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Abstract

Genetic mutations can cause improper protein folding, leading to a loss of function or unwanted interference with cellular processes. To address these issues, cells have developed a sophisticated protein quality control (QC) network. Protein QC operates in different cellular compartments, and the handling of cytosolic misfolded proteins remains underexplored. We screened the human ORFeome collection to identify several cytosolic proteins with disease-associated missense mutations that are unstable in cells. A fluorescence-based reporter assay showed that their degradation depends on the ubiquitin–proteasome system (UPS). BioID proximity-labelling revealed that the co-chaperones DNAJA1 and DNAJA2 interact with one of the assessed mutants, but not the corresponding wild-type protein. DNAJA2 knockout increased turnover of the mutant indicating that, while dispensable for wild-type stability, it becomes essential for the misfolded client. Assessment of a broader panel of cytosolic misfolded proteins shows that the co-chaperone DNAJA2 is required to “balance” some of the additional unstable mutants and the corresponding wild-types. This suggests that DNAJA2 may have specific cellular clients, expanding our understanding of its function in maintaining protein stability. We next asked whether E3 ligases previously implicated in cytosolic QC contribute to the turnover of cytosolic misfolded proteins. Knockouts of these candidate E3s failed to stabilize the assessed substrates, indicating that either multiple ligases act redundantly or that recognition may involve a broader network of UPS factors. To more precisely probe recognition elements and potential E3 interaction sites, we generated peptide-tiles spanning the cytosolic enzyme glycine N-methyltransferase (GNMT). Several GNMT peptides conferred instability when expressed as reporters, mapping degron activity to discrete sequence elements. Scanning mutagenesis of these regions revealed motifs resembling those recognized by known QC factors, including BAG6, which is required for efficient degradation of the C-terminal GNMT degron. TurboID with strong degrons identified additional UPS and autophagy proteins potentially involved in protein QC. This degron-centric approach highlights how short linear motifs exposed upon misfolding can recruit UPS components. Together, these findings demonstrate that co-chaperones such as DNAJA2 can selectively stabilize mutant proteins. Moreover, degron mapping provides a framework to uncover sequence features underlying recognition and proteasomal degradation of misfolded cytosolic clients.