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Abstract

Many proteins have intrinsically disordered regions or are entirely disordered. Despite their lack of a stable three-dimensional structure, they are important regulators of a variety of cellular processes. The small intrinsically disordered protein Roq1 regulates SHRED, a protein quality control pathway in the budding yeast, Saccharomyces cerevisiae. Upon stress, Roq1 is cleaved, resulting in an exposed N-terminal arginine residue, namely R22. Via R22, Roq1 can bind to the E3 ubiquitin ligase Ubr1 as a pseudosubstrate, thereby outcompeting regular substrates. Ubr1 is the main N-recognin in yeast and recognizes proteins with destabilizing N-terminal residues through two distinct sites, the type-1 site for basic residues, and the type-2 site for hydrophobic residues. Binding of Roq1 shifts the substrate spectrum of Ubr1 from type-1 N- degron substrates to misfolded proteins, while promoting the turnover of type-2 substrates. However, the mechanism through which Roq1 reprograms Ubr1 is unclear. In this study, I built on a Roq1 mutagenesis screen that was performed prior to this study to identify SHRED- critical residues in Roq1. Analysis of the hits yielded fourteen SHRED-defective Roq1 point mutants. In addition, I identified a hydrophobic motif in the middle of the protein sequence (residues 55-58) that is necessary for Roq1 function. Specifically, these residues are required for both binding and regulating Ubr1 in vivo and in vitro. In vitro studies showed that the hydrophobic motif is important for promoting ubiquitination of misfolded substrates. In contrast, the hydrophobic motif is dispensable for the ubiquitination of regular N-degron substrates or substrates with internal degrons. Furthermore, this study demonstrates that Roq1 promotes ubiquitin chain initiation on misfolded substrates by Ubr1 dependent on the hydrophobic motif. Finally, I identified R22 and the hydrophobic motif connected by a short generic flexible linker as the minimally required building blocks of Roq1 to regulate Ubr1. Overall, this work provides mechanistic insights into how an IDP can control the substrate specificity of an E3 ligase.