UBE2O remodels the proteome during terminal erythroid differentiation

Abstract

During terminal differentiation, the global protein complement is remodeled, as epitomized by erythrocytes, whose cytosol is ~98% globin. The erythroid proteome undergoes a rapid transition at the reticulocyte stage; however, the mechanisms driving programmed elimination of preexisting cytosolic proteins are unclear. We found that a mutation in the murine Ube2o gene, which encodes a ubiquitin-conjugating enzyme induced during erythropoiesis, results in anemia. Proteomic analysis suggested that UBE2O is a broad-spectrum ubiquitinating enzyme that remodels the erythroid proteome. In particular, ribosome elimination, a hallmark of reticulocyte differentiation, was defective in Ube2o^-/- mutants. UBE2O recognized ribosomal proteins and other substrates directly, targeting them to proteasomes for degradation. Thus, in reticulocytes, the induction of ubiquitinating factors may drive the transition from a complex to a simple proteome.

Fig. 1. Ube2o^−/− reticulocytes are deficient in eliminating ribosomal proteins

(A) Proteins from reticulocyte lysates were resolved by SDS-PAGE and immunoblotted with an anti-ubiquitin antibody. Each sample is from a different mouse whose reticulocytes were induced by serial bleeding. (B) Ubiquitination was reconstituted in Ube2o^−/− reticulocyte lysates using recombinant UBE2O. Reactions were supplemented with biotin-tagged ubiquitin and ubiquitin activating enzyme (UBE1), and incubated for 45 min at 37°C. Samples were resolved by SDS-PAGE. Proteins were electroblotted and visualized using streptavidin-HRP. The catalytically inactive C1037A mutant (UBE2O-CA) was used as a control. UBE2O-CA did not have conjugating activity in lysates, as the only bands evident in this case were biotin-ubiquitin and autoubiquitinated E1. In the absence of lysate, UBE2O was autoubiquitinated, as previously reported (27). W, UBE2O-WT; C, UBE2O-CA. (C) UBE2O ubiquitinates ribosomal proteins in reconstituted Ube2o^−/− lysates. Ubiquitin conjugates (see Fig. 2A) purified by NeutrAvidin-biotin pulldown were digested on beads with trypsin. Peptides containing di-glycine modified lysines (ubiquitination sites) were identified and mapped by LC-MS/MS. All ribosomal protein ubiquitination events were unique to the wild-type UBE2O sample. Δ spectral counts denotes subtraction of all spectral counts collected in the UBE2O-CA sample from those of the UBE2O-WT reaction for a given di-glycine peptide over six replicate experiments. Ub sites, unique ubiquitination sites identified. (D), Levels of ribosomal proteins from Ube2o^−/− and wild-type reticulocytes assessed by immunoblotting. GAPDH and β-spectrin are loading controls. 100 μg of protein was loaded per lane. (E) Volcano plot of quantitative proteomics analysis, representing the relation of the log₁₀ of the p-value adjusted using Benjamini-Hochberg correction [-log (adj. p-value)] and the log₂ of the fold change [log2 (Ube2o^−/−/Ube2o^+/+)]. Proteins significantly (adj. p-value < 0.05) upregulated or downregulated at least 25% in Ube2o^−/− samples were displayed in red and blue respectively. Highly significant enrichment of ribosomes (adj. p-value=3.23 x 10⁻⁶) was found by KEGG pathway enrichment analysis of all proteins upregulated significantly and by more than 25% in Ube2o^−/− reticulocytes. Brackets, number of proteins per group.

Fig. 2. Ube2o^−/− reticulocytes have elevated levels of ribosomes

(A) Polysome profiles were generated by fractionating Ube2o^−/− and WT reticulocyte lysates on 20–50% sucrose gradients. The X-axis represents position in the gradient, the Y-axis OD260. Left, freshly-isolated Ube2o^−/− reticulocytes showed an 80S monosome peak that is elevated in comparison to WT. Right, as above but after 31 hours of ex vivo differentiation. (B) Flow cytometry analysis after ex vivo differentiation. WT CD71+ reticulocytes showed progressive loss of both MitoTracker Deep Red and thiazole orange staining. Ube2o^−/− CD71+ reticulocytes retained thiazole orange staining after 72 hours of ex vivo differentiation (upper panels), with mitochondrial elimination unaffected (lower panels).

Fig. 3. UBE2O is sufficient to drive the elimination of ribosomes in HEK293 cells

(A) UBE2O induction by doxycycline treatment in Flp-In T-REx 293 cells was assessed by immunoblotting, using antibodies to UBE2O. Full length human UBE2O-WT and UBE2O-CA genes were genomically integrated in an untagged form, and induced with doxycycline for 24 hr. 20 μg of cell lysate was loaded per lane. GAPDH is a loading control. (B, C) Mass spectrometry (TMT) quantification of 7,808 proteins after UBE2O (WT or CA) induction for 0, 12, 24, 48 and 72 hours. All 685 proteins that were downregulated more than 50% after 72h of UBE2O-WT induction, in comparison to UBE2O-CA, are shown in (B). Coloring reflects Pearson correlation (r) to the median pattern from high (red) to low (black). The bar graph represents results from KEGG pathway enrichment analysis, which indicated highly significant enrichment (adj. p-value=2.77 x 10⁻⁶) of ribosomes, with 18 proteins downregulated (shown in C). (D) Isoelectric point (pI) comparison of all quantified proteins (red), and all downregulated proteins (blue), based on data from panel B. pI values were obtained from the proteome pI database (46). (E) After induction of UBE2O (WT or CA) for the indicated times (48 and 72 hours), ribosomal protein levels were analyzed by immunoblotting. GAPDH is a loading control. 20 μg of cell lysate was loaded per lane, and all samples were from the same experiment. (F) Sucrose gradient analysis of cells overexpressing UBE2O-WT showed a reduced 80S monosome peak after 72 hours of doxycycline treatment.

Fig. 4. UBE2O recognizes substrates directly

(A) Immunoblotting of Ube2o^−/− and WT reticulocyte lysates showed an elevation of putative non-ribosomal UBE2O substrates. GAPDH and β-spectrin are loading controls. 100 μg of protein was loaded per lane. (B) Schematic representation of domains within UBE2O. The T1 construct lacks conserved region 1 (CR1), whereas T2 lacks both CR1 and CR2. In the CA mutant, the active-site Cys1037 is substituted with Ala. Domain assignment is described in the Methods. (C) In vitro ubiquitination assays were performed in a purified system with UBE1, HA-ubiquitin, UBE2O (WT or CA, W and C respectively) and the indicated candidate substrates for 4 hours at 37°C. Histone H2B is a model substrate of UBE2O (27). N-terminal truncations (T1 and T2) abrogate the ubiquitination of ribosomal proteins and of H2B, but do not prevent modification of AHSP. Samples were analyzed by immunoblotting with either anti-HA or anti-AHSP antibodies, after SDS-PAGE. The left and right panels represent different exposures from the same gel. (D) Left, UBE2O can directly recognize and ubiquitinate putative non-ribosomal substrates DDX56, NOL12, NOP16, and PTRF, which are elevated in either Ube2o^−/− reticulocytes or decreased in 293-E2O cells. Right, purified UBE2O can ubiquitinate individual ribosomal proteins outside of the 80S complex. Samples were analyzed by immunoblotting with anti-HA antibodies, after gradient SDS-PAGE. W, UBE2O-WT; C, UBE2O-CAThe apparent molecular masses of substrates (in kDa) were determined by SDS-PAGE followed by Coomassie staining (data not shown): calmodulin, 17; DDX56, 64; NOL12, 27; NOP16, 25; PTRF, 55–65; RPL35, 17; RPL36a, 16; RPL37, 15. (E) CR1 and CR2 provide substrate-recognition with distinct specificities. The CR1 and CR2 fragments were expressed in E. coli in a biotin-tagged form, and loaded onto streptavidin resin, using amounts sufficient to saturate the resin’s binding capacity. Purified UBE2O substrates were mixed with resin at 20-fold excess in the presence of carrier. The resin was washed in 50 mM NaCl, 20 mM Tris-HCl (pH 7.4). Bound material was eluted and 25% of the eluate was resolved by SDS-PAGE followed by immunoblotting. The input represents 2.5% of the total sample; consequently CR1 and CR2 are not visualized in the input lanes, though present. Note, rows 6–8 are from the same experiment.

Fig. 5. The ubiquitin-proteasome system in late erythroid differentiation

(A) WT and Ube2o^−/− reticulocytes were differentiated ex vivo at 37°C for 48 hours with proteasome inhibitors (50nM epoxomicin and 50nM PS-341) or DMSO vehicle control, then analyzed by FACS. (B) Reconstitution of ribosomal protein degradation in Ube2o^−/− reticulocyte lysates. Ubiquitin levels were supplemented to prevent depletion of free ubiquitin upon proteasome inhibition. Proteasomes were inhibited by adding PS-341 (50 nM) and epoxomicin (50 nM) together. GAPDH is a loading control. 100 μg of protein was loaded per lane. (C) WT reticulocytes were treated with proteasome inhibitors (50 nM epoxomicin and 50 nM PS-341) and differentiated ex vivo for 48 hours. Quantitative metabolomic profiling of treated and untreated WT reticulocytes showed a depletion of multiple free amino acids due to proteasome inhibition, with Lys and Arg most strongly affected. Significance was calculated by two-sample t-test. *p<0.05, **p<0.01, ***p<0.001. (D) An ensemble of ubiquitin ligases and ubiquitin conjugating enzymes is induced in late erythroid differentiation. Stages are arranged in a temporal progression. Stage 1 includes proerythroblasts and early basophilic erythroblasts; stage 2, early and late basophilic erythroblasts; stage 3, polychromatophilic and orthochromatophilic erythroblasts; and stage 4, late orthochromatophilic erythroblasts and reticulocytes. The induction of globin mRNA is shown for comparison. Raw RNA-seq data are taken from (6).