The data presented above provide a framework of autophagosome-associated proteins and will help in characterizing underlying cellular processes regulating the biogenesis and cargo selection of autophagosomes. Employing PCP-SILAC, we grouped the identified proteins in three clusters per organelle preparation for cells treated with concanamycin A, rapamycin, and HBSS. The clusters consisted of autophagosomal candidate proteins (cluster A); proteins found both in autophagosomal fractions and in other organelles such as endolysosomes, ER, and Golgi apparatus (cluster B); and nonautophagosomal proteins such as ER and nuclear proteins (cluster C). For further investigations, we concentrated on proteins observed in cluster A.
The most comprehensive proteomic analysis of autophagosomes prior to this study identified 39 and 101 proteins localizing to autophagosomal membranes in starved rat hepatocytes and human cell lines, respectively (33
). We chose a different approach characterizing not only autophagosomal membrane proteins but also autophagosomal cargo proteins to learn more about cellular proteome dynamics during autophagy. With this approach, we identified 728 autophagosomal candidate proteins, of which 94 were found in the autophagosome cluster regardless of the stimulus. Our data suggest that 31 proteins previously linked to autophagy are also associated with the autophagosome, among them the proteasome.
Comparison of the 94 common proteins with the other two reported data sets revealed seven human ortholog proteins shared with the autophagosomal rat proteins identified by Øverbye et al.
) and a single protein shared with the 101 identified by Gao et al.
). The rather small overlap might reflect differences in stimuli, isolation procedures, cell types, and MS methods. Whereas Øverbye et al.
did not identify any of the known ATG proteins, we determined complete protein profiles of the two human ATG8 homologs, MAP1LC3B and GABARAPL2. Gao et al.
identified MAP1LC3B, ATG7 and ATG9. We identified ATG3 and ATG4B and detected single peptides from ATG9A and ATG5. The abundance of the latter proteins was too low for the extraction of complete protein profiles, and therefore they were omitted from further analysis. However, the partial profiles of these proteins closely resemble the profiles of proteins in cluster A (data not shown), indicating that these ATGs may also localize to autophagosomes as suggested previously (43
). Taken together, ATG proteins appear to be low abundant and are not commonly identified by large scale proteomics studies. For their analyses, dedicated affinity purification protocols appear to be more promising (32
By analyzing autophagosomes from cells undergoing stress-induced autophagy by amino acid starvation, rapamycin-induced autophagy, and basal autophagy, which was blocked by concanamycin A, we compared autophagosomes from cells under fundamental different conditions. These analyses revealed proteins that always appear to associate with autophagosomes and others that appear to associate in a cell condition-dependent manner. Hence, stress-induced macroautophagy appears to differ from basal macroautophagy, and further studies are needed to understand stimulus-dependent cargo recruitment to autophagosomes.
An interesting finding in our study was the association between the proteasome and the autophagosome. In the past, the proteasomal and the autophagosomal/lysosomal pathways have been regarded as discrete degradation routes. Lately, a closer connection became more likely (4
), and it has been described that the proteasome can be degraded in lysosomes (45
). However, no detailed analyses of the interplay between these two degradation pathways have been reported. Here, we show that the proteasome localizes to autophagosomes regardless of the three stimuli and that the amount and activity of the proteasome are significantly decreased by functional autophagy. It is surprising that the proteasome associates with autophagosomes as the overall protein degradation increases during autophagy. However, several possible reasons might exist. First, autophagy induction is often associated with cellular stress and decreased cell growth. Thus, the removal of the proteasome and the subsequent accumulation of proteasomal substrates such as cyclins may contribute to cell cycle arrest. We tested this hypothesis but were not able to detect increased levels of cyclin D in cell lysate after autophagy induction (data not shown). This might be due to several compensatory mechanisms: 1) inhibition of protein synthesis because of inhibition of mTORC1 during autophagy activation or 2) autophagy might degrade proteins normally removed by the proteasome, e.g.
via removal of p62-ubiquitinated protein aggregates (22
). Second, one of the major functions of autophagy is to preserve cellular energy and thus degradation of the excess proteasome, which is not needed in circumstances where autophagy is activated, and de novo
protein synthesis minimized via the inhibition of mTORC1 may serve as an important source of energy and amino acids. Furthermore, the degradation of proteasome substrates in autolysosomes may be less energy demanding than proteolysis by the proteasome. Last but not least, the proteasome could “help” to degrade ubiquitinated/unfolded cargo in the autophagosome. In favor of this theory, several subunits of PSMA (subunits 2, 3, 5, 6, and 7) and PSMB (subunits 1, 2, 3, 4, 5, 6 and 7) proteins were detected in the autophagosome cluster. Because these subunits form the “active proteasome,” the functional proteasome complex appears to be autophagosomal cargo. This is further supported by the detection of protein-conjugated ubiquitin in autophagosomes by PCP-SILAC, immunofluoresence, and Western blot (data not shown) and by the fact that the proteasome activity is not decreased in concanamycin A-treated cells even though autophagosomes containing proteasomes accumulate in these cells.
Our data suggest that there is a balance between the two degradation systems that can be shifted in favor of one or the other, in our case active autophagy leading to reduced proteasome activity. In support of this notion, we observed increased levels of rapamycin-induced autophagy in yeast when the proteasome was inactivated by temperature-sensitive pre1-1 or pre2-1 mutants. The pre1-1/2-1 double mutant displayed synergistic effects (supplemental Fig. S7
). Enhanced autophagy upon proteasome impairment suggests a compensatory mechanism as reported previously (21
Among the new autophagosomal candidate proteins, we identified six autophagy regulators by a functional yeast screen that were not identified in the original screens characterizing Atg
genes in yeast (46
). This difference might be due to different screening methods. The original paper identified mutants defective in nitrogen starvation-induced autophagy as clones that lost viability faster than wild-type cells and were defective in accumulation of autophagic bodies in the vacuole. In contrast, we used the ALP assay. The orthologs of mammalian CAP1, EEF1G, RHEB, NP, and VPS35 were required for starvation-induced autophagy, whereas the yeast strain lacking the ortholog of mammalian GNB2L1 showed an increased autophagic response compared with the wild-type strain. Interestingly, testing additional subunits of the retromer complex revealed the same phenotype initially observed for the VPS35 subunit. In addition, the subunit VPS26 associated with autophagosomes in starved cells as identified by PCP-SILAC and anti-eGFP pulldowns. These data suggest that the retromer complex and retrograde transport play a role in autophagy, possibly by delivering vesicles or recycling autophagosomal membrane proteins.
Applying siRNA knockdown in human cells, we observed a change in the autophagic response after depleting cells for EEF1G or RHEB. Although we did not observe any major changes in the level of autophagy in cells depleted for CAP1, VPS35, and GNB2L1, we cannot exclude that these might have an effect if a more efficient down-regulation was achieved. As expected, the mTORC1 activator RHEB constitutively blocked autophagy in human cells. The fact that we identify it as an autophagosomal protein fits with our recent data showing that starvation induces its degradation (19
). Therefore, removal of RHEB by autophagy might serve as a positive feedback mechanism to ensure a massive autophagy response. Surprisingly, the ortholog of RHEB in yeast was found to be required for both starvation- and rapamycin-induced autophagy. This controversy could be explained by the fact that contrary to mammals, which have two RHEB isoforms, yeast only has one RHEB isoform (RHB), which is only distantly related to mammalian RHEBs and whose knock-out does not affect the activity of TOR (48
). Interestingly, EEF1G was found to be a universal autophagy regulator. Similar to LC3 it locates in/at the autophagosomes and is required for the process. However, contrary to LC3, it functions upstream of mTORC1. The exact molecular mechanism whereby EEF1G regulates mTORC1 activity remains to be revealed.
In summary, the analysis of organellar protein composition in our study highlights the complex character of autophagy shedding light on an elemental metabolic pathway that we are only starting to comprehend. The catalog of autophagosome-related proteins assembled from PCP-SILAC data and cross-examined by autophagosome immunoprecipitations revealed interplay between the autophagosome and the proteasome systems. In addition, we identified novel autophagosomal components of which EEF1G was found to be an autophagy regulator in both yeast and human cells.