncations. Points of C-terminal truncations for SEA proteins were selected based on the secondary structure prediction and PAL data (11Dokudovskaya S. Waharte F. Schlessinger A. Pieper U. Devos D.P. Cristea I.M. Williams R. Salamero J. Chait B.T. Sali A. Field M.C. Rout M.P. Dargemont C. A conserved coatomer-related complex containing Sec13 and Seh1 dynamically associates with the vacuole in Saccharomyces cerevisiae.Mol. Cell. Proteomics. 2011; 10 (M110.006478)Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The C-terminal deletions carried a human rhinovirus 3C protease site (GLEVLFQGPS) between a SEA protein and PrA tag and were constructed essentially as described in Ref. 22Fernandez-Martinez J. Phillips J. Sekedat M.D. Diaz-Avalos R. Velazquez-Muriel J. Franke J.D. Williams R. Stokes D.L. Chait B.T. Sali A. Rout M.P. Structure-function mapping of a heptameric module in the nuclear pore complex.J. Cell Biol. 2012; 196: 419-434Crossref PubMed Scopus (90) Google Scholar. Affinity purifications of SEA complex protein complexes from whole cell lysates using magnetic beads were performed as described previously (11Dokudovskaya S. Waharte F. Schlessinger A. Pieper U. Devos D.P. Cristea I.M. Williams R. Salamero J. Chait B.T. Sali A. Field M.C. Rout M.P. Dargemont C. A conserved coatomer-related complex containing Sec13 and Seh1 dynamically associates with the vacuole in Saccharomyces cerevisiae.Mol. Cell. Proteomics. 2011; 10 (M110.006478)Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The extraction and washing buffers used for immunoprecipitations are listed in supplemental Table S2. Protein bands appearing after Coomassie staining were cut from the gel. Gel bands were washed first with 100 μl of 25 mm ammonium bicarbonate (Sigma, 11204)/acetonitrile (Sigma, 34967) 50/50 v/v over 10 min at room temperature and then with 100 μl of 100% acetonitrile for 10 min at room temperature. These washes were repeated twice. Samples were dried in a SpeedVac for 2 min; then 20 μl of 11.55 ng/μl trypsin (Calbiochem, 650279) was added to each gel piece, and gels were incubated at room temperature for 15 min. Samples were further incubated overnight at 37 °C with 20 μl of 50 mm ammonium bicarbonate. Supernatants were separated from gel pieces and transferred to analysis vials. 20 μl of 5% formic acid (Sigma, 33015)/acetonitrile 30/70 v/v was added to each piece of a gel to extract remaining peptides. Supernatants were combined together and dried in a SpeedVac. 10 μl of 3% acetonitrile, 0.1% formic acid solution in water was added to solubilize peptides. The peptide mixture obtained from tryptic digestion of gel bands was analyzed via nano-HPLC (Agilent Technologies 1200, Santa Clara, CA) directly coupled to an ion trap mass spectrometer (Bruker 6300) equipped with a nano-electrospray source. 4 μl of peptide mixture were separated on a ProtID-Chip-43 II 300A C18 43-mm column (Agilent Technologies, G4240–62005) with a 3% to 97% acetonitrile gradient over 30 min. The acquisition was performed as follows: one full MS scan over the range of 200–2200 m/z, followed by three data-dependent MS/MS scans on the three most abundant ions in the full scan. The data were analyzed using Spectrum Mill MS Proteomics Workbench Rev A.03.03.084 SR4, with the following settings: Data Extractor, MH+ 200 to 4400 Da; scan range, 0 to 30 min; MS/MS search, Swiss-Prot database; S. cerevisiae; trypsin; two missed cleavages; oxidized methionine (M), phosphorylated S, T, Y: monoisotopic masses; cutoff score/expectation value for accepting individual MS/MS spectra, 17; precursor mass tolerance, ±2 Da; and product mass tolerance, ±0.8 Da. The lists of putative proteins were obtained by searching against the Swiss-Prot protein database (updated weekly; last version used for the analysis was from October 15, 2013), and the number of protein entries was 526,969. 5 to 20 g of cryo-grindate obtained from ySD227 strain (Sea1-ppx-PrA, supplemental Table S1) were used for immunopurification of native SEA complex. 20 mm K/HEPES, pH 7.4, 110 mm KOAc, 300 mm NaCl, 0.1% CHAPS, 2 mm MgCl2, 1 mm DTT, 1/500 protease inhibitors was used as extraction and washing buffer. The complex was released from magnetic beads by protease digestion through incubation with 1 μg of protease per 1 μg of complex in extraction buffer (without protease inhibitors) for 1 h at 4 °C. The recovered sample was centrifuged at 20,000g for 10 min. 100 to 150 μl of supernatant was loaded on top of a 5%–20% sucrose gradient in a buffer containing 20 mm K/HEPES, pH 7.4, 110 mm KOAc, 150 mm NaCl, 0.01% CHAPS, 0.2 mm MgCl2, 0.1 mm DTT, 1/1000 protease inhibitors. Gradients were centrifuged on an SW 55 Ti rotor (Beckman Coulter) at 35,000 rpm and 5 °C for 6 h. Gradients were manually unloaded from the top in 12 fractions of 410 μl. Fractions were precipitated using 90% methanol. Pellets were resuspended in protein loading buffer, and the proteins were separated in 4–12% Bis-Tris gels (Novex/LifeTechnologies, Grand Island, NY) and visualized with Coomassie stain. For stoichiometry, quantification gels were stained with SYPRO Ruby (Molecular Probes/Life Technologies, Grand Island, NY) and visualized on an LAS-3000 system (linear detection range; Fujifilm). The SEA complex protein band intensities were measured using ImageJ software (National Institutes of Health), with values normalized for protein molecular weight. The copy number of SEA members was calculated as relative to Sea1 (the handle used for the affinity purification). As a control, the same procedure was applied to affinity-purified Nup84 complex samples showing the expected 1:1 stoichiometry for all Nup84 complex members (22Fernandez-Martinez J. Phillips J. Sekedat M.D. Diaz-Avalos R. Velazquez-Muriel J. Franke J.D. Williams R. Stokes D.L. Chait B.T. Sali A. Rout M.P. Structure-function mapping of a heptameric module in the nuclear pore complex.J. Cell Biol. 2012; 196: 419-434Crossref PubMed Scopus (90) Google Scholar). ∼10 to 20 μg of the SEA complex purified from ySD227 strain were cleaved off from the affinity beads via protease treatment (see above) and eluted in 250 μl of elution buffer. The complex was cross-linked by incubation with 0.1 mm disuccinimidyl suberate at room temperature for 30 min with constant agitation at 750 rpm and quenched by the addition of ammonium bicarbonate at a final concentration of 50 mm. The cross-linked complex was subsequently reduced with 5 mm tris(2-carboxyethyl)phosphine and alkylated in the dark with 20 mm iodoacetamide for 20 min. After cross-linking with disuccinimidyl suberate, the SEA complex was digested either in-solution or in-gel with trypsin to identify the cross-linked peptides. For in-solution digestion, ∼20 μg of purified complex was digested with 1 μg of trypsin (Promega) in 1 m urea and ∼2% acetonitrile at 37 °C. After 12 to 16 h of incubation, an additional 0.5 μg of trypsin was added to the digest, which was then incubated for an additional 4 h. The resulting proteolytic peptide mixture was purified using a C18 cartridge (Sep-Pak, Waters, Milford, MA), lyophilized, and fractionated via peptide size exclusion chromatography (23Leitner A. Reischl R. Walzthoeni T. Herzog F. Bohn S. Forster F. Aebersold R. Expanding the chemical cross-linking toolbox by the use of multiple proteases and enrichment by size exclusion chromatography.Mol. Cell. Proteomics. 2012; 11 (M111.014126)Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). For in-gel digestion, ∼10 μg of purified complex was precipitated by methanol, resuspended, and heated at 95 °C in 1× SDS loading buffer. The sample was cooled to room temperature for cysteine alkylation and separated via electrophoresis in a 4–12% SDS-PAGE gel. The gel region above ∼160 kDa was sliced, crushed into small pieces, and digested in-gel by trypsin. After extraction and purification, the resulting proteolytic peptide mixture was dissolved in 20 μl of a solution containing 30% acetonitrile and 0.2% formic acid and fractionated via peptide size exclusion chromatography (Superdex Peptide PC 3.2/30, GE Healthcare) using off-line HPLC separation with an autosampler (Agilent Technologies). Three size exclusion chromatography fractions in the molecular mass range of ∼2.5 kDa to 8 kDa were collected and analyzed via LC/MS. Purified peptides were dissolved in the sample loading buffer (5% MeOH, 0.2% formic acid) and loaded onto a self-packed PicoFrit® column with an integrated electrospray ionization emitter tip (360 outer diameter, 75 inner diameter, 15-μm tip; New Objective, Woburn, MA). The column was packed with 5 cm of reverse-phase C18 material (3-μm porous silica, 200-Å pore size, Dr. Maisch GmbH, Ammerbuch-Entrigen, Germany). Mobile phase A consisted of 0.5% acetic acid, and mobile phase B of 70% acetonitrile with 0.5% acetic acid. The peptides were eluted in a 150-min LC gradient (8% B to 46% B, 0–118 min; 46% B to 100% B, 118–139 min; equilibrated with 100% A until 150 min) using an HPLC system (Agilent Technologies) and analyzed with an LTQ Velos Orbitrap Pro mass spectrometer (Thermo Fisher). The flow rate was ∼200 nl/min. The electron-spray voltage was set at 1.7–2.2 kV. The capillary temperature was 275 °C, and ion transmission on Velos S lenses was set at 35%. The instrument was operated in the data-dependent mode, where the top eight most abundant ions were fragmented by means of higher energy collisional dissociation (24Olsen J.V. Macek B. Lange O. Makarov A. Horning S. Mann M. Higher-energy C-trap dissociation for peptide modification analysis.Nat. Methods. 2007; 4: 709-712Crossref PubMed Scopus (720) Google Scholar) (dissociation energy 27–33, 0.1-ms activation time) and analyzed in the Orbitrap mass analyzer. The target resolution for MS1 was 60,000, and that for MS2 was 7500. Ions (370–1700 m/z) with charge states of ≥3 were selected for fragmentation. A dynamic exclusion of 15/2/55 s was used. Other instrumental parameters included the following: lock mass at 371.1012 Da, monoisotopic mass selection off, minimal threshold of 5000 to trigger an MS/MS event, and ion trap accumulation limits of 105 and 106, respectively, for the linear ion trap and Orbitrap. The maximum ion injection times for the LTQ and Orbitrap were, respectively, 100 ms and 500 ms. The raw data were transformed to Mascot generic format and searched by pLink software (25Yang B. Wu Y.J. Zhu M. Fan S.B. Lin J. Zhang K. Li S. Chi H. Li Y.X. Chen H.F. Luo S.K. Ding Y.H. Wang L.H. Hao Z. Xiu L.Y. Chen S. Ye K. He S.M. Dong M.Q. Identification of cross-linked peptides from complex samples.Nat. Methods. 2012; 9: 904-906Crossref PubMed Scopus (406) Google Scholar) with a database containing sequences of the eight protein subunits of SEA complex together with BSA. Other search parameters included mass accuracy of MS1 ≤ 10 ppm and MS2 ≤ 20 ppm for the initial database search, cysteine carboxymethylation as a fixed modification, methionine oxidation as a variable modification, and a maximum of two trypsin miscleavages allowed. The results were filtered at a 5% false discovery rate, which led to a total of 295 unique cross-linked peptides. We noticed that false discovery rate estimation of cross-linking might not have been accurate (even with a high mass accuracy measurement), presumably because of the greatly expanded database search space and the fact that many cross-linked peptides are of low abundance, leading their identification to be complicated by other low-abundant peptide species resulting from, for example, trypsin miscleavages, nonspecific cleavages, chemically modified peptides, or combinations of these. We treated the 5% false discovery rate (a default parameter of the pLink software) as a rough initial filter of the raw data (albeit quite permissive). Next, we applied additional stringent filters (including high mass accuracy, large enough individual chain lengths, and extensive fragmentation information) to remove potential false positive identifications from our dataset. We applied the following criteria for verification of cross-linked peptides: (i) only identifications with mass accuracy ≤ 5 ppm for MS1 and ≤ 10 ppm at MS/MS were considered, as 94% of the identifications have an MS1 mass accuracy of ≤±2 ppm; and (ii) for positive identifications, both peptide chains had to contain at least four amino acids (in 97% of the identifications, each peptide chain contained at least five amino acids). For both peptide chains, the major MS/MS fragmentation peaks had to be assigned and follow a pattern that contained a continuous stretch of fragmentations. The appearance of dominant fragment ions N-terminal to proline and C-terminal to aspartic acid and glutamic acid for arginine-containing peptides was generally expected (26Qin J. Chait B.T. Preferential fragmentation of protonated gas-phase peptide ions adjacent to acidic amino-acid-residues.J. Am. Chem. Soc. 1995; 117: 5411-5412Crossref Scopus (141) Google Scholar, 27Michalski A. Neuhauser N. Cox J. Mann M. A systematic investigation into the nature of tryptic HCD spectra.J. Proteome Res. 2012; 11: 5479-5491Crossref PubMed Scopus (82) Google Scholar). 188 of the high-confidence cross-linked peptides (selected from 295 of 5% false discovery rate–filtered cross-links) passed these criteria and were used as restraints for determination of the SEA complex architecture. Thus, we did not allow uncertainty of the cross-linking data for our integrative modeling approach. Our integrative approach to determining the SEA complex structure proceeds through four stages (22Fernandez-Martinez J. Phillips J. Sekedat M.D. Diaz-Avalos R. Velazquez-Muriel J. Franke J.D. Williams R." @default.
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• W2067109110 date "2014-11-01" @default.
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• W2067109110 title "Molecular Architecture and Function of the SEA Complex, a Modulator of the TORC1 Pathway" @default.
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• W2067109110 doi "https://doi.org/10.1074/mcp.m114.039388" @default.
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