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Special Issue: Prospects in Space and Time
Open Access

Identification of Autophagosome-associated Proteins and Regulators by Quantitative Proteomic Analysis and Genetic Screens

Jörn Dengjel, Maria Høyer-Hansen, Maria O. Nielsen, Tobias Eisenberg, Lea M. Harder, Søren Schandorff, Thomas Farkas, Thomas Kirkegaard, Andrea C. Becker, Sabrina Schroeder, Katja Vanselow, Emma Lundberg, Mogens M. Nielsen, Anders R. Kristensen, Vyacheslav Akimov, Jakob Bunkenborg, Frank Madeo, Marja Jäättelä and Jens S. Andersen
Molecular & Cellular Proteomics March 1, 2012, First published on February 6, 2012, 11 (3) M111.014035; https://doi.org/10.1074/mcp.M111.014035
Jörn Dengjel
From the ‡Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark, the §Freiburg Institute for Advanced Studies-LifeNet, University of Freiburg, 79104 Freiburg, Germany, the ¶Center for Biological Systems Analysis (ZBSA), University of Freiburg, 79104 Freiburg, Germany, the ‖Centre for Biological Signaling Studies (BIOSS), University of Freiburg, 79104 Freiburg, Germany,
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Maria Høyer-Hansen
the §§Apoptosis Department and Center for Genotoxic Stress Research, Danish Cancer Society, 2100 Copenhagen, Denmark,
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Maria O. Nielsen
From the ‡Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark,
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Tobias Eisenberg
the ¶¶Institute of Molecular Biosciences, University of Graz, 8010 Graz, Austria, and
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Lea M. Harder
From the ‡Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark,
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Søren Schandorff
From the ‡Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark,
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Thomas Farkas
the §§Apoptosis Department and Center for Genotoxic Stress Research, Danish Cancer Society, 2100 Copenhagen, Denmark,
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Thomas Kirkegaard
the §§Apoptosis Department and Center for Genotoxic Stress Research, Danish Cancer Society, 2100 Copenhagen, Denmark,
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Andrea C. Becker
the §Freiburg Institute for Advanced Studies-LifeNet, University of Freiburg, 79104 Freiburg, Germany, the ¶Center for Biological Systems Analysis (ZBSA), University of Freiburg, 79104 Freiburg, Germany,
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Sabrina Schroeder
the ¶¶Institute of Molecular Biosciences, University of Graz, 8010 Graz, Austria, and
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Katja Vanselow
From the ‡Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark,
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Emma Lundberg
the ‖‖Royal Institute of Technology, Roslagstullsbacken 21, SE-10691 Stockholm, Sweden
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Mogens M. Nielsen
From the ‡Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark,
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Anders R. Kristensen
From the ‡Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark,
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Vyacheslav Akimov
From the ‡Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark,
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Jakob Bunkenborg
From the ‡Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark,
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Frank Madeo
the ¶¶Institute of Molecular Biosciences, University of Graz, 8010 Graz, Austria, and
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Marja Jäättelä
the §§Apoptosis Department and Center for Genotoxic Stress Research, Danish Cancer Society, 2100 Copenhagen, Denmark,
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Jens S. Andersen
From the ‡Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark,
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    Fig. 1.

    PCP-SILAC analyses of autophagosomes. A, schematic outline of experiments performed to identify and characterize proteins associated with autophagosomes. B, accumulation of autophagosomes in MCF7-eGFPLC3 cells was tested by fluorescent microscopy. Representative confocal images (scale bars, 20 μm) are shown for cells left untreated (control, Ctrl) or treated with 1 μm Rapa and 2 nm ConA or starved in HBSS for 7 h. Notably, the cells expressing a mutated form of eGFP-LC3-G120A unable to become lipid-conjugated did not form dots upon stimulation (data not shown), strongly suggesting that the dots observed are autophagosomes rather than unspecific aggregates. C, to identify autophagosome-associated proteins using the PCP-SILAC methods, the cells were isotope-labeled and subsequently treated for 7 h with 2 nm ConA. Autophagosomes were purified by gradient centrifugation, six fractions were collected, and the Lys0/Arg0 fractions were combined, yielding an internal standard mixture of proteins over the gradient. This internal standard was distributed in a 1:1 ratio to the original Lys4/Arg6 labeled fractions, and the combined samples were separated by SDS-PAGE, in-gel digested by trypsin, and analyzed by MS. The experiment was repeated using 100 nm rapamycin and starvation in HBSS as stimuli. Applying this setup, we were able to identify 7935 proteins by 180 LC-MS/MS experiments of 140-min length each. D, mass spectra of the LC3 peptide FLVPDHVNMSELIK showing isotope envelopes of signal doublets, which represent its relative enrichment over the gradient in the PCP-SILAC experiment. The colored circles represent the respective SILAC label. E, comparison between PCP-SILAC protein enrichment profiles and anti-eGFP Western blot analyses of biological replicates from cells starved (HBSS) or treated with ConA or rapamycin for 7 h. Shown are eGFP-LC3-I (upper row) and eGFP-LC3-II (lower row) bands. Both experiments indicate that autophagosomes peak in fractions 2 and 3. Fr., fraction.

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    Fig. 2.

    Cluster analysis of PCP-SILAC data. A, cluster analysis of protein enrichment profiles obtained from the PCP-SILAC experiment of ConA-treated cells. Three clusters were generated consisting of 238, 273, and 285 proteins, respectively, using the fuzzy c-means algorithm. Cluster A contains all identified proteins known to be associated with autophagosomes. Cluster membership values of protein enrichment profiles are indicated by the color scale. Cluster analysis of protein enrichment profiles from the PCP-SILAC experiments of HBSS- and Rapa-treated cells are shown in supplemental Fig. S2. B, Venn diagram of the cluster A proteins identified from the HBSS-, Rapa-, and ConA-treated cells. Whereas 94 proteins were in common for the three stimuli, the majority of proteins were detected by only one or two stimuli. Shown are three representative data sets of five biological replicates. C, subcellular localization of common proteins in clusters A–C based on Gene Ontology.

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    Fig. 3.

    Identification of proteins in immunopurified autophagosomes. A, schematic diagram of the SILAC-based immunoprecipitation experiment performed to identify and quantify autophagosome associated proteins in HBSS- and ConA-treated (20 nm) cells (3 h) as compared with untreated cells. The lysate was centrifuged to enrich autophagosome-associated LC3 and to deplete the free pool of LC3 prior to affinity purification using anti-GFP antibody coated magnetic beads. B, graph showing the relative ratios of proteins identified in the affinity experiments (HBSS and ConA versus control, GFP-IP). Common proteins identified in clusters A–C from the PCP-SILAC experiment displayed nonrandom ratio distributions. C, common cluster A proteins are significantly enriched in immunopurified autophagosomes (HBSS and ConA versus control) as compared with common cluster B and C proteins (one-way analysis of variance, Tukey post hoc test). Ctrl, control.

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    Fig. 4.

    Validation of autophagosomal localization of p62, RHEB and FKBP1A. MCF7 cells expressing eGFP-LC3 (A), DsRed-RHEB (B), or FKBP1A-DsRed (C) were treated for 7 h with 2 nm concanamycin A and stained with anti-p62 (A) or anti-LC3-antibody (B and C). Yellow staining indicating co-localization of LC3 puncta and candidate proteins was observed in all three cases (scale bars, 20 μm). Fluorophore profiles of co-localization are shown in supplemental Figs. S4 and S5. PCP-SILAC profiles of the respective proteins closely follow the MAP1LC3B profiles shown in Fig. 1E.

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    Fig. 5.

    Autophagosome-associated protein dynamics. A, comparison of cluster A proteins identified by PCP-SILAC indicates stimuli-dependent autophagosome-protein association for Rapa- and ConA-treated and starved cells based on Gene Ontology analysis. B, direct comparison of autophagosome-associated proteins by SILAC-based quantitative analysis of autophagosomes isolated from MCF7 cells treated with 100 nm Rapa or 2 nm ConA or starved in HBSS for 7 h and mixed in a ratio of 1:1:1. The relative abundance of proteins per autophagosome is normalized to the ConA isotope signal. Shown are the combined results from two biological replicates. C, p62 levels in MCF7 cells treated with 1 μm Rapa or 2 nm ConA or starved in HBSS for the indicated time periods were analyzed by immunoblotting using GAPDH as loading control. D, relative abundances of indicated annexins in autophagosomes from cells treated and analyzed as in B. Shown are the combined results from two biological replicates. E, autophagosome-associated protein dynamics. Autophagosomes were isolated from SILAC labeled cells and combined in a ratio of 1:1:1 after starvation for 3, 6, and 12 h. Protein-dependent targeting dynamics was observed. Shown are the combined results from two biological replicates.

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    Fig. 6.

    Targeting of the proteasome to autophagosomes. A–D, 20 S core proteasome subunits were visualized using a polyclonal antibody in MCF7-eGFP-LC3 cells left untreated (control) or treated for 7 h with 2 nm ConA or 100 nm Rapa or starved for amino acids in HBSS. Whereas untreated control cells show an evenly distributed staining (A), autophagosome-protein co-localization can be detected in autophagy-induced cells (B–D). Scale bars, 20 μm. E–G, partial co-localization of proteasomes and autophagosomes after induction of autophagy were observed from profiles of relative intensities of the two fluorophores along the respective white lines marked in B–D. H–J, PCP-SILAC profiles of proteasomal proteins were validated by Western blot analyses of biological replicates (ConA, Rapa, and HBSS). Shown are bands for the 20 S core subunits, which follow the MS profiles in all three stimuli. K, the relative abundance of proteasomal subunits were determined by SILAC-based mass spectrometry of cells left untreated or starved for 12 h with or without 10 mm 3-methyladenine combined in a ratio of 1:1. Shown are the relative changes compared with control cells (average ratio of detected PSMA, PSMB, PSMC, and PSMD proteins; the error bars indicate standard deviations). *, p < 0.01 as analyzed by a one-sample t test. L, changes in proteasome activity in response to autophagy were analyzed in lysates of MCF7 cells left untreated (control) or treated for 24 h with 2 nm ConA or 1 μm Rapa or starved for amino acids in HBSS. The values are percentages of proteasome activity/protein concentration as compared with untreated control samples and represent the averages ± S.D. from four independent experiments. *, p < 0.01 as analyzed by a one-sample t test. M, proteasome association with LC3 affinity-purified autophagosomes was analyzed by SILAC-based mass spectrometry using MCF7-eGFP-LC3 cells left untreated (control) or stimulated with 2 nm ConA for 7 h. Anti-GFP immunoprecipitations were performed in lysis buffer with or without 1% Nonidet P-40. Without detergent, intact autophagosomes were purified. Under these conditions, enrichment of proteasomal proteins (average ratio of detected PSMA, PSMB, PSMC, and PSMD proteins) was observed similar to p62/SQSTM1. In the presence of detergent, autophagosomes were destroyed, and the proteasomal proteins were no longer enriched in contrast to proteins binding directly to LC3 such as SQSTM1. The values represent the averages from two independent experiments ± S.D. Ctrl, control.

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    Fig. 7.

    Genetic screens for novel autophagy regulators in yeast and mammalian cells. A and B, ALP screen in S. cerevisiae. The ALP activity was measured in indicated S. cerevisiae knock-out strains starved for nitrogen (A) or treated with 500 ng/ml rapamycin (B) for 3 h. A histidine-dependent strain and an ATG7 knock-out strain were used as negative and positive controls, respectively. The values are percentages of ALP activity/μg protein as compared with the wild-type control strain and represent the averages ± S.D. from three independent experiments. *, p < 0.05; **, p < 0.005 as analyzed by one-way analysis of variance followed by a least significant difference post hoc test. C, functional analysis of yeast retromer subunits. The indicated retromer yeast knock-out strains were treated as in A and B. The values are percentages of ALP activity/μg protein as compared with the wild-type control strain and represent the averages ± S.D. from three independent experiments. An ATG7 knock-out strain was used as a positive control. D, immunoblot analysis of p62 and GAPDH (loading control) from lysates of MCF7-eGFP-LC3 cells 64 h after transfection with indicated siRNAs, which had been validated by QRT-PCR (data not shown). The cells were left untreated or treated with 50 μm etoposide or starved for amino acids for 16 or 8 h before harvesting, respectively. The experiment was repeated a minimum of three times with essentially same results. E, functional analysis of human candidate proteins. MCF7 cells stably expressing RLuc-LC3wt or RLuc-LC3G120A were plated in separate wells of a 96-well dish and transfected with indicated siRNAs. In the upper graph, 50 nm EnduRenTM was added 17 h after the transfection, and the luciferase activities were measured at the indicated time points. For the lower graph, 50 nm EnduRenTM was added 54 h after transfection. Two hours later the luciferase activities were measured (T = 0), the cells were left untreated (control) or treated with 50 μm etoposide, and the luciferase activities were measured at the indicated time points. The experiment was repeated five times with similar results. F, functional analysis of human candidate proteins. MCF7-eGFP-LC3 cells were left untreated or treated with 50 μm etoposide for 6 h. Histograms with percentages of green cellular cross-sections with over five LC3-positive dots are shown. The values represent the means ± S.D. from three to six independent experiments. *, p value < 0.05 analyzed by a two-tailed unpaired t test. Ctrl, control; WT, wild type; MM, mock treated control.

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    Table I Autophagic activity screen in yeast strains defective of the orthologs of the genes encoding for the common autophagosome associated proteins

    The common proteins localizing to autophagosomes of MCF7 cells independent of the stimuli.

    ProteinGene ID (human)Yeast orthologAutophagic activityaReferencedeGFP-IPe
    Nitrogen starvationbRapamycinc
    Alanyl tRNA synthetaseAARSALA1f+
    Annexin A4ANXA432g
    Annexin A5ANXA532g++
    Archain 1ARCN1RET2f++
    ADP-ribosylation factor 1ARF1ARF2126.1 ± 16.032g++
    Argininosuccinate synthase 1ASS1ARG1115.8 ± 11.6++
    5-Aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolaseATICADE1799.7 ± 13.532g
    Biliverdin reductase BBLVRB++
    γ-Glutamyl cyclotransferaseC7orf24
    Calcyclin-binding proteinCACYBP32g+
    Calpain, small subunit 1CAPNS132g+
    Chaperonin containing TCP1, subunit 2CCT2CCT2f++
    Chaperonin containing TCP1, subunit 5CCT5CCT5f++
    Chaperonin containing TCP1, subunit 8CCT8CCT8f++
    Cofilin 1CFL1COF1f++
    Chromatin modifying protein 6CHMP6
    Coatomer protein complex, subunit ζ1COPZ1RET3f+
    Copine IIICPNE3
    Cellular retinoic acid-binding protein 2CRABP2+
    Cystatin BCSTB
    DestrinDSTN++
    Eukaryotic translation elongation factor 1γEEF1GTEF456.2 ± 16.384.3 ± 19.1++
    Eukaryotic translation elongation factor 2EEF2EFT2104.5 ± 23.2++
    Enolase 1ENO1ENO195.8 ± 38.034h++
    Fatty acid synthaseFASNCEM1101.4 ± 14.2++
    Fructose-1,6-bisphosphatase 1FBP1FBP199.2 ± 16.934h+
    FK506-binding protein 4, 59 kDaFKBP4FPR186.2 ± 17.032+
    Glucose-6-phosphate dehydrogenaseG6PDZWF1108.3 ± 12.8++
    GABAA receptor-associated protein-like 2GABARAPL2ATG826.1 ± 21.444.7 ± 26.358+
    GDP dissociation inhibitor 2GDI232g++
    Guanine nucleotide-binding protein, β polypeptide 2-like 1GNB2L1ASC1148.0 ± 21.5210.7 ± 43.1+
    Glucose phosphate isomeraseGPIPGI1f++
    Glutathione reductaseGSRGLR190.7 ± 9.1
    Hypoxanthine phosphoribosyltransferase 1HPRT1+
    Heat shock protein 90-kDa α, class A member 1HSP90AA1HSC82100.0 ± 15.9++
    Heat shock protein 90-kDa α, class B member 1HSP90AB1HSP8290.6 ± 6.8++
    Heat shock 27-kDa protein 1HSPB1++
    KIAA1609KIAA1609++
    LIM and SH3 protein 1LASP132g+
    Lectin, galactoside-binding, soluble, 3LGALS3++
    Microtubule-associated protein 1 light chain 3βMAP1LC3BATG826.1 ± 21.444.7 ± 26.358
    Malate dehydrogenase 1MDH134h
    Macrophage migration inhibitory factorMIF++
    Purine nucleoside phosphorylaseNPPNP146.0 ± 5.930.1 ± 10.6
    Aminopeptidase, puromycin-sensitiveNPEPPSAAP1101.4 ± 34.432g+
    Platelet-activating factor acetylhydrolase 1b, catalytic subunit 3PAFAH1B3
    Poly(rC)-binding protein 1PCBP1PBP2103.4 ± 8.833++
    Phosphatidylethanolamine-binding protein 1PEBP132g+
    Profilin 1PFN1PFY1f++
    Profilin 2PFN2
    Phosphogluconate dehydrogenasePGDGND1111.5 ± 25.2
    Phosphoglycerate dehydrogenasePHGDHSER3398.7 ± 31.8++
    Pyruvate kinase, musclePKM2CDC19f++
    Peptidylprolyl isomerase APPIACPR194.9 ± 16.334h++
    Protein phosphatase 3, catalytic subunit, α isoformPPP3CACMP2102.7 ± 11.1+
    Peroxiredoxin 1PRDX1TSA196.8 ± 11.634h++
    Peroxiredoxin 2PRDX2TSA196.8 ± 11.634h++
    Peroxiredoxin 6PRDX6PRX190.9 ± 6.034h++
    Proteasome subunit, α type, 2PSMA2PRE8f45g+
    Proteasome subunit, α type, 3PSMA3PRE10f45g
    Proteasome subunit, α type, 5PSMA5PUP2f45g+
    Proteasome subunit, α type, 6PSMA6SCL1f45g+
    Proteasome subunit, α type, 7PSMA7PRE6f45g+
    Proteasome subunit, β type, 1PSMB1PRE7f45g
    Proteasome subunit, β type, 2PSMB2PRE1f45g+
    Proteasome subunit, β type, 3PSMB3PUP3f45g
    Proteasome subunit, β type, 4PSMB4PRE4f45g
    Proteasome subunit, β type, 5PSMB5PRE2f45g
    Proteasome subunit, β type, 6PSMB6PRE3f45g
    Proteasome subunit, β type, 7PSMB7PUP1f45g
    Proteasome 26 S subunit, ATPase, 6PSMC6RPT4f45g
    Proteasome activator subunit 1PSME145g++
    Proteasome activator subunit 2PSME245g++
    Ras homolog enriched in brain 1RHEB1RHB136.3 ± 11.137.2 ± 4.259g
    Ribosomal protein S21RPS21RPS21B85.5 ± 4.3
    Ribosomal protein SARPSARPS0A90.8 ± 32.7+
    S100 calcium-binding protein A11S100A11++
    S100 calcium-binding protein A13S100A13
    SH3 domain binding glutamic acid-rich protein likeSH3BGRL
    Sequestosome 1SQSTM141++
    Spermidine synthaseSRMSPE3119.1 ± 23.132g
    Stress-induced-phosphoprotein 1STIP1STI1113.4 ± 24.3++
    Tumor protein D52-like 2TPD52L2++
    Triosephosphate isomerase 1TPI1TPI1f++
    Tubulin, βTUBBTUB2f++
    Tubulin, β 2CTUBB2C++
    ThioredoxinTXNTRX2101.2 ± 29.7+
    Thioredoxin reductase 1TXNRD132g
    Ubiquitin-like modifier activating enzyme 1UBE1UBA1f
    Ubiquitin-conjugating enzyme E2NUBE2NUBC13106.5 ± 19.4++
    Tryptophanyl-tRNA synthetaseWARSWRS1f32g+
    Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, β polypeptideYWHAB32g++
    Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, ζ polypeptideYWHAZBMH298.7 ± 20.7++
    Zinc and ring finger 2ZNRF2
    Additional proteins associated to autophagosomal clusters
        CAP, adenylate cyclase-associated protein 1CAP1SRV250.4 ± 11.142.4 ± 10.4++
        Vacuolar protein sorting 35 homologVPS35VPS3512.4 ± 3.236.3 ± 21.1++
    • ↵a As measured by the alkaline phosphatase (ALP) activity assay. The values represent triplicates, and the standard deviations are indicated. The numbers marked in bold type indicate significant changes in the value obtained by ALP assay compared with randomly chosen control KO strains (p < 0.05, one-way analysis of variance).

    • ↵b Nitrogen starvation (3 h).

    • ↵c Rapamycin treatment (0.5 μg/ml; 3 h).

    • ↵d Proteins previously described in autophagosomes are referenced.

    • ↵e Identified in one (+) or two (++) eGFP-IPs of cells treated with HBSS or rapamycin in addition to concanamycin A.

    • ↵f Knockout strain not viable.

    • ↵g Autophagy-related proteins but so far not linked to the autophagosome are referenced.

    • ↵h Rat ortholog.

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Identification of Autophagosome-associated Proteins and Regulators by Quantitative Proteomic Analysis and Genetic Screens
Jörn Dengjel, Maria Høyer-Hansen, Maria O. Nielsen, Tobias Eisenberg, Lea M. Harder, Søren Schandorff, Thomas Farkas, Thomas Kirkegaard, Andrea C. Becker, Sabrina Schroeder, Katja Vanselow, Emma Lundberg, Mogens M. Nielsen, Anders R. Kristensen, Vyacheslav Akimov, Jakob Bunkenborg, Frank Madeo, Marja Jäättelä, Jens S. Andersen
Molecular & Cellular Proteomics March 1, 2012, First published on February 6, 2012, 11 (3) M111.014035; DOI: 10.1074/mcp.M111.014035

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Identification of Autophagosome-associated Proteins and Regulators by Quantitative Proteomic Analysis and Genetic Screens
Jörn Dengjel, Maria Høyer-Hansen, Maria O. Nielsen, Tobias Eisenberg, Lea M. Harder, Søren Schandorff, Thomas Farkas, Thomas Kirkegaard, Andrea C. Becker, Sabrina Schroeder, Katja Vanselow, Emma Lundberg, Mogens M. Nielsen, Anders R. Kristensen, Vyacheslav Akimov, Jakob Bunkenborg, Frank Madeo, Marja Jäättelä, Jens S. Andersen
Molecular & Cellular Proteomics March 1, 2012, First published on February 6, 2012, 11 (3) M111.014035; DOI: 10.1074/mcp.M111.014035
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Molecular & Cellular Proteomics: 11 (3)
Molecular & Cellular Proteomics
Vol. 11, Issue 3
1 Mar 2012
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