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

Abstract

Summary 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.

The human GID complex engages two independent modules for substrate recruitment

“…In addition to Gid4, yeast cells express two alternative substrate receptors, Gid10 and Gid11, which all interact with the GID E3 ligase complex through Gid5/ARMC8 (Melnykov et al , 2019; Kong et al , 2021). Gid4 and Gid10 bind substrates containing nonproline degron motif (Dong et al , 2020), and further systematic screening identified many candidates that do not fulfill the Pro/N‐end degron criteria (Kong et al , 2021). Similarly, human GID4 may also recognize substrates such as ZMYND19 that lack Pro/N‐end degron motifs.…”

“…For example, Cullin‐RING ligases (CRL) engage one out of a large family of substrate receptors, and their assembly is regulated by substrate availability and the exchange factor CAND1 (Pierce et al , 2013). In addition to Gid4, yeast cells express two alternative substrate receptors, Gid10 and Gid11, which all interact with the GID E3 ligase complex through Gid5/ARMC8 (Melnykov et al , 2019; Kong et al , 2021). Gid4 and Gid10 bind substrates containing nonproline degron motif (Dong et al , 2020), and further systematic screening identified many candidates that do not fulfill the Pro/N‐end degron criteria (Kong et al , 2021).…”

“…Gid4 is partially redundant with Gid10, which is upregulated by heat and osmotic stress conditions (Melnykov et al , 2019; Qiao et al , 2019). Moreover, Gid11/Ylr149c was recently identified as a GID substrate receptor recognizing proteins with N‐terminal threonine residues (Kong et al , 2021), thus expanding the specificity of the GID complex. Interestingly, these substrate receptors are recruited to the GID complex by binding to Gid5, which, in turn, interacts with the catalytic core composed of Gid8 and the RING domain‐containing subunits Gid2 and Gid9.…”

Variation in Ubiquitin System Genes Creates Substrate-Specific Effects on Proteasomal Protein Degradation

“…TFTs are fusions of a rapidly maturing green fluorescent protein (GFP) and a slower maturing red fluorescent protein (RFP) 58, 59 . The TFT’s ouput, expressed as the -log 2 RFP / GFP ratio, is directly proportional to its degradation rate and, when fused to N-degrons, measures UPS N-end rule activity 39, 55, 58 . Because the TFT is expressed as a single protein construct, the output of the TFT is also independent of its expression level 39, 55, 58, 60 , enabling its use in genetically diverse populations.…”

“…Most traits are genetically complex, shaped by many loci of small effect and few loci of large effect throughout the genome 36, 37 , suggesting that large-effect variants in UPS genes may represent only one extreme of a continuum of genetic effects on UPS activity. Moreover, variation in UPS activity can differentially affect the degradation of distinct UPS sub-strates 38, 39 . Whether genetic effects on UPS activity affect the turnover of distinct proteins consistently or in a substrate-specific manner remains a fundamentally open question.…”