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

Abstract

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.

Keywords: GID ubiquitin ligase; N-degron pathways; fluorescent timers; protein quality control; proteostasis; selective protein degradation; ubiquitin-proteasome system.

Graphical abstract

Figure 1

Turnover of the yeast proteome (A) Cartoon of the mCherry-sfGFP timer (top). Because of different maturation kinetics of mCherry (slow, m_S) and sfGFP (fast, m_F), the mCherry/sfGFP ratio reports the stability of timer-tagged proteins in the steady state (bottom). (B) Median-centered distribution of mCherry/sfGFP ratios in the tFT library, representing protein stability in the yeast proteome. Shown are fluorescence measurements of colonies, median of 2 biological replicates, each with 4 technical replicates per protein. Dashed lines, quantiles used in downstream analyses (Figures S1M and S1N). (C) Relationship between sfGFP intensities (protein abundance) and mCherry/sfGFP ratios in the tFT library. Example protein complexes are highlighted. (D) Median mCherry/sfGFP ratios of proteins in the tFT library mapped to Gene Ontology (GO) terms. GO term 5975, carbohydrate metabolic process; 51603, proteolysis involved in cellular protein catabolic process; 55086, nucleobase-containing small molecule metabolic process. Similar GO terms are closer in semantic space (Supek et al., 2011). (E and F) Distributions of median absolute deviations (MADs) of sfGFP intensities (E) or mCherry/sfGFP ratios (F) for complexes in the tFT library. Random samples of the proteome, drawn in sets of N (where N is the number of complex subunits), are shown for comparison (n = 100 random draws). Dashed lines, medians of the distributions. ^∗∗∗p < 0.001 in a Wilcoxon rank-sum test. See also Figure S1 and Table S1.

Figure 2

Influence of UPS components on proteome abundance and stability (A) Cartoon of screens to profile the yeast UPS. Each strain in the tFT library (tFT query) was crossed with an array of mutants in UPS components (UPS array), followed by mCherry and sfGFP fluorescence measurements of colonies. (B) Summary of phenotypic outcomes (changes of protein abundance and stability; n.a., not affected) across all tested mutant-tFT pairs at 1% FDR (false discovery rate). The percentage of mutant-tFT pairs with each phenotype is indicated. (C) Number of mutants affecting protein stability or abundance for the 3,806 tested tFT queries. Only significant interactions (1% FDR, absolute stability or abundance score > 4) were considered (C–E). (D) Number of proteins affected in terms of stability or abundance in 132 mutants in the UPS array. Centerlines mark the medians, box limits indicate the 25^th and 75^th percentiles, and whiskers extend to minimum and maximum values. (E) Number of proteins destabilized or stabilized in UPS mutants grouped by function (Table S2). (F) Overlap between turnover interactions, grouped according to change in protein stability at 1% FDR and external datasets. ^∗p < 0.05 and ^∗∗p < 0.01 in a Fisher’s exact test. See also Figures S2–S4 and Tables S2 and S3.

Figure 3

Ubr1-dependent protein turnover (A) Scheme of the Arg/N-degron pathway, which targets proteins with the indicated N-terminal residues for degradation. Φ, large hydrophobic residues (W, L, F, Y, I). (B) Heatmap of protein stability changes in the absence of Ubr1 (screens in Figure 2). Changes in mCherry/sfGFP ratios are color coded from green (decrease) to magenta (increase). Only proteins stabilized in the ubr1Δ mutant (1% FDR, stability score >4) are shown; their behavior in E2 mutants is included for comparison. Proteins localized to mitochondria based on GFP tagging (Huh et al., 2003) or mapped to the GO term mitochondrion are marked. ^∗, Ubi4-tFT is not stabilized in the ubr1Δ mutant. Processing of the Ubi4-tFT fusion by DUBs releases free tFT with an N-terminal asparagine, which is the substrate of the Arg/N-degron pathway (B and C). (C) mCherry/sfGFP ratios of colonies expressing tFT fusions and lacking components of the Arg/N-degron pathway (mean ± SD, n = 4). Hereafter, red dashed lines mark mCherry/sfGFP ratios in the wild type (WT). ^∗∗, protein stability measurements in the rad6Δ mutant are confounded by its fitness defect; this effect is partially corrected for in the screen (B). (D) Immunoblots of strains expressing Mcr1-TAP (left) or Mcr1-3xHA (right). Samples were collected from log-phase cultures or after 48 h of growth in low-glucose medium (glucose starvation). (E) Quantification of Mcr1(32) and Mcr1(34) relative abundance by immunoblotting of strains expressing Mcr1-3xHA (mean ± SD, n = 2 biological replicates each with 3 technical replicates). ^∗p < 0.04 in a one-sided unpaired t test. See also Figure S4.

Figure 4

Correlations of proteome turnover profiles (A) Heatmap of correlations of proteome turnover profiles for all tested mutants in the UPS array (screens in Figure 2). For each pair of mutants, a shrunken correlation was calculated based on the set of proteins with altered stability (1% FDR, absolute stability score >4) in at least one of the mutants. Select clusters of correlating mutants are highlighted. (B) Overlap between shrunken correlations of proteome turnover profiles and external datasets. Correlations of proteome turnover profiles from (A) were grouped according to significance (1% FDR) and sign (pos, positive; ns, not significant; neg, negative). ^∗∗p < 0.01 in a Fisher’s exact test. (C) Magnitude of shrunken correlations of proteome turnover profiles grouped according to significance and sign as in (B) (left) and number of significant correlations between UPS mutants grouped by function (Table S2) (right). See also Figures S5 and S6 and Table S4.

Figure 5

Protein turnover by the GID complex (A) Heatmap of protein stability changes in the absence of GID components (screens in Figure 2). Only proteins stabilized in at least one of the mutants (gid2Δ, gid9Δ, and ubc8Δ; 1% FDR, stability score >4) are shown; their behavior in the ubp14Δ mutant is included for comparison. (B) Differences in mCherry/sfGFP ratios between gid2Δ and WT cells for tFT-tagged proteins from (A). Flow cytometry measurements of log-phase cultures and plate reader measurements of colonies (mean, n = 4). Proteins stabilized in the gid2Δ background in cultures are indicated. (C and D) mCherry/sfGFP ratios of colonies expressing tFT fusions and lacking GID components (mean ± SD, n = 4) and cartoon of the GID complex; SR, substrate receptor (right; adapted from Qiao et al., 2020). (E) Frequency of residues at the second position in potential GID substrates from (A) (GID hits). See also Figure S7 and Table S5.

Figure 6

Gid11-dependent turnover of proteins with an N-terminal threonine (A) Genome-wide screens for factors involved in turnover of Cpa1 and Phm8. Only gene deletions with positive Z scores are shown. (B) Immunoblot of strains expressing chromosomally tagged Gid11-HA (top) and quantification of relative Gid11-HA expression levels (bottom, mean ± SD, n ≥ 3). Samples were collected from log-phase cultures in synthetic complete medium with glucose as a carbon source (SC glucose) from colonies or after a 3-h shift from SC glucose into the indicated environment (red). (C) mCherry/sfGFP ratios of colonies expressing tFT fusions and lacking GID2 and/or GID11 (mean ± SD, n = 4). (D) Co-immunoprecipitation of overexpressed HA-Gid11 and chromosomally tagged Gid1-tFT. The relative amount of co-immunoprecipitated HA-Gid11, normalized to precipitated Gid1-tFT, was reduced to 0.43 ± 0.15 (mean ± SD, n = 3) in gid5Δ cells compared with the WT. (E) C termini of GID receptors from different organisms. Sc, S. cerevisiae; Hs, Homo sapiens; Mm, Mus musculus; Dr, Danio rerio; Ag, Anopheles gambiae; Sp, Schizosaccharomyces pombe. (F and G) mCherry/sfGFP ratios of colonies expressing tFT fusions (mean ± SD, n = 3 [F] or n = 4 [G]). Dashed lines mark mCherry/sfGFP ratios in gid11Δ or gid5Δ mutants complemented with WT GID11 or GID5, respectively. (H) mCherry/sfGFP ratios of colonies expressing tFT-tagged proteins, either WT or with the second residue X mutated to alanine (X2A) or glycine (X2G) (mean ± SD, n ≥ 3). (I) Model of Gid11 as a receptor for substrates with an N-terminal threonine, exposed after removal of the initiator methionine by MetAPs. Gid11-dependent protein turnover requires all core GID subunits but not Gid7. See also Figure S7 and Tables S6 and S7.