A Conserved Coatomer-related Complex Containing Sec13 and Seh1 Dynamically Associates With the Vacuole in Saccharomyces cerevisiae
2011; Elsevier BV; Volume: 10; Issue: 6 Linguagem: Inglês
10.1074/mcp.m110.006478
ISSN1535-9484
AutoresSvetlana Dokudovskaya, François Waharte, Avner Schlessinger, Ursula Pieper, Damien P. Devos, Ileana M. Cristea, Rosemary Williams, Jean Salamero, Brian T. Chait, Andrej Šali, Mark C. Field, Michael P. Rout, Catherine Dargemont,
Tópico(s)Nuclear Structure and Function
ResumoThe presence of multiple membrane-bound intracellular compartments is a major feature of eukaryotic cells. Many of the proteins required for formation and maintenance of these compartments share an evolutionary history. Here, we identify the SEA (Seh1-associated) protein complex in yeast that contains the nucleoporin Seh1 and Sec13, the latter subunit of both the nuclear pore complex and the COPII coating complex. The SEA complex also contains Npr2 and Npr3 proteins (upstream regulators of TORC1 kinase) and four previously uncharacterized proteins (Sea1–Sea4). Combined computational and biochemical approaches indicate that the SEA complex proteins possess structural characteristics similar to the membrane coating complexes COPI, COPII, the nuclear pore complex, and, in particular, the related Vps class C vesicle tethering complexes HOPS and CORVET. The SEA complex dynamically associates with the vacuole in vivo. Genetic assays indicate a role for the SEA complex in intracellular trafficking, amino acid biogenesis, and response to nitrogen starvation. These data demonstrate that the SEA complex is an additional member of a family of membrane coating and vesicle tethering assemblies, extending the repertoire of protocoatomer-related complexes. The presence of multiple membrane-bound intracellular compartments is a major feature of eukaryotic cells. Many of the proteins required for formation and maintenance of these compartments share an evolutionary history. Here, we identify the SEA (Seh1-associated) protein complex in yeast that contains the nucleoporin Seh1 and Sec13, the latter subunit of both the nuclear pore complex and the COPII coating complex. The SEA complex also contains Npr2 and Npr3 proteins (upstream regulators of TORC1 kinase) and four previously uncharacterized proteins (Sea1–Sea4). Combined computational and biochemical approaches indicate that the SEA complex proteins possess structural characteristics similar to the membrane coating complexes COPI, COPII, the nuclear pore complex, and, in particular, the related Vps class C vesicle tethering complexes HOPS and CORVET. The SEA complex dynamically associates with the vacuole in vivo. Genetic assays indicate a role for the SEA complex in intracellular trafficking, amino acid biogenesis, and response to nitrogen starvation. These data demonstrate that the SEA complex is an additional member of a family of membrane coating and vesicle tethering assemblies, extending the repertoire of protocoatomer-related complexes. A hallmark of eukaryotic cells is the presence of distinctive internal membrane compartments, dynamically connected via selective transport pathways. Various intracellular transport complexes regulate exchange of material between these compartments and maintain their distinct composition. Recent analyses have suggested that the last common eukaryotic ancestor (LCEA) 1The abbreviations used are:LCEAlast common eukaryotic ancestorNPCnuclear pore complexCOPIcoatomer complex ICOPIIcoatomer complex IIHOPShomotypic fusion and protein sortingCORVETclass C core vacuole/endosome tetheringTIRFMtotal internal reflection fluorescence microscopyCVcoated vesicleERendoplasmic reticulumMALDImatrix-assisted laser desorption ionizationSEASeh-1 associatedDTTdithiotreitolPICprotease inhibitor cocktailGFPgreen fluorescent protein., a hypothetical lineage that gave rise to all modern eukaryotes, and evolved from the first common eukaryotic ancestor by gene duplication and divergence, possessed a highly complex membrane-trafficking system (1Dacks J.B. Field M.C. Evolution of the eukaryotic membrane-trafficking system: origin, tempo and mode.J. Cell Sci. 2007; 120: 2977-2985Crossref PubMed Scopus (212) Google Scholar, 2DeGrasse J.A. DuBois K.N. Devos D. Siegel T.N. Sali A. Field M.C. 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The coat also defines the vesicle type. For example, clathrin, in association with one of four distinct adaptin (AP) complexes, is involved in endocytosis and trafficking between the Golgi apparatus, vacuole and lysosome, and endosomes. Coatomer complex I (COPI) coated vesicles mediate intra-Golgi movement and are responsible for retrograde transport between the Golgi and endoplasmic reticulum (ER), whereas coatomer complex II (COPII) coated vesicles function in anterograde transport from the ER to Golgi apparatus (6Bonifacino J.S. Glick B.S. The mechanisms of vesicle budding and fusion.Cell. 2004; 116: 153-166Abstract Full Text Full Text PDF PubMed Scopus (1296) Google Scholar). The common evolutionary origin of these three types of vesicles is supported by the presence of structurally similar elements and mechanisms of vesicle formation, as well as clear common ancestry of multiple subunits within these complexes (5Lee C. Goldberg J. 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These structures (∼50 MDa in yeast) contain multiple copies of ∼30 different nucleoporins or nups. The structural scaffold of the NPC, comprising ∼50% of the total NPC mass, is formed almost entirely from proteins consisting of only two folds—α-solenoid-like and β-propellers (4Devos D. Dokudovskaya S. Williams R. Alber F. Eswar N. Chait B.T. Rout M.P. Sali A. Simple fold composition and modular architecture of the nuclear pore complex.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2172-2177Crossref PubMed Scopus (226) Google Scholar, 10Alber F. Dokudovskaya S. Veenhoff L.M. Zhang W. Kipper J. Devos D. Suprapto A. Karni-Schmidt O. Williams R. Chait B.T. Rout M.P. Sali A. Determining the architectures of macromolecular assemblies.Nature. 2007; 450: 683-694Crossref PubMed Scopus (440) Google Scholar, 11Alber F. Dokudovskaya S. Veenhoff L.M. Zhang W. Kipper J. Devos D. Suprapto A. Karni-Schmidt O. Williams R. Chait B.T. Sali A. Rout M.P. 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The compartmentalized bacteria of the planctomycetes-verrucomicrobia-chlamydiae superphylum have membrane coat-like proteins.PLoS Biol. 2010; 8e1000281Crossref PubMed Scopus (116) Google Scholar). In both coated vesicles and NPCs these structural folds likely fulfill a similar function, namely to form and stabilize curved membranes. In addition, the β-propeller protein Sec13 is a constituent of both the NPC scaffold Nup84 subcomplex and COPII vesicle coats, in the latter forming a heterodimer with Sec31 (13Stagg S.M. Gürkan C. Fowler D.M. LaPointe P. Foss T.R. Potter C.S. Carragher B. Balch W.E. Structure of the Sec13/31 COPII coat cage.Nature. 2006; 439: 234-238Crossref PubMed Scopus (243) Google Scholar, 14Stagg S.M. LaPointe P. Razvi A. Gürkan C. Potter C.S. Carragher B. Balch W.E. Structural basis for cargo regulation of COPII coat assembly.Cell. 2008; 134: 474-484Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 15Fath S. Mancias J.D. Bi X. Goldberg J. Structure and organization of coat proteins in the COPII cage.Cell. 2007; 129: 1325-1336Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). The similarity between the NPC scaffold nups and vesicle coat proteins extends to the atomic level (reviewed in 16Brohawn S.G. Partridge J.R. Whittle J.R. Schwartz T.U. The nuclear pore complex has entered the atomic age.Structure. 2009; 17: 1156-1168Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). The presence of shared components, folds and fold arrangements, overall architecture and functions in membrane curvature are the key evidence supporting the hypothesis that CVs and NPCs evolved from a common ancestral protocoatomer (3Devos D. Dokudovskaya S. Alber F. Williams R. Chait B.T. Sali A. Rout M.P. Components of coated vesicles and nuclear pore complexes share a common molecular architecture.PLoS Biol. 2004; 2: e380Crossref PubMed Scopus (323) Google Scholar, 4Devos D. Dokudovskaya S. Williams R. Alber F. Eswar N. Chait B.T. Rout M.P. Sali A. Simple fold composition and modular architecture of the nuclear pore complex.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2172-2177Crossref PubMed Scopus (226) Google Scholar, 9Field M.C. Dacks J.B. First and last ancestors: reconstructing evolution of the endomembrane system with ESCRTs, vesicle coat proteins, and nuclear pore complexes.Curr. Opin. Cell Biol. 2009; 21: 4-13Crossref PubMed Scopus (97) Google Scholar, 10Alber F. Dokudovskaya S. Veenhoff L.M. Zhang W. Kipper J. Devos D. Suprapto A. Karni-Schmidt O. Williams R. Chait B.T. Rout M.P. Sali A. Determining the architectures of macromolecular assemblies.Nature. 2007; 450: 683-694Crossref PubMed Scopus (440) Google Scholar, 11Alber F. Dokudovskaya S. Veenhoff L.M. Zhang W. Kipper J. Devos D. Suprapto A. Karni-Schmidt O. Williams R. Chait B.T. Sali A. Rout M.P. The molecular architecture of the nuclear pore complex.Nature. 2007; 450: 695-701Crossref PubMed Scopus (821) Google Scholar). If much of the endomembrane system did indeed evolve from an ancestral protocoatomer, it might be expected that additional complexes, structurally related to the coated vesicles and NPC, are present elsewhere within the cell. Indeed, such complexes have been found, and are also predicted to play roles in intracellular transport and/or membrane deformation. One example is the coatomer-related intraflagellar transport complex, required for the assembly and maintenance of cilia and flagella (17Jékely G. Arendt D. Evolution of intraflagellar transport from coated vesicles and autogenous origin of the eukaryotic cilium.Bioessays. 2006; 28: 191-198Crossref PubMed Scopus (178) Google Scholar). Another complex contains a number of conserved Bardet-Biedl Syndrome proteins (BBSome) and is required for sorting of membrane proteins to primary cilia (18Jin H. White S.R. Shida T. Schulz S. Aguiar M. Gygi S.P. Bazan J.F. Nachury M.V. The conserved Bardet-Biedl syndrome proteins assemble a coat that traffics membrane proteins to cilia.Cell. 2010; 141: 1208-1219Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar). Two additional complexes containing components with characteristic protocoatomer-like fold arrangements are the multisubunit membrane tethering complexes HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering). These two complexes, collectively termed Vps-C complexes, are associated with vacuoles (lysosomes) and endosomes respectively, and have as yet ill-defined roles in cell control of growth, nutrient transport, autophagy, as well as endosomal and vacuolar assembly and trafficking (19Nickerson D.P. Brett C.L. Merz A.J. Vps-C complexes: gatekeepers of endolysosomal traffic.Curr. Opin. Cell Biol. 2009; 21: 543-551Crossref PubMed Scopus (179) Google Scholar). Here, we describe a new complex, the SEA (Seh1-associated) complex, in the yeast S. cerevisiae, which contains Seh1, Sec13, and evolutionarily conserved proteins with predicted secondary structure similarities to components of HOPS and CORVET. The SEA complex is dynamically associated with the vacuole membrane and functional and genetic analyses are consistent with a role for the members of the SEA complex in membrane trafficking and autophagy. Yeast strains used in this study are listed in the 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 nitrogen base 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 (SD - N:0.17% yeast nitrogen base without amino acids, 2% glucose). SEA complex proteins were genomically tagged with PrA as previously described (20Rout M.P. Aitchison J.D. Suprapto A. Hjertaas K. Zhao Y. Chait B.T. The yeast nuclear pore complex: composition, architecture, and transport mechanism.J. Cell Biol. 2000; 148: 635-651Crossref PubMed Scopus (1152) Google Scholar). Affinity purification of SEA complex protein complexes from whole cell lysates using magnetic beads was performed as described previously (10Alber F. Dokudovskaya S. Veenhoff L.M. Zhang W. Kipper J. Devos D. Suprapto A. Karni-Schmidt O. Williams R. Chait B.T. Rout M.P. Sali A. Determining the architectures of macromolecular assemblies.Nature. 2007; 450: 683-694Crossref PubMed Scopus (440) Google Scholar). The extraction buffer used in immunoprecipitation of Seh1-PrA (see Fig. 1) and Sec31-PrA (see Fig. 3B, #2) was 20 mm K/HEPES, pH 7.4, 1% Triton, 0.5% sodium deoxycholate, 0.3% sodium N-lauroyl-sarcosine, 0.1 mm MgCl2, 1 mm dithiotreitol (DTT), 1:200 dilutions of solution P (2% PMSF, 0.04% pepstatin A in absolute ethanol) and protease inhibitor cocktail (PIC solution) (Sigma, P8340). Beads were washed with 20 mm K/HEPES, pH 7.4, 1 mm EDTA, 0.1% Triton, 0.05% sodium deoxycholate, 0.03% sodium N-lauroyl-sarcosine. PrA-tagged Sea1-Sea4, Npr2 and Npr3 (see Fig. 1) were extracted with 20 mm K/HEPES, pH 7.4, 110 mm KOAc, 2 mm MgCl2, 0.1% Tween 20 (TBT buffer), 1% Triton, 75 mm NaCl, 1 mm DTT, as well as 1:200 dilutions of solutions P and PIC. Beads were washed with the TBT buffer, containing 1 mg/ml heparin. Immunoprecipitation of Sea4-PrA in sea3Δ strain (see Fig. 3B, #1) was performed with the same extraction and wash buffer (TBT, 1% Triton, 1 m NaCl, 1 mm DTT, 1:200 dilutions of solutions P and PIC). After wash proteins were eluted with 0.5 m NH4OH, 0.5 mm EDTA by incubation for 20 min at room temperature. The eluant was lyophilized, resuspended in SDS-PAGE sample buffer, separated on a 4–20% Tris-glycine gel (Invitrogen), and visualized with Coomassie blue (R-250) staining.Fig. 3SEA complex proteins have evolutionarily conserved structural characteristics similar to membrane coats. A, Protease accessibility laddering (PAL) analysis of SEA complex proteins. PAL readily detects domain boundaries and flexible loops within proteins (23Dokudovskaya S. Williams R. Devos D. Sali A. Chait B.T. Rout M.P. Protease accessibility laddering: a proteomic tool for probing protein structure.Structure. 2006; 14: 653-660Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Protein A-tagged SEA proteins were purified on magnetic beads in their natively folded state. While attached to the beads, proteins were probed with proteases (Asp-D, Lys-C and Trypsin). Proteolytic fragments, containing C-terminal PrA tag were eluted and detected by immunoblotting with IgG-HRP. Shown are immunoblots of PAL digests for PrA-tagged versions of Sea1, Sea2, Sea3, and Sea4. Full-length proteins are indicated with a dot and proteolytic fragments with a star and a letter. Sites of proteolysis are marked with arrows on a secondary structure prediction map (shown to the right of each gel). Uncertainties in the precise cleavage positions are indicated by lines to the left of the map (see also supplemental Table S4). B, Sea4 forms a dimer with Seh1 similar to the COPII coat complex Sec31-Sec13. Note, that in this experiment Sea4-PrA was expressed in the cells deleted for Sea3 and immunoprecipitated under stringent conditions with 1 m NaCl present both in the extraction and washing buffers (see Experimental procedures). Therefore the resulting complex is different than the one shown on Fig. 1, lane #5. Sec31-PrA expressed in wild type yeast (lane #2) was immunoprecipitated as described under "Experimental Procedures." Eluted proteins were resolved on SDS-PAGE gels, stained with Coomassie blue and identified by mass spectrometry (supplemental Table S2). Arrows indicate predicted folds. Seh1 and Sec13 are indicated as 6-blade β-propeller, according to their x-ray structures (15Fath S. Mancias J.D. Bi X. Goldberg J. Structure and organization of coat proteins in the COPII cage.Cell. 2007; 129: 1325-1336Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 76Brohawn S.G. Leksa N.C. Spear E.D. Rajashankar K.R. Schwartz T.U. 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Fluorescent proteins as proteomic probes.Mol. Cell Proteomics. 2005; 4: 1933-1941Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) or on a MALDI linear trap quadrupole (LTQ) Orbitrap XL (Thermo) (22Luo Y. Li T. Yu F. Kramer T. Cristea I.M. Resolving the composition of protein complexes using a MALDI LTQ Orbitrap.J. Am. Soc. Mass Spectrom. 2010; 21: 34-46Crossref PubMed Scopus (29) Google Scholar). MALDI LTQ Orbitrap MS analyses were acquired for a mass range of m/z 700–4000 with the following parameters: resolution setting, 60,000 at m/z 400; automated spectrum filter off; 50 scans/step; automated gain control on; allowing storage of 5e5 ions. The list of singly charged monoisotopic masses was generated using Xtract within Qual Browser (XCalibur, version 2.0.7) with the following parameters: MH+, m/z 700–4000 mass range; resolution, 60,000 at m/z 400; and signal-to-noise threshold of peak picking, 2. The lists of putative proteins were obtained by database searching against the National Center for Biotechnology Information nonredundant protein database, version October 16, 2006, using the computer algorithm XProteo, version 1.2 (http://www.xproteo.com). Search parameters for MS data were: species, Saccharomyces cerevisiae (11105 sequences); protein mass, 0–300 kDa; protein pI, 1–14; mixture search, auto; number of candidates displayed, 20; enzyme, trypsin; miscleavages, 1; mass type, monoisotopic; charge state, MH+; mass errors, 0.06 Da for analyses performed on the MALDI Q-ToF and 5ppm for those performed on the MALDI LTQ Orbitrap; fixed modification, carbamidomethylation of Cys; and variable modification, oxidation of Met. Gi numbers of identified proteins, protein description, kDa, number of matched versus observed masses, protein sequence coverage (%), XProteo scores (d') following database search are listed in supplemental Table S2. An XProteo d' score of 4 reflects a positive rate of 0.99 and false positive rate of 0.05. Only the proteins that were identified with d' > 4 are reported. PAL analysis of PrA-tagged Sea proteins was performed as described previously (23Dokudovskaya S. Williams R. Devos D. Sali A. Chait B.T. Rout M.P. Protease accessibility laddering: a proteomic tool for probing protein structure.Structure. 2006; 14: 653-660Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Secondary structure of a query sequence was predicted by PSIPRED (24McGuffin L.J. Bryson K. Jones D.T. The PSIPRED protein structure prediction server.Bioinformatics. 2000; 16: 404-405Crossref PubMed Scopus (2737) Google Scholar) from the multiple sequence alignment constructed by two iterations of PSI-BLAST. For fold assignment, the protein sequences were divided into domains based on the PAL data, predicted secondary structure, and the output from the disorder predictions by IUPred (25Dosztányi Z. Csizmók V. 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