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BaginskyNot availablePlant CellRootRosetteThe proteomes for the import mutants ppi2 and tic56-1 as well as those for spectinomycin treated plants (Spec) and the according wildtype controls were analyzed in three biological replicates and several technical replicates (tic56-1: 19, wt [tic56-1]: 18/ ppi2: 9, wt [ppi2]: 9/ Spec: 10, wt [Spec]: 11). Therefor total protein was extracted from plant material cultivated on ½ MS-medium containing 1% agar and 0.8% sucrose, for spectinomycin-treatment the medium contains additionally 60 µg/ml spectinomycin. The plant material was cultivated under short day conditions (8 h light/ 16 h dark). For extraction of total protein, plant material was frozen in liquid nitrogen and crushed with a pestle. The material was incubated with extraction buffer (100mM sodium chloride, 50 mM Tris, pH7.5, 4% [w/v] SDS, 0.1% [v/v] plant protease inhibitor cocktail [Sigma-Aldrich], and 1 mM phenylmethylsulfonyl fluoride) at 70°C for 10 min under shaking. The extract was clarified by centrifugation at maximum speed in a tabletop centrifuge for 10 min at room temperature. For mass spectrometry 100 µg of total protein were digested in the presents of 0.005% (w/v) RapiGest SF (Waters) with sequencing grade modified trypsin (Promega, protein:trypsin ratio 50:1) and prepared for mass spectrometry as described by Helm et al. (2014). Before mass spectrometry measurements samples were spiked with 10 fmol rabbit glycogen phosphorylase B (P00489). LC separation and HD-MSE data acquisition was performed as previously described (Helm et al., 2014) using 1 μl from each of the in solution digested samples on a nanoACQUITY UPLC System coupled to a Synapt G2-S mass spectrometer (Waters, Eschborn, Germany). MS acquisition was set to 50–5000 Da. Helm, S., Dobritzsch, D., Rodiger, A., Agne, B., and Baginsky, S. (2014). Protein identification and quantification by data-independent acquisition and multi-parallel collision-induced dissociation mass spectrometry (MS(E)) in the chloroplast stroma proteome. Journal of proteomics 98, 79-89.PrideSacha BaginskySynapt MSPARTIALInstitute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Weinbergweg 22, 06120 Halle (Saale), Germany27733515 Köhler D, Helm S, Agne B, Baginsky S. Importance of Translocon Subunit Tic56 for rRNA Processing and Chloroplast Ribosome Assembly. Plant Physiol. 2016 Dec;172(4):2429-2444Institut fuer Biochemie und Biotechnologiesacha.baginsky@biochemtech.uni-halle.deHDMSEMass SpectrometryShotgun proteomicsDisease FreeHi3 quantificationPlastidic translationHdmseTic56-1Toc-tic-import machinerySpectinomycinQuantitative mass spectrometryPlastidShotgun proteomicsPpi2http://www.ebi.ac.uk/pride/archive/projects/PXD004125Spectral Abundance Factor - SAFiodoacetamide derivatized residuemonohydroxylated residueData analysis was carried out by ProteinLynx Global Server (PLGS 3.0.1, Apex3D algorithm v. 2.128.5.0, 64 bit, Waters, Eschborn, Germany) with automated determination of chromatographic peak width as well as MS TOF resolution. Lock mass value for charge state 2 was defined as 785.8426 Da/e and the lock mass window was set to 0.25 Da. Low/high energy threshold was set to 180/15 counts, respectively. Elution start time was 5 min, intensity threshold was set to 750 counts. Databank search query (PLGS workflow) was carried out as follows: Peptide and fragment tolerances was set to automatic, two fragment ion matches per peptide, five fragment ions for protein identification, and two peptides per protein. Maximum protein mass was set to 250 kDa. Primary digest reagent was trypsin with one missed cleavage allowed. According to the digestion protocol fixed (carbamidomethyl on Cys) as well as variable (oxidation on Met) modifications were set. The false discovery rate (FDR) was set to 4% at the protein level. MSE data were searched against the modified A. thaliana database (TAIR10, ftp://ftp.arabidopsis.org) containing common contaminants such as keratin (ftp://ftp.thegpm.org/fasta/cRAP/crap.fasta) and glycogen phosphorylase B from rabbit (Uniprot ID: P00489). Quantification was performed based on the intensity of the three most abundant proteotypic peptides (Silva et al., 2006). Silva, J.C., Gorenstein, M.V., Li, G.Z., Vissers, J.P., and Geromanos, S.J. (2006). Absolute quantification of proteins by LCMSE: a virtue of parallel MS acquisition. Molecular & cellular proteomics : MCP 5, 144-156.ProteomicsMultiomicsArabidopsis Thaliana (mouse-ear Cress)pflanzen@biochemtech.uni-halle.deBiologicalGermanyToc159-containing complexes at the outer chloroplast envelope membrane form stable supercomplexes with a 1-MD translocon at the inner chloroplast envelope membrane of which Tic56 is one essential subunit. While the single mutants tic56-1 and ppi2 (toc159) have an albino phenotype and are able to grow heterotrophically, we find the double mutant to be embryo lethal. Comprehensive quantitative proteome profiling with both single mutants in combination with GeneChip analyses identified a posttranscriptional defect in the accumulation of plastid ribosomal proteins and diminished expression of plastid encoded proteins. In the tic56-1 mutant, the assembly of functional ribosomes is furthermore hampered by a processing defect of the plastid 23S rRNA. Spectinomycin-treatment of wild-type plants phenocopies the molecular phenotype of plastid proteome accumulation in tic56-1 and to a smaller degree also ppi2 plastids, suggesting that a defect in plastid translation is largely responsible for the phenotype of both import mutants. Import experiments with the tic56-3 mutant revealed no significant defect in the import of small ribosomal protein 16 in the absence of full-length Tic56, suggesting that the defect in ribosome assembly in tic56-1 may be independent of a function of Tic56 in protein import. Our data establish a previously unknown link between plastid protein import, the processing of plastid rRNAs, and the assembly of plastid ribosomes and provide further knowledge on the function of the translocon components and the molecular basis for their albino phenotype.Importance of Translocon Subunit Tic56 for rRNA Processing and Chloroplast Ribosome Assembly.Köhler Daniel D, Helm Stefan S, Agne Birgit B, Baginsky Sacha S6beta, Hac-1/Dark, BBE, IPP2A2, rabbits, 9alpha, Pflanze, DmelCG6829, alpha-Spe, Visible Light, SDH, protein, rabbit, Apaf-1, alpha-Spc, extracted material, viridiplantae, 5730420M11Rik, SDS, l(3)04276, protein polypeptide chains, halite, hnu, 9-trihydroxy-2-methyl-6, Phenylmethylsulfonyl, Min, Apaf1, Analysis, 7, protein aggregate, ARK, foton, DmelCG42273, alpha-spec, Mass Spectrum Analysis, SET, kus, hac-1, Analyses, TAF-I, Ferkel Spectam, N, phosphatase 2A inhibitor I2PP2A, (24)NaCl, arc, Fluoride, min, mAPC, plants, proteins, ark, 4abeta, alpha-spectrin, T1, Stanilo, AI047805, DmelCG4299, cloruro sodico, Benzenemethanesulfonyl Fluoride, salt, set, IGAAD, Spectam, SOLO DANCERS, DmelCG10574, dApaf1, Lepus cuniculus, gamma, ratio, phapii, Salmosan T, rock salt, Radiation, European rabbit, D-Apaf-1, land plants, 8-bis(methylamino)-, anon-53Fa, Dmel_CG7826, l(2)SH0173, AI836084, StF-IT-1, Light, beta-D-fructofuranosyl, Spectrum Analysis, L-ornithine carboxy-lyase activity, collisionally activated dissociation, CID, Spectroscopy, shortened, alphaSpec, common salt, LIGHT, l(1)G0108, Stickstoff, Dm1, Pentahydrate, spectrin, CG6829, Dmel_CG7835, dark/hac-1/dapaf-1, Visible Radiations, Mnb, MNB, Visible Radiation, table sugar, Ferkel, HVEML, HLA-DR-associated protein II, DI-2, 1-alpha-D-Glucopyranosyl-2-beta-D-fructofuranoside, Skeletal muscle enolase, I-2Dm, alpha-D-Glucopyranoside, proportionality, Plant, Spectrometry, fmol, rate, alpha, hac1, CG4299, inhibitor of granzyme A-activated DNase, AW124434, natrii chloridum, beta Trypsin, Adspec, organ system, I-2PP1, dapaf-1S, higher plants, apaf-1, light quantum, dapaf-1L, nitrogen, TAF-IBETA, i6, TAF-Ibeta, GSD13, 7N, Spectinomycin Hydrochloride, short, i2pp2a, Proteomes, 10abeta))-, Specbeta, F10B6_15, 3-b)(1, Sucrose, Prospec, anon-EST:fe2E2, l(3)alpha-Spec, Dapaf-1/HAC-1, alpha-Spect, Eno-3, beta-D-Fruf-(2<->1)-alpha-D-Glcp, decahydro-4a, Spectrum Analyses, protein-containing complex, body system, F10B6.15, CG7826, PHAPII, Ly113, Buffer, Dark/Hac-1/dApaf1, Hac1, (2R-(2alpha, Dark/Hac-1/dApaf-1, B-spec, polypeptide chain, template-activating factor I, Phenylmethanesulfonyl Fluoride, betaspec, klo, CG7835, Mass, CG42273, Sacharose, system, alpha-Sp, light, CG1977, Kempi, Mass Spectroscopy, Pro-Mega, 4.2.1.11, plantae, Japanese white rabbit, Actinospectacin, 9aalpha, proportion, anatomical systems, 5abeta, Saccharose, Lichtquant, (22)Na, ipp2a2, dapaf-1, beta-Trypsin, dre3, stubby, chlorure de sodium, 4733401P19Rik, 2pp2a, dapaf, Visible, dark, Trobicin, CG10574, MSE, Solution, anon-EST:fe1B3, Sodium Chloride, Benzenemethanesulfonyl fluoride, 2PP2A, DARK, SPEC8, taf-ibeta, 8beta, Kochsalz, photon, dSET, dSet, liquid, TR2, CG5870, Salmosan-T, SUCROSE, dArk, Dapaf-1, Anhydrous, l(3)dre3, Peptidomics., Spec-beta, dark/dapaf-1/hac-1, 4)benzodioxin-4-one, DYRK1, data, alpha-Spectrin, 3A9, protein complex, DmelCG1977, dApaf-1, igaad, 7beta, 2-phospho-D-glycerate hydro-lyase, total expressed protein, apaf1, i168, CD258, buffer, SpeC, L-ornithine carboxy-lyase (putrescine-forming), APAF1, CAD, native protein, sacarosa, Temperatures, natural protein, Natriumchlorid, Hac-1, I-2PP2A, sucrose, Protein, Dm I-2, I2PP2A, betaSpec, table salt, Dyrk1, Dark, Phenylmethanesulfonyl, connected anatomical system, NaCl, alphaSp, b-Spec, Dark/Apaf-I, Mass Spectrum Analyses, Radiations, AU020952, Mass Spectrum, DmelCG5870, l(1)G0074, l(1)G0198, HVEM-L, CC1, Photoradiation, Sodium chloride (NaCl), FCP-B, Tripcellim, b spectrin, Dark/Dapaf-1/HAC1, SWDS, azote, Muscle-specific enolase, CGI-97, 4H-Pyrano(2, beta-D-fructofuranosyl alpha-D-glucopyranoside, dApaf-1/DARK/HAC-1, beta, LTg, nitrogeno, ME-IV, Trypure, domestic rabbit, dSET/TAF-Ibeta, 2610030F17Rik, Photoradiations, Cane sugar, l(3)62Bd, beta-spec, velocity, Spectinomycin Dihydrochloride, quotient, alfa-Spec, AA407739, White sugar, Spec, dAPAF-1, Benzenemethanesulfonyl, Enolase 3, sodium chloride35S primary transcript processing.IPP2A2, BBE, rabbits, l(3)Ca, ion, Hgfr, A., mCRY, Brain Ig-like molecule with tyrosine-based activation motifs, FEZL, cleavage, 5730555F13Rik, 26S protease, d-titin, protein, rabbit, Long Term, Cardaminopsis, 5730420M11Rik, peptide, Polypeptides, protein polypeptide chains, large multicatalytic protease, Arabidopsis thalianas, Methionine, ClvPrd, peptido, HGFR, A. thalianas, Plgs, DmelCG1915, Min, Analysis, Work Flow, protein aggregate, Gpi-1, multicatalytic endopeptidase complex, Effect, Ket, WMS, DmelCG42273, ASD2, SET, me75, F, peptides, kDa, Analyses, TAF-I, MUB3_18, TACHD, Nlk, M, phosphatase 2A inhibitor I2PP2A, Mouse-ear Cress, min, mAPC, proteins, Inhibitory receptor SHPS-1, MUB3.18, ket, D17Mit170, T1, Mouse-ear, DmelCG4299, AI047805, set, IGAAD, Sirp-alpha-2, Sirp-alpha-3, DmelCG10574, Sirp-alpha-1, polyhedral organelle, SHP substrate 1, Lepus cuniculus, RG7MT1, AHUS2, GPHYSD2, Arabidopses, z, Racemethionine, TRA2.10, Long-Term Effects, multicatalytic proteinase, reagents, SGS, ratio, NK/GPI, phapii, Gpi, proto-oncogene c-Met, European rabbit, PRO, titin, 2-Amino-4-(methylthio)butyric acid, Longterm Effect, Dmel_CG7826, StF-IT-1, A. thaliana, threonine endopeptidase activity, 0020/01, Tl3, 9130215G10Rik, Tl2, CYS, ACMICD, Workflows, CFU-MC, mOC-X, Dm1, Ionen, SHPS1, Algorithm, Methionin, methionine, NK|GPI, prosome, Hmet, reactif, fixed, and GLY protein 2, Dmel_CG7835, tricorn proteinase, Mnb, MNB, HGF receptor, HGF/SF receptor, and GLY protein 1, ORG, Org, HLA-DR-associated protein II, DI-2, Skeletal muscle enolase, I-2Dm, proportionality, Phosphorylase ab, Gpi1-r, common, reagent, rate, Gpi1-s, MASS, CG4299, inhibitor of granzyme A-activated DNase, hCMT1c, AW124434, MIC10, Signal-regulatory protein alpha-1, study protocol, c-Met, Gpi1-t, Signal-regulatory protein alpha-2, beta Trypsin, CD172a, Signal-regulatory protein alpha-3, Crap, I-2PP1, TLX, CG1915, Arabidopsis, MyD-1 antigen, 1110030K05Rik, TAF-IBETA, Bit, BIT, multicatalytic proteinase (complex), Phosphorylase, MARP, tricorn protease, CG18242, TAF-Ibeta, Polypeptide, GSD13, CG18245., mSIRP-alpha1, i2pp2a, time, alkaline protease, CD172A, Work Flows, Bglap-rs1, Gpi1s, SHPS-1, CARP, breadth, 2-amino-4-(methylsulfanyl)butanoic acid, Effects, False, AI461847, Peptidomics, FBN, Eno-3, Gene, Virtue, protein-containing complex, CD172 antigen-like family member A, SLS, Sls, PHAPII, CG7826, period, ingensin, Carp, method, HGF, template-activating factor I, polypeptide chain, TOF, ECTOL1, HEL-S-66, method used in an experiment, AI835480, CG7835, thalianas, Gene Products, CG42273, SIRP, F15E12.6, Pgi, Low, Titin, Crry, RCCP2, anon-CREST, ZNF312, CFU-Mast, 4.2.1.11, l(3)j1D7, Japanese white rabbit, F15E12_6, proportion, reactivo, Longterm, Cresses, scatter factor receptor, Gpi-1r, AFCP, P84, iones, Gpi-1s, ipp2a2, Phi, beta-Trypsin, Gpi-1t, colony forming unit mast cell, 2pp2a, Long-Term, CG18857, BMC, ions, CG10574, AUTS9, MSE, OCTD, lens neutral proteinase, l(3)dre8, Glycogen, 2PP2A, 2-amino-4-(methylthio)butanoic acid, taf-ibeta, p84, KZ, dSET, dSet, peptidos, Cress, l(3)rL182, Long-Term Effect, AI838057, Mouse ear, metionina, FEZ, Mcp, MCP, DYRK1, data, PTPNS1, cou, Macrophage fusion receptor, DL-Methionine, AI447866, protein complex, Arabidopsis thaliana, Proteins, igaad, 2-phospho-D-glycerate hydro-lyase, l(3)62Ca, MF, Amf, Peptide, polypeptide, kettin, Lr, native protein, natural protein, I-2PP2A, Dm I-2, Long Term Effects, Protein, I2PP2A, MFS1, Dyrk1, WMS2, ATCMPG1, MET, Met, Data Base, NK, ATCMPG2, D-titin, AU020952, VSD1, MARP1, SHP-1, tyrosine-protein kinase Met, CC1, Tripcellim, thaliana, Alrp, MFR, Muscle-specific enolase, D-Titin, MYD-1, Ptpns1, Mouse-ear Cresses, Longterm Effects, sam, ME-IV, sal, plan specification, Protein Gene Products, Trypure, domestic rabbit, Gene Proteins, ZFP312, dSET/TAF-Ibeta, BAC, 2610030F17Rik, CT41299, Ion, Data, Macrophage membrane protein MFP150, SSKS, Bra, alpha-amino-gamma-methylmercaptobutyric acid, quotient, l(3)S002001, Peptid, Par4, variable, AA407739, 5730455C01Rik, Data Analyses, SF receptor, Enolase 3, proteasome endopeptidase complex6beta, 3-b)(1, BBE, Prospec, l(3)Ca, 9alpha, Peptidomics, mCRY, number, A4, Eno-3, Gene, 26S protease, d-titin, decahydro-4a, protein, Virtue, protein-containing complex, SLS, Sls, presence, TYPE, CG11121, DAGA4, ingensin, protein polypeptide chains, method, large multicatalytic protease, old, (2R-(2alpha, polypeptide chain, DmelCG11121, HEL-S-66, method used in an experiment, 9-trihydroxy-2-methyl-6, Gene Products, DmelCG1915, Titin, Crry, 7, MAM, protein aggregate, multicatalytic endopeptidase complex, SCG3, anon-CREST, Ket, Kempi, CFU-Mast, 4.2.1.11, l(3)j1D7, Actinospectacin, 9aalpha, somda, 5abeta, AFCP, Ferkel Spectam, colony forming unit mast cell, proteins, Trobicin, CG18857, BMC, ket, SO, 4abeta, MSE, Stanilo, lens neutral proteinase, l(3)dre8, Spectam, 8beta, KZ, polyhedral organelle, l(3)rL182, Salmosan-T, AHUS2, TRA2.10, Anhydrous, Mcp, MCP, So, multicatalytic proteinase, Drl, data, Salmosan T, 4)benzodioxin-4-one, protein complex, Proteins, titin, 8-bis(methylamino)-, 7beta, 2-phospho-D-glycerate hydro-lyase, l(3)62Ca, threonine endopeptidase activity, 0020/01, not genetically inherited, Mdu, LGMD2C, kettin, count in organism, CFU-MC, native protein, natural protein, mda, Protein, prosome, Pentahydrate, ami, tricorn proteinase, D-titin, Ferkel, DMDA1, Skeletal muscle enolase, med, Muscle-specific enolase, D-Titin, MIC10, 4H-Pyrano(2, Adspec, sam, sal, plan specification, Protein Gene Products, TLX, Gene Proteins, CG1915, BAC, DMDA, CT41299, multicatalytic proteinase (complex), SCARMD2, Spectinomycin Dihydrochloride, l(3)S002001, tricorn protease, CG18242, GSD13, CG18245., quantitative, Spectinomycin Hydrochloride, 10abeta))-, alkaline protease, Enolase 3, proteasome endopeptidase complex, presence or absence in organismForms, 6beta, 3-b)(1, Prospec, 9alpha, degree (angle), Amyloplast, plant germ, ribosomal subunit assembly, number, A4, Gene, ribosomal RNA, Ribosomal Protein, decahydro-4a, Leucoplast, presence, TYPE, Leukoplast, DAGA4, (2R-(2alpha, reduced, integral component of membrane, Gene Products, 9-trihydroxy-2-methyl-6, tiny, 7, MAM, SCG3, Kempi, Actinospectacin, Ribosomal, 9aalpha, 5abeta, Plastid, Ferkel Spectam, absent from organism, membrane region, hypoplasia, proteins, plants, Trobicin, 4abeta, ribosomal ribonucleic acid, Stanilo, embryonic organism, Spectam, Chromoplasts, 8beta, region of membrane, Salmosan-T, Chromoplast, plastid protein synthesis, Anhydrous, protein uptake, small, membrane, Salmosan T, 4)benzodioxin-4-one, data, embryo, land plants, Proteins, 8-bis(methylamino)-, Phenotypes., 7beta, integral to membrane, total expressed protein, plastid protein formation, function, developing organism, membranous organ component, plastid protein translation, plastid protein anabolism, LGMD2C, count in organism, INSDC_feature:rRNA, Protein, whole membrane, Pentahydrate, membrane bound ribosome, free ribosome, Ribosome, Epistemology, transmembrane, membrane of organ, absence, Ferkel, DMDA1, underdeveloped, ribosomal protein, Plant, pooled, INSDC_qualifier:unknown, arc degree, Leukoplasts, 4H-Pyrano(2, Adspec, Amyloplasts, Leucoplasts, Protein Gene Products, Phenotypes, Gene Proteins, higher plants, DMDA, developmental tissue, SCARMD2, Spectinomycin Dihydrochloride, quantitative, Spectinomycin Hydrochloride, Proteomes, 10abeta))-, plastid protein biosynthesis, presence or absence in organism6beta, Salmosan T, 3-b)(1, 4)benzodioxin-4-one, Prospec, 9alpha, land plants., A., Arabidopsis thaliana, number, 8-bis(methylamino)-, 7beta, total expressed protein, A. thaliana, decahydro-4a, Spectrum Analyses, Spectrum Analysis, presence, Cardaminopsis, Spectroscopy, count in organism, (2R-(2alpha, Arabidopsis thalianas, A. thalianas, Mass, 9-trihydroxy-2-methyl-6, thalianas, Analysis, Pentahydrate, 7, Mass Spectrum Analyses, Kempi, Mass Spectroscopy, Mass Spectrum Analysis, Mass Spectrum, Actinospectacin, 9aalpha, Ferkel, 5abeta, Analyses, Cresses, Ferkel Spectam, Mouse-ear Cress, Spectrometry, Plant, thaliana, plants, Trobicin, 4H-Pyrano(2, Mouse-ear Cresses, Adspec, 4abeta, Mouse-ear, Stanilo, Arabidopsis, higher plants, Spectam, 8beta, Spectinomycin Dihydrochloride, Cress, quantitative, Mouse ear, Salmosan-T, Spectinomycin Hydrochloride, Arabidopses, Proteomes, 10abeta))-, Anhydrous, presence or absence in organism300trueMass spectrometry based quantitative proteome analyses for the import mutants ppi2 and tic56-1 as well as for spectinomycin treated Arabidopsis thalian plantsWe acquired quantitative proteomics data from eight week old ppi2 and tic56-1 mutants as well as from wild type treated with 60 µg/ml spectinomycin and related them to their respective wildtype reference. Protein identification and quantification was performed by data-independent MSE acquisition on a Synapt G2S mass spectrometer (Waters). Quantification was based on the Hi3 method (Silva et al., 2006). Silva, J.C., Gorenstein, M.V., Li, G.Z., Vissers, J.P., and Geromanos, S.J. (2006). Absolute quantification of proteins by LCMSE: a virtue of parallel MS acquisition. Molecular & cellular proteomics : MCP 5, 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