We wish to better understand the mechanisms of ubiquitin chain buildup on misfolded substrates at the endoplasmic reticulum, which is a major protein quality control hub in the cell. In yeast, two ubiquitylation complexes are present at the endoplasmic reticulum membrane and they are named by the E3 ligases involved, HRD1 and doa10. Each of the complexes recognize a distinct set of substrates, with minor overlaps. The common E2 enzyme, Ubc7, operates with both E3 complexes Doa10 and Hrd1, while Ubc6 operates only with the Doa10 E3 ligase. How these enzymes co-operate during ubiquitin conjuration is yet unknown but our current studies shade light on the subject.
We found that the activity of Ubc7, the major conjugating enzyme at the endoplasmic reticulum, requires covalent and non-covalent interactions with ubiquitin. Besides the covalent thioester bond between ubiquitin and Ubc7, interactions between ubiquitin and the α2 helix of Ubc7 are also critical for the enzyme activity (Cohen et al., 2015). Importantly, amino acids that stabilize non-covalent interactions between Ubc7 and ubiquitin are only essential for the degradation of substrates of Doa10 but not of Hrd1. According to the emerging model, non-covalent interactions between helix α2 of Ubc7 and Ub are essential for efficient ubiquitin transfer during the buildup of poly-ubiquitin chains. The differential activation of Ubc7 by its cognate E3 ligases represents a previously unidentified mechanism for regulating protein ubiquitylation.
Another important mechanistic question is why two E2 enzymes are required for ubiquitin conjugation by Doa10? In collaboration with the Thomas Sommer laboratory from the Max Delbruck Center for Molecular Medicine, Berlin, we address the mechanistic basis for this unique requirement for two E2 Ub conjugating enzymes. we find that poly ubiquitylation in the doa10 pathway is accomplished by two sequential steps:
- Initially, Ubc6 attaches a single Ub at multiple sites on the protein substrate but cannot synthesize poly-Ub chains (priming).
- Ubc7 then forms a poly-Ub chain by successive formation of lysine 48-linked Ub-Ub bonds, thereby generating the signal for degradation by the proteasome (Elongation).
Besides lysine (K) residues on the substrate, our results implies that Ubc6 can also attach ubiquitin on serine (S) and probably threonine (T) residues via an ester bond. The ability of Ubc6 to prime polyubiquitylation on various amino acid residues may thus serve to increase the target range of quality control substrates available for Doa10 and consequently the degradation capacity of the quality control system.