The p62/sequestosome 1 binds irreversibly to protein aggregates prior to autophagy clearance in ARPE‐19 cells

Abstract

AbstractPurpose The pathogenesis of AMD involves impaired protein degradation in RPE cells. The ubiquitin‐proteasome pathway and the lysosomal pathway including autophagy are the major proteolytic systems in eukaryotic cells. Recently, p62/sequestosome 1 (p62) has been shown to be a key player linking the proteasomal and lysosomal clearance systems. In this study, expression and trafficking of p62 was examined.Methods To study the effect of autophagy activator (AICAR) and inhibitor (bafilomycin) on p62 expression levels, the ARPE‐19 cells were treated with proteasome inhibitor (MG‐132, 5µM) with or without AICAR (2mM) or bafilomycin (50nM) for 24h hours. The protein levels of p62 were evaluated by western blotting. The localization and movement of p62 were analyzed by live confocal microscopy.Results MG‐132 increased the p62 protein levels, while AICAR robustly decreased the p62 levels. When autophagy was inhibited with bafilomycin the p62 was highly accumulated. We show that p62 binds irreversibly to protein aggregates that are finally degraded in autophagy.Conclusion The p62/sequestosome 1 function as a linker protein between proteasomes and autophagy and can be used as a autophagy flux marker. Autophagy is effective clearance machine that may be disturbed in aged RPE cells.