Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3316729&req=5

Figure 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.

Mentions: To distinguish autophagosomal candidate proteins from nonspecific co-purifying proteins, we applied the PCP-SILAC method (35, 36). This method is based on the assumption that proteins residing in the same organelle will have similar distributions through a density gradient. As outlined in Fig. 1C, two preparation of autophagosomes were isolated in parallel from MCF7-eGFP-LC3 cells labeled with different combinations of isotope-encoded lysine and arginine (37). Fractions collected after the final iodixanol density gradient centrifugation (1.05–1.25 g/ml iodixanol) were tested for the presence of organellar markers and known autophagosomal proteins by Western blot analysis (Fig. 1E and supplemental Fig. S1). For peptide isotope ratio determination, six fractions containing autophagosomal markers from the unlabeled cells were combined to generate a common internal standard that was equally distributed into the corresponding six fractions from the labeled cells. Proteins in these six samples were then analyzed by LC-MS, and the resulting data were used to determine the relative enrichment of proteins in each fraction (Fig. 1D). The protein enrichment profile of LC3 and Western blot analyses of eGFP-LC3 indicated that the majority of autophagosomes appeared in fractions 2 and 3 regardless of the stimulus (Fig. 1E). In contrast, marker proteins for other organelles showed clearly distinct profiles (supplemental Fig. S1B). In total, 7935 proteins were identified in single representative PCP-SILAC experiments for each of the three stimuli (from a total set of one concanamycin A experiment, two rapamycin experiments, and two starvation experiments; supplemental Fig. S2). Complete protein enrichment profiles were obtained for 4516 proteins detected in all six fractions (supplemental Fig. S2 and Tables S1–S3). These 4516 proteins were grouped into three clusters per experiment based on their profiles using the noise robust fuzzy c-means algorithm (38). Cluster A proteins peaked in fractions 2 and 3 and included known autophagosomal proteins such as LC3, p62 (SQSTM1), and GABARAPL2 (Figs. 2A and supplemental Fig. S2). Cluster B contained proteins that also peaked in fractions 2 and 3 but showed an additional rise in fraction 6. Cluster C included proteins that peaked in fraction 5 and 6 and were regarded as nonspecific co-purifying proteins. Autophagosomal clusters (cluster A) from concanamycin A-treated, rapamycin-treated, and starved cells contained 238, 359, and 482 protein profiles, respectively (supplemental Tables S1–S3). These profiles represent 728 different proteins, of which 94 were found in the autophagosomal cluster for all three stimuli (Table I and Fig. 2B). In support of the PCP-SILAC method, we identified 42 of the 94 proteins in cluster A that were known autophagosomal proteins or recently reported interaction partners of autophagy-related proteins (Table I and supplemental Table S4). Identification of autophagy regulators such as FK506-binding protein 1 (FKBP1A) and Ras homolog enriched in brain (RHEB) suggests that they operate at the autophagosome scaffold.


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3316729&req=5

Figure 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.
Mentions: To distinguish autophagosomal candidate proteins from nonspecific co-purifying proteins, we applied the PCP-SILAC method (35, 36). This method is based on the assumption that proteins residing in the same organelle will have similar distributions through a density gradient. As outlined in Fig. 1C, two preparation of autophagosomes were isolated in parallel from MCF7-eGFP-LC3 cells labeled with different combinations of isotope-encoded lysine and arginine (37). Fractions collected after the final iodixanol density gradient centrifugation (1.05–1.25 g/ml iodixanol) were tested for the presence of organellar markers and known autophagosomal proteins by Western blot analysis (Fig. 1E and supplemental Fig. S1). For peptide isotope ratio determination, six fractions containing autophagosomal markers from the unlabeled cells were combined to generate a common internal standard that was equally distributed into the corresponding six fractions from the labeled cells. Proteins in these six samples were then analyzed by LC-MS, and the resulting data were used to determine the relative enrichment of proteins in each fraction (Fig. 1D). The protein enrichment profile of LC3 and Western blot analyses of eGFP-LC3 indicated that the majority of autophagosomes appeared in fractions 2 and 3 regardless of the stimulus (Fig. 1E). In contrast, marker proteins for other organelles showed clearly distinct profiles (supplemental Fig. S1B). In total, 7935 proteins were identified in single representative PCP-SILAC experiments for each of the three stimuli (from a total set of one concanamycin A experiment, two rapamycin experiments, and two starvation experiments; supplemental Fig. S2). Complete protein enrichment profiles were obtained for 4516 proteins detected in all six fractions (supplemental Fig. S2 and Tables S1–S3). These 4516 proteins were grouped into three clusters per experiment based on their profiles using the noise robust fuzzy c-means algorithm (38). Cluster A proteins peaked in fractions 2 and 3 and included known autophagosomal proteins such as LC3, p62 (SQSTM1), and GABARAPL2 (Figs. 2A and supplemental Fig. S2). Cluster B contained proteins that also peaked in fractions 2 and 3 but showed an additional rise in fraction 6. Cluster C included proteins that peaked in fraction 5 and 6 and were regarded as nonspecific co-purifying proteins. Autophagosomal clusters (cluster A) from concanamycin A-treated, rapamycin-treated, and starved cells contained 238, 359, and 482 protein profiles, respectively (supplemental Tables S1–S3). These profiles represent 728 different proteins, of which 94 were found in the autophagosomal cluster for all three stimuli (Table I and Fig. 2B). In support of the PCP-SILAC method, we identified 42 of the 94 proteins in cluster A that were known autophagosomal proteins or recently reported interaction partners of autophagy-related proteins (Table I and supplemental Table S4). Identification of autophagy regulators such as FK506-binding protein 1 (FKBP1A) and Ras homolog enriched in brain (RHEB) suggests that they operate at the autophagosome scaffold.

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