Identifying the key elements involved in cytosolic protein quality control

Efficient cellular function depends on the proteome’s ability to balance protein folding, refolding and degradation. Misfolded proteins, arising from genetic mutations, stress, age and metabolic shifts, constantly challenge eukaryotic cells. To address this, cells have inherent quality control systems to maintain the protein homeostasis balance. Key proteins in the quality control network operate in a compartmentalized manner, with them having specific roles in different cellular compartments. While the mechanism of quality control in the endoplasmic reticulum is widely understood, cytosolic quality control mechanisms remain less clear. To identify the key cytosolic quality control proteins, we used a cytosolic mutant substrate – GNMT H177N as a model substrate for this study. This substrate has a missense mutation that compromises the GNMT enzyme’s stability, impacting its function in glycine metabolism, resulting in a genetic condition marked by elevated blood methionine levels. When expressed in cells, GNMT H177N is less stable and is likely degraded as compared to its wildtype counterpart. Inhibiting the 26S proteasome and E1-activating enzyme led to an accumulation of GNMT H177N, highlighting the ubiquitin-proteosome system’s (UPS) role in substrate turnover. Moreover, our findings indicate HSP70’s role in this turnover, while HSP90 seems to prevent degradation, likely through refolding of the misfolded substrate. We first explored the function of a cytosolic E3 ligase – HERC1 and conclude that it does not play a pivotal role in this turnover, suggesting another E3 ligase might be involved. Therefore, we used proximity-based labelling screens with BioID and TurboID to identify potential elements of the protein homeostasis network that may regulate the turnover of the model substrate, as compared to the wildtype protein. Our screens identified a diverse, interconnected network of stress-related protein quality control proteins, including proteins related to the UPS and chaperone proteins, that often act at the forefront of defense against a misfolded protein. We shortlisted several proteins from the BioID screen for follow up experiments. Notably, knockdown of DNAJA1 and DNAJA2 led to a significant increase in the levels of GNMT H177N. While these HSP70 co-chaperones impact cytosolic mutant protein turnover, identifying the specific E3(s) involved requires further investigation.