Inactive proteasomes routed to autophagic turnover are confined within the soluble fraction of the cell
2Microbiology and Molecular Genetics, Hebrew University, Israel
3Microbiology and Immunology, National University of Singapore (NUS), Singapore
Ubiquitin-mediated proteasomal degradation of many proteins plays a key role in the protein quality control (PQC) machinery. However, the proteasome itself can become dysfunctional as a result of transcriptional and translational failures, genomic mutations, or diverse stress conditions. We previously demonstrated that among the multilayered PQC mechanisms available to yeast, proteasome-mediated degradation of its own impaired subunits is the favored pathway, and that upon proteasome inhibition, the small heat shock protein, Hsp42, mediates an alternative pathway, characterized by accumulation of these subunits in the insoluble protein deposit (IPOD), a cytoprotective cytoplasmic aggregates, that reside in the juxtavacuolar. Interestingly, it was later shown that this step also mediates the ubiquitination of inactive proteasomes, which is then targeted to autophagy (proteophagy).
In this study we examined the solubility state proteasomes subjected to autophagy as a result of their inactivation, or under nitrogen starvation. The results clearly show that in both cases only soluble proteasomes could serve as a substrate to autophagy, and that the deposition in the IPOD serves as an alternative pathway, when the system becomes overloaded. Altogether, our study shows that the substrates for proteophagy are dysfunctional proteasomes in their near-native soluble state, and not as previously believed, those sequestered at the IPOD, and that the insoluble fraction that accumulates in the IPOD may represent an alternative and secondary pathway for inactive proteasomes that escaped proteophagy when the system became saturated.