A mutational atlas for Parkin

More than half of disease-causing missense variants are thought to lead to protein degradation, but the molecular mechanism of how these variants are recognized by the cell remains enigmatic. To approach this issue we have applied deep mutational scanning experiments to test the degradation of thousands of missense protein variants in large multiplexed experiments in cultured human cells. As a model protein we selected the ubiquitin-protein ligase Parkin, where known missense variants result in an autosomal recessive early onset Parkinsonism. The resulting mutational map comprises 9219 out of the 9300 (>99%) possible single-amino-acid substitution and nonsense Parkin variants. With a few notable exceptions, the majority of the destabilizing mutations are located within the structured domains of the protein, while the flexible linker regions are more tolerant to mutations. The cellular abundance data correlate with Parkin structural stability, evolutionary conservation, and separates known disease-linked variants from benign variants. Systematic mapping of degradation signals (degrons) shows that inherent primary degrons in Parkin largely overlap with regions that are highly sensitive to mutations. We identify a degron region proximal to the ACT element, which is enhanced by substitutions to hydrophobic residues. The vast majority of unstable Parkin variants are degraded through the ubiquitin-proteasome system and are stabilized at lowered temperatures. In conclusion, in addition to providing a diagnostic tool for rare genetic disorders, deep mutational scanning technologies have the potential to reveal both protein specific and general information on the specificity of the protein quality control network and the ubiquitin-proteasome system. Overall design: To investigate cellular abundance levels of parkin single amino acid variants, a stie saturation library was GFP labeled and FACS sorted in 3 replicates and 4 biological replicates