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Identification of autophagosome-associated proteins and regulators by quantitative proteomic analysis and genetic screens.

Dengjel J, Høyer-Hansen M, Nielsen MO, Eisenberg T, Harder LM, Schandorff S, Farkas T, Kirkegaard T, Becker AC, Schroeder S, Vanselow K, Lundberg E, Nielsen MM, Kristensen AR, Akimov V, Bunkenborg J, Madeo F, Jäättelä M, Andersen JS - Mol. Cell Proteomics (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark. joern.dengjel@frias.uni-freiburg.de

ABSTRACT
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.

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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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Figure 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.

Mentions: Our findings suggest that the autophagic machinery and the proteasome are interconnected (Figs. 2C and 5A). Interplay between the two degradation systems has been proposed lately (24). However, because a detailed description of the connection of these two major cellular degradation pathways is still missing, we investigated their interplay in more detail. First, we confirmed the stimulus-independent partial co-localization of proteasomal proteins and LC3 by fluorescence microscopy and Western blot analyses (Fig. 6, A–J). This evidence of proteasomal 20 S subunits associated with autophagosomes led us to speculate whether autophagy might decrease the proteasome level in cells. Indeed, the abundance of proteasomal proteins in whole cell lysates decreased upon amino acid starvation, and importantly this decrease could be blocked by the addition of 3-methyladenine, an inhibitor of autophagy (40) (Fig. 6K). Accordingly, induction of autophagic flux by rapamycin or starvation led to a significant decrease in proteasomal activity (Fig. 6L). Notably, concanamycin A, which inhibits the degradation of autophagosomal cargo (Fig. 5C), did not influence the proteasomal activity (Fig. 6L). Furthermore autophagosomes purified by immunoprecipitation of eGFP-LC3 with an anti-GFP antibody contained proteasomal subunits, supporting their association with autophagosomes. Interestingly, incubation of these purified autophagosomes with detergent abolished the pulldown of the proteasomal subunits. This suggests that the proteasomal subunits do not directly interact with LC3 but rather associate with the autophagosome. Contrary, p62 was pulled down with eGFP-LC3 independent of detergent treatment as expected (Fig. 6M). Taken together, these data suggest that the proteasome associates with autophagosomes independently of LC3 and that functional autophagy leads to a decrease in proteasome amount and activity.


Identification of autophagosome-associated proteins and regulators by quantitative proteomic analysis and genetic screens.

Dengjel J, Høyer-Hansen M, Nielsen MO, Eisenberg T, Harder LM, Schandorff S, Farkas T, Kirkegaard T, Becker AC, Schroeder S, Vanselow K, Lundberg E, Nielsen MM, Kristensen AR, Akimov V, Bunkenborg J, Madeo F, Jäättelä M, Andersen JS - Mol. Cell Proteomics (2012)

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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Figure 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.
Mentions: Our findings suggest that the autophagic machinery and the proteasome are interconnected (Figs. 2C and 5A). Interplay between the two degradation systems has been proposed lately (24). However, because a detailed description of the connection of these two major cellular degradation pathways is still missing, we investigated their interplay in more detail. First, we confirmed the stimulus-independent partial co-localization of proteasomal proteins and LC3 by fluorescence microscopy and Western blot analyses (Fig. 6, A–J). This evidence of proteasomal 20 S subunits associated with autophagosomes led us to speculate whether autophagy might decrease the proteasome level in cells. Indeed, the abundance of proteasomal proteins in whole cell lysates decreased upon amino acid starvation, and importantly this decrease could be blocked by the addition of 3-methyladenine, an inhibitor of autophagy (40) (Fig. 6K). Accordingly, induction of autophagic flux by rapamycin or starvation led to a significant decrease in proteasomal activity (Fig. 6L). Notably, concanamycin A, which inhibits the degradation of autophagosomal cargo (Fig. 5C), did not influence the proteasomal activity (Fig. 6L). Furthermore autophagosomes purified by immunoprecipitation of eGFP-LC3 with an anti-GFP antibody contained proteasomal subunits, supporting their association with autophagosomes. Interestingly, incubation of these purified autophagosomes with detergent abolished the pulldown of the proteasomal subunits. This suggests that the proteasomal subunits do not directly interact with LC3 but rather associate with the autophagosome. Contrary, p62 was pulled down with eGFP-LC3 independent of detergent treatment as expected (Fig. 6M). Taken together, these data suggest that the proteasome associates with autophagosomes independently of LC3 and that functional autophagy leads to a decrease in proteasome amount and activity.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark. joern.dengjel@frias.uni-freiburg.de

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus