FACS-based Degron Screen to Investigate How Protein Quality Control Systems Cooperate to Protect the Cell from Misfolded Proteins.

Protein Quality Control (PQC) systems protect the cell from abnormal protein accumulation. Each protein is thought to contain a cryptic quality control degradation signal (degron) that would be exposed upon structural perturbation. Consequently, the ubiquitin conjugation system recognizes the degron and eventually misfolded proteins are targeted for degradation by the proteasome. Despite their central role in proteostasis maintenance, PQC degrons remain largely elusive.

Our lab has previously identified PQC degrons within the entire yeast proteome. Results of this study are consistent with previous studies showing that hydrophobic regions are key PQC degron features. However, the principles of misfolded substrate selection by a specific E3 ligase were not revealed in these studies. Recently, a research, using FACS for analyzing the stability of the human peptidome, has revealed specific sequence requirement of C-terminal degron and identified their cognate E3 ubiquitin ligases. We have adopted this technique to the yeast research, in order to identify a large set of misfolded degrons and ultimately assign them to specific E3s. Our screen covers the majority of PQC E3s in distinct yeast compartments. Understanding the individual and overlapping functions of these E3 ligases provides insight into how ubiquitin ligases cooperate in the clearance of misfolded proteins.