Substrate degradation by the Tul1 ubiquitin ligase complex is directed by ubiquitin chains.
We performed deep mutational scanning of Saccharomyces cerevisiae Tul1 (transmembrane ubiquitin ligase 1). We focused on Golgi-localized Tul1 function, which targets proteins for degradation by either the cytosolic proteasome (surveyed by monitoring degradation of Orm2*-RFP) or the vacuole (surveyed by monitoring degradation of GFP-Tlg1*). Yeast cells expressing both functional reporters also expressed one of eight Tul1 variant libraries, which sequentially spanned the protein and were created by tiling-primer PCR, and were sorted using fluorescence activated cell sorting (FACS) based on their ability to degrade (expected functional, 'P3'), or not degrade (expected nonfunctional, 'P2') both types of substrates. Yeast cells expressing Library 8 were also sorted into an off-axis population expected to degrade only vacuolar substrate and not proteasomal substrate ('P1').