Quality control-associated proteolysis (QCAP) is a fundamental mechanism that maintains cellular homeostasis by eliminating improperly folded proteins. In QCAP, the exposure of normally hidden cis-acting protein sequences, termed degrons, triggers misfolded protein ubiquitination, resulting in their elimination by the proteasome. To identify the landscape of QCAP degrons and learn about their unique features we have developed an unbiased screening method in yeast, termed yGPS-P, which facilitates the determination of thousands of proteome-derived sequences that enhance proteolysis. Here we describe the fundamental features of the yGPS-P method and provide a detailed protocol for its use as a tool for degron search. This includes the cloning of a synthetic peptidome library in a fluorescence-based reporter system, and data acquisition, which entails the combination of Fluorescence-Activated Cell Sorting (FACS) and Next-Generation Sequencing (NGS). We also provide guidelines for data extraction and analysis and for the application of a machine-learning algorithm that established the evolutionarily conserved amino acid preferences and secondary structure propensities of QCAP degrons.

• PMID: 37371568
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Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, Non-P.H.S.

Authors

Mashahreh B, Armony S, Ravid T

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