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

2012; Elsevier BV; Volume: 11; Issue: 3 Linguagem: Inglês

10.1074/mcp.m111.014035

ISSN

1535-9484

Autores

Jörn Dengjel, Maria Høyer-Hansen, Maria Overbeck Nielsen, Tobias Eisenberg, Lea Mørch Harder, Søren Schandorff, Thomas Farkas, Thomas Kirkegaard, Andrea C. Becker, Sabrina Schroeder, Katja Vanselow, Emma Lundberg, Mogens M. Nielsen, Anders Riis Kristensen, Vyacheslav Akimov, Jakob Bunkenborg, Frank Madeo, Marja Jäättelä, Jens Andersen,

Tópico(s)

Ubiquitin and proteasome pathways

Resumo

Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome-associated proteins were dependent on stimulus, but a core set of proteins was stimulus-independent. Remarkably, proteasomal proteins were abundant among the stimulus-independent common autophagosome-associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome-associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection. Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome-associated proteins were dependent on stimulus, but a core set of proteins was stimulus-independent. Remarkably, proteasomal proteins were abundant among the stimulus-independent common autophagosome-associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome-associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection. Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved lysosomal pathway involved in the turnover of long-lived proteins, cytoplasm, and whole organelles (1Kroemer G. Jäättelä M. Lysosomes and autophagy in cell death control.Nat. Rev. Cancer. 2005; 5: 886-897Crossref PubMed Scopus (1052) Google Scholar, 2Mizushima N. Levine B. Cuervo A.M. Klionsky D.J. Autophagy fights disease through cellular self-digestion.Nature. 2008; 451: 1069-1075Crossref PubMed Scopus (5234) Google Scholar, 3Nakatogawa H. Suzuki K. Kamada Y. Ohsumi Y. 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The autophagic process begins with the nucleation of a flat membrane cistern that enwraps cytoplasmic organelles and/or a portion of the cytosol. The membrane elongates until the edges of the membrane fuse, thereby forming a double membrane structure called an autophagosome, which fuses with endosomes forming an amphisome (9Gordon P.B. Seglen P.O. Prelysosomal convergence of autophagic and endocytic pathways.Biochem. Biophys. Res. Commun. 1988; 151: 40-47Crossref PubMed Scopus (214) Google Scholar) and subsequently matures to an autolysosome by fusing with lysosomal vesicles. The final degradation of the cargo takes place within autolysosomes, where lysosomal hydrolases digest the luminal content, allowing the recycling of amino acids, nucleotides, and fatty acids (10Finn P.F. Dice J.F. Proteolytic and lipolytic responses to starvation.Nutrition. 2006; 22: 830-844Crossref PubMed Scopus (244) Google Scholar). The process is controlled by a set of evolutionarily conserved autophagy-related proteins (Atg proteins) initially identified in yeast (11Klionsky D.J. Cregg J.M. Dunn Jr., W.A. Emr S.D. Sakai Y. Sandoval I.V. Sibirny A. Subramani S. Thumm M. Veenhuis M. Ohsumi Y. A unified nomenclature for yeast autophagy-related genes.Dev. Cell. 2003; 5: 539-545Abstract Full Text Full Text PDF PubMed Scopus (1024) Google Scholar, 12Ohsumi Y. Molecular dissection of autophagy: Two ubiquitin-like systems.Nat. Rev. Mol. Cell Biol. 2001; 2: 211-216Crossref PubMed Scopus (1052) Google Scholar). The protein complex consisting of phosphatidylinositol 3-phosphate kinase class 3, p150 myristylated protein kinase, and beclin 1 (Atg6) is essential for the initial membrane assembly, whereas the following membrane elongation depends on two ubiquitin-like conjugation systems. One of them converts microtubule-associated protein 1 light chain 3 (LC3/Atg8) 1The abbreviations used are:LClight chainERendoplasmic reticulummTORCmammalian target of rapamycin complexPCPprot lation profilingSILACstable isotope labeling by amino acids in cell cultureGFPgreen fluorescent proteineGFPenhanced GFPALPalkaline phosphataseHBSSHanks' balanced salt solutionsiRNAsmall interfering RNAConAconcanamycin ARaparapamycinRHEBRas homolog enriched in brainGABARAPL2GABAA receptor-associated protein-like 2EEF1Geukaryotic translation elongation factor 1γPSMproteasome subunit. from its free form (LC3-I) to a phosphatidylethanolamine-conjugated form (LC3-II), which associates with both membranes of the autophagosome (13Yang Z. Klionsky D.J. Mammalian autophagy: Core molecular machinery and signaling regulation.Curr. Opin. Cell Biol. 2010; 22: 124-131Crossref PubMed Scopus (1549) Google Scholar). This process is frequently used as an autophagy marker because the change in the LC3 staining pattern from diffuse to dotted can be readily visualized. light chain endoplasmic reticulum mammalian target of rapamycin complex prot lation profiling stable isotope labeling by amino acids in cell culture green fluorescent protein enhanced GFP alkaline phosphatase Hanks' balanced salt solution small interfering RNA concanamycin A rapamycin Ras homolog enriched in brain GABAA receptor-associated protein-like 2 eukaryotic translation elongation factor 1γ proteasome subunit. Autophagy is generally considered an unselective bulk degradation pathway. However, under certain conditions, autophagosomes have been suggested to selectively remove, for example, damaged mitochondria (14Kim I. Rodriguez-Enriquez S. Lemasters J.J. Selective degradation of mitochondria by mitophagy.Arch. Biochem. 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Cell. 2009; 33: 517-527Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar, 23Kraft C. Peter M. Hofmann K. Selective autophagy: Ubiquitin-mediated recognition and beyond.Nat. Cell Biol. 2010; 12: 836-841Crossref PubMed Scopus (512) Google Scholar, 24Lamark T. Johansen T. Autophagy: Links with the proteasome.Curr. Opin. Cell Biol. 2010; 22: 192-198Crossref PubMed Scopus (112) Google Scholar). The aim of this study is to identify proteins associated with the mature autophagosome and to compare the protein composition of autophagosomes induced by different stimuli. For this purpose, we analyzed autophagosomes isolated from MCF7 breast cancer cells following amino acid starvation or treatment with either rapamycin (an inhibitor of the mammalian target of rapamycin complex 1 (mTORC1)) or concanamycin A (an inhibitor of the lysosomal H+-ATPase) by quantitative MS-based proteomics (25Zimmermann A.C. Zarei M. Eiselein S. Dengjel J. Quantitative proteomics for the analysis of spatio-temporal protein dynamics during autophagy.Autophagy. 2010; 6: 1009-1016Crossref PubMed Scopus (27) Google Scholar) relying on protein correlation profiling (PCP) (26Andersen J.S. Wilkinson C.J. Mayor T. Mortensen P. Nigg E.A. Mann M. Proteomic characterization of the human centrosome by protein correlation profiling.Nature. 2003; 426: 570-574Crossref PubMed Scopus (1063) Google Scholar) and stable isotope labeling by amino acids in cell culture (SILAC) (27Dengjel J. Kratchmarova I. Blagoev B. Receptor tyrosine kinase signaling: A view from quantitative proteomics.Mol. Biosyst. 2009; 5: 1112-1121Crossref PubMed Scopus (50) Google Scholar, 28Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4625) Google Scholar). We identified 728 putative autophagosome-associated proteins from a background of co-purifying proteins. Of these, only a total of 94 proteins were common to all stimuli. To validate that the co-migrating proteins were derived from autophagosomes, we performed GFP pulldowns directed against a GFP-tagged version of the autophagosomal marker LC3 and tested subcellular localization by fluorescent microscopy for selected candidate proteins. To test whether the common autophagosome-associated proteins functioned as autophagy regulators, we turned to yeast genetics and screened the available yeast strains with mutations in the corresponding genes for changes in autophagy by the alkaline phosphatase (ALP) assay (29Klionsky D.J. Monitoring autophagy in yeast: The Pho8Delta60 assay.Methods Mol. Biol. 2007; 390: 363-371Crossref PubMed Google Scholar) identifying inter alia the retromer complex. Human cells depleted for the novel autophagy regulators identified in the yeast screen were subsequently analyzed for changes in autophagic flux by cell assays detecting the turnover of autophagosome-associated proteins (30Farkas T. Hoyer-Hansen M. Jaattela M. Identification of novel autophagy regulators by a luciferase-based assay for the kinetics of autophagic flux.Autophagy. 2009; 5: 1018-1025Crossref PubMed Scopus (78) Google Scholar, 31Klionsky D.J. Klionsky D.J. Abeliovich H. Agostinis P. Agrawal D.K. Aliev G. Askew D.S. Baba M. Baehrecke E.H. Bahr B.A. Ballabio A. Bamber B.A. Bassham D.C. Bergamini E. Bi X. Biard-Piechaczyk M. Blum J.S. Bredesen D.E. Brodsky J.L. Brumell J.H. Brunk U.T. Bursch W. Camougrand N. Cebollero E. Cecconi F. Chen Y. Chin L.S. Choi A. Chu C.T. Chung J. Clarke P.G. Clark R.S. Clarke S.G. Clavé C. Cleveland J.L. Codogno P. Colombo M.I. Coto-Montes A. Cregg J.M. Cuervo A.M. Debnath J. Demarchi F. Dennis P.B. Dennis P.A. Deretic V. Devenish R.J. Di Sano F. 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Vaquero E.C. Vellai T. Vogel M.W. Wang H.G. Webster P. Wiley J.W. Xi Z. Xiao G. Yahalom J. Yang J.M. Yap G. Yin X.M. Yoshimori T. Yu L. Yue Z. Yuzaki M. Zabirnyk O. Zheng X. Zhu X. Deter R.L. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.Autophagy. 2008; 4: 151-175Crossref PubMed Scopus (1980) Google Scholar). The data presented provide a quantitative proteomic characterization of autophagosomes identifying a set of autophagosome-associated proteins and regulators. For SILAC experiments MCF7-eGFP-LC3 cells (49Høyer-Hansen M. Bastholm L. Szyniarowski P. Campanella M. Szabadkai G. Farkas T. Bianchi K. Fehrenbacher N. Elling F. Rizzuto R. Mathiasen I.S. Jäättelä M. Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-β, and Bcl-2.Mol. Cell. 2007; 25: 193-205Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with penicillin/streptomycin (100 units/ml, 100 μg/ml), glutamine, and 10% dialyzed fetal calf serum (Invitrogen) and labeled with either l-lysine and l-arginine (Lys0, Arg0), l-lysine-2H4 and l-arginine-13C6 (Lys4, Arg6), or l-lysine13C6-15N2 and l-arginine13C6-15N4 (Lys8, Arg10) (Cambridge Isotope Laboratories, Andover, MA; Sigma-Aldrich). Otherwise the cells were grown in Dulbecco's modified Eagle's medium or RPMI (Invitrogen) with 10% FCS and penicillin/streptomycin. The cells were treated with concanamycin A, rapamycin, and etoposide (all from Sigma-Aldrich) or washed three times in PBS before they were amino acid-starved in HBSS containing calcium, magnesium, and 1 g of glucose/liter (Invitrogen). Fusion constructs of DsRed and RHEB or FKBP1A were generated using vectors pDsRed-C1 and -N1, respectively, (Clontech). RHEB and FKBP1A cDNAs were purchased from imaGenes (Berlin, Germany), and fusion constructs were sequence checked. Stable cell lines were generated by liposomes transfection using MetafecteneTM (Biontex, Martinsried, Germany). During transient transfections FuGENE 6 (Roche Applied Science) was used as a transfection agent according to the manufacturer's instructions. siRNA sequences corresponding to the human cDNA sequence are listed in the supplemental material. The cells were transfected with 50 nm siRNA employing OligofectamineTM (Invitrogen). The primary antibodies used for immunoblot analysis are listed in the supplemental "Experimental Procedures." The appropriate peroxidase-conjugated secondary antibodies were from Dako A/S (Glostrup, Denmark) and Vector Laboratories (Burlingame, CA), and the ECL immunoblotting reagents were from Amersham Biosciences. The cells were fixed and stained applying standard procedures (49Høyer-Hansen M. Bastholm L. Szyniarowski P. Campanella M. Szabadkai G. Farkas T. Bianchi K. Fehrenbacher N. Elling F. Rizzuto R. Mathiasen I.S. Jäättelä M. Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-β, and Bcl-2.Mol. Cell. 2007; 25: 193-205Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar). For detection of DsRed fusion proteins, the cells were fixed and permeabilized in 3.7% paraformaldehyde for 7 min followed by methanol overnight. The primary antibodies are listed in supplemental "Experimental Procedures." The respective fluorescent secondary antibodies were from Molecular Probes or Jackson Immunolabs. The images were obtained using either a Zeiss LSM 510 META confocal laser scanning microscope or Zeiss Axiovert 100M confocal laser scanning microscope (Carl Zeiss, Jena, Germany). Percentages of cells with eGFP-LC3 translocation into dots (a minimum of 100 cells/sample) were counted blindly in cells fixed in 3.7% formaldehyde and 0.19% picric acid (v/v) applying a Zeiss Axiovert 100M confocal laser scanning microscope (Carl Zeiss). The autophagic flux was measured applying a luciferase-based assay as described previously (30Farkas T. Hoyer-Hansen M. Jaattela M. Identification of novel autophagy regulators by a luciferase-based assay for the kinetics of autophagic flux.Autophagy. 2009; 5: 1018-1025Crossref PubMed Scopus (78) Google Scholar). The proteasome activity was measured using the 20 S proteasome activity assay kit from Chemicon International (catalog number APT280; Temecula, CA), and the protein levels were measured using the Bio-Rad Dc protein assay kit (Bio-Rad) according to the manufacturer's instructions (supplemental "Experimental Procedures"). The experiments were carried out with BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and respective null mutants, obtained from Euroscarf (growth conditions described in the supplemental "Experimental Procedures"). For induction of autophagy by nitrogen starvation, the cells were inoculated from fresh overnight cultures to 0.4 A600 (∼4 × 106 cells/ml) in Synthetic Complete Dextrose, grown for 4–5 h to mid-log phase reaching ∼1.5 A600, washed twice in double distilled H2O, and incubated in SD-N medium at 1 A600 for 3 h. For induction of autophagy by rapamycin treatment, the cells were grown to mid-log phase (∼1 A600) as above, and rapamycin (AG Scientific) was added to the culture to a final concentration of 0.5 μg/ml preceding 3 h of incubation. Autophagy was determined in cellular extracts of 1-ml culture aliquots by ALP activity according to Ref. 50Kissová I. Deffieu M. Manon S. Camougrand N. Uth1p is involved in the autophagic degradation of mitochondria.J. Biol. Chem. 2004; 279: 39068-39074Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar using respective strains transformed with and selected for stable insertion of pTN9 HindIII fragment containing the cytosolic form of Pho8p (Pho8pΔN60). For details see the supplemental "Experimental Procedures". Autophagosome purification was modified from the protocol described in Ref. 51Strømhaug P.E. Berg T.O. Fengsrud M. Seglen P.O. Purification and characterization of autophagosomes from rat hepatocytes.Biochem. J. 1998; 335: 217-224Crossref PubMed Scopus (139) Google Scholar (for details see the supplemental "Experimental Procedures"). The PCP-SILAC background fractions (Lys0/Arg0) were combined and spiked in a 1:1 ratio to the respective (Lys4/Arg6) PCP SILAC fractions (52Dengjel J. Jakobsen L. Andersen J.S. Organelle proteomics by label-free and SILAC-based protein correlation profiling.Methods Mol. Biol. 2010; 658: 255-265Crossref PubMed Scopus (9) Google Scholar). The resulting fractions were dilluted 1:1 in Homogenization Medium and pelleted at 31,000 × g. The pellets were resuspended in SDS loading buffer and reduced and alkylated. Protein mixtures were separated by SDS-PAGE (4–12% Bis-Tris gradient gel, NuPAGE; Invitrogen) and in-gel digested with trypsin. In the GFP tag enrichment experiment, vesicles were enriched by centrifugation followed by GFP antibody pulldown (supplemental Fig. S3). The proteins were digested in solution with trypsin and the peptides separated by ion exchange chromatography. The resulting peptide mixtures were analyzed by online C18 reversed phase nanoscale liquid chromatography tandem mass spectrometry essentially as described (53Zarei M. Sprenger A. Metzger F. Gretzmeier C. Dengjel J. Comparison of ERLIC-TiO2, HILIC-TiO2, and SCX-TiO2 for global phosphoproteomics approaches.J. Proteome Res. 2011; 10: 3474-3483Crossref PubMed Scopus (81) Google Scholar) (for details see the supplemental "Experimental Procedures"). Raw MS2 spectra were centroided and merged into a single peak list file using the in-house-developed software DTASupercharge (default values; version 1.19, msquant.sourceforge.net) and searched against the human MSIPI database (v. 3.34 with 68404 entries) (54Schandorff S. Olsen J.V. Bunkenborg J. Blagoev B. Zhang Y. Andersen J.S. Mann M. A mass spectrometry-friendly database for cSNP identification.Nat. Methods. 2007; 4: 465-466Crossref PubMed Scopus (60) Google Scholar) using MASCOT 2.0 (Matrix Science, London, UK) basically as described (19Kristensen A.R. Schandorff S. Høyer-Hansen M. Nielsen M.O. Jäättelä M. Dengjel J. Andersen J.S. Ordered organelle degradation during starvation-induced autophagy.Mol. Cell. Proteomics. 2008; 7: 2419-2428Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) and the following parameters: carbamidomethyl-cysteine was set as fixed modification, methionine oxidation, deamidation of asparagine and glutamine, and protein amino-terminal acetylation were set as variable modifications. Double or triple SILAC were chosen as quantification mode. Two miss cleavages were allowed, enzyme specificity was trypsin, precursor mass accuracy had to be within 30 ppm for FT-data and 7 ppm for Orbitrap data, and the fragment spectra mass accuracy was set at 0.6 Da. The identified peptides were recalibrated using MSQuant (55Mortensen P. Gouw J.W. Olsen J.V. Ong S.E. Rigbolt K.T. Bunkenborg J. Cox J. Foster L.J. Heck A.J. Blagoev B. Andersen J.S. Mann M. MSQuant, an open source platform for mass spectrometry-based quantitative proteomics.J. Proteome Res. 2010; 9: 393-403Crossref PubMed Scopus (222) Google Scholar), and the results were combined using MGFcombiner (version 1.05) and searched again using MASCOT 2.0 with the above mentioned parameters, except that the precursor mass tolerance was set to 5 ppm. To determine the number of false positive peptide hits, the data were searched against a full-length human MSIPI decoy database, and the MASCOT peptide score was adjusted to yield a number of false positive peptide identifications of less than 1% (calculated as follows: false positive rate (%) = reverse hits × 2 × 100/forward hits). For a protein to be counted as identified, a minimum of two unique peptides (bold red hits, minimum length of 7 amino acids) had to be sequenced and to fulfill the determined criteria. To yield the maximum number of peptides per identified protein, the data were researched against decoy databases consisting of the identified proteins only considering proteins that were identified using a full-length decoy database and that did pass the stringent identification criteria stated above. Thus, the peptide MASCOT identification score yielding less than 1% false positive could be dropped to 15, resulting in considerably more peptide IDs/protein. PCP SILAC profiles were calculated using all bold red and parenthesized peptides. The proteins were only included if respective peptides were sequenced in each of the six fractions yielding six data point profiles. In a few exceptions, the peptides were inserted based in on elution time predicted from the analyses of neighbor fractions. The peptides were quantified by MSQuant (55Mortensen P. Gouw J.W. Olsen J.V. Ong S.E. Rigbolt K.T. Bunkenborg J. Cox J. Foster L.J. Heck A.J. Blagoev B. Andersen J.S. Mann M. MSQuant, an open source platform for mass spectrometry-based quantitative proteomics.J. Proteome Res. 2010; 9: 393-403Crossref PubMed Scopus (222) Google Scholar) using extracted ion chromatograms of the monoisotopic signals. For cluster analyses, the protein ratios were normalized using the base 2 logarithm. The GFP pulldown data was processed and analyzed using MaxQuant (version 1.0.13.13) (56Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9467) Google Scholar) and Mascot (version 2.3). The Quant module of MaxQuant was used with parameters set as follows: Arg6/Lys4 and Arg10/Lys8 as triple SILAC labels, a maximum of two missed cleavages, filtering of MS/MS spectra to retain only the six most intense peaks/100 Da, and MS/MS mass tolerance of 0.5 Da. Variable modifications were methionine oxidation and protein amino-terminal acetylation. Cysteine carbamidomethylation was a fixed modification. Protein and peptide FDR of 0.01, PEP based on Mascot score, minimum peptide length of 6, minimum score of 7, minimum unique sequence of 1, minimum peptides of 1, use of both unmodified and modified peptides for protein quantification, and use of both razor and unique peptides for quantification with a minimum ratio count of 2. The protein profiles were clustered using GProX (57Rigbolt K.T. Vanselow J.T. Blagoev B. GProX, a user-friendly platform for bioinformatics analysis and visualization of quantitative proteomics data.Mol. Cell. Proteomics. 2011; 10.1074/mcp.O110.007450Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) to identify proteins with profiles similar to known autophagosomal proteins. Clustering was done using the fuzzy c-means algorithm (38Futschik M.E. Carlisle B. Noise-robust soft clustering of gene expression time-course data.J. Bioinform. Comput. Biol. 2005; 3: 965-988Crossref PubMed Scopus (276) Google Scholar), which is a soft clustering algorithm being noise robust and indicating how well protein profiles are represented by respective clusters. The Fuzzy c-means parameter m varied between 1.25 (Rapa), 2.5 (HBSS), and 3.0 (ConA) for the three stimuli to compensate for differences in resolution of the data and to ensure minimal size of autophagosomal clusters. Prot

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