Abstract
Cellular protein quality control consists of multiple, networked systems that survey and maintain a healthy eukaryotic proteome. In Saccharomyces cerevisiae, the transmembrane ubiquitin ligase 1 (Tul1) complex is an integral membrane protein quality control system that functions within the Golgi-endosomal system. Golgi-localized Tul1 complexes target proteins for degradation by either the cytosolic proteasome or the vacuole. To understand how the complex directs substrates for degradation, we developed high-throughput functional assays for deep mutational scanning analysis of the Tul1 ubiquitin ligase. We identified mutations that disrupted Tul1 interactions with the complex or altered complex specificity by disrupting substrate polyubiquitination. This work demonstrates that Tul1 plays an important role in directing substrate degradation by influencing polyubiquitin chain length and provides tools for future study of the complex.