UBE2O is a quality control factor for orphans of multiprotein complexes

Abstract

Many nascent proteins are assembled into multiprotein complexes of defined stoichiometry. Imbalances in the synthesis of individual subunits result in orphans. How orphans are selectively eliminated to maintain protein homeostasis is poorly understood. Here, we found that the conserved ubiquitin-conjugating enzyme UBE2O directly recognized juxtaposed basic and hydrophobic patches on unassembled proteins to mediate ubiquitination without a separate ubiquitin ligase. In reticulocytes, where UBE2O is highly up-regulated, unassembled α-globin molecules that failed to assemble with β-globin were selectively ubiquitinated by UBE2O. In nonreticulocytes, ribosomal proteins that did not engage nuclear import factors were targets for UBE2O. Thus, UBE2O is a self-contained quality control factor that comprises substrate recognition and ubiquitin transfer activities within a single protein to efficiently target orphans of multiprotein complexes for degradation.

Fig. 1. UBE2O associates with an aberrant nascent protein in the cytosol.

(A) Experimental strategy to identify quality control factors by cross-correlation of native size, physical interactors, and ubiquitination activity. (B) ³⁵S-labeled Sec61β(3R) was translated reticulocyte lysate (RRL) for 15 min, which is just long enough to synthesize the proteins but before appreciable downstream ubiquitination. The reaction was separated by native size on a sucrose gradient, and each fraction analysed for ubiquitination competence (middle panel) or physical interactions (bottom panel). Red asterisks indicate fractions with peak ubiquitination activity, and red arrowheads indicate crosslinked proteins with a peak in these same fractions. (C) Sec61β (WT) or mutants lacking the transmembrane domain (ΔTM) or containing 3R were translated in RRL, immunoprecipitated under native conditions, and analysed by immunoblotting for either BAG6 or UBE2O. BAG6 interacts with the intact TM domain, while UBE2O preferentially interacts with the 3R-disrupted TM domain.

Fig. 2. Reconstitution of UBE2O-mediated client ubiquitination with purified factors.

(A) Experimental strategy to test UBE2O interaction and ubiquitination in a defined system. (B) ³⁵S-labeled 3R in complex with Calmodulin (CaM) was treated with either Ca²⁺ or EGTA in the presence or absence of UBE2O and subjected to the amine-reactive crosslinker DSS. Positions of the starting 3R substrate and its crosslinks to CaM (xCaM) and UBE2O (xUBE2O) are indicated. (C) ³⁵S-labeled 3R in complex with CaM was mixed with E1, ATP, ubiquitin, and the indicated E2 (UBCH5 or UBE2O), then treated with EGTA to induce substrate release from CaM. One sample was incubated with Ca²⁺ instead of EGTA. (D) Ubiquitination reactions as in panel c with the indicated UBE2O variants. The purified UBE2O proteins before and after the ubiquitination reaction are also shown to indicate auto-ubiquitination of UBE2O in all reactions except with the C1040S mutant.

Fig. 3. Unassembled α-globin is a target for UBE2O ubiquitination.

(A) Wild type α-globin (HBA1) or two assembly mutants (H104R and F118S) were translated in RRL supplemented with His-tagged Ubiquitin and either analysed directly (bottom panel; total) or after ubiquitin-pulldown (Ub-PD) via the His tag (top panel). (B) HA-tagged HBA1 or the H104R mutant were translated in RRL, subjected to sulhydryl-reactive crosslinking with BMH, and affinity purified via the HA tag. The purified products were denatured and analysed directly (input) or further immunoprecipitated with antibodies to either UBE2O or GFP. The positions of ubiquitinated H104R and its crosslink to UBE2O are indicated. (C) ³⁵S-labeled HBA1 synthesized in the PURE translation system was incubated with E1, ATP, Ubiquitin, and either UBCH5 or UBE2O, and analysed for ubiquitination. (D) ³⁵S-labeled wild type (WT) or H104R mutant HBA1 synthesized in the PURE system was incubated with or without UBE2O and AHSP as indicated. The positions of unmodified and ubiquitinated HBA1 are indicated.

Fig. 4. UBE2O facilitates degradation of cytosolic orphan ribosomal proteins.

(A) Wild type KRAS or RPL8 was translated in RRL supplemented with His-tagged Ubiquitin and either analysed directly (total) or after ubiquitin-pulldown (Ub-PD) via the His tag. (B) The indicated FLAG-tagged proteins were translated in RRL, affinity purified under native conditions, and analysed for co-purifying proteins by SYPRO Ruby stain. The asterisks indicate the primary translation product, and the arrowhead indicates UBE2O (verified by immunoblotting and mass spectrometry). (C) ³⁵S-labeled RPL8 was synthesized in the PURE system and incubated with recombinant E1, ATP, ubiquitin, and UBE2O. Aliquots of the reaction after different times of incubation at 37°C are shown in the left panel. In the right panel, RPL8 was incubated for 60 min at 32°C with E1, ATP, ubiquitin, and the indicated UBE2O variant. (D) HEK293 cells stably expressing GFP-RPL24 were treated with three different siRNAs against UBE2O and analysed by flow cytometry. The relative GFP-RPL24 level, normalized to an internal expression control (see fig. S10D), is plotted as a histogram. The grey histogram is from cells treated with a control siRNA. (E) GFP-RPL24 cells were transiently transfected with plasmids encoding UBE2O, a catalytically inactive mutant (C1040S), or BFP (a negative control) and analysed for GFP-RPL24 levels by flow cytometry. (F) GFP-RPL24 cells were treated with the indicated siRNAs and analysed for GFP-RPL24 levels by flow cytometry.