Proteins are degraded via two main pathways in eukaryotic cells. Short-lived proteins are degraded by the proteasome, whereas long-lived proteins are degraded by autophagy. Protein aggregates that form during oxidative stress and other conditions, leading to misfolding and aggregation of proteins such as polyglutamine and polyalanine expansion mutations, are degraded by autophagy (Ravikumar et al., 2002
). Autophagy is generally thought of as a nonspecific bulk degradation mechanism. It is presently unclear whether there is a specific recognition or targeting of polyubiquitinated protein aggregates by the autophagic machinery.
Our data suggest that p62 may link polyubiquitinated proteins to the autophagic machinery. This function seems dependent on both the polymerization of p62 via the NH2
-terminal PB1 domain and polyubiquitin binding via the COOH-terminal UBA domain of p62. Both endogenous and ectopically expressed p62 could be copurified with the autophagy marker LC3. Both p62 and LC3 colocalized with mutant huntingtin aggregates. Such aggregates were recently shown to be degraded by autophagy (Ravikumar et al., 2002
). Very recently, studies of conditional knockout mice of Atg7 demonstrated that autophagy is needed for clearance of ubiquitin-positive aggregates (Komatsu et al., 2005
). We found that p62 formed a shell surrounding huntingtin aggregates. Cell death induced by the expression of aggregation-prone mutant huntingtin was increased both in HeLa and SHSY-5Y neuroblastoma cells after antisense RNA–mediated depletion of p62 levels or by interfering with p62 function by expressing a p62 deletion mutant lacking the UBA domain.
We found that p62 was located in two different types of bodies in the cytosol. The first type of structure appears as large protein aggregates (sequestosomes) that are not surrounded by a membrane and have very low mobility in living cells. However, the majority of p62 bodies in S–GFP-p62 cells were generally smaller structures with a much higher mobility that colocalized poorly with early endosomal markers but strongly with coexpressed myc- or GFP-tagged LC3. The finding that a high number of p62 bodies colocalized with LysoTracker and the lysosomal marker CD63 is consistent with p62 being localized to autophagosomes. By detergent extraction of live cells, we observed two populations of p62 bodies. LysoTracker-positive p62 bodies were rapidly lost after extraction, whereas LysoTracker-negative structures were not dissolved by 1% Triton X-100. This is consistent with p62 being partly located to membrane-enclosed autophagosomes and partly in cytosolic sequestosomes. Induction of autophagy by amino acid starvation led to a clear increase in the number of GFP-LC3–labeled autophagosomes, and all of these were positive for endogenous p62. The idea that a large fraction of p62 bodies are autophagosomes was also supported by the extensive accumulation of p62-LC3–positive structures observed in cells upon treatment with bafilomycin A1. Bafilomycin A1 inhibits the autophagosome–lysosome fusion step leading to accumulation of autophagosomal vacuoles. EM experiments confirmed that p62 is found both in autophagosomes and in sequestosomes and that p62 is colocalized with CD63 in cytoplasmic membrane-enclosed autophagosomal structures. Interestingly, we found p62 and LC3 to be components of the same protein complex by coimmunoprecipitation. It should be noted that coexpression of p62 with GFP-LC3 did not increase the total amount of p62 that was copurified with LC3. This suggests that the interaction might not be direct but may depend on a limiting third cellular factor. This notion is consistent with our failure to detect any interaction between GST-p62 and in vitro translated LC3 in a GST pull-down assay (unpublished data). Furthermore, the p62 D69A mutant that inhibits polymerization of p62 was very inefficiently coimmunoprecipitated with GFP-LC3, suggesting that polymeric p62 is important for interaction with LC3. Our data suggest that p62 is needed for the accumulation of GFP-LC3 in dots in HeLa cells in response to amino acid starvation. No GFP-LC3 dots were formed in response to amino acid starvation in cells depleted for endogenous p62 after transfection with siRNA. Similarly, no GFP-LC3 dots were formed in cells coexpressing mutants of p62, resulting in diffuse localization of endogenous p62. These results indicate that p62 polymerization is important for autophagosome formation in HeLa cells.
Previous studies have established p62 as a stress response protein induced by oxidative stress (Ishii et al., 1997
). The protein has also been identified as a common component in protein aggregates that was found in a wide range of protein aggregation diseases (Zatloukal et al., 2002
). Recently, expression of mutant huntingtin was shown to induce p62 (Nagaoka et al., 2004
). Interestingly, reactive oxygen species are produced in response to proteasomal inhibition (Ling et al., 2003
), and aggregation-prone mutant proteins with expanded polyglutamine stretches inhibit proteasomal activity (Bence et al., 2001
). Thus, the induction of p62 in aggregation diseases might also be caused by reactive oxygen species. Prostaglandin J2 is known to induce oxidative stress by causing decreases in glutathione, glutathione peroxidase, and mitochondrial membrane potential as well as increases in the production of protein-bound lipid peroxidation products (Kondo et al., 2001
). In human neuroblastoma cells, p62 is needed for the sequestration of ubiquitinated proteins into bodies in response to treatment with the inflammatory agent prostaglandin J2 (Wang and Figueiredo-Pereira, 2005
). Our present results provide a molecular mechanism of how p62 might recognize ubiquitinated protein bodies and present these to the autophagic machinery.
Mutant huntingtin is found to be both diffuse in some cells and aggregated in others. It seems clear that aggregation is a mechanism for cell survival (Arrasate et al., 2004
). In line with this, Steffan et al. (2004)
found that ubiquitination of mutant huntingtin is an important way to detoxify the protein, whereas sumoylation of the same residues prevents aggregation and leads to cell death. Autophagy is important for the clearance of huntingtin aggregates, and induced autophagy leads to increased cell survival of cells expressing mutant huntingtin (Ravikumar et al., 2002
). Similar to Nagaoka et al. (2004)
, we found that mutant huntingtin could also form aggregates in the absence of p62. Thus, we believe that the protective role of p62 may be to recruit autophagosomal components to the polyubiquitinylated protein aggregates rather than to help or facilitate the formation of these aggregates. Given the essential role of autophagy in preventing protein aggregate–induced neurodegeneration, p62 and proteins with related functions could be attractive targets for the development of neuroprotective drugs.