Timer-based proteomic profiling of the ubiquitin-proteasome system reveals a substrate receptor of the GID ubiquitin ligase

Selective protein degradation by the ubiquitin-proteasome system (UPS) is involved in all cellular processes. However, the substrates and specificity of most UPS components are not well understood. Here we systematically characterized the UPS in Saccharomyces cerevisiae. Using fluorescent timers, we determined how loss of individual UPS components affects yeast proteome turnover, detecting phenotypes for 76% of E2, E3, and deubiquitinating enzymes. We exploit this dataset to gain insights into N-degron pathways, which target proteins carrying N-terminal degradation signals. We implicate Ubr1, an E3 of the Arg/N-degron pathway, in targeting mitochondrial proteins processed by the mitochondrial inner membrane protease. Moreover, we identify Ylr149c/Gid11 as a substrate receptor of the glucose-induced degradation-deficient (GID) complex, an E3 of the Pro/N-degron pathway. Our results suggest that Gid11 recognizes proteins with N-terminal threonines, expanding the specificity of the GID complex. This resource of potential substrates and relationships between UPS components enables exploring functions of selective protein degradation. [Display omitted] •Global analysis of proteome turnover in budding yeast•Proteome turnover profiles for most components of the ubiquitin-proteasome system•Substrates and functions for Ubr1 and GID, two E3s of the N-degron pathways•Identification of a GID receptor for substrates with a threonine N terminus The ubiquitin-proteasome system (UPS) is key for selective protein degradation in eukaryotes. To understand its specificity, Kong et al. systematically analyze the effect of UPS mutants on yeast proteome turnover and explore the resulting resource of potential substrates and relationships between UPS components to gain insights into N-degron pathways.